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    28 November 2023, Volume 27 Issue 33 Previous Issue    Next Issue
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    Epidermal neural crest stem cells of hair follicles regulate the local expression of inflammatory factors after facial nerve injury
    Tang Li, Pan Yao, Zhu Guochen
    2023, 27 (33):  5249-5255.  doi: 10.12307/2023.750
    Abstract ( 252 )   PDF (3978KB) ( 59 )   Save
    BACKGROUND: Previous studies had shown that hair follicle epidermal neural crest stem cells have the great potential in facilitating peripheral nerve rehabilitation, but the role in regulating inflammation after facial nerve injury had rarely been reported.  
    OBJECTIVE: To investigate whether epidermal neural crest stem cells can modify the expression of pro-inflammatory factor tumor necrosis factor α and anti-inflammatory factor interleukin-4 during facial nerve regeneration to promote the functional and morphological recovery of the facial nerve.
    METHODS: Epidermal neural crest stem cells were extracted from one 4-day-old SD rat, cultured and identified. Fifty-four adult SD rats were used to construct the bridge model of facial nerve trunk defect with the autologous venous catheter. Models were randomly and equally divided into the normal saline group, Dulbecco's modified eagle medium group and epidermal neural crest stem cell group. Tumor necrosis factor α and interleukin-4 expression levels were detected at postoperative 4 to 14 days using western blotting, immunohistochemistry, and immunofluorescence. The facial nerve function of rats was scored 12 weeks after the operation and the morphology of the facial nerve was observed by hematoxylin-eosin staining.  
    RESULTS AND CONCLUSION: (1) Double positive expression of Nestin and p75NTR could be observed in epidermal neural crest stem cells and the cell purity was over 90%. (2) Compared with the other two groups, tumor necrosis factor α expression in the epidermal neural crest stem cell group turned weaker 7 days after surgery (P < 0.05), and interleukin-4 expression in the epidermal neural crest stem cell group was enhanced on postoperative 3, 7 and 14 days, respectively (P < 0.05). (3) The recovery score of facial nerve function in the epidermal neural crest stem cell group was significantly better than that in the other two groups (P < 0.05). (4) There were abundant nutrient vessels and regenerative axons in the graft segment. Compared with the other two groups, the arrangement of nerve fibers was more orderly and the wrapped myelin sheath was thicker in the graft and distal segment in the epidermal neural crest stem cell group. (5) The data inferred that epidermal neural crest stem cells regulate the local expression of tumor necrosis factor α and interleukin-4 at the early stage of facial nerve injury, inhibit the local inflammatory reaction, and improve facial nerve function and morphology of the regenerated nerve.
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    Aerobic exercise intervenes with beta-amyloid 1-42 induced changes in hippocampal synaptic structure and proteins in a rat model of Alzheimer’s disease
    Du Jia, Fu Yan, Fan Jia, Zhou Miaorong, Zhang Yeting
    2023, 27 (33):  5256-5262.  doi: 10.12307/2023.497
    Abstract ( 272 )   PDF (3926KB) ( 75 )   Save
    BACKGROUND: Beta-amyloid (Aβ) accumulation and deposition in the hippocampus can affect synaptic morphology and function, resulting in impaired synaptic plasticity, which is considered to be the root cause of learning and memory deficits of Alzheimer’s disease. It is not yet clear whether aerobic exercise can alleviate Aβ-induced synaptic damage and improve the learning and memory abilities of patients with Alzheimer’s disease. 
    OBJECTIVE: To observe the effect of aerobic exercise on the hippocampal synaptic structure and synapse marker proteins, synaptophysin and postsynaptic dense 95, in a rat model of Alzheimer’s disease induced by Aβ1-42, so as to investigate the mechanism by which aerobic exercise influences learning and memory ability of patients with Alzheimer’s disease. 
    METHODS: Eighty healthy Sprague-Dawley rats were randomly divided into four groups (n=20 per group): control group, exercise group, Aβ1-42 model group, and Aβ1-42 exercise group. Rats in the latter two groups were injected with 10 μL of Aβ1-42 (1 μg/μL) into the bilateral hippocampi, while those animals in the former two groups were injected 10 μL of normal saline in the same way. Rats in the two exercise groups began aerobic exercise training on the 2nd day after injection and the training lasted for 5 weeks, 6 days per week. Morris water maze test was conducted to test the spatial learning and memory ability of rats. Then brain tissue samples of rats were taken. The hippocampal synaptic structure and the expression of synaptophysin and postsynaptic dense 95 in the hippocampus were detected by electron microscopy, immunofluorescence, and western blot assay. 
    RESULTS AND CONCLUSION: (1) In the Morris water maze test, the average escape latency of all rats was gradually shortened during the location-based navigation training. The average escape latency decline rate of the Aβ1-42 exercise group was slower than that of the control group but showed a faster trend compared with that of the Aβ1-42 model group. (2) In the space exploration experiment, the frequency of platform crossing and retention time in the target quadrant were significantly lower in the Aβ1-42 exercise and Aβ1-42 model groups than the control group (P < 0.01, P < 0.05), but were significantly increased in the Aβ1-42 exercise than the Aβ1-42 control group (P < 0.05). Moreover, there was no significant difference between the exercise and control groups (P > 0.05). (3) Under the electron microscope, compared with the control group, the number of synapses decreased (P < 0.01) and the thickness of postsynaptic compacts became thinner in the Aβ1-42 model group (P < 0.05). Compared with the Aβ1-42 model group, the Aβ1-42 exercise group had an increase in the number of synapses (P < 0.05) and the thickness of postsynaptic compacts (P < 0.05). (4) Results from the immunofluorescence and western blot detections showed that the expressions of postsynaptic dense 95 and synaptophysin in the hippocampus were significantly lower in the Aβ1-42 model group than the control group (P < 0.01, P < 0.05), while the expression levels in the Aβ1-42 exercise group was significantly higher than those in the Aβ1-42 model group (P < 0.01, P < 0.05). (5) These findings indicate that aerobic exercise can effectively reduce Aβ1-42-induced damage to the hippocampal synaptic structure and promote the expression of hippocampal postsynaptic dense 95 and synaptophysin in rats, which may be one of the mechanisms by which aerobic exercise alleviates the learning and memory impairment in Alzheimer’s disease rats caused by Aβ1-42 toxicity.
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    Aspartate-glutamate carrier 1 regulates myelination in preterm rats with periventricular leukomalacia
    Wang Lizhen, Yang Caijin, Chen Rong, Li Ling
    2023, 27 (33):  5263-5269.  doi: 10.12307/2023.485
    Abstract ( 276 )   PDF (6289KB) ( 74 )   Save
    BACKGROUND: Periventricular leukomalacia is one of the most serious medical complications in preterm infants and there is yet no effective treatment for periventricular leukomalacia.
    OBJECTIVE: To investigate the effects of aspartate-glutamate carrier 1 (AGC1) on myelination in preterm rats with periventricular leukomalacia. 
    METHODS: Sixty male Sprague-Dawley rat pups on postnatal day 2 were randomly divided into sham group (n=30) and periventricular leukomalacia group (n=30). For in vitro studies, periventricular white matter tissues were isolated from five rat pups on postnatal day 2 to prepare white matter progenitor cells and establish an experimental oxygen-glucose deprivation model. Before the establishment of periventricular leukomalacia and oxygen-glucose deprivation models, pcDNA3-AGC1 plasmids were used to treat rats or cells. RT-qPCR was used to detect the expression of AGC1 in periventricular white matter tissue and cells at different time points after modeling. The expression of myelin basic protein and allophycocyanin was detected by immunofluorescence, and the ultrastructure of the corona radiate and corpus callosum was observed by electron microscopy at 14 days after periventricular leukomalacia. 
