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Title: [Effect of electroacupuncture on colonic autophagy and AMPK/mTOR signaling pathway in rats with acute ulcerative colitis].
Author: Wu RL, Xu HC, Jiang ZW, Wang HJ, Cao YX, Kong FM, Dong AA, Li RQ, Ji LX.
Journal: Zhen Ci Yan Jiu; 2023 Aug 25; 48(8):818-24. PubMed ID: 37614141.
Abstract:
OBJECTIVE: To observe the effect of electroacupuncture （EA） at "Zhongwan" （CV12）, "Tianshu" （ST25） and "Shangjuxu" （ST37）（an acupoint prescription "Changbingfang" for treatment of intestinal disorders） on autophagy and expression of AMPK/mTOR signaling pathway in rats with ulcerative colitis （UC）, so as to explore its mechanism underlying improvement of UC. METHODS: Thirty-two male SD rats were randomly divided into control, model, medication and EA groups, with 8 rats in each group. The UC model was established by free drinking of 5% dextran sulfate sodium salt solution for 7 days. EA stimulation （10 Hz/50 Hz） was delivered to CV12, ST25 and ST37 for 20 min, once a day for 3 consecutive days. Rats of the medication group received gavage of mesalazine suspension （200 mg/kg） once a day, 3 times in total. The rats' general conditions were recorded for calculating the disease activity index （DAI） score （0-4 points）. Histomorphological changes of colon were observed via HE staining. The levels of serum interleukin 6 （IL-6）, tumor necrosis factor-α （TNF-α） and IL-10 were measured by ELISA. The mRNA expressions of LC3B and p62 were tested by fluorescence quantitative PCR. Western blot was used to detect the expression levels of LC3B, p62 and AMPK/mTOR pathway related proteins in colon tissues. RESULTS: Compared with the control group, the DAI score, contents of serum IL-6 and TNF-α, the expression levels of p62 protein and mRNA, ratio of p-mTOR/mTOR were significantly increased （P<0.01）； while the content of serum IL-10, the expression levels of LC3B mRNA, ratio of LC3BⅡ/LC3BⅠ and p-AMPK/AMPK were decreased （P<0.01, P<0.05） in the model group. Relevant to the model group, modeling-induced increases of DAI score, serum IL-6, TNF-α and IL-10 contents, expressions of p62 protein and mRNA, LC3B mRNA, ratio of p-mTOR/mTOR, LC3BⅡ/LC3BⅠ and p-AMPK/AMPK were reversed in both medication and EA groups （P<0.01, P<0.05）. The effect of EA was apparently superior to that of mesalazine in up-regulating ratio of LC3BⅡ/LC3BⅠ and p-AMPK/AMPK, p62 mRNA expression （P<0.01, P<0.05）, and in down-regulating ratio of p-mTOR/mTOR （P<0.05）. H.E. staining showed severe damage of the colonic mucosal barrier with infiltration of a large number of inflammatory cells in the model group, which was milder in medication and EA groups. CONCLUSION: EA of acupoint recipe "Changbingfang" can improve the symptoms in UC rats, which may be related to its functions in promoting colonic autophagy, increasing AMPK phosphorylation level, and decreasing mTOR phosphorylation level. 目的：观察电针“肠病方”对溃疡性结肠炎（UC）大鼠自噬水平和腺苷酸激活蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路相关蛋白表达的影响，探讨其治疗UC的作用机制。方法：雄性SD大鼠随机分为空白组、模型组、西药组和电针组，每组8只。采用自由饮用5%葡聚糖硫酸钠溶液7 d复制UC大鼠模型。电针组予双侧“天枢”“上巨虚”和“中脘”电针治疗，每次20 min，每日1次，连续3 d；西药组予美沙拉嗪悬液（200 mg/kg）灌胃，每日1次，连续3 d。观察并记录各组大鼠一般情况，记录粪便形态并进行隐血试验以评估疾病活动指数（DAI）；HE染色法观察大鼠结肠形态及病理变化；ELISA法检测血清白细胞介素（IL）-6、肿瘤坏死因子（TNF-α）、IL-10的含量；荧光定量PCR法检测结肠组织自噬标志物微管相关蛋白3B（LC3B）、p62 mRNA的表达水平；Western blot法检测结肠组织中p62蛋白表达量及LC3BⅡ/LC3BⅠ、p-AMPK/AMPK、p-mTOR/mTOR比值。结果：与空白组比较，模型组大鼠一般情况较差，结肠黏膜屏障破坏，炎性细胞浸润，腺体萎缩或消失，隐窝数量减少，DAI评分升高（P<0.01），血清IL-6、TNF-α含量升高（P<0.01），IL-10含量降低（P<0.01），结肠组织中p62蛋白及mRNA表达水平、p-mTOR/mTOR比值均升高（P<0.01），LC3B mRNA表达水平及LC3BⅡ/LC3BⅠ、p-AMPK/AMPK比值均降低（P<0.01 ，P<0.05）。与模型组比较，电针组和西药组大鼠一般情况好转，结肠黏膜屏障缺损好转，炎性细胞浸润减少，腺体排列相对整齐，隐窝数量增加，DAI评分降低（P<0.01），血清IL-6、TNF-α含量降低（P<0.05，P<0.01），IL-10含量升高（P<0.01，P<0.05），结肠组织中p62蛋白及mRNA表达水平、p-mTOR/mTOR比值均降低（P<0.01），LC3B mRNA表达水平及LC3BⅡ/LC3BⅠ、p-AMPK/AMPK比值均升高（P<0.01）。与西药组比较，电针组结肠组织中p62 mRNA表达水平及LC3BⅡ/LC3BⅠ、p-AMPK/AMPK比值均升高（P<0.01，P<0.05），p-mTOR/mTOR比值降低（P<0.05）。结论：电针“肠病方”能改善急性UC大鼠症状，其作用机制可能与促进结肠细胞自噬、提高AMPK磷酸化水平、降低mTOR磷酸化水平相关。.

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