槲皮素通过调节SIRT1/NLRP3信号通路对实验性巨结肠大鼠肠损伤的影响

Effects of quercetin on intestinal injury in experimental megacolon rats by regulating SIRT1/NLRP3 signaling pathway

目的探究槲皮素通过调节沉默信息调控因子1(SIRT1)/NOD样受体蛋白3(NLRP3)信号通路对实验性巨结肠大鼠肠损伤的影响。方法采用苯扎溴铵滴注法诱导建立大鼠实验性巨结肠模型,并随机分为模型组,槲皮素低(5 mg/kg)、高(50 mg/kg)剂量组,SIRT1抑制剂(EX527)组,槲皮素+EX527组,每组15只,另取15只作为假手术组。各组大鼠连续给药2周,1次/d,末次给药12 h后,观察大鼠排便和腹胀情况,开腹观察结肠变化。免疫荧光法检测肽能神经递质蛋白基因产物9.5(PGP9.5)、M2型巨噬细胞标记抗体-CD206表达水平。苏木精–伊红(HE)观察结肠病理变化,免疫组化法检测乙酰胆碱酯酶(AChE)、NLRP3阳性表达变化。Western blotting检测结肠组织SIRT1、NLRP3、炎性因子[肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β]、促神经恢复相关蛋白[骨形成蛋白-2(BMP-2)、骨形成蛋白受体(BMPR)]、神经节细胞相关因子[乙酰胆碱(ACh)、神经巢蛋白(Nestin)]表达。结果与假手术组相比,模型组大鼠处理段结肠组织神经节细胞减少、结肠黏膜炎症损伤加重,SIRT1以及M2型巨噬细胞介导的抗炎和促神经恢复蛋白表达降低,NLRP3炎性反应升高(P<0.05)。与模型组相比,槲皮素5、50 mg/kg组大鼠结肠组织炎症损伤缓解,SIRT1以及M2型巨噬细胞介导的抗炎和促神经恢复蛋白表达升高,NLRP3炎性反应降低(P<0.05),且槲皮素高剂量组改善效果优于低剂量组(P<0.05);EX527组可减弱槲皮素的上述作用(P<0.05)。结论槲皮素可通过上调SIRT1表达,抑制NLRP3促炎反应,提高M2型巨噬细胞介导的抗炎和促神经恢复作用,改善实验性巨结肠大鼠结肠炎性损伤。

Objective To investigate the effect of quercetin on intestinal injury in experimental hirscholon rats through regulating silencing message modulator 1(SIRT1)/Nod-like receptor protein 3(NLRP3)signaling pathway.Methods Benzalkonium bromide infusion was used to induce the establishment of rat experimental megacolon models,and they were randomly divided into model group,quercetin low(5 mg/kg)and high(50 mg/kg)dose groups,SIRT1 inhibitor(EX527)group,quercetin+EX527 group,15 per group,another 15 rats in the sham operation group.Rats in each group were administered for 2 weeks,once daily,12 h after the last administration,the rat's defecation and abdominal distension were observed,and the abdomen was opened to observe changes in the colon.Immunofluorescence method was used to detect the expression levels of peptidergic neurotransmitter protein gene product 9.5(PGP9.5)and M2 macrophage marker antibody-CD206.HE staining was used to observe the pathological changes of colon,immunohistochemical method was used to detect the positive expression of acetylcholinesterase(AChE)and NLRP3.Western blotting was used to detect the expression of SIRT1,NLRP3,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,neurorestoration-related protein(BMP-2,BMPR),acetylcholine(ACh)and Nestin in colon tissues.Results Compared with the sham operation group,the rats in the model group had fewer ganglion cells in the treated colon tissue and more severe colonic mucosal inflammation,the activity of SIRT1 and the expression of anti-inflammatory and neurorestorative proteins mediated by M2 macrophages decreased,and the inflammatory response of NLRP3 increased(P<0.05).Compared with the model group,the inflammation in the colon tissue of rats in quercetin 5 and 50 mg/kg groups was alleviated,the activity of SIRT1 and the expression of anti-inflammatory and neurorestorative proteins mediated by M2 macrophages increased,and the inflammatory response of NLRP3 decreased(P<0.05),and the improvement effects of the high-dose quercetin group were better than those of the low-dose group(P<0.05).EX527 could attenuate the abovementioned effects of quercetin(P<0.05).Conclusion Quercetin can up-regulate the expression of SIRT1,inhibit the proinflammatory response of NLRP3,enhance the anti-inflammatory and neurorestorative effects mediated by M2 macrophages,and improve the intestinal inflammatory injury in experimental hirscholon rats.