    RESULTS AND CONCLUSION: (1) Compared with the sham group, the expression of myelin basic protein and the number of allophycocyanin-positive oligodendrocytes and myelinated axons were significantly decreased in the periventricular leukomalacia group at 14 days after periventricular leukomalacia (P < 0.01). (2) AGC1 mRNA expression began to significantly increase in the white matter tissue at 12-24 hours (P < 0.05) and decreased significantly at 72 hours-14 days (P < 0.05) after periventricular leukomalacia in vivo, compared with 0 hour. (3) Under control conditions in vitro, AGC1 mRNA expression was increased significantly at 24-48 hours after oxygen-glucose deprivation (P < 0.05), and the AGC1 mRNA expression and the formation of myelin basic protein+/AGC1+ oligodendrocytes decreased significantly at 7-14 days after oxygen-glucose deprivation (P < 0.05), compared to 0 hour in the control group. (4) The myelin basic protein staining of the myelin sheath and the number of allophycocyanin-positive oligodendrocytes and myelinated axons in the pcDNA3-AGC1 treated group were significantly higher than those in the pcDNA3-NC group at 14 days after periventricular leukomalacia (P < 0.05). (5) In vitro, enhanced AGC1 expression further promoted the differentiation of oligodendrocyte precursor cells into myelin basic protein+/AGC1+ oligodendrocytes at 72 hours, 7 days, and 14 days after oxygen-glucose deprivation (P < 0.05). In conclusion, the expression of AGC1 is down-regulated in the preterm rat model of periventricular leukomalacia and the cell model of oxygen-glucose deprivation in vitro. Up-regulation of AGC1 can promote oligodendrocyte differentiation and myelination.
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    Schnurri3 regulates osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2
    Xie Yingchun, Xu Wenjuan, Li Yuwan
    2023, 27 (33):  5270-5276.  doi: 10.12307/2023.707
    Abstract ( 225 )   PDF (21534KB) ( 98 )   Save
    BACKGROUND: This study is based on the evidence that bone morphogenetic protein 2 plays an important role in osteogenesis and bone repair, and Schnurri3 can regulate the transforming growth factor-β signal transduction.  

    OBJECTIVE: To investigate the effect of inhibiting Schnurri3 on bone morphogenetic protein 2-induced osteogenic differentiation of C3H10T1/2 cells. 

    METHODS: Mouse mesenchymal stem cell line C3H10T1/2 cells were used as research materials. Four groups were set in this study: adenovirus-green fluorescent protein, adenovirus-bone morphogenetic protein 2, adenovirus-Simshn3 and adenovirus-bone morphogenetic protein 2+adenovirus-Simshn3. The expression of bone morphogenetic protein 2 was enhanced and the expression of Schnurri3 was inhibited. Alkaline phosphatase staining was used to detect the expression of alkaline phosphatase. The mRNA transcriptional levels of bone morphogenetic protein 2, Schnurri3, osteogenic and angiogenic markers were determined by RT-qPCR. Immunohistochemical staining was used to detect the expression of type I collagen, vascular endothelial growth factor and endothelial mucin. Alizarin red staining and semiquantitative analysis were used to detect calcium salt deposition levels. Subcutaneous stem cell implantation in nude mice was used to measure ectopic bone. 

    RESULTS AND CONCLUSION: (1) Recombinant adenovirus-bone morphogenetic protein 2 could induce osteogenic differentiation of C3H10T1/2 cells. Compared with the adenovirus-bone morphogenetic protein 2 group, mRNA transcription levels of osteogenic markers alkaline phosphatase, type I collagen, osteoblast-specific transcription factor, osteocalcin, Runt related transcription factor 2 and angiogenic markers neurite guidance factor, vascular endothelial growth factor, von Willebrand factor, angiopoietin and endothelial mucin were significantly increased in the adenovirus-bone morphogenetic protein 2+adenovirus-Simshn3 group (P < 0.001). Simultaneously, it significantly enhanced the expression of type I collagen, vascular endothelial growth factor and endothelial mucin at the protein level (P < 0.05), and the calcium salt deposition level (P < 0.05). (2) The application of bone morphogenetic protein 2 and Schnurri3 alone could act on C3H10T1/2 cells to form ectopic bone blocks subcutaneously in nude mice. Bone morphogenetic protein 2 combined with Schnurri3 could form more significant ectopic bone blocks. (3) The results showed that inhibition of Schnurri3 significantly enhanced the osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2, and enhanced the expression of angiogenesis factors mRNA and protein to regulate angiogenesis. 
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    Exosomes derived from bone marrow mesenchymal stem cells with chondromodulin-1 overexpression affect the proliferation of chondrocytes in osteoarthritis
    Wu Zhiwen, Shen Enpu, Li Beibei, Liu Danping, Qi Hui
    2023, 27 (33):  5277-5282.  doi: 10.12307/2023.754
    Abstract ( 243 )   PDF (3615KB) ( 78 )   Save
    BACKGROUND: Chondromodulin-1 (Chm-1), which is highly expressed in normal cartilage, can inhibit angiogenesis, prevent chondrocyte hypertrophy and alleviate the development of osteoarthritis. Chm-1 plays an important role in the occurrence and development of osteoarthritis.  
    OBJECTIVE: To investigate the effect of exosomes (Exos) derived from bone marrow mesenchymal stem cells with high expression of Chm-1 on the proliferation of chondrocytes in osteoarthritis.
    METHODS: Rat bone marrow mesenchymal stem cells were isolated, cultured and identified. Bone marrow mesenchymal stem cells were transfected with Chm-1 lentivirus and no-load lentivirus. NC-Exos and Chm-1-Exos in the supernatant of cell culture were extracted by ultra-high speed centrifugation. The model of osteoarthritis in vitro was established by treating chondrocytes with interleukin-1 β for 24 hours. Then osteoarthritis chondrocytes were treated with NC-Exos and Chm-1-Exos for 7 days, respectively, and PBS-treating osteoarthritis chondrocytes were used as a control group. The Exos uptake of osteoarthritis chondrocytes was observed by a confocal microscope. The proliferation of osteoarthritis chondrocytes was evaluated by CCK-8 assay. Expression levels of Chm-1 in NC-Exos and Chm-1-Exos were detected by western blot assay. The protein and mRNA expression levels of Col II, SOX9 and AGG in osteoarthritis chondrocytes were detected by western blot assay and RT-qPCR, respectively.  
    RESULTS AND CONCLUSION: (1) Bone marrow mesenchymal stem cells showed a typical fusiform shape and had the ability to differentiate into three lines. (2) Lentivirus with green fluorescence was successfully transfected into bone marrow mesenchymal stem cells. (3) Exos, whose size was between 40-150 nm, could be absorbed by osteoarthritis chondrocytes and expressed their specific marker proteins CD63, CD81 and TSG101. (4) Compared with NC-Exos, Chm-1-Exos expressed higher proteins of Chm-1 (P < 0.05). (5) At 24 hours after treatment, compared with the NC-Exos group, the Chm-1-Exos group presented higher cell viability. (6) At 7 days after treatment, compared with the NC-Exos group, the Chm-1-Exos group showed higher protein and mRNA expression levels of Col II, SOX9 and AGG (P < 0.05). (7) The above experimental results have shown that Chm-1-Exos can promote the proliferation of osteoarthritis chondrocytes.
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    Differential expression of RNA binding protein Lin28A regulates osteoblastic differentiation of periodontal ligament stem cells
    He Qin, Bu Yan, Lin Guanglei, Luo Jing, Yong Min, Huang Yongqing
    2023, 27 (33):  5283-5291.  doi: 10.12307/2023.713
    Abstract ( 214 )   PDF (10710KB) ( 59 )   Save

    BACKGROUND: The cellular activity of Lin28A can be influenced in several ways, including the self-renewal of embryonic stem cells, somatic cell reprogramming, individual growth and development, tissue metabolism, and carcinogenic consequences. However, neither domestically nor internationally have its effects on periodontal ligament stem cell ability to differentiate into osteoblasts been well explored; the underlying mechanisms are not known.

    OBJECTIVE: To investigate the impact of Lin28A on periodontal ligament stem cell capacity for osteogenic differentiation.

    METHODS: By using conventional enzyme digestion, periodontal ligament stem cells were extracted and cultured. The distribution of Lin28A in these cells and the differential expression of Lin28A at different time points of osteogenic induction were detected by qRT-PCR. Lentiviral vectors with Lin28A overexpression and interference expression were constructed and transfected into periodontal ligament stem cells. Fluorescence microscopy was used to observe the transfection effect. qRT-PCR was used to verify the expression difference of Lin28A after lentivirus transfection. The expression levels of osteoblastic genes alkaline phosphatase, Runt-related transcription factor 2 and osteopontin, alkaline phosphatase staining and alizarin red staining results were further detected from positive and negative aspects. The protein expression level of Runt-related transcription factor 2 was detected by western blot assay. Bioinformatics techniques and dual luciferase were used to predict and verify the relationship between Lin28A-related let-7 family members. The periodontal ligament stem cells differentially expressing Lin28A were cultured through osteoblastic induction, and the miRNA with the most obvious expression change during osteoblastic differentiation was determined. 
