Mild hypothermia combined with adipose-derived mesenchymal stem cells protects hepatic function in hepatic ischemia-reperfusion injury

doi:10.3969/j.issn.2095-4344.1691

Abstract

Abstract:

BACKGROUND: Hepatic ischemia-reperfusion injury is the bottleneck restricting liver surgeries as its complex and diverse mechanisms. How to reduce liver ischemia-reperfusion injury has always been one of the hotspots and difficulties in medical research.
OBJECTIVE: To explore the protective mechanism of mild hypothermia pretreatment combined with adipose-derived mesenchymal stem cells transplantation for hepatic ischemia reperfusion injury in rats.
METHODS: Sixty male Sprague-Dawley rats (provided by the Experimental Animal Center of Kunming Medical University in China) were randomly divided into six groups with 10 rats in each group: sham operation group (Sham), hepatic ischemia-reperfusion injury group (HIRI), mild hypothermia group (MH), adipose-derived mesenchymal stem cells group (ADMSCs), mild hypothermia+ADMSCs group (MH+ADMSCs) and mild hypothermia+ADMSCs+ ERK inhibitor group (MH+ADMSCs+PD98059). The rats were given mild hypothermia, adipose-derived mesenchymal stem cells, or ERK inhibitor intervention at 30 minutes before ischemia. The serum and liver tissue samples were collected 12 hours after hepatic ischemia-reperfusion.
RESULTS AND CONCLUSION: The activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase in the serum of groups MH, ADMSCs and MH+ADMSCs were significantly higher than those in the HIRI group (P < 0.05). The activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase in group MH+ADMSCs were significantly lower than those in groups MH and ADMSCs (P < 0.05). Subsequently, compared with group HIRI, the activities of superoxide dismutase and glutathione peroxide were significantly higher in groups MH, ADMSCs and MH+ADMSCs (P < 0.05), whereas the levels of malondialdehyde were significantly lower in the three groups (P < 0.05). The activities of superoxide dismutase and glutathione peroxide in group MH+ADMSCs were significantly higher than those in groups MH and ADMSCs (P < 0.05), whereas the level of malondialdehyde in group MH+ADMSCs was lower than that in groups MH and ADMSCs (P < 0.05). The relative expression level of p-ERK1/2 in the liver tissue was significantly increased in groups MH, ADMSCs and MH+ADMSCs as compared with group HIRI (P < 0.05), but there were no significant differences in the relative expression level of ERK1/2 among the groups. Furthermore, the apoptotic cell proportion of liver tissues in groups HIRI and MH+ADMSCs+PD98059 were higher than that in groups MH, ADMSCs and MH+ADMSCs. These results were in consistent with the findings of western blot detection. To conclude, mild hypothermia combined with adipose-derived mesenchymal stem cells transplantation has a protective effect against hepatic ischemia-reperfusion injury, and the mechanism may be through activating ERK pathway to downregulate the percentage of apoptotic hepatocytes in vivo.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Liver, Reperfusion Injury, Adipose Tissue, Mesenchymal Stem Cell Transplantation, Hypothermia, Induced, Tissue Engineering

CLC Number:

Liu Jian, Li Li, Wang Xiaochuan, Zhang Shengning, Li Laibang, Mang Yuanyi, Gao Yang, Chen Yonglin, Ren Gang, Li Wang. Mild hypothermia combined with adipose-derived mesenchymal stem cells protects hepatic function in hepatic ischemia-reperfusion injury[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2081-2087.

Figures/Tables 3

2.2 亚低温联合脂肪间充质干细胞减轻肝缺血再灌注损伤诱导的氧化应激采用试剂盒检测各组大鼠肝组织中超氧化物歧化酶、丙二醛和谷胱甘肽过氧化物酶水平。从表2中可发现，肝缺血再灌注损伤组、亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组肝组织中超氧化物歧化酶和谷胱甘肽过氧化物酶水平显著低于假手术组(P < 0.05和P < 0.01)，而丙二醛含量显著高于假手术组(P < 0.05和P < 0.01)。同时，亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组肝组织中超氧化物歧化酶和谷胱甘肽过氧化物酶水平显著高于肝缺血再灌注损伤组 (P < 0.05和P < 0.01)，而丙二醛含量低于肝缺血再灌注损伤组(P < 0.01)。亚低温+脂肪间充质干细胞组肝组织中超氧化物歧化酶和谷胱甘肽过氧化物酶水平显著高于亚低温组和脂肪间充质干细胞组(P < 0.05)，而丙二醛含量低于亚低温组和脂肪间充质干细胞组(P < 0.05和P < 0.01)。由此可知，亚低温联合脂肪间充质干细胞可缓解肝缺血再灌注损伤诱导的氧化应激。 2.3 亚低温联合脂肪间充质干细胞对ERK信号通路的影响采用Western blotting检测各组肝组织中ERK1/2的磷酸化水平，见图1。肝缺血再灌注损伤组中p-ERK1/2的相对表达量明显低于假手术组(P < 0.01)；亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组肝组织中p-ERK1/2的相对表达量显著高于肝缺血再灌注损伤组 (P < 0.01)，且亚低温+脂肪间充质干细胞组肝组织中p-ERK1/2的相对表达量显著高于亚低温组和脂肪间充质干细胞组(P < 0.05)，但ERK蛋白的表达水平在各组间差异无显著性意义。由此可知，亚低温联合脂肪间充质干细胞处理后激活了ERK信号通路。"

References

[1] 吴勤荣,时军,王永刚. 细胞凋亡与肝移植缺血再灌注损伤[J]. 中国组织工程研究与临床康复, 2011,15(18):3371-3375.

