基于let-7f-2-3p/SCN1A轴探究加味肾气丸对泻剂结肠大鼠肠动力障碍的影响

Effects of Jiawei Shenqi Pills on Intestinal Motility Disorder in Cathartic Colon Rats Based on let-7f-2-3p/SCN1A Axis

目的:探讨加味肾气丸对泻剂结肠大鼠肠动力障碍的影响,并探讨其对let-7f-2-3p/SCN1A轴的调控作用。方法:将40只雄性SD大鼠随机分为正常对照组、模型组、普卢卡必利组、加味肾气丸组;采用生大黄免煎剂混悬液递增灌胃建立泻剂结肠大鼠模型,造模成功后分别给予相应药物。统计大鼠治疗后1 w首粒黑便排出时间;取大鼠结肠标本,HE染色观察大鼠肠黏膜损伤情况;免疫荧光标记观察ICC数量;免疫组化染色检测结肠组织中AChE、NOS表达;采用试剂盒测定大鼠结肠组织SOD、MDA、NO、VIP、SP、Na^(+)-K^(+)-ATPase、Ca^(2+)-ATPase水平;RT-qPCR检测大鼠结肠组织let-7f-2-3p、SCN1A的表达;通过生物信息学在线软件预测let-7f-2-3p与SCN1A互补结合位点,双荧光素酶报告基因实验验证二者靶向关系。结果:与模型组比较,加味肾气丸组首粒黑便排出时间显著缩短(P<0.05),结肠黏膜损伤明显改善,结肠ICC数量显著增加,NO、VIP、MDA水平及NOS表达显著降低,SP、SOD、Na^(+)-K^(+)-ATPase、Ca^(2+)-ATPase水平及AChE表达显著升高,let-7f-2-3p表达显著降低,SCN1A表达显著升高(P<0.05或P<0.01)。双荧光素酶报告实验证实SCN1A是let-7f-2-3p的靶向调控分子。结论:加味肾气丸可能通过let-7f-2-3p/SCN1A轴缓解肠动力障碍,进而对泻剂结肠大鼠产生保护作用。

Objective:To investigate the effects of Jiawei shenqi pills on intestinal motility disorder in cathartic colon rats,and to explore its regulation on let-7 f-2-3 P/SCN1 A axis.Methods:40 male SD rats were randomly divided into normal control group,model group,prucalopride group and Jiawei shenqi pills group.The cathartic colon rat model was established by increasing gavage administration of free decoction suspension of Rhei Radix et Rhizoma,and the corresponding drugs were given after the model was successfully established.The excretion time of the first black stool was counted one week after treatment.Colon specimens of rats were taken and HE staining was used to observe the damage of intestinal mucosa.The number of ICC was observed by immunofluorescence labeling.The expressions of AchE and NOS in colon tissues were detected by immumohistochemical staining.The levels of SOD,MDA,NO,VIP,SP,Na^(+)-K^(+)-ATPase and Ca^(2+)-ATPase in colon tissus of rats were determined by the kit.RT-qPCR was used to detect the expressions of let-7 f-2-3 p and SCN1 A in colon tissues of rats.The complementary binding sites of let-7 f-2-3 P and SCN1 A were predicted by bioinformatics online software,the targeting relationship between the two was verified by double luciferase reporter gene assay.Results:Compared with model group,the excretion time of the first black stool in Jiawei shenqi pills group was significantly shortened(P<0.05),the intestinal mucosal injury was significantly improved,the number of ICC was significantly increased,the levels of NO,VIP,MDA and the expression of NOS were significantly reduced,the levels of SP,SOD,Na^(+)-K^(+)-ATPase,Ca^(2+)-ATPase and the expression of AChE were significantly increased,the expression of let-7 f-2-3 p was down-regulated and the expression of SCN1 A was up-regulated(P<0.05 or P<0.01).The dual luciferase report assay confirmed that SCN1 A was a targeted regulatory molecule of let-7 f-2-3 p.Conclusion:Jiawei shenqi pills may relieve intestinal motility disorder via let-7 f-2-3 p/SCN1 A axis and then have protective effects on purgative colon rats.