    RESULTS AND CONCLUSION: (1) Lin28A was enriched in the nucleus of periodontal ligament stem cells, and the expression of Lin28A increased at different time points after osteogenic induction. (2) In the periodontal ligament stem cell models with Lin28A overexpression, alkaline phosphatase staining and alizarin red staining were significantly deeper than those in the blank control group; the expression levels of alkaline phosphatase, Runt-related transcription factor 2 and osteopontin were increased 3.3, 5.1 and 2.2 times, respectively, compared with the blank ones (P < 0.01). In cell models that interfered with Lin28A expression, alkaline phosphatase staining and alizarin red staining were also decreased compared with the control group. The levels of alkaline phosphatase, Runt-related transcription factor 2 and osteopontin decreased 0.19-fold, 0.61-fold and 0.1-fold, respectively (P < 0.01). (3) The protein level of Runt-related transcription factor 2 was consistent with the mRNA difference tendency. The expression of Runt-related transcription factor 2 was increased in the overexpression group and decreased in the interference group. (4) Bioinformatics predicted that Lin28A was associated with eight let-7 family members, which were let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g and let-7i, respectively. After screening, the expression levels of let-7a and let-7c showed significant changes with Lin28A expression, and let-7a showed the most significant trend. Double luciferase binding assay confirmed that let-7a was directly bound to the 3'-UTR of Lin28A and was involved in the osteogenic differentiation of periodontal ligament stem cells. (5) The results suggested that Lin28A was involved in the regulation of osteogenic differentiation of periodontal ligament stem cells, with a positive correlation. That was, overexpression of Lin28A promoted the osteogenic ability of periodontal ligament stem cells, and the osteogenic ability of periodontal ligament stem cells was weakened after interference of Lin28A expression. The osteogenic differentiation of periodontal ligament stem cells was regulated by Lin28A .
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    Analysis of risk factors for varicella-zoster virus infection after allogeneic hematopoietic stem cell transplantation
    Qin Jing, Zhang Suping, Li Li, Zhang Ran, Peng Yingnan, Gao Siyu, Fan Jinpeng, Bian Zhilei, Wan Dingming
    2023, 27 (33):  5292-5297.  doi: 10.12307/2023.728
    Abstract ( 275 )   PDF (1531KB) ( 99 )   Save
    BACKGROUND: Allogeneic hematopoietic stem cell transplantation is an important or even the only method to cure a variety of hematological diseases. Herpes zoster virus infection after transplantation seriously affects the quality of life.  
    OBJECTIVE: To explore the risk factors of varicella-zoster virus infection in patients after allogeneic hematopoietic stem cell transplantation, in order to reduce the incidence of varicella-zoster virus infection after allogeneic hematopoietic stem cell transplantation and the severity of the disease.
    METHODS: Clinical data of 654 patients who underwent allogeneic hematopoietic stem cell transplantation in the First Affiliated Hospital of Zhengzhou University from November 2016 to June 2021 were retrospectively analyzed to explore the related factors of the varicella-zoster virus infection after allogeneic hematopoietic stem cell transplantation.  
    RESULTS AND CONCLUSION: 66 of the 654 patients developed varicella-zoster virus infection after allogeneic hematopoietic stem cell transplantation, and the morbidity was 10.1%. The average age of patients who infected with varicella-zoster virus was 21.5 (4-62) years old, and the average time of varicella-zoster virus infection was 149 days (19-1 179 days). The main manifestations were regional skin herpes, including 7 cases of herpes zoster encephalitis, 17 cases of post herpetic neuralgia, and 1 case of right ear deafness. The main treatments were topical use of ganciclovir gel, intravenous infusion of penciclovir, application of immunoglobulin to improve immune function and reduce the dosage of immunosuppressant. The median time of treatment was 11 days (3-38 days), and the herpes was scabbed or improved. Incidence of varicella-zoster virus infection after allogeneic hematopoietic stem cell transplantation was related to age (χ2= 7.816, P=0.005), whether fludarabine was used in the pretreatment scheme (χ2=5.004, P=0.025), whether there was cytomegalovirus infection (χ2=5.983, P=0.014), and the primary diseases (χ2=4.860, P=0.027). It was not related to the type of transplantation and the source of stem cells. The varicella-zoster virus infection is a common complication of allogeneic hematopoietic stem cell transplantation. It usually occurs within one year after transplantation. Patients with risk factors should pay more attention to prevention and treatment. Preventive application of antiviral drugs can reduce the incidence of varicella-zoster virus infection.
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    Ngn2 effects on brain microstructure and keratinocyte activity in cerebral ischemia rats via regulating Nrf2/HO-1
    Wang Mingsheng, Cui Huanxi, Cui Hongkai, Pei Guanhui, Li Daoguang, Yan Haiqing, Zhang Ping
    2023, 27 (33):  5298-5303.  doi: 10.12307/2023.514
    Abstract ( 168 )   PDF (1979KB) ( 69 )   Save
    BACKGROUND: Heme oxidase-1 (HO-1) plays an important role in cerebral ischemia-reperfusion injury. Nuclear factor-erythroid 2-related factor 2 (Nrf2) can reduce cerebral ischemia-reperfusion injury via regulating downstream antioxidant proteins. Therefore, it is speculated that Nrf2/HO-1 has a certain regulatory role in brain diseases.
    OBJECTIVE: To explore the effects of Neurogenin 2 (Ngn2) on brain microstructure and keratinocyte activity in cerebral ischemia rats by regulating Nrf2/HO-1.
    METHODS: Fifty-five SPF male Sprague-Dawley rats were selected, 10 of which were randomly selected as healthy group and the remaining rats were used to establish animal models of cerebral ischemia. After modeling, 40 model rats were randomly divided into 4 groups: rats in model group were intracerebrally injected with normal saline; rats in Ngn2 group were intracerebrally injected with Ngn2 10 mg/kg; rats in Nrf2/HO-1 group was intracerebrally injected with Ho-1 and Nrf2 agonist sulforaphane 10 mg/kg; and rats in combined group received intracerebral injection of Ngn2 combined with Nrf2/HO-1 10 mg/kg. Administration in each group lasted for 3 consecutive days. Fractional anisotropy imaging was used to observe the brain microstructure. Hematoxylin-eosin staining was used to observe the pathological morphology of brain tissue. TUNEL method was used to detect glial cell apoptosis. Western blot and PCR were used to detect Nrf2 and HO-1 protein and mRNA expressions, respectively.
    RESULTS AND CONCLUSION: (1) Compared with the healthy group, the relative fraction anisotropy values of the peri-infarct cortex and infarct core were lower in the model group (P < 0.05). Compared with the model group, the relative fraction anisotropy values of peri-infarct cortex and infarct core were increased in the Ngn2 group and Nrf2/HO-1 group (P < 0.05). Compared with the Ngn2 group and Nrf2/HO-1 group, the relative fraction anisotropy values of peri-infarct cortex and infarct core were increased in the combined group (P < 0.05). (2) In the model group, a large number of neurons were damaged, and the cells were sparse, disordered and infiltrated. In the Ngn2 group and Nrf2/HO-1 group, the neurons on the injured side were improved, and the loss, disorder, and adhesion of nerve cells were still observed. In the combined group, the necrosis of nerve cells was reduced and cell infiltration was improved. (3) Compared with the healthy group, the apoptosis of glial cells was higher in the model group (P < 0.05). Compared with the model group, the apoptosis of glial cells was decreased in the Ngn2 group and Nrf2/HO-1 group (P < 0.05). Compared with the Ngn2 group and Nrf2/HO-1 group, the apoptosis of glial cells was decreased in the combined group (P < 0.05). (4) Compared with the healthy group, the expressions of Ngn2 mRNA and Nrf2 and HO-1 at protein and mRNA levels were lower in the model group (P < 0.05). Compared with the model group, the expressions of Ngn2 mRNA and Nrf2 and HO-1 at protein and mRNA levels were increased in the Ngn2 group and Nrf2/HO-1 group (P < 0.05). Compared with the Ngn2 group and Nrf2/HO-1 group, the expressions of Ngn2 mRNA and Nrf2 and HO-1 at protein and mRNA levels were increased in the combined group (P < 0.05). (5) To conclude, Ngn2 can protect rats from cerebral ischemia by regulating Nrf2/HO-1, and the mechanism may be related to improving brain microstructure, keratinocyte activity and expression of Nrf2 and HO-1.
