針灸對克羅恩病大鼠低氧環境下結腸DNA甲基轉移酶的影響

祁琴 王曉梅 吳煥淦 包春輝 馬曉芃 趙琛 朱毅 劉慧榮 施茵 黃艷 劉雅楠

摘要 目的：觀察針灸對克羅恩病（CD）大鼠結腸組織DNA甲基轉移酶的影響，初步探討針灸對CD的表觀遺傳調控機制。方法：將SD大鼠隨機分為空白組、模型組、隔藥餅灸組、電針組和柳氮磺吡啶組。采用5% 2，4，6-三硝基苯磺酸灌腸的方法制備CD大鼠模型。隔藥餅灸組和電針組均采用天樞（雙側）和氣海穴進行干預，柳氮磺吡啶組采用柳氮磺吡啶腸溶片進行灌胃干預。治療結束后，采用ELISA檢測血清中C反應蛋白（CRP）、IL-6、白細胞介素-1β（IL-1β）、腫瘤壞死因子-α（TNF-α）的含量;實時熒光定量PCR（RT-qPCR）檢測結腸組織中缺氧誘導因子-1α（HIF-1α）的mRNA表達;Western Blotting檢測結腸組織中DNMT1、DNMT3a、DNMT3b蛋白的表達。結果：與正常組比較，模型組血清中CRP、IL-6、IL-1β、TNF-α的蛋白含量均顯著升高（P<0.01）;與模型組比較，隔藥餅灸組、電針組和柳氮磺吡啶組上述炎癥介質的蛋白含量均顯著降低（P<0.01）。與正常組比較，模型組結腸組織中HIF-1α mRNA和DNMT1、DNMT3a、DNMT3b蛋白表達均顯著升高（P<0.01）;與模型組比較，隔藥餅灸組和柳氮磺吡啶組HIF-1α mRNA和DNMT1、DNMT3a、DNMT3b蛋白表達均顯著降低（P<0.05或P<0.01），電針組HIF-1α mRNA和DNMT1蛋白表達均顯著降低（P<0.05或P<0.01）。結論：隔藥餅灸和電針均能抑制CD大鼠CRP、IL-6、IL-1β、TNF-α等炎癥介質以及HIF-1α、DNA甲基轉移酶DNMT1的表達，其中隔藥餅灸還能抑制DNMT3a、DNMT3b表達，可能是針灸減輕CD腸道炎癥的機制之一。

關鍵詞 克羅恩病;針灸;DNA甲基轉移酶;表觀調控;缺氧誘導因子

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

QI Qin1，WANG Xiaomei1，WU Huangan1，2，BAO Chunhui1，MA Xiaopeng1，2，ZHAO Chen3，ZHU Yi1，LIU Huirong1，2，SHI Yin1，HUANG Yan1，LIU Yanan1

（1 Shanghai Research Institute of Acupuncture and Meridian，Shanghai 200030，China; 2 Yueyang Hospital of Integrated Traditional Chinese and Western Medicine，Shanghai University of Traditional Chinese Medicine，Shanghai 200437，China; 3 School of Acupuncture-Moxibustion and Tuina，Shanghai University of Traditional Chinese Medicine，Shanghai 201203，China）

Abstract 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，DNMT3a，and DNMT3b 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，DNMT3a，and DNMT3b 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 DNMT3b，which may be one of the mechanisms of acupuncture and moxibustion in reducing intestinal inflammation in CD.

Keywords Crohn′s disease; Acupuncture and moxibustion; DNA methyltransferase; Epigenetic regulation; Hypoxia-inducible factor

中圖分類號：R245文獻標識碼：Adoi：10.3969/j.issn.1673-7202.2022.03.005

克羅恩?。–rohn′s Disease，CD）屬于炎癥性腸?。↖nflammatory Bowel Disease，IBD）的一種，常表現為消化道管壁全層性炎癥，可并發腸狹窄、腸梗阻，多數患者終生反復發作。近年來，國內發病率逐年升高，至今仍缺乏理想的治療方法，是一種難治性腸腑病癥?！?br>