[2] Martikos G, Kapelouzou A, Peroulis M, et al. Remote Ischemic Preconditioning Decreases the Magnitude of Hepatic Ischemia-Reperfusion Injury on a Swine Model of Supraceliac Aortic Cross-Clamping. Ann Vasc Surg. 2018; 48:241-250.

[3] Li Y, Yang Y, Feng Y, et al. A review of melatonin in hepatic ischemia/reperfusion injury and clinical liver disease. Ann Med. 2014;46(7):503-511.

[4] 陈茂松,王军,欧雷. 促红细胞生成素对大鼠肝脏缺血再灌注损伤的抗炎作用[J]. 广东医学,2016,37(6):828-830.

[5] Hernandez-Alejandro R, Zhang X, Croome KP, et al. Reduction of liver ischemia reperfusion injury by silencing of TNF-α gene with shRNA. J Surg Res. 2012;176(2):614-620.

[6] Behrends M, Hirose R, Serkova NJ, et al. Mild hypothermia reduces the inflammatory response and hepatic ischemia/ reperfusion injury in rats. Liver Int. 2006;26(6):734-741.

[7] Sia D, Hoshida Y, Villanueva A, et al. Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes. Gastroenterology. 2013;144(4): 829-840.

[8] Yin TC, Wu RW, Sheu JJ, et al. Combined Therapy with Extracorporeal Shock Wave and Adipose-Derived Mesenchymal Stem Cells Remarkably Improved Acute Ischemia-Reperfusion Injury of Quadriceps Muscle. Oxid Med Cell Longev. 2018;2018:6012636.

[9] Zhang JB, Wang XQ, Lu GL, et al. Adipose-derived mesenchymal stem cells therapy for acute kidney injury induced by ischemia-reperfusion in a rat model. Clin Exp Pharmacol Physiol. 2017;44(12):1232-1240.

[10] Lin KC, Yip HK, Shao PL, et al. Combination of adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes for protecting kidney from acute ischemia-reperfusion injury. Int J Cardiol. 2016;216:173-185.

[11] Yin TC, Wu RW, Sheu JJ, et al. Combined Therapy with Extracorporeal Shock Wave and Adipose-Derived Mesenchymal Stem Cells Remarkably Improved Acute Ischemia-Reperfusion Injury of Quadriceps Muscle. Oxid Med Cell Longev. 2018;2018:6012636.

[12] Tanzi MC, Farè S. Adipose tissue engineering: state of the art, recent advances and innovative approaches. Expert Rev Med Devices. 2009;6(5):533-551.

[13] Choi JW, Shin S, Lee CY, et al. Rapid Induction of Osteogenic Markers in Mesenchymal Stem Cells by Adipose-Derived Stromal Vascular Fraction Cells. Cell Physiol Biochem. 2017; 44(1):53-65.

[14] Zhou L, Song Q, Shen J, et al. Comparison of human adipose stromal vascular fraction and adipose-derived mesenchymal stem cells for the attenuation of acute renal ischemia/ reperfusion injury. Sci Rep. 2017;7:44058.

[15] Saito K, Fukuda N, Matsumoto T, et al. Moderate low temperature preserves the stemness of neural stem cells and suppresses apoptosis of the cells via activation of the cold-inducible RNA binding protein. Brain Res. 2010;1358: 20-29.

[16] 邱季,方芳,李珍,等. ERK-CREB信号通路在白藜芦醇预处理对大鼠局灶性脑损伤缺血再灌注损伤神经保护中的作用[J].安徽医科大学学报, 2013, 48(10):1152-1155.

[17] 刘琴,林亚平,陈文,等.针刺联合亚低温对脑损伤缺血再灌注损伤大鼠脑组织p-Raf1、p-ERK1/2的影响[J].湖南中医药大学学报, 2016, 36(1):58-62.

[18] Choi DE, Jeong JY, Choi H, et al. ERK phosphorylation plays an important role in the protection afforded by hypothermia against renal ischemia-reperfusion injury. Surgery. 2017; 161(2):444-452.

[19] Chen Y, Ba L, Huang W, et al. Role of carvacrol in cardioprotection against myocardial ischemia/reperfusion injury in rats through activation of MAPK/ERK and Akt/eNOS signaling pathways. Eur J Pharmacol. 2017;796:90-100.

