Effects of Wenhua Juanbi Recipe on m6A methylation in collagen⁃induced arthritis rats

ZHOU Haili; HUANG Liuyun; LIU Xide

doi:10.16305/j.1007-1334.2022.2205023

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Effects of Wenhua Juanbi Recipe on m6A methylation in collagen⁃induced arthritis rats

更新时间：2022-10-26

- Effects of Wenhua Juanbi Recipe on m6A methylation in collagen⁃induced arthritis rats
- Shanghai Journal of Traditional Chinese Medicine Vol. 56, Issue 10, Pages: 65-70(2022)
- 作者机构：
  
  1.扬州市中医院（江苏　扬州　225127）
  2.浙江中医药大学第二临床医学院（浙江杭州 310053）
  3.浙江中西医结合医院中医科（浙江杭州 310003）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2022.2205023
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周海丽,黄柳云,刘喜德.温化蠲痹方对胶原诱导性关节炎大鼠N6⁃甲基腺苷甲基化的影响[J].上海中医药杂志,2022,56(10):65-70.

ZHOU Haili,HUANG Liuyun,LIU Xide.Effects of Wenhua Juanbi Recipe on m6A methylation in collagen⁃induced arthritis rats[J].Shanghai Journal of Traditional Chinese Medicine,2022,56(10):65-70.
周海丽,黄柳云,刘喜德.温化蠲痹方对胶原诱导性关节炎大鼠N6⁃甲基腺苷甲基化的影响[J].上海中医药杂志,2022,56(10):65-70. DOI： 10.16305/j.1007-1334.2022.2205023.

ZHOU Haili,HUANG Liuyun,LIU Xide.Effects of Wenhua Juanbi Recipe on m6A methylation in collagen⁃induced arthritis rats[J].Shanghai Journal of Traditional Chinese Medicine,2022,56(10):65-70. DOI： 10.16305/j.1007-1334.2022.2205023.

摘要

目的,2,研究温化蠲痹方对胶原诱导性关节炎（CIA）大鼠炎症及N6-甲基腺苷（m6A）甲基化的影响。,方法,2,选用雌性Wistar大鼠50只，随机分为正常组（,n,=10）和造模组（,n,=40）。采用尾部皮内多点注射乳化剂的方法建立CIA模型。采用容积法（排水体积）和关节炎指数（AI）评分法对模型进行评估，足肿胀体积,>,1.6 mL、AI评分≥4分视为造模成功。将造模成功的30只CIA大鼠随机分为模型组（,n,=10）、温化蠲痹方组（中药组，,n,=10）、甲氨蝶呤（MTX）组（,n,=10）。中药组给予温化蠲痹方，剂量为22.9 g·kg,-1,·d,-1 ,；MTX组给予甲氨蝶呤混悬液，剂量为0.78 mg·kg,-1,，每周1次；正常组和模型组给予等量质量分数为0.9%的氯化钠溶液，每日1次。治疗30 d取各组大鼠膝关节滑膜。采用HE染色法检测滑膜病理情况，以realtime PCR法检测滑膜中甲基转移酶样蛋白3（,Mettl3,）、甲基转移酶样蛋白14（,Mettl14,）、肥胖相关蛋白（,Fto,）、Alkb同系物5（,Alkbh5,）、Yth N6-甲基腺苷RNA结合蛋白1（,Ythdf1,）、Yth N6-甲基腺苷RNA结合蛋白2（,Ythdf2,）基因表达及关节炎性因子肿瘤坏死因子-,α,（,TNF,-,α,）、白介素-1β（,IL,-,1β,）、白介素-6（,IL,-,6,）基因表达水平，并采用Western blot法检测Mettl3、Fto蛋白表达。,结果,2,与正常组比较，模型组大鼠足肿胀体积、AI评分明显升高，,Mettl3,、,Mettl14,、,Fto,、,Alkbh5,、,Ythdf1,基因表达增加，,Ythdf2,基因表达降低，,TNF,-,α、IL,-,1β、IL,-,6,基因表达增加，Mettl3、Fto蛋白表达增加，差异均有统计学意义（,P,<,0.05）。与模型组比较，中药组和MTX组足肿胀体积、AI评分均随时间延长逐渐降低，,Mettl3,、,Mettl14,、,Fto,、,Alkbh5,、,Ythdf1,基因表达降低，,Ythdf2,基因表达增加，,TNF,-,α、IL,-,1β、IL,-,6,基因表达降低，Mettl3、Fto蛋白表达降低，差异均有统计学意义（,P,<,0.05）。与MTX组比较，中药组,Mettl3,、,Mettl14,、,Fto,、,Alkbh5,、,Ythdf1,基因表达降低，,Ythdf2,基因表达增加，,TNF,-,α、IL,-,1β、IL,-,6,基因表达降低，Mettl3、Fto蛋白表达降低，差异均有统计学意义（,P,<,0.05）。,结论,2,中药温化蠲痹方可能通过影响m6A甲基化来发挥抗炎作用，从而对CIA起到治疗效果。

