半夏泻心汤对小鼠Cajal间质细胞表型维持及相关钙离子通道蛋白的影响

Effects of Banxia Xiexin decoction on phenotype maintenance of cajal mesenchymal cells in mice and their related calcium channel proteins

目的通过观察小鼠胃Cajal间质细胞(interstitial cells of Cajal,ICC)表型变化及相关蛋白,讨论半夏泻心汤调节胃肠运动的细胞分子机制。方法制备半夏泻心汤药物血清,以去离子水灌胃所得血清为对照。取对数生长期的ICC,随机分为空白组、对照组、低浓度组(5%半夏泻心汤药物血清)、中浓度组(10%半夏泻心汤药物血清)和高浓度组(20%半夏泻心汤药物血清),分别给予相应药液,于12小时、24小时和48小时后进行指标检测。采用免疫荧光双染法检测ICC标记蛋白c-kit/α-SMA、c-kit/desmin和c-kit/SMMHC表达,镜下观察半夏泻心汤对小鼠ICC表型维持的影响;采用免疫印迹和实时荧光定量PCR法检测ICC中钙离子通道蛋白三磷酸肌醇受体(inositol 1,4,5-triphosphate receptors,IP3R)、兰尼碱受体(ryanodine receptor,RyR)、磷脂酶C(phospholipases C,PLC)及其mRNA的表达。结果与低浓度和中浓度组相比,高浓度组c-kit阳性表达显著增加(P<0.05),α-SMA阳性表达明显减少(P<0.01),SMMHC表达和desmin表达差异不显著(P>0.05)。与药物干预24小时、48小时组相比,12小时组c-kit阳性表达显著增加(P<0.05);与48小时组相比,12小时组α-SMA阳性表达明显降低(P<0.05);与24小时相比,12小时组α-SMA、desmin和SMMHC平均灰度值无明显差异(P>0.05)。与空白组、对照组比,药物组IP3R、RyR、PLC蛋白及mRNA表达均明显升高(P<0.05)。结论20%半夏泻心汤药物血清干预12小时能显著增强ICC表型的维持,加强c-kit表达,其机制可能与调节钙库受体蛋白IP3R、RyR、PLC表达水平,进而影响相关离子通道蛋白有关。

Objective To discuss the cell and molecular mechanism of Banxia Xiexin decoction regulating gastrointestinal motility, by observing the phenotype changes of gastric interstitial cells of Cajal and their related proteins in mice. Methods The serum containing Banxia Xiexin decoction was prepared, and the serum obtained by deionized water gavage was used as the control. The logarithmic phase of the ICC, were randomly divided into blank group, control group, low concentration (5% serum containing Banxia Xiexin decoction), medium concentration group (10% serum containing Banxia Xiexin decoction) and high concentration group (20% serum containing Banxia Xiexin decoction), given the corresponding solution, respectively in 12 hours, 24 hours a day and 48 hours after the test index. The expression of ICC labeled proteins C-KIT/-SMA, C-Kit/DESmin and C-Kit/SMMHC were detected by immunofluorescence double-staining. The effect of Banxia Xiexin decoction on the maintenance of ICC phenotype in mice was observed under a microscope. Western blot and real-time fluorescent quantitative PCR were used to detect the expression of calcium channel protein inositol 1,4,5-triphosphate (IP3R), Ryanodine receptor (RyR), phospholipases C (PLC) and their mRNA. Results Compared with the low and medium concentration groups, the c-kit positive expression was significantly increased in the high concentration group ( P <0.05), the α-SMA positive expression was significantly decreased ( P <0.01), and the SMMHC expression and desmin expression were not significant difference in different concentration groups ( P <0.05 ). Compared with the 24h drug intervention group and the 48h drug intervention group, the c-kit positive expression was significantly increased in the 12h drug intervention group group ( P <0.05). Compared with the 48h drug intervention group, the α-SMA positive expression was significantly decreased in the 12h drug intervention group ( P <0.05). The mean gray values of α-SMA, desmin and SMMHC in the 24h drug intervention group were not significantly different from those in the 24h drug intervention group ( P >0.05). Compared with the blank group and the control group, the protein and mRNA expression levels of IP3R, RyR and PLC in the drug group were significantly increased ( P <0.05). Conclusion The 20% serum containing Banxia Xiexin decoction can significantly enhance the maintenance of ICC phenotype and enhance c-kit expression after 12h intervention. The mechanism may be related to regulating the expression levels of calcium library receptor proteins IP3R, RyR and PLC, thus affecting related ion channel proteins.