针灸对克罗恩病大鼠低氧环境下结肠DNA甲基转移酶的影响

Effects of Acupuncture and Moxibustion on Colonic DNA Methyltransferase Under Hypoxic Environment in Rats with Crohn's Disease

目的:观察针灸对克罗恩病(CD)大鼠结肠组织DNA甲基转移酶的影响,初步探讨针灸对CD的表观遗传调控机制。方法:将SD大鼠随机分为空白组、模型组、隔药饼灸组、电针组和柳氮磺吡啶组。采用5%2,4,6-三硝基苯磺酸灌肠的方法制备CD大鼠模型。隔药饼灸组和电针组均采用天枢(双侧)和气海穴进行干预,柳氮磺吡啶组采用柳氮磺吡啶肠溶片进行灌胃干预。治疗结束后,采用ELISA检测血清中C反应蛋白(CRP)、IL-6、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的含量;实时荧光定量PCR(RT-qPCR)检测结肠组织中缺氧诱导因子-1α(HIF-1α)的mRNA表达;Western Blotting检测结肠组织中DNMT1、DNMT3a、DNMT3b蛋白的表达。结果:与正常组比较,模型组血清中CRP、IL-6、IL-1β、TNF-α的蛋白含量均显著升高(P<0.01);与模型组比较,隔药饼灸组、电针组和柳氮磺吡啶组上述炎症介质的蛋白含量均显著降低(P<0.01)。与正常组比较,模型组结肠组织中HIF-1α mRNA和DNMT1、DNMT3a、DNMT3b蛋白表达均显著升高(P<0.01);与模型组比较,隔药饼灸组和柳氮磺吡啶组HIF-1α mRNA和DNMT1、DNMT3a、DNMT3b蛋白表达均显著降低(P<0.05或P<0.01),电针组HIF-1α mRNA和DNMT1蛋白表达均显著降低(P<0.05或P<0.01)。结论:隔药饼灸和电针均能抑制CD大鼠CRP、IL-6、IL-1β、TNF-α等炎症介质以及HIF-1α、DNA甲基转移酶DNMT1的表达,其中隔药饼灸还能抑制DNMT3a、DNMT3b表达,可能是针灸减轻CD肠道炎症的机制之一。

Objective:To observe the effects of acupuncture and moxibustion on DNA methyltransferase in the colon tissues of rats with Crohn's disease(CD)and explore the mechanism of acupuncture and moxibustion in the epigenetic regulation of CD.Methods:SD rats were randomly divided into a normal group,a model group,an indirect moxibustion group,an electroacupuncture(EA)group,and a sulfasalazine group.The CD model was induced by enema with 5% 2,4,6-trinitrobenzene sulfonic acid in rats.The rats in the indirect moxibustion group and the EA group were treated correspondingly at bilateral“Tianshu”(ST 25)and“Qihai”(CV 6),and those in the sulfasalazine group received sulfasalazine enteric-coated tablets by gavage.ELISA was used to detect the protein content of CRP,IL-6,IL-1β,and TNF-α in the serum.RT-qPCR was used to detect the expression of HIF-1α mRNA in the colon tissues.Western Blotting was used to detect the protein expression of DNMT1,DNMT3a,and DNMT3b in the colon tissues.Results:Compared with the normal group,the model group showed increased protein content of CRP,IL-6,IL-1β,and TNF-α in the serum(P<0.01).Compared with the model group,the indirect moxibustion group,the EA group,and the sulfasalazine group showed decreased protein content of the above-mentioned inflammatory factors(P<0.01).Compared with the normal group,the model group displayed increased expression of HIF-1α mRNA and DNMT1,DNMT3 a,and DNMT3 b proteins in the colon tissues(P<0.01).Compared with the model group,the indirect moxibustion group and the sulfasalazine group exhibited decreased expression of HIF-1α mRNA and DNMT1,DNMT3 a,and DNMT3 b proteins(P<0.05 or P<0.01),and the EA group showed reduced expression of HIF-1α mRNA and DNMT1 protein(P<0.05 or P<0.01).Conclusion:The indirect moxibustion and EA can both inhibit the expression of inflammatory factors such as CRP,IL-6,IL-1β,TNF-α,as well as HIF-1α and DNMT1 in CD rats,and the indirect moxibustion can also inhibit the expression of DNMT3a and DNMT3 b,which may be one of the mechanisms of acupuncture and moxibustion in reducing intestinal inflammation in CD.