复方血竭对结肠炎模型大鼠Toll样受体4/核因子κB及树突状细胞表型的影响

doi:10.3969/j.issn.2095-4344.2015.05.017

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (5): 752-758.doi: 10.3969/j.issn.2095-4344.2015.05.017

• 器官移植动物模型 organ transplantation and animal model • 上一篇下一篇

复方血竭对结肠炎模型大鼠Toll样受体4/核因子κB及树突状细胞表型的影响

李楠¹，王雪明²，稽杨³，石玉玲¹，王欣¹，李娜¹，苏丽¹，沙丽娜¹

解放军总参谋部总医院(309)，¹消化科，²药剂科，北京市 100091；³解放军总后勤部药品仪器检验所药理室，北京市 100071

修回日期:2014-12-17 出版日期:2015-01-30 发布日期:2015-03-02
通讯作者: 李楠，解放军总参谋部总医院，北京市 100091
作者简介:李楠，男，1957年生、山东省青岛市人、1996年于解放军第三军医大学毕业，博士、主任医师、博士生导师。主要从事炎症性肠病的基础与临床研究。
基金资助:
北京市自然科学基金(717329)

Effects of resina draconis on Toll-like receptor-4/nuclear factor-kappaB and dendritic cell phenotypes in colitis rats

Li Nan¹, Wang Xue-ming², Ji Yang³, Shi Yu-ling¹, Wang Xin¹, Li Na¹, Su Li¹, Sha Li-na¹

1 Department of Gastroenterology, the 309th Hospital of Chinese PLA, Beijing 100091, China
2 Department of Pharmacy, the 309th Hospital of Chinese PLA, Beijing 100091, China
3 Department of Pharmacology, Institute of Drug and Instrument Control, General Logistics Department of Chinese PLA, Beijing 100071,
China

Revised:2014-12-17 Online:2015-01-30 Published:2015-03-02
Contact: Li Nan, Department of Gastroenterology, the 309th Hospital of Chinese PLA, Beijing 100091, China
About author:Li Nan, M.D., Chief physician, Doctoral supervisor, Department of Gastroenterology, the 309th Hospital of Chinese PLA, Beijing 100091, China
Supported by:
the Natural Science Foundation of Beijing, No. 717329

摘要/Abstract

摘要：

背景：树突状细胞可调节肠道内环境的免疫反应，这一功能缺陷会导致炎症性肠病，Toll样受体4/核因子κB通路与上述反应密切相关。
目的：构建实验性结肠炎模型大鼠，观察复方血竭对其树突状细胞及Toll样受体4/核因子κB表达的影响并探讨其作用机制。
方法：将大鼠随机抽样法分为空白对照组、模型组、复方血竭组和5-氨基水杨酸治疗组。除空白对照组外，其他3组大鼠建立2，4，6-三硝基苯磺酸诱导的实验性结肠炎模型。造模成功后，复方血竭组和5-氨基水杨酸治疗组分别给予复方血竭[0.75 g/(kg•d)]和5-氨基水杨酸 [(100 mg/(kg•d)]灌胃治疗10 d。
结果与结论：复方血竭组较模型组大鼠的疾病活动指数评分、大体形态损伤评分、病理组织学评分均显著降低(P < 0.05)，5-氨基水杨酸治疗组、复方血竭组的症状、结肠组织损害均较模型组明显减轻。模型组大鼠肠系膜淋巴结CD80和CD86的表达率，Toll 样受体4，核因子κB表达较其他3组均显著升高(P < 0.05或P < 0.01)。复方血竭组Toll样受体4及核因子κB表达明显低于5-氨基水杨酸治疗组。结果证实，复方血竭可部分缓解实验性结肠炎模型大鼠的症状，有效抑制其肠系膜淋巴结中树突状细胞的活化。复方血竭可能通过抑制大鼠肠黏膜细胞Toll 样受体4和核因子κB的表达而缓解肠道的炎症反应。

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关键词: 实验动物, 消化系统损伤模型, 复方血竭, 2, 4, 6-三硝基苯磺酸, 结肠炎, 树突状细胞, Toll 样受体4, 核因子κB, 流式细胞仪, 大鼠, 免疫组织化学, RT-PCR, 病理, 北京市自然科学基金

