痛泻要方通过miR-199/TRPV1信号通路改善腹泻型肠易激综合征大鼠内脏高敏感

Tongxie Yaofang mproves Visceral Hypersensitivity in Diarrhea-predominant Irritable Bowel Syndrome Through MiR-199a/TRPV1 Signaling Pathway

目的 探究痛泻药方对腹泻型肠易激综合征(IBS-D)内脏高敏感大鼠miR-199a/TRPV1通路的改善作用。方法 将40只SD大鼠束缚1 h+1 ml 4%乙酸灌肠建立IBS-D模型,随机分为模型组、中药组、西药组,未造模SD大鼠6只作为空白组,中药组予痛泻药方,西药组予匹维溴铵,其余组予等量蒸馏水灌胃。给药2周后,检测各组大鼠粪便含水量和腹壁回撤反射评分(AWR),处死大鼠后取远端结肠组织,用实时荧光定量聚合酶链反应技术(RT-qPCR)检测远端结肠miR-199a-3p和miR-199-5p表达水平,用蛋白质印迹(Western Blot)、免疫组化(Immunohistochemistry)检测大鼠结肠TRPV1、p-PKC、p-CaMKII表达水平。结果 与正常组比较,模型组大鼠粪便含水量、AWR评分明显升高(P<0.05,P<0.01),miR-199a-3p和miR-199a-5p相对含量升高(P<0.05),TRPV1、p-PKC、p-CaMKII蛋白水平降低(P<0.05,P<0.01);与模型组比较,中药组miR-199a-3p和miR-199a-5p相对含量降低(P<0.05),TRPV1、p-PKC、p-CaMKII蛋白水平降低(P<0.05,P<0.01)。结论 痛泻药方可能通过抑制miR-199a/TRPV1信号通路改善IBS-D内脏高敏感。

Objective To explore the improving effect of Tongxie Yaofang on visceral hypersensitivity in diarrhea-predominant irritable bowel syndrome(IBS-D)by regulating the miR-199a/TRPV1 pathway.Methods The IBS-D model was induced by binding for 1 h and 1 ml of 4%acetic acid enema in 40 SD rats and the model mice were randomly divided into a model group,a Chinese medicine group(Tongxie Yaofang),and a western medicine group(pinaverium bro-mide).Six SD rats free from model induction were assigned to the blank group.Rats were treated with corresponding drugs,while those in the model group and the blank group received the same volume of distilled water by gavage.After two weeks of intervention,the fecal water content and abdominal withdrawal reflex(AWR)score of each group were detected.Distal colon tissues were collected from the rats after sacrifice.The expression levels of miR-199a-3p and miR-199-5p in distal colon tissues were detected by Real-time fluorescence-based quantitative polymerase chain reaction(RT-qPCR).The expression levels of TRPV1,p-PKC,and p-CaMKII were detected by Western blot and immunohistochem-istry.Results Compared with the blank group,the model group showed increased fecal water content and AWR score(P<0.05,P<0.01),increased relative content of miR-199a-3p and miR-199a-5p(P<0.05),and reduced protein levels of TRPV1,p-PKC,and p-CaMKII(P<0.05,P<0.01).Compared with the model group,the Chinese medicine group showed reduced relative content of miR-199a-3p and miR-199a-5p(P<0.05)and decreased protein levels of TRPV1,p-PKC,and p-CaMKII(P<0.05,P<0.01).Conclusion Tongxie Yaofang can improve visceral hypersensi-tivity in IBS-D by inhibiting the miR-199a/TRPV1 signaling pathway.