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    Ultrastructural features and clinical significance of autophagosome in benign meningiomas
    Zhang Qi, Li Guilin, He Yanjiao
    2023, 27 (33):  5304-5308.  doi: 10.12307/2023.752
    Abstract ( 236 )   PDF (3347KB) ( 55 )   Save
    BACKGROUND: Meningiomas are one of the most common benign tumors in the intracranial field, and clinical treatment is mainly surgery. However, the postoperative recurrence rate is high, and other treatment modalities do not yet have a definite effect, so the exploration of new targeted drugs is promising for meningioma treatment. At present, the pathogenesis of autophagy in benign meningiomas is not very clear, and a large number of experiments are still needed for in-depth study.  
    OBJECTIVE: To investigate the ultrastructural features and clinical significance of autophagy in benign meningiomas.
    METHODS: Meningiomas were diagnosed by light microscopy with different subtyping. Diagnosis and typing of meningiomas were aided by transmission electron microscopy, and the ultrastructural morphology and number of autophagosomes in the cytoplasm of meningioma cells of different grades were observed. Western blot assay was used to detect the expression levels of autophagy-related proteins Beclin1 and LC3 in different grades of meningiomas. The altered expression of Vimentin, EMA, PR, S-100 and GFAP in meningiomas was evaluated by immunohistochemical staining.  
    RESULTS AND CONCLUSION: 176 meningiomas were identified, including 98 males and 78 females. (1) Histopathological types CNS WHO grade 1 benign meningiomas comprised a total of 168 cases, including 62 cases of meningocortical type, 36 cases of fibrous type, 58 cases of transitional type, 12 cases of angiomatous type, 4 cases of CNS WHO grade 2 clear cell type, 2 cases of atypical type, and 2 cases of WHO grade 3 anaplastic lesions. (2) Transmission electron microscopy revealed autophagic bodies in 27 cases of WHO grade 1 meningiomas, and no autophagic bodies were detected in WHO grades 2 and 3 meningiomas. (3) LC3 and Beclin1 were highly expressed in WHO grade 1 benign meningiomas by western blot assay and were negatively expressed in WHO grades 2 and 3 meningiomas. (4) The immunohistochemical markers Vimentin, EMA, and PR, were expressed to various degrees, while GFAP and S-100 were negative. (5) A combination of electron microscopy, immunohistochemical staining and western blot assay showed that autophagy only plays an important role in the occurrence and development of WHO grade 1 benign meningiomas.
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    Effect of formononetin on glucose oxidase-induced oxidative stress in rat hepatic stellate cells
    Xie Na, Ma Run, Wang Lian, Shu Yuanhui, He Ping, Zhou Yan, Xiang Yining, Wang Yuping
    2023, 27 (33):  5309-5313.  doi: 10.12307/2023.753
    Abstract ( 242 )   PDF (1941KB) ( 32 )   Save
    BACKGROUND: Studies have shown that formononetin possesses potent anti-inflammatory, oxidative stress and immunomodulatory properties. However, it is still unclear whether formononetin can regulate the oxidative stress induced by glucose oxidase in rat hepatic stellate cells.
    OBJECTIVE: To investigate the possible mechanism and the effect of formononetin on oxidative stress in rat hepatic stellate cells HSC-T6 induced by glucose oxidase. 
    METHODS: The experiment contained control group, model group, low-, medium- and high-dose formononetin groups and curcumin group. The control group was not subjected to any treatment; the model group was treated with glucose oxidase for 2 hours; the formononetin and curcumin groups were treated with different concentrations of formononetin or curcumin for 24 hours, followed by glucose oxidase for 2 hours. CCK8 assay was used to investigate the effect of formononetin on HSC-T6 cell viability. Chemical kits were used to measure the contents of superoxide dismutase and malondialdehyde in each group. DCFH-DA fluorescence staining was performed to determine reactive oxygen species expression in each group. ELISA was performed to measure interleukin 1β and tumor necrosis factor α in each group. Immunofluorescence staining was used to detect the expression of type I collagen in each group. Western blot assay was used to measure the protein level of type I collagen, NOX4 and Nrf2 in each group.   
    RESULTS AND CONCLUSION: (1) Compared with the model group, formononetin could inhibit the activation and proliferation of HSC-T6 cells (P < 0.05). (2) Compared with the control group, superoxide dismutase activity and Nrf2 protein expression of HSC-T6 cells in the model group were significantly down-regulated (P < 0.05), while malondialdehyde, reactive oxygen species, interleukin 1β, tumor necrosis factor α, type I collagen and NOX4 protein expression levels were significantly up-regulated (P < 0.05). (3) Compared with the model group, superoxide dismutase activity and Nrf2 protein expression of HSC-T6 cells in formononetin treatment groups and curcumin group were significantly increased (P < 0.05), while malondialdehyde, reactive oxygen species, interleukin 1β, tumor necrosis factor α, type I collagen and NOX4 protein expression levels were markedly decreased (P < 0.05). (4) The results suggested that formononetin could inhibit glucose oxidase-induced activation and proliferation of hepatic stellate cells by regulating the Nrf2/NOX4 signaling pathway.
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    Lymphatic endothelial cells-derived exosomes promote axonal regeneration following peripheral nerve injury
    Huang Jinsheng, Zhang Geyi, Li Senrui, Li Jiangnan, Lu Laijin, Zhou Nan
    2023, 27 (33):  5314-5319.  doi: 10.12307/2023.748
    Abstract ( 265 )   PDF (12363KB) ( 77 )   Save
    BACKGROUND: The lymphatic system is involved in the regulation of nerve regeneration. Exosomes have intercellular communication functions and various biological characteristics. Exosomes derived from the lymphatic system have great potential in the treatment of peripheral nerve injury diseases.  
    OBJECTIVE: To investigate the role and mechanism of lymphatic endothelial cells-derived exosomes on axonal regeneration after peripheral nerve injury. 
    METHODS: (1) The effect of lymphatic endothelial cells-derived exosomes on the proliferation ability of Schwann cells was detected by EdU cell proliferation assay. (2) Twenty-four male SD rats were randomly divided into three groups with 8 rats in each group, including the sham operation group, peripheral nerve injury group and lymphatic endothelial cells-derived exosomes treatment group. The right sciatic nerves of rat models in the sham operation group were exposed without injury. For the rat models in the peripheral nerve injury group and lymphatic endothelial cells-derived exosomes treatment group, the right sciatic nerves were injected with PBS and lymphatic endothelial cells-derived exosomes under the epineurium of the nerve respectively following sciatic nerve crush. All left sciatic nerves of rats were not treated. The rats in each group were sacrificed 28 days following the operation. The bilateral gastrocnemius muscles were removed to measure the muscle wet-weight ratio. Right sciatic nerves were removed. Histopathological changes and axon arrangement of the sciatic nerve were evaluated by hematoxylin-eosin staining and Masson staining, and axonal regeneration was analyzed by immunofluorescence staining.
    RESULTS AND CONCLUSION: (1) Compared with the control group, lymphatic endothelial cells-derived exosomes significantly enhanced the proliferation ability of Schwann cells, and the difference was statistically significant (P < 0.05). (2) At 28 days following the operation, the wet-weight ratio of muscles was significantly higher in the lymphatic endothelial cells-derived exosomes treatment group than that in the peripheral nerve injury group (P < 0.05). Axons in the lymphatic endothelial cells-derived exosomes treatment group were arranged more closely and orderly than those in the peripheral nerve injury group and the axon disintegration and vacuolation caused by injury were less. The NF200 and S100β fluorescence intensities in the lymphatic endothelial cells-derived exosomes treatment group were significantly higher than that in the peripheral nerve injury group, and the difference was statistically significant (P < 0.05). Our findings indicated that lymphatic endothelial cells-derived exosomes could enhance the proliferation ability of Schwann cells to promote axonal regeneration following peripheral nerve injury by elevating the protein expression of NF200 and S100β. 
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    Irisin/peroxisome proliferator-activated receptor alpha signaling pathway mediates vascular smooth muscle cell proliferation in mice
    Wang Yao, Liu Danan, Zhao Guangjian, Zhou Bo, Wang Xiang
    2023, 27 (33):  5320-5326.  doi: 10.12307/2023.493
    Abstract ( 236 )   PDF (4142KB) ( 101 )   Save
    BACKGROUND: Irisin is released from fibronectin type III domain containing 5 (FNDC5) after hydrolysis and modification. In addition to regulating the main physiological functions of glucolipid metabolism, irisin also has the ability to regulate the proliferation and apoptosis of a variety of cells.