Abstract

Objective,2,To study the effects of Wenhua Juanbi Recipe （WJR） on inflammation and m6A methylation in rats with collagen-induced arthritis （CIA）.,Methods,2,Fifty female Wistar rats were randomly divided into two groups： a normal group （,n,=10） and a model group （,n,=40）. CIA model was established by intracaudal multi-point injection of emulsifier. The model was evaluated by volumetric method （drainage volume） and arthritis index （AI） scoring method to assess the degree of toe swelling and joint inflammation， and the model with toe swelling,>, 1.6 mL and AI score≥4 was considered as successful. Thirty successfully modeled CIA rats were randomly divided into a model group （,n,=10）， a WJR group （TCM group， ,n,=10） and a MTX group （,n,=10）. The TCM group was administered with WJR at a dose of 22.9 g·kg,-1,·d,-1,； and the MTX group was given methotrexate suspension （MTX） at a dose of 0.78 mg·kg,-1, once a week. The normal group and the CIA group were given equal amounts of 0.9% NaCl solution once a day. The synovial membrane of the knee of rats was taken from each group after 30 days of treatment. The synovial pathology was examined by HE staining. The gene expressions of ,Mettl3,， ,Mettl14,， ,Fto,， ,Alkbh5,， ,Ythdf1, and ,Ythdf2 ,and the expression levels of joint inflammatory factors ,TNF,-,α,， ,IL,-,1β, and ,IL,-,6 ,genes in synovial membrane were detected by real-time PCR. Western blot was used to detect the expressions of Mettl3 and Fto proteins.,Results,2,Compared with the condition in the normal group， the toe swelling degree and AI score of rats in the model group increased significantly， and in the model group the expressions of ,Mettl3,， ,Mettl14,， ,Fto,， ,Alkbh5, and ,Ythdf1, genes increased， the expression of ,Ythdf2 ,gene decreased， the expressions of ,TNF,-,α,， ,IL,-,1β, and ,IL,-,6 ,genes increased， and the expressions of Mettl3 and Fto proteins increased （,P,<,0.05）. Compared with the condition in the model group， the toe swelling degree and AI score of rats in the WJR group and the MTX group decreased gradually with time， and in the WJR group and the MTX group the expressions of ,Mettl3,， ,Mettl14,， ,Fto,， ,Alkbh5, and ,Ythdf1, decreased， the expression of ,Ythdf2 ,increased， the expressions of ,TNF,-,α,， ,IL,-,1β, and ,IL,-,6 ,genes decreased， and the expressions of Mettl3 and Fto proteins decreased （,P,<,0.05）. Compared with the condition in the MTX group， the expressions of ,Mettl3,， ,Mettl14,， ,Fto,， ,Alkbh5, and ,Ythdf1, decreased， the expression of ,Ythdf2, gene increased， the expressions of ,TNF,-,α,， ,IL,-,1β, and ,IL,-,6, genes decreased， and the expressions of Mettl3 and Fto proteins decreased in the WJR group （,P,<,0.05）.,Conclusion,2,Wenhua Juanbi Recipe may have a therapeutic effect on CIA by affecting the m6A methylation to exert anti-inflammatory effects.

关键词

类风湿关节炎胶原诱导性关节炎温化蠲痹方N6-甲基腺苷大鼠模型中药研究

Keywords

rheumatoid arthritiscollagen induced arthritisWenhua Juanbi Recipem6Arat modeltraditional Chinese herbal medicine research

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