Abstract:

BACKGROUND: Dendritic cells can regulate the immunological reaction in the intestinal tract, this functional deficit may induce inflammatory bowel disease. Toll-like receptor-4/nuclear factor-κB pathway is highly involved in this reaction.
OBJECTIVE: To establish experimental colitis model in rats, to observe effects of resina draconis on dendritic cells and Toll-like receptor-4/nuclear factor-κB expression in rats with experimental colitis, and to explore its action mechanism.
METHODS: A total of 44 male Sprague-Dawley rats were randomly assigned to four groups (n = 11): blank control group, model group, resina draconis group, 5-aminosalicylic acid treatment group. With the exception of blank control group, 2,4,6-trinitrobenzenesulfonic acid-induced ulcerative colitis models were established in the model group, resina draconis group and 5-aminosalicylic acid treatment group. After the models were successfully established, the rats in the resina draconis and 5-aminosalicylic acid treatment groups were intragastrically treated with resina draconis [(0.75 g(kg•d)] and 5-aminosalicylic acid [100 mg(kg•d)] respectively for 10 days.
RESULTS AND CONCLUSION: Disease activity index, macroscopic colonic damage score and histopathological score were significantly decreased in the resina draconis group compared with the model group (P < 0.05). Symptoms and tissue damages were obviously lessened in the 5-aminosalicylic acid treatment and resina draconis groups compared with the model group. Expression rates of CD80 and CD86, as well as expression levels of Toll-like receptor-4 and nuclear factor-κB were significantly higher in the model group compared with the blank control group, resina draconis group and 5-aminosalicylic acid treatment group (P < 0.05, P < 0.01). Toll-like receptor-4 and nuclear factor-κB expression was significantly lower in the resina draconis group than that in the 5-aminosalicylic acid treatment group. Experimental findings indicate that, resina draconis can partially relieve experimental colitis symptoms in rats and effectively inhibit the activation of dendritic cells in the mesenteric lymph node. Resina draconis can relieve enteric inflammatory reaction by suppressing the expression of Toll-like receptor-4 and nuclear factor-κB in rats.

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Key words: Tissue Engineering, Colitis, Trinitrobenzenesulfonic Acid, Mesentery

中图分类号:

R318

李楠，王雪明，稽杨，石玉玲，王欣，李娜，苏丽，沙丽娜. 复方血竭对结肠炎模型大鼠Toll样受体4/核因子κB及树突状细胞表型的影响[J]. 中国组织工程研究, 2015, 19(5): 752-758.

Li Nan, Wang Xue-ming, Ji Yang, Shi Yu-ling, Wang Xin, Li Na, Su Li, Sha Li-na. Effects of resina draconis on Toll-like receptor-4/nuclear factor-kappaB and dendritic cell phenotypes in colitis rats[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(5): 752-758.

图/表（结果） 7

Quantitative analysis of experimental animals

All the involved 44 rats entered the final analysis without any dropout. The process of model establishment is shown in Figure 1.

General condition following model induction

In the TNBS model group, rat colitis specimens exhibited mucosal hyperemia, edema, erosions, necrosis, thickening intestinal wall and ulcer. Ulcer, inflammatory exudates and lymphoid hyperplasia were visible in the mucosa, submucosa and muscular layer. In the blank control group, intact smooth mucosa and clear structure of glandular organ were observed, without erosion or ulcer, with the presence of slight neutrophil infiltration. In the resina draconis and 5-aminosalicylic acid treatment groups, there were slight ulcer and partially intact glandular organ (Figure 2).

Disease activity index scoring

Ulcer size was observed according to colonic mucosal damage index. Significant differences in weight changes were detected between the resina draconis and blank control groups (P=0.017, P=0.022, P=0.027), but there was no significant difference as compared TNBS model group with resina draconis and 5-aminosalicylic acid treatment groups (P=0.067, P=0.077). Disease activity index, macroscopic colonic damage score and histopathological score were significantly decreased in the resina draconis group compared with the model group (P < 0.05). Symptoms and tissue damages were obviously lessened in the 5-aminosalicylic acid treatment and resina draconis groups compared with the model group. Within 10 days, disease activity index scores were higher in the TNBS model group than those in the resina draconis and 5-aminosalicylic acid treatment groups (P=0.008, P=0.001; Table 1).