    OBJECTIVE: To investigate the role and mechanism of irisin/peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway mediated heme oxygenase-1 in regulating the balance between proliferation and apoptosis of vascular smooth muscle cells. 
    METHODS: FNDC5 overexpression and silencing lentivirus vector were used to transfect vascular smooth muscle cells to increase or inhibit irisin production, and an in vitro cell model was constructed. There were seven groups: blank control group, overexpressed empty vector group, FNDC5 overexpression group, overexpressed empty vector+PPARα inhibitor (GW6471) group, FNDC5 overexpression+GW6471 group, silencing empty vector group, and sh-FNDC5 group. The mRNA and protein expression levels of FNDC5, PPARα, heme oxygenase-1, Bax, and Bcl-2 were detected by real-time PCR and western blot assay, respectively. Apoptosis of vascular smooth muscle cells was detected by flow cytometry and proliferation activity of vascular smooth muscle cells was detected by cell counting kit-8 assay. 
    RESULTS AND CONCLUSION: (1) Compared with the blank control group and overexpressed empty vector group, the expressions of FNDC5, PPARα, heme oxygenase-1, and Bax were increased, the expressions of Bcl-2 were decreased, and the ratio of Bax/Bcl-2 was increased in the FNDC5 group and FNDC5+GW6471 group (all P < 0.05). (2) Compared with the blank control group and silencing empty vector group, the expressions of FNDC5, PPARα, heme oxygenase-1, and Bax were decreased, the expressions of Bcl-2 were increased, and the ratio of Bax/Bcl-2 was decreased in the sh-FNDC5 group (all P < 0.05). (3) Compared with the FNDC5 group, the expressions of PPARα, heme oxygenase-1, and Bax were significantly decreased, Bcl-2 expression was increased, and Bax/Bcl-2 ratio was decreased in the FNDC5+GW6471 group (all P < 0.05). (4) Compared with the blank control group and overexpressed empty vector group, the proliferation activity of vascular smooth muscle cells was significantly decreased in the FNDC5 group and FNDC5+GW6471 group, while the apoptotic rate was significantly increased (all P < 0.05). (5) Compared with the blank control group and silencing empty vector group, the proliferation activity of vascular smooth muscle cells was increased in the sh-FNDC5 group, while the apoptotic rate was significantly decreased (both P < 0.05). (6) Compared with the FNDC5 group, the proliferation activity of vascular smooth muscle cells was significantly increased, while the apoptotic rate was significantly decreased in the FNDC5+GW6471 group (both P < 0.05). (7) Therefore, irisin/PPARα signaling pathway can inhibit proliferation activity and accelerate apoptosis of vascular smooth muscle cells by inducing heme oxygenase-1 expression.
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    Molecular mechanism of total flavonoids of hawthorn leaves regulating astrocytes to repair spinal cord injury
    Ma Ruixin, Liu Pan, Zhang Qiong, Zeng Gaofeng, Zong Shaohui
    2023, 27 (33):  5327-5333.  doi: 10.12307/2023.724
    Abstract ( 217 )   PDF (2728KB) ( 36 )   Save
    BACKGROUND: Astrocytes play an important role in neuronal repair after spinal cord injury. Studies have shown that total flavonoids of hawthorn leaves can promote motor function recovery after spinal cord injury in rats. Whether total flavonoids of hawthorn leaves can affect astrocytes to promote neuronal repair remains unclear.  
    OBJECTIVE: To explore the molecular mechanism of total flavonoids of hawthorn leaves regulating astrocytes to repair spinal cord injury.
    METHODS: Transwell was used to construct the co-culture model of injured spinal cord neurons and spinal cord astrocytes. The control group consisted of normal spinal astrocytes and normal spinal neurons. The injured group consisted of normal astrocytes and injured spinal neurons. The drug group consisted of 125 μg/mL total flavonoids of hawthorn leaves-intervened normal spinal astrocytes and injured spinal neurons. The generation of reactive oxygen species in spinal cord neurons was detected by the fluorescence probe DCFH-DA. Real-time fluorescent quantitative PCR was used to detect the expression of brain-derived neurotrophic factor, nerve growth factor and transcription factor-4. The expression levels of brain-derived neurotrophic factor, nerve growth factor, Wnt/β-catenin pathway key proteins GSK-3β, phsopho-GSK-3β (ser9), β-catenin and transcription factor-4 were detected by western blot assay. The expression of key protein β-catenin of the Wnt/β-catenin pathway was observed by immunofluorescence method.  
    RESULTS AND CONCLUSION: (1) The production of reactive oxygen species in the injury group was much higher than that in the control group (P < 0.001). The production of reactive oxygen species in the drug group was lower than that in the injury group (P < 0.01) and higher than that in the control group (P < 0.001). (2) The gene expression of brain-derived neurotrophic factor and nerve growth factor in the drug group was higher than that in the control group and injury group (P < 0.01). (3) Compared with the control group and the injury group, the protein expression of phsopho-GSK-3β(ser9) increased (P < 0.001); β-catenin protein expression increased (P < 0.001); the protein and gene expression of transcription factor 4 was increased (P < 0.01) in the drug group. (4) The results showed that the total flavonoids of hawthorn leaves could alleviate the oxidative damage of spinal cord neurons, activate the Wnt/β-catenin pathway of astrocytes, combine with transcription factor 4 to start transcription, promote its secretion of neurotrophic factors, and realize the repair of spinal cord neurons.
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    Rougan Jiangmei Formula medicated serum inhibits activation and autophagy of human hepatic stellate cells
    Tao Bonan, Wang Yonglan, Jiang Lu, Zhang Zongxing, Liu Daozhong, Wan Xing, Huang Debin, Yuan Lin
    2023, 27 (33):  5334-5341.  doi: 10.12307/2023.762
    Abstract ( 229 )   PDF (2286KB) ( 143 )   Save
    BACKGROUND: Autophagy can play an important role in liver fibrosis by inducing the progression of hepatic stellate cell activation. Previous studies have shown that Rougan Jiangmei Formula could protect liver function, anti-inflammation, and anti-oxidative stress response. The mechanism of fighting liver fibrosis through autophagy has not been elucidated.  
    OBJECTIVE: To investigate the effect of Rougan Jiangmei Formula medicated serum on activation and autophagy of human hepatic stellate cells induced by lipopolysaccharide and its underlying mechanism.
    METHODS: Human hepatic stellate cell activation models were established using lipopolysaccharide and divided into various treatment groups: (1) Blank group: human hepatic stellate cells+blank serum; (2) model group: human hepatic stellate cells+blank serum+lipopolysaccharide; (3) low-, medium- and high-dose Rougan Jiangmei Formula medicated serum groups: human hepatic stellate cells+low-, medium- and high-dose Rougan Jiangmei Formula medicated serum+lipopolysaccharide; (4) high-dose Rougan Jiangmei Formula medicated serum combined with rapamycin group: based on high-dose group+rapamycin; (5) high-dose Rougan Jiangmei Formula medicated serum combined with PI3K inhibitor group: based on high-dose group+LY294002. Cell proliferation was observed by CCK-8 assay. The autophagy was observed by monodansylcadaverine staining. The effects of Rougan Jiangmei Formula medicated serum on the expression levels of cell pathway-related proteins (PI3K, P-PI3K, AKT, P-AKT, mTOR, P-mTOR), autophagy-related proteins (P62, Beclin-1, LC3-I, LC3-II), the activation marker protein of hepatic stellate cells (α-smooth-muscle actin and collagen I) were determined by western blot assay.  
    RESULTS AND CONCLUSION: (1) Rougan Jiangmei Formula medicated serum significantly reduced the proliferation of activated human hepatic stellate cells, down-regulated the protein expression of α-smooth-muscle actin and collagen I, up-regulated the expression of autophagy protein P62, and decreased the expression of Beclin-1 and LC3-II in a dose-dependent manner. (2) Monodansylcadaverine staining results exhibited that Rougan Jiangmei Formula medicated serum significantly inhibited the fluorescence intensity of the cell green spots. (3) After the addition of autophagy agonist rapamycin, the expression of autophagy protein P62 was reduced and the expression levels of Beclin-1 and LC3-II were increased. (4) After Rougan Jiangmei Formula medicated serum treatment, the phosphorylation levels of PI3K, Akt, and mTOR proteins were increased in a dose-dependent manner. This effect could be inhibited by the PI3K inhibitor LY294002. (5) It is indicated that Rougan Jiangmei Formula medicated serum may inhibit autophagy via the PI3K/Akt/mTOR signaling pathway and then reduce lipopolysaccharide-induced human hepatic stellate cell activation.
