目的:从P2X7R-NLRP3炎性小体通路角度探讨针灸干预溃疡性结肠炎(UC)的作用机制.方法:将Sprague-Dawley大鼠随机分为正常组、模型组、隔药灸组和电针组.造模方法为4%右旋糖酐硫酸钠连续饮用7 d.隔药灸组和电针组分别接受连续7 d隔药灸或电针双侧天枢穴干预.苏木素-伊红(HE)染色法观察结肠组织病理学变化;免疫荧光法检测结肠组织相关分子的蛋白表达;酶联免疫吸附法检测血清和结肠组织相关分子的浓度或含量.采用野生型(WT)和P2X7R基因敲除(KO)小鼠构建UC模型,HE染色观察结肠组织病理学变化;免疫组化法检测结肠组织NLRP3蛋白表达.结果:与正常组比较,模型组结肠组织病理学评分,结肠组织P2X7R、NLRP3、ASC、Caspase-1、IL-1β及IL-18蛋白表达,血清IL-1β及IL-18的蛋白水平均显著升高(P<0.05).与模型组比较,隔药灸组及电针组结肠组织病理学评分,结肠组织P2X7R、NLRP3、ASC、Caspase-1、IL-1β及IL-18蛋白表达,以及血清IL-18蛋白水平均显著降低(P<0.05).经UC造模后,P2X7R KO小鼠结肠黏膜上皮损伤及炎性细胞浸润程度较WT小鼠减轻,结肠NLRP3蛋白表达也较WT小鼠降低(P<0.05).结论:隔药灸、电针天枢穴可能通过抑制UC大鼠结肠组织P2X7R、NLRP3、ASC及Caspase-1的蛋白活性,降低P2X7R-NLRP3炎性小体通路中下游分子IL-1β及IL-18的表达达到缓解UC炎症的目的.
Objective:To explore the mechanism of acupuncture and moxibustion for ulcerative colitis(UC)from the perspective of the P2X7 receptor(P2X7R)-nucleotide-binding oligomerization domain(NOD)-like receptor protein 3(NLRP3)inflammasome pathway. Methods:Sprague-Dawley rats were randomly assigned to a normal(N)group,a model(M)group,a herb-partitioned moxibustion(HM)group,and an electroacupuncture(EA)group.For modeling,the rats drank 4%dextran sulfate sodium for 7 d.Rats in the HM group and EA group received 7 consecutive days of HM or EA at bilateral Tianshu(ST25),respectively.The histopathological change in colon tissue was observed by hematoxylin-eosin(HE)staining;immunofluorescence staining was used to detect the protein expression of related molecules in the colon tissue,and enzyme-linked immunosorbent assay was used to detect the concentrations or contents of related molecules in the serum and colon tissue.Wild-type(WT)and P2X7R gene knockout(KO)mice were used to construct UC models,histopathological changes in the colon tissue were observed by HE staining,and the NLRP3 protein expression in the colon tissue was observed by immunohistochemistry. Results:Compared with the N group,the colon histopathological score in the M group was significantly increased,and the protein expression of P2X7R,NLRP3,apoptosis-associated speck-like protein containing CARD(ASC),Caspase-1,interleukin-1β(IL-1β),and interleukin-18(IL-18)in the colon tissue and the protein levels of IL-1β and IL-18 in the serum were significantly increased(P<0.05).Compared with the M group,the histopathological scores of the colon in the HM group and the EA group were significantly decreased,and the protein expression levels of P2X7R,NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in the colon tissue and the protein level of IL-18 in the serum were significantly decreased(P<0.05).After UC modeling,the colonic mucosal epithelial damage and inflammatory cell infiltration in P2X7R KO mice were less than those in WT mice,and the NLRP3 protein expression in the colon was also decreased compared with that in WT mice(P<0.05). Conclusion:HM and EA at Tianshu(ST25)may inhibit the protein activities of P2X7R,NLRP3,ASC,and Caspase-1 in the colon tissue of rats with UC,thereby reducing the downstream molecules IL-1β and IL-18 in the P2X7R-NLRP3 inflammasome pathway to relieve UC inflammation.