二陈汤通过调控SCF/C-kit通路介导的痰湿型脑出血急性期胃肠功能障碍的研究

Study on the gastrointestinal dysfunction in acute stage of phlegm dampness type cerebral hemorrhage mediated by Erchen Decoction by regulating the SCF/C-kit pathway

目的通过观察二陈汤对痰湿证脑出血急性期大鼠胃肠组织干细胞因子(stemcellfactor,SCF)/干细胞因子受体(C-kit)信号通路的动态变化的影响,探讨痰湿型体质与脑出血急性期胃肠功能障碍的关系及二陈汤的作用机制。方法将60只SD大鼠随机均分为空白组、假手术组、模型组、莫沙必利组(0.27 mg/d)、二陈汤低剂量组和二陈汤高剂量组(217、434 mg/d),每组10只。除空白组、假手术组外其余各组均采用自体血注入法制备大鼠脑出血模型,各组按剂量给予灌胃,空白组、假手术组、模型组给予生理盐水灌胃,每天2次,于给药7天处死大鼠,采用苏木精—伊红(hematoxylin-eosin,HE)染色法观察大鼠胃肠黏膜细胞形态变化,采用实时荧光定量PCR(Real-time PCR,RT-PCR)法和蛋白印迹(Western blot,WB)法检测大鼠胃、小肠组织中的SCF、C-kit蛋白及mRNA的表达水平。结果光镜下见空白组、假手术组大鼠胃肠黏膜细胞结构完整、形态规则,偶见轻微充血现象;模型组见组织细胞充血、水肿、炎性细胞浸润明显、组织糜烂;莫沙必利组及二陈汤低、高剂量组胃肠黏膜各层次欠清晰、排列不规则,部分组织可见组织细胞充血、水肿、炎性细胞浸润,此3组中以二陈汤低剂量组胃肠黏膜表现最为严重。模型组大鼠胃肠组织中SCF、C-kit mRNA表达水平较空白组、假手术组显著降低(P<0.05),莫沙必利组、二陈汤低剂量组、二陈汤高剂量组的表达增加(P<0.05),莫沙必利组和二陈汤高剂量组大鼠胃组织中的SCF mRNA表达显著增加(P<0.05),二陈汤高剂量组C-kit mRNA表达显著增加(P<0.05);小肠组织中的SCF、C-kit mRNA含量随二陈汤浓度提高而增加(P<0.05)。与模型组相比,莫沙必利组、二陈汤低、高剂量组大鼠胃肠组织中SCF、C-kit蛋白表达水平较模型组显著升高(P<0.05),三组中以莫沙必利及高剂量二陈汤效果更佳(P<0.05)。结论脑出血急性期痰湿型胃肠黏膜损伤及功能障碍的作用机制与SCF/C-kit信号通路相关蛋白及mRNA的表达降低有关,二陈汤可通过此机制进行有效干预,上调相关蛋白及mRNA的表达,有利于脑出血急性期胃肠功能障碍恢复。

Objective To observe the effects of Erchen Decoction on the dynamic changes of SCF/C-kit signaling pathway in the gastrointestinal tissue of rats with acute stage of cerebral hemorrhage caused by phlegm dampness syndrome and to explore the relationship between phlegm dampness constitution and gastrointestinal dysfunction during acute stage of cerebral hemorrhage and the mechanism of action of Erchen Decoction.Methods Sixty SD rats were randomly divided into blank group,sham surgery group,model group,mosapride group(0.27 mg/d),Erchen Decoction low-dose group and high-dose group(217 mg/d,434 mg/d),with 10 rats in each group.Except the blank group and the sham surgery group,autologous blood injection method was used to prepare the rat cerebral hemorrhage model.Each group was given gastric perfusion according to the measurement.Rats in the blank group,the sham surgery group,and the model group were given normal saline twice a day.The rats were killed 7 days after administration.Hematoxylin-eosin staining method was used to observe the morphological changes of the gastrointestinal mucosa cells of the rats.Real-time PCR(RT-PCR)and Western blot(WB)methods were used to detect the expression levels of SCF、C-kit protein and mRNA.Results Under the light microscope,the gastrointestinal mucosa cells of the blank group and the sham surgery group were complete in structure and regular in shape,and slight congestion was occasionally seen;the model group showed tissue cell congestion,edema,significant infiltration of inflammatory cells,and tissue erosion;gastrointestinal mucosa of mosapride group and Erchen Decoction low and high dose groups were not clear and arranged irregularly,and some tissues could see congestion,edema and inflammatory cell infiltration of histocytes.Gastrointestinal mucosa of Erchen Decoction low dose group was the most serious in these three groups.The content of SCF and C-kit mRNA in the gastrointestinal tissue of the model group rats was significantly lower than that in the blank group and the sham surgery group(P<0.05),and the content of SCF and C-kit mRNA in the mosapride group,the Erchen Decoction low-dose group,and the Erchen Decoction high-dose group were increased(P<0.05).The content of SCF mRNA in the gastric tissue of the rats in the Mosapride group and Erchen Decoction high-dose group were increased significantly(P<0.05),and the expression of C-kit mRNA in the Erchen Decoction high-dose group were increased most significantly(P<0.05);The content of SCF and C-kit mRNA in small intestine tissue were increased with the up-regulation of Erchen Decoction concentration(P<0.05).The expression levels of SCF and C-kit protein in gastrointestinal tissues of rats in mosapride group,Erchen Decoction low-dose group and high-dose group were significantly higher than those in model group(P<0.05);Among the three groups,mosapride and Erchen Decoction high-dose group had the best effect(P<0.05).Conclusion The mechanism of phlegm dampness type gastrointestinal mucosal injury in the acute stage of cerebral hemorrhage and dysfunction are related to the reduced expression of SCF/C-kit signaling pathway related proteins and mRNA.Erchen Decoction can effectively intervene through this mechanism,upregulate the expression of related proteins and mRNA,which is beneficial for the recovery of gastrointestinal dysfunction in the acute stage of cerebral hemorrhage.