基于IL-23/IL-17轴探讨消脂化纤汤联合BM-MSCs移植对实验性NASH肝硬化小鼠的作用效应

Effect of Xiaozhi Huaxian Decoction Combined with BM-MSCs Transplantation on Experimental NASH Cirrhosis Mice Based on IL-23/IL-17 Axis

目的:基于IL-23/IL-17轴探讨消脂化纤汤联合骨髓间充质干细胞(BM-MSCs)移植对实验性非酒精性脂肪性肝炎(NASH)肝硬化小鼠的作用。方法:56只健康SPF级C57BL/6小鼠随机选出8只用于骨髓间充质干细胞的分离与培养,8只小鼠作为空白对照组,其余40只小鼠采用蛋氨酸胆碱缺乏饮食(MCD)联合10%四氯化碳(CCl4∶橄榄油=1∶9)腹腔注射共8周制备实验性NASH肝硬化小鼠模型。造模成功后将其分为模型组、自然恢复组、中药干预组、BM-MSCs移植组和中药干预联合BM-MSCs移植组。BM-MSCs移植组经尾静脉注射BM-MSCs,中药干预组给予消脂化纤汤方灌胃,中药干预联合BM-MSCs移植组予以消脂化纤汤方灌胃联合尾静脉注射BM-MSCs。BM-MSCs移植在第9周进行,灌胃均为每日2次,连续4周,12周末处死小鼠并采集血液及标本。采用全自动生化分析仪检测血清生化指标(ALT、AST、ALB),ELISA法测定血清及肝组织炎性因子IL-23、IL-17水平,ELISA法测定血清及肝组织中肝细胞再生增强因子(ALR)表达,RT-qPCR法检测肝脏淋巴细胞中IL-17、IL-23mRNA的表达。结果:小鼠一般状态观察,与空白对照组比较,模型组小鼠精神萎靡,活动减少,毛色暗,进食减少,体质量逐渐降低;各干预组较模型组均有不同程度改善;与空白对照组比较,模型组和自然恢复组小鼠血清ALT及AST活性、血清及肝组织炎性因子(IL-23、IL-17)水平、肝脏淋巴细胞中IL-17和IL-23 mRNA相对表达水平明显升高(P<0.05),血清及肝组织ALR水平、ALB含量降低(P<0.05);与模型组比较,中药干预组、BM-MSCs移植组、中药干预联合BM-MSCs移植组小鼠血清ALT及AST活性、血清及肝组织炎性因子(IL-23、IL-17)水平、肝脏淋巴细胞中IL-17和IL-23 mRNA相对表达水平明显降低(P<0.01),血清及肝组织ALR水平、ALB含量升高(P<0.01);与BM-MSCs移植组和中药干预组比较,中药干预联合BM-MSCs移植组改善最为明显(P<0.05)。结论:消脂化纤汤联合BM-MSCs移植对改善实验性NASH肝硬化小鼠肝脏功能、促进BM-MSCs移植转/分化及肝脏细胞再生具有良好效应,其机制可能与抑制机体IL-23/IL-17炎症轴过度激活,改善肝脏免疫微环境相关。

Objective:To investigate the effect of Xiaozhi Huaxian Decoction combined with bone marrow mesenchymal stem cells(BM-MSCs) transplantation on experimental cirrhosis mice with non-alcoholic steatohepatitis(NASH) based on IL-23/IL-17 axis.Methods:8 mice were randomly selected from a total of 56 healthy SPF C57BL/6 mice for BM-MSCs culture,another 8 mice were used as the blank control,and the rest 40 mice were established the NASH cirrhosis model by intraperitoneal injection of methionine choline deficiency diet(MCD) combined with 10% carbon tetrachloride(CCl4∶olive oil = 1∶9) for 8 weeks.The successful modeling mice were randomly divided into the model group,the natural recovery group,the Chinese medicinal intervention group,the BM-MSCs transplantation group,and the Chinese medicinal intervention combined with BM-MSCs transplantation group.The BM-MSCs transplantation group was injected with BM-MSCs through the tail vein,the Chinese medicinal intervention group was given Xiaozhi Huaxian Decoction by gavage,and the Chinese medicinal intervention combined with BM-MSCs transplantation group was given Xiaozhi Huaxian Decoction by gavage combined with tail vein transplantation of BM-MSCs.BM-MSCs transplantation was performed once at the 9th week,and gavage was performed twice a day from the 9th week for 4 consecutive weeks.The mice were sacrificed at the end of the 12th week,and blood and samples were collected.The serum biochemical indexes(ALT,AST,ALB) were detected by automatic biochemical analyzer.The levels of inflammatory factors of IL-23 and IL-17 in serum and liver tissues were measured by ELISA method.The expression of augmenter of liver regeneration(ALR) in serum and liver tissues was also measured by ELISA method.The mRNA expressions of IL-17 and IL-23 in liver lymphocytes were detected by RT-qPCR.Results:In terms of the general state of the mice,the mice in the model group gradually showed mental malaise,decreased activity,lackluster hair,decreased food intake and decreased body weight compared with those in the blank control group;after 4 weeks of medical interventions,the general state was improved in the mice.Compared with those in the blank group,the activities of serum ALT and AST,the levels of IL-23 and IL-17 in serum and liver tissues,as well as the mRNA expressions of IL-17 and IL-23 in liver lymphocytes were significantly increased(P<0.05),and the levels of ALR and ALB in serum and liver tissues were decreased in the model group and in the natural recovery group(P<0.05).Compared with those in the model group,the activities of serum ALT and AST,the levels of IL-23and IL-17 in serum and liver tissues,as well as the mRNA expressions of IL-17 and IL-23 in liver lymphocytes were significantly decreased(P<0.01),and the levels of ALR and ALB in serum and liver tissues were increased in the Chinese medicinal intervention group,the BM-MSCs transplantation group and the Chinese medicinal intervention combined with BM-MSCs group(P<0.01).In terms of the improvements of the above indexes,they were more significant in the Chinese medicinal combined with BMMSCs transplantation group than those in the BM-MSCs transplantation group and in the Chinese medicinal intervention group(P<0.05).Conclusion:Xiaozhi Huaxian Decoction combined with BM-MSCs transplantation has a good effect on improving liver function,promoting BM-MSCs transplantation/differentiation and liver cell regeneration in mice with experimental NASH cirrhosis.The mechanism may be related to inhibiting excessive activation of IL-23/IL-17 inflammatory axis and improving liver immune microenvironment.