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    Triptolide protects damaged neurons by regulating microglial polarization
    Zhang Huiyu, Yu Jingwen, Bai Zhenjun, Li Liang, Mu Bingtao, Zhang Jinfeng, Xie Jiawei
    2023, 27 (33):  5342-5347.  doi: 10.12307/2023.723
    Abstract ( 264 )   PDF (15804KB) ( 110 )   Save
    BACKGROUND: Transforming microglia from the M1 phenotype to the M2 phenotype is considered to be a promising strategy for the treatment of neurodegenerative diseases. Many studies have shown that triptolide can inhibit neuroinflammation and alleviate a variety of neurodegenerative diseases, but its mechanism is still unclear.  
    OBJECTIVE: To investigate the protective effect of triptolide on SH-SY5Y cell injury induced by lipopolysaccharide-activated microglia and its mechanism.
    METHODS: CCK8 assay was used to detect the viability of BV2 cells and screen the best concentration of triptolide. The BV2 cells were divided into three groups: control group, model group (1 μg/mL lipopolysaccharide), and triptolide group (1 nmol/L triptolide + 1 μg/mL lipopolysaccharide). The supernatant was collected and the content of nitric oxide was detected by Griess assay. The levels of proinflammatory cytokines interleukin 6, tumor necrosis factor α, interleukin-1β and anti-inflammatory factor interleukin 10 were measured by ELISA. The expression levels of inducible nitric oxide synthase and arginase 1 in BV2 cells were determined by immunofluorescence staining and western blot assay. Three groups of microglia-conditioned medium were collected and treated on SH-SY5Y cells respectively: control-microglia-conditioned medium group, model-microglia-conditioned medium group and triptolide-microglia-conditioned medium group. TUNEL staining was utilized to detect the cell apoptosis rate in SH-SY5Y cells. Immunofluorescence staining and western blot assay were used to examine the expression of caspase 3, Bax and Bcl-2.  
    RESULTS AND CONCLUSION: (1) Compared with the control group, the levels of nitric oxide, interleukin-6, tumor necrosis factor α and interleukin-1β were significantly increased, and the level of interleukin-10 was significantly decreased, and the expression of inducible nitric oxide synthase protein increased, the expression of arginase 1 protein decreased in the supernatant of BV2 cells in the model group. Compared with the model group, the levels of nitric oxide, interleukin-6, tumor necrosis factor α and interleukin-1β were significantly decreased, and the level of interleukin-10 was significantly increased, and the expression of inducible nitric oxide synthase protein was decreased; the expression of arginase 1 protein was increased in the supernatant of BV2 cells in the triptolide group. (2) Compared with the control-microglia-conditioned medium group, the apoptosis rate increased, the expression of caspase 3 and Bax increased, and the expression of Bcl-2 decreased in SH-SY5Y cells of the model-microglia-conditioned medium group. Compared with the model-microglia-conditioned medium group, the cell apoptosis rate decreased, the expression of caspase 3 and Bax decreased, and the expression of Bcl-2 increased in the triptolide-microglia-conditioned medium group. (3) The results indicate that triptolide can reduce the neurotoxicity induced by lipopolysaccharide-activated microglia, and its mechanism may be related to promoting the polarization of microglia to M2 type.
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    Macrophage migration inhibitory factor promotes the differentiation of human embryonic stem cells into ventral midbrain dopaminergic neural progenitor cells
    Zheng Xiaohan, Feng Xiaoli, Hu Lan, Gao Shijun, Wei Yanzhao, Huang Ting, Sun Shengtong, Wei Xufang, Wang Tan, Zhao Zhenqiang
    2023, 27 (33):  5348-5356.  doi: 10.12307/2023.714
    Abstract ( 252 )   PDF (3124KB) ( 55 )   Save
    BACKGROUND: Cell replacement therapy is a very promising approach to treating Parkinson’s disease. The current main regimen for neural differentiation of human pluripotent stem cells is the “dual SMAD” inhibition regimen, which uses blockade of glycogen synthase kinase 3β to activate WNT pathway signaling, followed by a combination of hedgehog signaling and bone morphogenetic protein signaling regulation to precisely control the specific fate of human pluripotent stem cells from the basal to the parietal regions. Macrophage migration inhibitory factor (MIF), a class of pleiotropic immunomodulatory cytokines with a unique structure, has been shown to be important for neural development and differentiation in several studies. However, whether MIF is involved in the induction of stem cell differentiation is unknown.
    OBJECTIVE: On the basis of the “dual SMAD” inhibition regimen, different concentrations of MIF and its inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isothiazoleacetic acid methyl ester (ISO-1) were added to the culture medium to observe its effect on the differentiation of human embryonic stem cells into midbrain dopaminergic progenitor cells, to find a new way to solve the problem of heterogeneity during differentiation of human embryonic stem cells into midbrain dopaminergic progenitor cells.
    METHODS: CCK8 assay was performed to detect the survival of human embryonic stem cells cultured with the addition of different concentrations of MIF and ISO-1. The expression of MIF receptors CD74, CD44 and CXCR7 on human embryonic stem cells was detected by immunofluorescence. The expression levels of ventral midbrain markers LMX1A, FOXA2, EN1, forebrain marker FOXG1, hindbrain marker HOXA2, midbrain substrate marker PAX6 and transcription factor SOX6 of Wnt1/β-catenin signaling pathway were determined by RT-qPCR and western blotting, respectively, 16 days after differentiation of embryonic stem cells levels. Meanwhile, the expression levels of LMX1A, FOXA2, EN1 and MAP2 were measured by immunofluorescence assay. The expression of LMX1A, FOXA2, TH and GIRK2 in dopaminergic neurons was determined by immunofluorescence assay after continuing the differentiation of embryonic stem cells until day 42. 
    RESULTS AND CONCLUSION: (1) MIF was expressed in human embryonic stem cells, and its associated receptors CD44 and CXCR7 were also expressed, but CD74 expression did not occur. (2) The addition of MIF promoted the protein expression of LMX1, FOXA2, and EN1, and promoted the gene expression of LMX1 and FOXA2. It also significantly inhibited the gene and protein expression of FOXG1 and HOXA2 and PAX6, effectively suppressing the heterogeneity of differentiation. (3) The addition of MIF did not result in high expression of SOX6 in dopaminergic neural progenitors as expected, but rather decreased expression at the gene level, while its inhibitor ISO-1 caused a mild increase in SOX6 expression at the gene level. (4) The present study demonstrates that MIF plays an important role in the dopaminergic differentiation potential and ventral midbrain fate determination of human embryonic stem cells and that MIF intervention further optimizes the regional localization of neural induction.
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    Molecular characteristic of immune cells in ulcerative colitis patients with spleen deficiency and dampness syndrome based on single-cell RNA sequencing
    Zhang Shijing, Huang Zhuojian, Tao Quyuan, Hou Jiangtao, Peng Bin, Luo Yongxin, Li Yanwu, Li Huibiao, Zhong Jiamin, Chen Xinlin
    2023, 27 (33):  5357-5362.  doi: 10.12307/2023.740
    Abstract ( 287 )   PDF (6311KB) ( 73 )   Save
    BACKGROUND: Immune-mediated tissue destruction is essential for the development and deterioration of inflammation in ulcerative colitis, but their roles remain unclear. Simultaneously, ulcerative colitis patients with spleen deficiency and dampness syndrome lack objective biomarkers.  
    OBJECTIVE: To investigate the molecular characteristics of immune cells in ulcerative colitis patients with spleen deficiency and dampness based on single-cell RNA sequencing so as to provide a basis for its clinical diagnosis and treatment.
    METHODS: Colon tissues of two ulcerative colitis patients and two healthy controls were obtained. Single-cell RNA sequencing was performed. The following analyses were carried out for the single-cell RNA sequencing data: cluster analysis, dimensionality reduction analysis, cell type annotation, differential expression analysis and cell communication analysis.  