Dendritic cells surface markers major histocompatibility complex class II and CD86 expression in the mesenteric lymph node

The expression of major histocompatibility complex class II and CD86 of dendritic cells surface markers in the mesenteric lymph node was significantly lower in the resina draconis group than those in the TNBS model group (Table 2).

Expression rates of major histocompatibility complex class II and CD86 were the lowest in the blank control group. Expression rates of major histocompatibility complex class II and CD86 were greater in the TNBS model group than those in the blank control group (P=0.006, P=0.007). Expression rates of major histocompatibility complex class II and CD86 in the resina draconis group were significantly lower than those in the TNBS model group (P=0.021, P= 0.019), but still higher than those in the blank control group (P=0.006, P=0.001). Expression rates of major histocompatibility complex class II and CD86 in the 5-aminosalicylic acid treatment group were significantly lower than those in the TNBS model group (P=0.015, P= 0.014), but higher than those in the blank control group (P= 0.077, P=0.032) (Table 2).

Effects of resina draconis on nuclear factor-κB p65 expression in rats with ulcerative colitis

Yellow particles in the cytoplasm and/or nuclei were positive cells (Figure 3). Compared with the control group, nuclear factor-κB p65 expression was significantly higher in the TNBS model group, resina draconis group and 5-aminosalicylic acid treatment group (P < 0.01, P < 0.05). Compared with the TNBS model group, nuclear factor-κB p65 expression was significantly lower in the resina draconis group and 5-aminosalicylic acid treatment group (P < 0.01). Compared with the 5-aminosalicylic acid treatment group, nuclear factor-κB p65 expression was lower in the resina draconis group (P < 0.01; Table 3).

Effects of resina draconis on Toll-like receptor-4 mRNA expression in rats with ulcerative colitis

Compared with the blank control group, Toll-like receptor-4 mRNA expression was significantly higher in the TNBS model group (P < 0.01). Compared with the TNBS model group, Toll-like receptor-4 mRNA expression was significantly lower in the 5-aminosalicylic acid treatment group and resina draconis group (P < 0.01). Compared with the 5-aminosalicylic acid treatment group, Toll-like receptor-4 mRNA expression was significantly lower in the resina draconis group (P < 0.01) (Table 4).

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引言

Ulcerative colitis is a cryptogenic chronic non-specificity inflammatory bowel disease, which associates with infection, heredity, intestinal microecology and inflammation^[1]. The effects of dendritic cells have been a hot point in recent studies^[2].

Studies have shown that dendritic cells are antigen presenting cells, and regulate immune response in the intestinal environment (antigen enriched). This regulatory functional defect would cause inflammatory bowel disease, but Toll-like receptor-4/nuclear factor-κB signaling pathway plays an important role in above-mentioned regulation^[3]. Previous studies demonstrated that, humoral immunity mediated by Th2 is the dominant position in UC^[4]. The expression of mRNA in cytokines interleukin-6 and interleukin-8 in patients with activities of UC were obviously higher than the inactive period, which play a key role in the development of inflammation^[5]. Dendritic cells is the most potent and the only antigen-presenting cell which can activate T lymphocytes^[6]. So far, the change in the signal of Toll-like receptor-4/nuclear factor-κB on the treatment of ulcerative colitis is rarely reported. Resina draconis, which is selected by this study team from clinical practice, is an effective prescription in treatment of ulcerative colitis.

A previous study has confirmed that resina draconis has a certain effect on improving the balance of inflammatory factors in rats with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis^[7].

This study observed the effects of resina draconis on the changes in dendritic cell phenotypes and Toll-like receptor, investigated immunological pathogenesis of ulcerative colitis and intervention mechanisms of resina draconis, and provided a basis for seeking a novel Chinese medicine for treatment of ulcerative colitis in a rat model of TNBS-induced inflammatory bowel disease.

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材料方法

Design

A randomized controlled experiment of animal models.

Time and setting

The experiment was completed in the 309^th Hospital of PLA and the Institute of Drug and Instrument Control, General Logistics Department of Chinese People’s Liberation Army from August 2010 to March 2012.

