芍药甘草汤对Oddi括约肌功能障碍豚鼠自噬相关蛋白Beclin1和LC3-B的影响

Effects of Shaoyao Gancao Decoction(芍药甘草汤)on Autophagy Related Proteins Beclinl and LC3-B in Guinea Pigs with Sphincter of Oddi Dysfunction

目的探讨芍药甘草汤对豚鼠Oddi括约肌功能障碍的治疗作用及其对自噬相关蛋白Beclin1和LC3-B表达的影响。方法30只豚鼠随机分为正常组、模型组和芍药甘草汤治疗组,每组10只。模型组和芍药甘草汤组按0.6 mg/kg体质量静脉注射吗啡注射液以诱导Oddi括约肌功能障碍模型,正常组豚鼠予等体积0.9%氯化钠溶液静脉注射,每周三次;从第一次造模起,芍药甘草汤组按12.5 g/kg灌胃治疗;正常组和模型组分别予等体积0.4%羧甲基纤维素钠(CMC-Na)灌胃,1次/d,共计4周。末次给药后禁食,收集血清和Oddi括约肌组织。全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平;H&E染色检测Oddi括约肌病理变化;qRT-PCR检测c-Kit、Beclin1、LC3-B mRNA表达;Western blot检测c-Kit、Beclin1、LC3-B蛋白表达。结果(1)与对照组相比,模型组豚鼠血清ALT、AST水平显著增高(P<0.01);与模型组相比,芍药甘草汤组豚鼠血清ALT、AST水平均明显下降(P<0.05)。(2)H&E染色显示,模型组豚鼠Oddi括约肌纤维排列不规则、部分紊乱,并可见炎性细胞浸润,黏膜下纤维组织增生明显、平滑肌层显著增厚;与模型组相比,芍药甘草汤组豚鼠Oddi括约肌组织中纤维排列紊乱、炎性细胞浸润及黏膜下纤维组织增生明显改善。(3)与正常组相比,模型组豚鼠Oddi括约肌中c-Kit的mRNA和蛋白表达显著降低(P<0.01,P<0.05),自噬相关Beclin1和LC3-B的mRNA和蛋白表达明显增加(P<0.01,P<0.05);与模型组相比,芍药甘草汤组c-Kit的mRNA和蛋白表达明显升高(P<0.01),Beclin1和LC3-B的mRNA和蛋白表达则显著减少(P<0.01)。结论芍药甘草汤可有效治疗吗啡诱导的豚鼠Oddi括约肌功能障碍,其作用机制可能是与减少细胞过度自噬、提升Cajal细胞活性有关。

Objective To investigate the therapeutic effects of Shaoyao Gancao Decoction(苟药甘草汤)on sphincter of Oddi dysfuction and explore its influence on the expression of autophagy-related proteins Beclinl and LC3-II.Methods Thirty guinea pigs were randomly divided into normal group,model group and Shaoyao Gancao Decoction group,with 10 guinea pigs in each group.The guinea pigs in model group and Shaoyao Gancao Decoction group were intravenously injected with morphine injection at O.6 mg/kg body weight to induce the sphincter of Oddi dysfuction.The normal group was intravenously injected with normal saline,with three times a week.From the first day of experiment,Shaoyao Gancao Decoction group was treated with Shaoyao Gancao Decoction[12.5 g/(kg d)] by intragastric administration.The normal group and model group were given 0.4%sodium carboxymethyl ellulose(CMC-Na)by intragastric administration once a day for 4 weeks.ll guinea pigs were sacrificed at the end of 4th week,and the related serum and sphincter of Oddi were collected.Serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by automatic biochemical analyzer.H&E staining was used to observe the pathological changes of sphincter of Oddi.The mRNA and protein expressions of c-Kit,Beclinl and LC3-B were respectively detected by qRT-PCR and Western blot.Results①Compared with those of the normal group,serum levels of ALT and AST were significantly increased in the model group(P<0.01);which were obviously decreased in Shaoyaogao Decoction group(P<0.05).②H&E staining showed that the sphincter of Oddi fibers were irregular and partially disordered,and the inflammatory cells were infiltrated.The submucosal fiber tissue hyperplasia was obvious,and the smooth muscle layer was significantly thickened in the guinea pigs of the model group.While the fibrin arrangement disorder,inflammatory cell infiltration and submucosal fibrin hyperplasia of Oddi sphincter tissue were significantly improved in the Shaoyao Gancao Decoction group.③Compared with those of the normal group,the mRNA and protein expressions of c-Kit in the sphincter of Oddi in the model group were significantly decreased(P<O.O01,P<0.05),and Autophagy-associated Beclinl and LC3-B were significantly increased(P<0.01,P<0.05).But the mRNA and protein expressions of c-Kit in the Shaoyao Gancao Decoction group were significantly increased(P<0.01),and the expressions of Beclinl and LC3-B were significantly decreased(all P<0.01).Conclusion Shaoyao Gancao Decoction can effectively treat sphincter of Oddi dysfunction induced by morphine in guinea pigs,and its mechanism may be related to reducing excessive autophagy and enhancing the interstitial cells of Cajal activity.