    RESULTS AND CONCLUSION: (1) A total of 29 cell sub-types were identified according to clustering analysis in single cell transcriptome, which consisted of 9 cell types. (2) Compared with healthy controls, the ulcerative colitis patients with spleen deficiency and dampness syndrome had higher expression levels of CD4+ cytotoxic T cells, naive CD8+ T cells, germinal center, activated B cells, S100Ahigh B cells, NK_C2 cells and monocytes (P < 0.05). Compared with healthy controls, Tfh cells, NKT cells and NK cells decreased in patients with ulcerative colitis (P < 0.05). (3) Functional enrichment of differentially expressed genes showed that there were hyperinflammatory activation in immune cells and higher metabolic reprogramming of NK cells, plasma cells and helper T cells for ulcerative colitis patients. (4) Cell communication analysis identified 14 interactions among different cell types, mainly from monocytes and endothelial cells. (5) There are significant differences in molecular characteristics of immune cells between ulcerative colitis patients and healthy controls. These cell subsets may be related to the occurrence and development of ulcerative colitis, which may be a biomarker for ulcerative colitis patients with spleen deficiency and dampness syndrome.
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    Tolerogenic dendritic cells can inhibit pyroptosis of spleen cells in collagen-induced arthritis rats
    Long Tiaoyu, Bao Lunmin, Wan Xiufang, Li Honghong, Zhang Yundong, Jiang Hongmei
    2023, 27 (33):  5363-5369.  doi: 10.12307/2023.498
    Abstract ( 216 )   PDF (6919KB) ( 89 )   Save
    BACKGROUND: In our previous study, we successfully constructed antigen-specific tolerogenic dendritic cells using nuclear factor-kappa B oligodeoxynucleotide decoy, and used them to effectively intervene a collagen-induced arthritis rat model. However, the therapeutic mechanism remains to be further studied.
    OBJECTIVE: To investigate the effect of tolerogenic dendritic cells on pyroptosis of spleen cells in collagen-induced arthritis rats.  
    METHODS: Bone marrow-derived dendritic cells were extracted and used to construct tolerogenic dendritic cells using nuclear factor-kappa B oligodeoxynucleotide decoy. Nine Sprague-Dawley rats were randomly divided into control group, model group, and treatment group, with three rats in each group. The latter two groups were used to establish rheumatoid arthritis models. Initial immunization was subcutaneous injection of bovine type II collagen emulsion on the left foot plantar; booster immunization was multiple injections of bovine type II collagen emulsion at the base of the tail on the 14th day after the initial immunization. Rats in the model and treatment groups were given normal saline and tolerogenic dendritic cells into the tail vein, respectively, on the 20th day after the initial immunization. Relevant measurements were performed on the 35th day after the initial immunization. 
    RESULTS AND CONCLUSION: (1) Compared with the model group, arthritis index score in the treatment group decreased significantly (P < 0.05). (2) Gross and pathological observations showed the ankle joints of the rats were severely swollen in the model group and slightly swollen in the treatment group. Hematoxylin-eosin staining results showed that compared with the control group, the synovium of the ankle joint had obvious hyperplasia, accompanied by inflammatory cell infiltration and bone erosion; compared with the model group, the treatment group had reduced synovial hyperplasia, bone erosion and inflammatory cell infiltration of the ankle joint. (3) Serum interleukin 1β level was higher in the model group than the control group (P < 0.01) and lower in the treatment group than the model group (P < 0.01). (4) Western blot results showed that compared with the control group, the protein expressions of NLR family pyrin domain containing 3, pro-caspase-1, caspase-1, GSDMD-NT, pro-interleukin 1β, and interleukin 1β in the spleen were significantly increased in the model group (P < 0.05). Compared with the model group, the protein expressions of spleen NLR family pyrin domain containing 3, pro-caspase-1, caspase-1, GSDMD-NT, pro-interleukin 1β, and interleukin 1β were all decreased in the treatment group (P < 0.05). (5) In conclusion, tolerogenic dendritic cells may alleviate inflammatory responses in collagen-induced arthritis rats by inhibiting the pyroptosis of spleen cells and the expression of interleukin-1β. 
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    Application of single-cell RNA sequencing in intervertebral disc degeneration
    Wu Hao, Ye Dongping
    2023, 27 (33):  5370-5371.  doi: 10.12307/2023.741
    Abstract ( 279 )   PDF (1618KB) ( 76 )   Save
    BACKGROUND: After over ten years of development, single-cell RNA sequencing has mellowed and in the last three years has been progressively adopted in the study of intervertebral disc degeneration.  
    OBJECTIVE: To overview the utilization and the progress of single-cell RNA technology within the field of intervertebral disc degeneration research during the last few years.
    METHODS: The first author searched PubMed, ScienceDirect, and Web of Science databases with the key words “single cell RNA sequencing, intervertebral disc degeneration, nucleus pulposus, annulus fibrosus”. The literature was screened for relevance by reading and 58 articles were included in the final analysis.  
    RESULTS AND CONCLUSION: As an emerging technology, single-cell RNA sequencing has been widely applied to the study of tumor heterogeneity, immune microenvironment, embryonic development, neuroscience, cell differentiation and many other fields. In recent years, an increasing number of scholars have applied single-cell RNA sequencing to research intervertebral disc degeneration. These studies have helped researchers to recognize the complex heterogeneity of the nucleus pulposus and fibrous rings: the newly discovered subpopulations of nucleus pulposus can be grouped into four groups: firstly, the nucleus pulposus cells that are more prevalent in normal nucleus pulposus tissue, often named “fibrous nucleus cells”, which can polydifferentiate to maintain fibrocartilage development; secondly, the “stable nucleus pulposus cells” that are more prevalent in mildly degenerated nucleus pulposus, which are responsible for regulating the homeostatic balance of chondrocyte metabolic activity in response to environmental signals and are highly expressed in collagen-related genes and extracellular matrix. Thirdly, the “inflammatory nucleus pulposus” is found only in degenerated nucleus pulposus tissue. In this subpopulation, the regulatory activity of NF-κB family transcription factors is enhanced. The fourth is the “adherent nucleus pulposus”, which is more abundant in severely degenerated nucleus pulposus tissue and has the FN1 marker gene. The discovery of these subpopulations adds to the understanding of the rich mechanisms of differentiation and degeneration within the tissue and the changes in intervertebral disc degeneration at the transcriptomic level. PROCR+PDGFRA+ nucleus pulposus cells, CD90+ nucleus pulposus cells and UTS2R+ nucleus pulposus cells have the strong growth and differentiation potential, and the discovery of these nucleus pulposus progenitor cells offers the possibility to explore therapeutic tools for intervertebral disc degeneration from regenerative medicine and tissue engineering perspectives in the future.
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    Action mechanism of traditional Chinese medicine combined with bone marrow mesenchymal stem cells in regulating blood-brain barrier after cerebral ischemia reperfusion injury
    Wang Shaona, Liu Feixiang, Ying Chunmiao, Gao Chen, Zhang Yunke
    2023, 27 (33):  5377-5384.  doi: 10.12307/2023.716
    Abstract ( 280 )   PDF (1655KB) ( 49 )   Save
    BACKGROUND: Ischemic stroke is one of the main causes of death and long-term disability, and the incidence is still rising year by year, seriously affecting the quality of life. Blood-brain barrier injury is an important pathological process that leads to a poor prognosis of cerebral ischemia reperfusion injury. However, due to the limited treatment methods and limitations of multiple factors, it is urgent to find new treatment methods to repair blood-brain barrier injury.
    OBJECTIVE: To review the relevant mechanisms of traditional Chinese medicine combined with bone marrow mesenchymal stem cells in regulating the blood-brain barrier after cerebral ischemia reperfusion injury, to further explore new ways to protect the blood-brain barrier and provide assistance for clinical treatment. 
    METHODS: The keywords were “traditional Chinese medicine, bone marrow mesenchymal stem cells, blood-brain barrier, cerebral ischemia reperfusion injury” in Chinese and English. WanFang, CNKI, PubMed, and Web of Science databases from 2012 to 2022 were retrieved for articles regarding the related mechanism of the blood-brain barrier damage and the related mechanism of Chinese medicine combined with bone marrow mesenchymal stem cells in regulating the blood-brain barrier. The obsolete and unqualified articles were excluded and 97 articles were included for analysis and summary. 
    RESULTS AND CONCLUSION: (1) The physiological structure of the blood-brain barrier and the pathological changes of the blood-brain barrier structure after cerebral ischemia reperfusion injury were combed. (2) This paper summarized the mechanism of traditional Chinese medicine combined with bone marrow mesenchymal stem cells in regulating the blood-brain barrier after cerebral ischemia reperfusion injury, including regulating the expression of tight junction protein, inhibiting the activity of aquaporin-4 and matrix metalloproteinase-9, promoting angiogenesis, inhibiting the release of inflammatory mediators, and regulating exosomes. (3) It is concluded that traditional Chinese medicine combined with bone marrow mesenchymal stem cells can improve the permeability of the damaged blood-brain barrier and provide assistance for clinical treatment. 