Materials

Animals

A total of 44 male Sprague-Dawley rats aged 6-8 weeks and weighing 180-220 g were provided by the Institute of Drug and Instrument Control, General Logistics Department of Chinese PLA (first class, certificate No. H00117). The rats were housed in the specific-pathogen free laboratory at Experimental Animal Center, Department of Pharmacology, Institute of Drug and Instrument Control, General Logistics Department of Chinese PLA. The rats were allowed to acclimate to their new surroundings for 1 week before experiments.

Drugs and reagents

Resina draconis (dragon’s blood, common bletilla tuber) is a compound Chinese medicine^[8]. Stock solution per milliliter contains resina draconis crude drug 2.0 g (adult dose: [1.45 g/(kg·d)], which is provided by the Manufacturing Laboratory, the 309^th Hospital of Chinese PLA. Resina draconis dose (5.0 g/kg) was equal to 3.5 times the daily dose of adult in clinic.

Methods

Grouping

The involved 44 rats were randomly assigned to four groups (n = 11): blank control group, TNBS model group, resina draconis group, and 5-aminosalicylic acid treatment group. The rats in the TNBS model group were injected with a mixture of TNBS 30 mg and 50% alcohol 0.25 mL. The resina draconis group was intragastrically treated with draconis [0.75 g(kg·d)] and the 5-aminosalicylic acid treatment group was given 5-aminosalicylic acid [100 mg(kg·d)] in the same way for 10 days. One rat was selected from each group in every experiment.

Establishment of colitis models and drug intervention

With the exception of blank control group, TNBS-induced ulcerative colitis models were established in the TNBS model group, resina draconis group, and 5-aminosalicylic acid treatment group in accordance with the methods of Sobczak et al ^[9]. The mixture of TNBS 30 mg and 50% alcohol 0.25 mL was injected into the rat intestine. The rats appeared any of the items can be successful model criteria, such as stools, occult blood positive, gross blood. On day 2 following model induction, the rats in the resina draconis and 5-aminosalicylic acid treatment groups were intragastrically treated with resina draconis [0.75 g(kg·d)] and 5-aminosalicylic acid [100 mg(kg·d)] respectively for 10 days. The rats in the blank control and TNBS model groups were administered a normal diet. 10 days later, the rats were intraperitoneally anesthetized with 2% sodium pentobarbital (3 mL/kg). An incision was made on the middle of sagittal line. The specimen of colon 8 cm above the anus was collected and the enteric cavity was incised along mesentery. Intestinal contents were washed with cold saline. Colon tissues were rapidly stored at -70 ℃ nitrogen canister for further use.

Evaluation of indexes in colitis rats following model establishment

In accordance with the method of Cooper et al ^[10], disease activity index was scored by recording rat weight and stool condition. Myeloperoxidase activity was measured within 1 week^[11]. One unit of myeloperoxidase activity was defined as the quantity of enzyme that degraded 1 mol H₂O₂ at 37 ℃ per g wet tissue. During the assessment of disease activity index, macroscopic colonic damage score, histopathological score and myeloperoxidase activity, the data of dead rats were excluded.

Detection of dendritic cell phenotypes in the mesenteric lymph node

Isolation of dendritic cells with immunomagnetic beads: Mesenteric lymph node was sterilely obtained from the rats to prepare single cell suspension. In accordance with the method of Brenan et al ^[12], dendritic cells were isolated from the rat mesenteric lymph node using a MiniMACS immunomagnetic separation system (MihenyiBiotec, Germany). Totally seven sets of rats received immunomagnetic bead separation technique.

Dendritic cells surface markers major histocompatibility complex class II and CD86 expression: After isolation, cells were quantified. Cell suspension of each rat was divided into two tubes, which was respectively incubated in PE-0X62 (Serotec, Oxford, UK), FITC-major histocompatibility complex class II (eBioscience, San Diego, CA, USA) and PE-0X62, FITC-CD86 (eBioscience) at 4°C for 30 minutes. The specimens were fixed in 1% paraformaldehyde, and measured using flow cytometry within 24 hours^[13].

Determination of nuclear factor-κB expression

In accordance with the method of Tanaka^[13], streptavidin-peroxidase immunohistochemical staining was used to detect nuclear factor-κB expression^[14]. Nuclear factor-κB p65 positive cells mainly contained mucosal cells, monocytes and macrophages. Brown particles were visible in the cytoplasm and nuclei. Image analysis technique was used to quantify the average number of positive cells within 1 mm × 1 mm so as to evaluate staining intensity.

