基于Nrf2-Keap1-ARE信号通路探讨疏肝健脾方对肠易激综合征大鼠内脏敏感性的影响

Based on Nrf2-Keap1-ARE signaling pathway to explore the effect of Shugan Jianpi Recipe on visceral sensitivity of irritable bowel syndrome rats

目的基于核因子E2相关因子2(Nrf2)-Kelch样环氧氯丙烷相关蛋白-1(Keap1)-抗氧化反应元件(ARE)信号通路观察疏肝健脾方对肠易激综合征(IBS)大鼠内脏敏感性的影响。方法取30只SPF级雄性Wistar大鼠采用4%醋酸灌肠复合慢性心理应激法建立IBS大鼠模型,并随机分为模型组、疏肝健脾方组以及阳性对照组(匹维溴铵组),另取10只不予任何处理作为空白组。疏肝健脾方组按5 mL/kg的剂量给予疏肝健脾方,阳性对照组按2 mg/kg的剂量灌胃给匹维溴铵,空白组和模型组按5 mL/kg的剂量给0.9%氯化钠溶液,造模结束后以粪便含水率进行模型评价。给药结束后当天,对粪便含水率进行检测;比较大鼠内脏的敏感性;HE染色检测大鼠结肠组织形态;比较血清胃肠激素水平;检测大鼠血清中氧化应激指标;免疫荧光法检测ZO-1、Occludin蛋白的损伤;Western blotting检测Nrf2-Keap1-ARE通路蛋白表达。结果与空白组相比,模型组大鼠的粪便含水量,血清中SP、SS、MDA水平,结肠组织中p-Nrf2、p-Keap1、p-ARE蛋白表达水平显著升高;腹部抬起和背部拱起程度,血清中GAS、MTL、SOD、NO水平,结肠黏膜中ZO-1、Occludin表达量显著降低。与模型组相比,疏肝健脾组和阳性对照组大鼠治疗后的粪便含水量,血清中SP、SS、MDA水平,结肠组织中p-Nrf2、p-Keap1、p-ARE蛋白表达水平显著下降;腹部抬起和背部拱起程度,血清中GAS、MTL、SOD、NO水平,结肠黏膜中ZO-1、Occludin表达量显著上升。结论疏肝健脾方能够有效改善大鼠IBS情况,降低氧化应激水平及结肠黏膜紧密连接蛋白的损伤程度,其机制可能与Nrf2-Keap1-ARE信号通路的调控有关。

Objective Observation of Shugan Jianpi Recipe′s visceral sensitivity to irritable bowel syndrome rats based on Nuclear factor E2 related factor 2(Nrf2)-kelch-like epichlorohydrin related protein-1(Keap1)-antioxidant response element(ARE)signaling pathway Sexual influence.Methods Thirty SPF male Wistar rats were selected to establish an IBS rat model using 4%acetic acid enema combined with chronic psychological stress,and were randomly divided into a model group,a Shugan Jianpi Recipe group,and a positive control group(pinaverium bromide group),Take another 10 as a blank group without any treatment.The blank group and the model group were given 0.9%sodium chloride solution,the Shugan Jianpi Recipe group was given Shugan Jianpi Recipe,and the positive control group was given pinaverium bromide.After the modeling,the fecal moisture content was used to evaluate the model.On the day after the end of the drug administration,the fecal moisture content was tested;the visceral sensitivity of rats was compared;HE staining was used to detect the colon tissue morphology of the rats;the serum gastrointestinal hormone levels were compared;the oxidative stress indexes in the rat serum were detected;the immunofluorescence method Detection of ZO-1,occludin protein damage;Western blotting detection of Nrf2-Keap1-ARE pathway protein expression.Results Compared with the blank group,the fecal water content,serum SP,SS levels,MDA content,colon tissue p-Nrf2,p-Keap1,and p-ARE protein expression levels in the model group were significantly increased,and the abdomen lifted and The degree of back arching,serum GAS,MTL levels,SOD,NO content,and colonic mucosal ZO-1 and Occludin expressions were significantly reduced;compared with the model group,rats in the Shugan Jianpi group and the positive control group were significantly reduced Stool water content,serum SP,SS levels,MDA content,colon tissue p-Nrf2,p-Keap1,p-ARE protein expression levels significantly decreased,the degree of abdominal lifting and back arching,serum GAS,MTL levels,The contents of SOD,NO,and the expression of ZO-1 and Occludin in colonic mucosa increased significantly.Conclusion Shugan Jianpi Decoction can effectively improve the IBS of rats,reduce the level of oxidative stress and the degree of damage to the colonic mucosa tight junction protein.The mechanism may be related to the regulation of the Nrf2-Keap1-ARE signaling pathway.Shugan Jianpi Recipe can effectively improve IBS in rats,reduce the level of oxidative stress and the degree of damage to the tight junction proteins of colonic mucosa.The mechanism may be related to the regulation of Nrf2-Keap1-ARE signaling pathway.