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    Bidirectional interaction between inflammatory factors and dental pulp stem cells during bone regeneration
    Xu Rongwei, Wang Hao, Fu Qiuyue, Lan Xingming, Yang Kun
    2023, 27 (33):  5385-5393.  doi: 10.12307/2023.687
    Abstract ( 232 )   PDF (2602KB) ( 134 )   Save
    BACKGROUND: Dental pulp stem cells have excellent characteristics of multi-directional differentiation. Among them, osteogenic differentiation potential has a good prospect in bone tissue engineering and regeneration therapy, while inflammatory factors are strongly associated with it.  
    OBJECTIVE: To review the research progress on the bidirectional effects of inflammatory factors and osteogenic differentiation of dental pulp stem cells during bone regeneration.
    METHODS: In the PubMed, CNKI, and Wanfang databases, the search terms were “dental pulp stem cells, inflammation, osteogenic differentiation, bone regeneration”in Chinese and “dental pulp stem cell*, dpsc*, inflam*, osteogenic differentiation, ossification, osteogenesis, bone formation” in English. Relevant articles published from 2012 to 2022 were retrieved, and 76 articles were finally included for review.  
    RESULTS AND CONCLUSION: (1) As dental mesenchymal stem cells, dental pulp stem cells have well-known multi-directional differentiation characteristics, among which the osteogenic differentiation potential is a research hotspot of bone tissue engineering. (2) Inflammatory factors are strongly associated with bone regeneration mediated by dental pulp stem cells whether they are the microenvironment where cells live, the adjustable immune inflammatory response, or the inflammatory disease with bone tissue defect that needs regeneration. In recent years, there has been no lack of related research. (3) This article selected relevant articles in the past decade, and found that even inflammatory dental pulp stem cells from inflamed pulp tissue still retain certain osteogenic differentiation potential. Further analysis of the bidirectional relationship between inflammatory factors and dental pulp stem cells during bone regeneration found that the effect of inflammatory factors on the osteogenic differentiation of dental pulp stem cells and the immune regulation of dental pulp stem cells on inflammation have both promoting and inhibiting effects. This is mainly related to the type, concentration and time of inflammatory mediators, but the specific mechanisms are not comprehensive. (4) Recently, scholars’ researches focus on exploring the promoting factors that mediate the osteogenic differentiation of dental pulp stem cells in an inflammatory environment, especially the exogenous factors in addition to inflammatory mediators, such as drugs. (5) Some animal experiments and a few clinical trials have verified the therapeutic effect of dental pulp stem cells on inflammatory bone defect diseases especially periodontitis and arthritis by using the anti-inflammatory and osteogenic properties of dental pulp stem cells. (6) At present, there is still a big gap in the research on the mechanism of the bidirectional interaction between inflammatory factors and osteogenic differentiation of dental pulp stem cells during bone regeneration, which needs further investigation and verification in the future.
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    Pretreatment methods improve the role of exosomes in spinal cord injury
    Zhou Heshan, Tan Longwang, Liu Chuang, Zhang Chi
    2023, 27 (33):  5394-5403.  doi: 10.12307/2023.711
    Abstract ( 291 )   PDF (1687KB) ( 70 )   Save
    BACKGROUND: Exosomes produced by paracrine secretion of different cell types can participate in many cellular processes and have shown good therapeutic effects in spinal cord injury. However, the use of natural exosomes alone to treat spinal cord injury suffers from poor organ targeting and inadequate therapeutic effects.
    OBJECTIVE: To review pretreatment methods to improve the role of exosomes in spinal cord injury.
    METHODS: The first author searched PubMed, Web of Science, and CNKI with English and Chinese search terms “exosomes, spinal cord injuries” and 91 articles were selected for review according to the inclusion and exclusion criteria.
    RESULTS AND CONCLUSION: (1) In the treatment of spinal cord injury using natural exosomes, the activity of exosomes is easily inhibited and suffers from a short half-life and poor targeting. (2) During the preparation of exosomes, the function and efficacy of natural exosomes in spinal cord injury can be further optimized and the possibility of adverse effects can be reduced using the corresponding pretreatment. (3) Three common pretreatment methods include the preculture of exosomes, material pre-packaging of exosomes, and preassociation of biological materials. Each of the three pretreatment methods has advantages and disadvantages and intervenes in exosomes from different dimensions. Among them, the drug delivery system constructed by exosomal substance pre-mounting has been the most used at present, and the technology is relatively mature, and clinical studies have been carried out in some fields, which have good prospects. (4) The optimal effect of other interventions on exosomes has also been confirmed experimentally, and these interventions are not independent of each other, combining them organically to achieve a multidimensional pretreatment to achieve repair after spinal cord injury. (5) Appropriate pretreatment measures can improve the targeting and stability of exosomes, prolong the half-life of exosomes, and enhance the colonization of exosomes at the site of spinal cord injury while ensuring the structure of exosomes, thus promoting the repair of spinal cord injury and improvement of neurological function for therapeutic purposes. (6) Taken together, exosomes have great potential and broad prospects in the field of spinal cord injury, and pretreatment and engineering transformation of exosomes are the only way to develop exosomes in the future, which should be gradually promoted from basic experiments into clinical research to realize the clinical application of exosomes in the field of spinal cord injury as soon as possible. 
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    Effect and mechanism of non-coding RNA in regulating neuronal apoptosis after spinal cord injury
    Xu Luchun, Yang Yongdong, Zhao He, Zhong Wenqing, Ma Yukun, Yu Xing
    2023, 27 (33):  5404-5412.  doi: 10.12307/2023.704
    Abstract ( 256 )   PDF (4976KB) ( 132 )   Save
    BACKGROUND: Neuronal cell apoptosis after spinal cord injury is a major cause of permanent spinal cord injury. It is of great significance to explore ways to prevent neuronal apoptosis after spinal cord injury. Existing animal experiments have shown that non-coding RNA can regulate neuronal apoptosis after spinal cord injury, which may provide a certain reference for clinical neuronal protective therapy after spinal cord injury.  
    OBJECTIVE: To review the role and mechanism of non-coding RNA in regulating neuronal apoptosis after spinal cord injury so as to provide a new target for protective treatment of spinal cord injury.
    METHODS: Search terms “microRNA, miR, lncRNA, circRNA, noncoding RNA, RNA, SCI, spinal cord injury, spinal cord, apoptosis” were searched in the PubMed database. The inclusion and exclusion criteria were formulated, and 87 relevant articles were included by reading the title, abstract and full text.  
    RESULTS AND CONCLUSION: (1) Neuronal cell apoptosis is an important pathophysiological change in secondary spinal cord injury, and it is an important mechanism of neuronal death and spinal cord dysfunction. (2) Animal experiments showed that miRNA, lncRNA and circRNA related to neuronal cell apoptosis were significantly differentially expressed in spinal cord tissues after spinal cord injury, and some miRNA, lncRNA and circRNA that promoted neuronal apoptosis were overexpressed. The expression of miRNA, lncRNA and circRNA that inhibited neuronal apoptosis was down-regulated. (3) By regulating the corresponding expression of specific miRNA, the excessive apoptosis of neuronal cells after spinal cord injury can be inhibited mainly by down-regulating the apoptotic gene Bax, inhibiting the TLR4/nuclear factor-κB signaling pathway, and activating the PI3K/Akt signaling pathway. (4) The regulation of lncRNA and circRNA expression mainly causes the silencing or overexpression of specific miRNA by targeting negative regulation, so as to exert its anti-neuronal apoptosis effect. (5) Among miRNAs, miR-125b can regulate Smurf1/KLF2/ATF2 axis and JAK1/STAT1 signaling pathway, while miR-21-5p can regulate programmed cell death factor 4 (PDCD4) and PI3K/AKT signaling pathway, showing multi-pathway anti-apoptotic effect. (6) Among lncRNAs and circRNAs, lncRNA-PTENP1, lncRNA-TCTN2 and circRNA-HIPK3 have targeted regulatory effects on a variety of miRNAs. These non-coding RNAs may become new targets for neuronal protective therapy after spinal cord injury. (7) At the moment, non-coding RNA regulation of neuronal apoptosis after spinal cord injury also only stays in animal cells. In the future, we need to continue to explore more targeted non-coding RNA drug carriers, joint biological tissue engineering as well as other emerging technologies, and strive for an early transition to clinical transformation.
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