Total RNA (Code: D9108; TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning Province, China) was extracted from about 100 mg tissues by one-step method. Absorbance values were measured with an ultraviolet spectrophotometer at 260 nm/280 nm. RNA purity was calculated. Using a PrimeScriptTMRT reagent Kit (Code: DRR037A; TaKaRa), RNA was reverse transcribed into cDNA. All procedures were performed strictly according to the instructions. A total of 25 μL of products were stored at -20 ℃ for polymerase chain reaction (PCR).

Real-time fluorescent quantitative PCR was conducted^[15] by using the high temperature method. Amplified specimens: glyceraldehyde phosphate dehydrogenase was considered as an internal reference (control)^[16]. Measurement results: normal specimens served as controls. Primers for amplification were synthesized by Beijing SBS Genetech Co., Ltd., Beijing, China. Rat Toll-like receptor-4: annealing temperature: 55 ℃; upstream primer: 5’-GTG GGT CAA GGA CCA GAA AA-3’, downstream primer: 5’-GAA ACT GCC ATG TCT GAG CA-3’; product size: 526 bp. Rat glyceraldehyde phosphate dehydrogenase (GAPDH): annealing temperature: 52 ℃; upstream primer: 5’-CAC CAT GGA GAA GGC CGG GG-3’, downstream primer: 5’-GAC GGA CAC ATT GGG GGT AG-3’, product size: 408 bp.

Reaction system of PCR was as follows^[17]: Taq DNA polymerase, dNTPs, dye, stabilizer, modifier, reaction buffer, reaction product, upstream and downstream primers. After shaking, the specimens were transiently centrifuged. A little mineral oil was added for amplification. Reaction conditions are as follows: 94 ℃ for 3 minutes, 30 cycles of 94 ℃ for 40 seconds, annealing temperature for 50 seconds and 72 ℃ for 90 seconds, followed by

72 ℃ for 10 minutes. A total of 6 μL of amplified products obtained from each specimen were electrophoresed in 1% agarose gel containing goldview nucleic acid stain^[18]. DNA Marker (DL2000) served as standard molecular weight marker. The specimens were observed with an ultraviolet transilluminator, photographed with a digital camera. The target electrophoresis strip was analyzed with BIO-PROFIF gel image analytical system (VL Co., France), and corresponding internal reference electrophoresis strip served as a reference. Results were represented by the ratio of their integral absorbance (A_{260 nm}).

Main outcome measures

Expression rates of major histocompatibility complex II and CD86, as well as expression levels of Toll-like receptor-4 and nuclear factor-κB, were observed.

Statistical analysis

Data were analyzed using SPSS 13.0 software, and measurement data were expressed as the mean±SD. Intergroup comparison was conducted using one-way analysis of variance. Intergroup comparison of numeration data was done using Chi-square test. A value of P < 0.05 was considered statistically significant.

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讨论

Recently, the incidence of ulcerative colitis has obviously increased. Ulcerative colitis has been a great clinical difficulty, because of its complicated etiopathogenesis, difficult to treat and high incidence. How to explore its pathogenesis and to choose effective therapeutic method are hot points to be studied in the field of digestion. This study observed the action mechanism of resina draconis in a rat model of TNBS-induced ulcerative colitis. In this study, erosion, ulcer and tissue adhesion were visible in the rat intestine. Moreover, histopathology displayed necrotic intestinal wall, inflammatory cell infiltration, showing success model induction^[19].

Draconis has anti-inflammatory and wound healing effect in the TNBS-induced experimental ulcerative colitis rats, and reduce the severity of colonic damage.