[20] Lin B, Yu H, Lin Y, et al. Suppression of GRASP65 phosphorylation by tetrahydrocurcumin protects against cerebral ischemia/reperfusion injury via ERK signaling. Mol Med Rep. 2016;14(5):4775-4780.

[21] 史光军,张亚东,胡音音,等. 脂肪间充质干细胞移植上调肝脏增殖细胞核抗原表达促进肝细胞的再生[J]. 中国组织工程研究, 2017, 21(17):2690-2695.

[22] Kohli V, Selzner M, Madden JF, et al. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver. Transplantation. 1999;67(8):1099-1105.

[23] Granger DN, Kvietys PR. Reperfusion injury and reactive oxygen species: The evolution of a concept. Redox Biol. 2015;6:524-551.

[24] Pell VR, Spiroski AM, Mulvey J, et al. Ischemic preconditioning protects against cardiac ischemia reperfusion injury without affecting succinate accumulation or oxidation. J Mol Cell Cardiol. 2018;123:88-91.

[25] Yang YF, Zhang MF, Tian QH, et al. SPAG5 interacts with CEP55 and exerts oncogenic activities via PI3K/AKT pathway in hepatocellular carcinoma. Mol Cancer. 2018;17(1):117.

[26] Dai HB, Xu MM, Lv J, et al. Mild Hypothermia Combined with Hydrogen Sulfide Treatment During Resuscitation Reduces Hippocampal Neuron Apoptosis Via NR2A, NR2B, and PI3K-Akt Signaling in a Rat Model of Cerebral Ischemia-Reperfusion Injury. Mol Neurobiol. 2016;53(7): 4865-4873.

[27] Xiao Q, Ye QF, Wang W, et al. Mild hypothermia pretreatment protects hepatocytes against ischemia reperfusion injury via down-regulating miR-122 and IGF-1R/AKT pathway. Cryobiology. 2017;75:100-105.

[28] Adachi N, Liu K, Motoki A, et al. A comparison of protective effects between L-histidine and hypothermia against ischemia-induced neuronal damage in gerbil hippocampus. Eur J Pharmacol. 2006;546(1-3):69-73.

[29] Erlinge D. A Review of Mild Hypothermia as an Adjunctive Treatment for ST-Elevation Myocardial Infarction. Ther Hypothermia Temp Manag. 2011;1(3):129-141.

[30] Zhu P, Zhao MY, Li XH, et al. Effect of low temperatures on BAX and BCL2 proteins in rats with spinal cord ischemia reperfusion injury. Genet Mol Res. 2015;14(3):10490-10499.

[31] Santos EB, Koff WJ, Grezzana Filho Tde J, et al. Oxidative stress evaluation of ischemia and reperfusion in kidneys under various degrees of hypothermia in rats. Acta Cir Bras. 2013; 28(8):568-573.

[32] Zhou T, Liang L, Liang Y, et al. Mild hypothermia protects hippocampal neurons against oxygen-glucose deprivation/reperfusion-induced injury by improving lysosomal function and autophagic flux. Exp Cell Res. 2017;358(2): 147-160.

[33] Kanagawa T, Fukuda H, Tsubouchi H, et al. A decrease of cell proliferation by hypothermia in the hippocampus of the neonatal rat. Brain Res. 2006;1111(1):36-40.

[34] 高小月,张玉泉,杨晓清,等. 人脐带间充质干细胞在组织损伤修复中的研究进展[J]. 生物医学工程与临床, 2018, 22(2):208-213.

[35] Lo CY, Weil BR, Palka BA, et al. Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs): Pilot validation in porcine ischemia-reperfusion model. Biomaterials. 2016;74:19-30.

[36] 王汝霖,林淼,黎力平,等. 骨髓间充质干细胞来源exosome对大鼠肾缺血再灌注损伤的保护作用[J]. 中华医学杂志,2014, 94(42): 3298-3303.

[37] Yu Y, Lu L, Qian X, et al. Antifibrotic effect of hepatocyte growth factor-expressing mesenchymal stem cells in small-for-size liver transplant rats. Stem Cells Dev. 2010;19(6): 903-914.

[38] Devey L, Mohr E, Bellamy C, et al. c-Jun terminal kinase-2 gene deleted mice overexpress hemeoxygenase-1 and are protected from hepatic ischemia reperfusion injury. Transplantation 2009;88(3):308-316.

[39] Kanazawa H, Fujimoto Y, Teratani T, et al. Bone marrow-derived mesenchymal stem cells ameliorate hepatic ischemia reperfusion injury in a rat model. PLoS One. 2011; 6(4):e19195.

[40] Pan GZ, Yang Y, Zhang J, et al. Bone marrow mesenchymal stem cells ameliorate hepatic ischemia/reperfusion injuries via inactivation of the MEK/ERK signaling pathway in rats. J Surg Res. 2012;178(2):935-948.