基于PI3K/Akt通路探讨滋阴润肠法治疗慢传输型便秘机制

Mechanism of nourishing yin and moistening intestine therapy for slow transit constipation based on PI3K/Akt pathway

关键词:  滋阴润肠方  慢传输型便秘  5-HT  Cajal间质细胞  PI3K/Akt

Key words:  Ziyin Runchang Recipe  slow transit constipation  5-HT  Cajal interstitial cells  PI3K/Akt

摘要:

[目的] 考察滋阴润肠法治疗慢传输型便秘（STC）的作用，并基于磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）通路探讨其机制。[方法] 采用盐酸洛哌丁胺灌胃建立STC小鼠模型，将50只雄性ICR小鼠随机分为正常组、模型组、滋阴润肠方低剂量组（1.3 g/kg）、滋阴润肠方高剂量组（2.6 g/kg）和阳性药组（莫沙比利，3 mg/kg），每组10只。各组小鼠按照相应药物浓度灌胃给药，正常组和模型组灌胃给予等剂量生理盐水，边造模边给药治疗，每日1次，连续14 d后检测各组小鼠粪便含水量、首次排黑便时间、肠道传输率；采用酶联免疫吸附法（ELISA）检测各组小鼠结肠组织中5-羟色胺（5-HT）含量；酶联实时荧光定量逆转录聚合酶链反应（RT-PCR）检测小鼠结肠组织中c-细胞因子受体（c-kit）与其干细胞因子（SCF） mRNA水平；蛋白免疫印迹（Western blot）法检测小鼠结肠组织中c-kit、PI3K、磷酸化磷脂酰肌醇3-激酶（p-PI3K）、Akt、磷酸化蛋白激酶B（p-Akt）的蛋白表达水平。[结果] 与正常组相比，模型组小鼠首次排黑便时间显著延长（P<0.05），粪便含水量及肠道传输率显著降低（P<0.05），结肠5-HT含量及c-kit、SCF mRNA水平显著降低（P<0.05），结肠c-kit、p-PI3K/PI3K和p-Akt/Akt蛋白表达量显著降低（P<0.05）。与模型组相比，滋阴润肠方高剂量组小鼠粪便含水量显著升高（P<0.05），滋阴润肠方低剂量组和阳性药组有升高的趋势；滋阴润肠方高剂量组和阳性药组小鼠首次排黑便时间显著缩短（P<0.05），滋阴润肠方低剂量组小鼠具有缩短的趋势；滋阴润肠方各剂量组和阳性药组肠道传输率均显著升高（P<0.05）；在结肠组织中，滋阴润肠方高剂量组c-kit、SCF mRNA水平显著上升（P<0.05），阳性药组具有上升的趋势；滋阴润肠方高剂量组和阳性药组5-HT含量和c-kit、p-PI3K/PI3K、p-Akt/Akt蛋白表达均显著升高（P<0.05）。[结论] 滋阴润肠方具有提高肠道传输率治疗便秘的作用，其作用机制与增加5-HT含量，上调PI3K/Akt表达进而增加Cajal间质细胞数量有关。

Abstract:

[Objective] To explore the effect and mechanism of nourishing yin and moistening intestine in the treatment of slow transit constipation（STC） mice based on PI3K/AKT pathway. [Methods] The STC mice model was established by intragastric administration of loperamide hydrochloride. The 50 male ICR mice were randomly divided into normal group，model group，Ziyin Runchang Recipe low-dose group（1.3 g/kg），Ziyin Runchang Recipe high-dose group（2.6 g/kg） and positive drug group（Mosapili，3 mg/kg），with 10 mice in each group. Mice in each group were administrated by intragastric administration according to the corresponding drug concentration；normal group and model group were given equal doses of physiological saline by gavage，once a day while modeling，and fecal water content，time of first black defecation and intestinal transmission rate of mice in each group were detected after 14 consecutive days. The content of 5-hydroxytryptamine（5-HT） in colon tissue of each group was detected by enzyme-linked immunosorbent assay（ELISA）. The mRNA levels of SCF and c-kit in colon tissues of mice were detected by RT-PCR. The protein expression levels of c-kit，PI3K，p-PI3K，Akt and p-Akt in colon tissues of mice were detected by Western blot. [Results] Compared with normal group，the first black defecation time of mice in model group was significantly extended（P<0.05），fecal water content and intestinal transmission rate were significantly decreased（P<0.05），and colon 5-HT content，c-kit and SCF mRNA levels were significantly decreased（P<0.05）. The expression levels of c-kit，P-PI3K /PI3K and P-Akt /Akt in colon were significantly decreased（P<0.05）. Compared with the model group，fecal water content of Ziyin Runchang Recipe high-dose group was significantly increased（P<0.05），while that of Ziyin Runchang Recipe low-dose group and positive drug group had an increasing trend. The time of first black defecation in Ziyin Runchang Recipe high-dose group and positive drug group was significantly shortened（P<0.05），and the time of first defecation in Ziyin Runchang Recipe low-dose group had a trend of shortening. The intestinal transmission rate of Ziyin Runchang Recipe in all dose groups and positive drug groups was significantly increased（P<0.05）. In colon tissues，the mRNA levels of c-kit and SCF in Ziyin Runchang Recipe high-dose group were significantly increased（P<0.05），and the positive drug group had an increasing trend. The content of 5-HT and the expression of c-kit，p-PI3K /PI3K and p-Akt /Akt protein in Ziyin Runchang Recipe high-dose group and positive drug group were significantly increased（P<0.05）. [Conclusion] Ziyin Runchang Recipe can significantly improve intestinal transmission rate in the treatment of constipation，and its mechanism is related to increasing the content of 5-HT and the up-regulation of PI3K/Akt expression level，and then increasing the number of interstitial cells of Cajal.