健脾消痞方联合推拿对厌食症胃肠动力、Cajal间质细胞自噬的影响

Effect of Jianpi Xiaopi Prescription Combined with Massage on Gastrointestinal Motility and Autophagy of Interstitial Cells of Cajal in Anorexia

目的:基于促分裂原活化的蛋白激酶(MAPK/MEK)/胞外信号调节激酶1/2(ERK1/2)信号通路分析健脾消痞方联合推拿疗法对厌食症幼鼠胃肠动力及胃窦Cajal间质细胞自噬的作用机制。方法:无特定病原体(SPF)级SD健康幼鼠50只随机分为健康组、模型组(小儿厌食症模型)、中药组(模型+健脾消痞方干预)、联合组(模型+健脾消痞方+推拿疗法干预),每组10只。另10只大鼠建立小儿厌食症模型给予MEK/ERK信号通路抑制剂PD98059进行注射作为抑制剂组。每2周测量幼鼠体质量和摄食量;比色法测血清淀粉酶水平;放射免疫法测血清胃泌素水平;苏木精-伊红(HE)染色观察病理形态;实时PCR检测Cajal间质细胞标志物氨酸蛋白激酶生长因子受体(c-kit)、微管相关蛋白轻链3(LC3)、自噬基因苄氯素1(Beclin1)基因表达。蛋白印迹检测MEK、ERK1/2、p-MEK、p-ERK1/2蛋白表达。结果:与健康组比较,模型组幼鼠体质量、体质量增长率、摄食量、c-Kit、小肠推进比、血清淀粉酶、血清胃泌素降低,MEK、ERK1/2、LC3、Beclin1、胃内残留水平升高(P<0.05)。在经过中药组及联合组干预后,幼鼠体质量、体质量增长率、摄食量、c-Kit、小肠推进比、血清淀粉酶、血清胃泌素升高,MEK、ERK1/2、LC3、Beclin1、胃内残留水平降低(P<0.05),且联合组较中药组效果更佳(P<0.05)。联合组与抑制剂组幼鼠蛋白表达比较,差异无统计学意义(P>0.05).结论:健脾消痞方联合推拿可能是通过抑制MEK-ERK1/2信号通路激活,从而抑制胃窦Cajal间质细胞自噬,改善胃肠道功能。

Objective:To analyze the mechanism of Jianpi Xiaopi Prescription combined with massage on gastrointestinal motility and autophagy of interstitial cells of Cajal in anorexia infant rats based on MEK-ERK1/2 signaling pathway.Methods:A total of 50 SPF healthy SD infant rats were randomly divided into healthy group,model group(infantile anorexia model),traditional Chinese medicine group(model+Jianpi Xiaopi Prescription intervention),and combined group(model+Jianpi Xiaopi Prescription+massage intervention),with 10 in each group.Another 10 rats of infantile anorexia model were injected with MEK/ERK signaling pathway inhibitor PD98059,which were used as the inhibitor group.The body weight and food intake of the rats were measured every 2 weeks.Serum amylase was measured by colorimetric method,and serum gastrin was measured by radioimmunoassay.HE staining was used to observe the pathological morphology.The mRNA expressions of c-kit,LC3 and Beclin1 in interstitial cells of Cajal were detected by RT-PCR.The protein expressions of MEK,ERK1/2,P-MEK and P-ERK1/2 were detected by western blot.Results:Compared with the conditions in healthy group,the body weight,growth rate of body weight,food intake,c-kit,intestinal propulsive ratio,serum amylase and serum gastrin were decreased in model group,while MEK,ERK1/2,LC3,Beclin1 and gastric residual volume were increased(P<0.05).After intervention,the body weight,growth rate of body weight,food intake,c-kit,intestinal propulsive ratio,serum amylase and serum gastrin were increased,while MEK,ERK1/2,LC3,Beclin1 and gastric residual volume were decreased(P<0.05),with the effect of combined group better than that of combined group(P<0.05).There was no significant difference in the protein expression between combined group and inhibitor group(P>0.05).Conclusion:Jianpi Xiaopi Prescription combined with massage may inhibit the autophagy of interstitial cells of Cajal in gastric antrum and improve gastrointestinal function by inhibiting the activation of MEK-ERK1/2 signaling pathway.