Dragon’s blood is a common Chinese medicine, contains various bioactive substances. Modern pharmacology verified that main ingredients of dragon’s blood are steroidal saponins and phytoalexin^[20-21]. Steroidal saponins can decrease capillary permeability, diminish prostaglandin and arachidonic acid, and promote the growth of granulation tissue. Phytoalexin can suppress bacterium and prevent corrosion. Resina draconis used in this study is composed of dragon’s blood and common bletilla tuber. Previous study has verified that resina draconis has obvious anti-inflammatory and repair effects. Following treatment, interleukin-1β and interleukin-4 levels were remarkably decreased, CD8⁺ contents increased, and the ratio of CD4⁺/CD8⁺ kept a certain proportion^[22-23]. Macroscopic colonic damage score, histopathological score and myeloperoxidase activity were decreased in the resina draconis group compared with the TNBS model group. There was no significant difference in above-mentioned indexes between resina draconis group and 5-aminosalicylic acid treatment group, suggesting that resina draconis and 5-aminosalicylic acid can be used as first-choice drugs in treatment of acute intestinal inflammation.

Intestinal dendritic cells play an important role in protecting intestinal mucosal barrier by regulating innate and acquired immunities of the intestine^[24]. Under the antigen stimulation, dendritic cells became mature, presented antigen and highly expressed major histocompatibility complex class II and CD86, induced and aggravated intestinal inflammation. Therefore, the expression of these surface molecules can reflect the severity of intestinal immune/inflammatory reaction. In addition, the detection of dendritic cells in the mesenteric lymph nodes can reflect the changes of intestinal dendritic cells. The present study used anti-rat (OX62) magnetic bead to rapidly separate dendritic cells from the mesenteric lymph node, and these cells were subjected to flow cytometry. Results revealed that expression rates of major histocompatibility complex class II and CD86 were significantly higher in the TNBS model group than those in the blank control group, suggesting that mature dendritic cells exert antigen presentation function. Following administration of resina draconis, expression rates of major histocompatibility complex class II and CD86 were diminished compared with the TNBS model group. Above-described results indicated that resina draconis exerts the effect of an immunomodulator, reduced antigen presentation of dendritic cells, inhibited immunological reaction, and lessened enteric inflammatory damage. 5-Aminosalicylic acid as a control drug has an identical effect. This study also confirmed that dendritic cells exert a crucial effect on immunoloregulation, which were consistent with the results from previous studies^[25-27], and it would be an important target molecule in treatment of inflammatory bowel disease.

The results show that the present study, blood and intracellular signals inhibit colitis by inhibiting Toll-like receptor-4 protein and activation of nuclear factor-κB, down inflammatory mediators and inflammatory cytokines. These molecular changes improve TNBS-induced colitis. Thus, blood can be used as a natural inhibition Toll-like receptor-4/ nuclear factor-κB signaling pathway.

This study measured nuclear factor-κB p65 expression in the colonic mucosa using immunohistochemical method. Results revealed that significant differences in nuclear factor-κB p65 expression were detected in each group. Toll-like receptor-4 and nuclear factor-κB expression in the submucosa and lamina propria was significantly enhanced in the TNBS model group than that in the blank control group, and its expression levels were identical to morphological score. Toll-like receptor-4 gene levels and nuclear factor-κB protein levels were reduced in the resina draconis group and 5-aminosalicylic acid treatment group, which indicated that Toll-like receptor-4 and nuclear factor-κB participated in immunoloregulation. Its mechanisms may be that: when Gram-positive and Gram-negative bacteria appeared in the rat intestinal tract, the activation of Toll-like receptor-4 and nuclear factor-κB signaling pathway led to the release of various inflammatory mediators and immunological reaction of the colon^[28-29]. Resina draconis reduced Toll-like receptor-4 mRNA transcriptional level, diminished Toll-like receptor-4 protein synthesis and expression, blocked the transduction of Toll-like receptor-4/nuclear factor-κB signaling pathway, decreased the release of proinflammatory factors, suppressed excessive activation of immune system, corrected abnormal immune/inflammatory reaction, resulting in treating ulcerative colitis. Moreover, resina draconis has double effects of hemostasis and blood flow promotion during intestinal bleeding. Resina draconis can protect intestinal mucosal barrier and relieve inflammatory reaction by improving microcirculation.

In summary, an appropriate dose of resina draconis can lessen TNBS-induced experimental colitis by regulating antigen presentation of intestinal dendritic cells.

复方血竭对结肠炎模型大鼠Toll样受体4/核因子κB及树突状细胞表型的影响

Effects of resina draconis on Toll-like receptor-4/nuclear factor-kappaB and dendritic cell phenotypes in colitis rats

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