基于AMPK/ULK1信号通路研究温针灸调控慢性疲劳综合征大鼠线粒体自噬的作用机制OA
Effect of warming needle moxibustion on mitochondrial autophagy in rats with chronic fatigue syndrome based on AMPK/ULK1 signaling pathway
目的:观察温针灸对慢性疲劳综合征(CFS)大鼠骨骼肌腺苷酸活化蛋白激酶(AMPK)/Unc-51样激酶1(ULK1)信号通路的影响,探讨温针灸干预CFS的作用机制.方法:雄性SD大鼠按随机数字表法分为空白组(10只)和造模组(30只).造模组采用力竭游泳、慢性束缚及隔日禁食复合因素造模方法复制CFS大鼠模型.将造模成功的30只大鼠按随机数字表法分为模型组、温针灸组、辅酶组,每组10只.温针灸组选取大鼠"足三里(双)""关元""中脘"进行温针灸治疗,每次15 min,每日1次,连续干预14 d;辅酶组给予辅酶Q10药液(1 mg/kg)进行灌服,每日1次,连续干预14 d.记录大鼠造模前后、干预后体质量与力竭游泳时间;旷场实验评价各组大鼠行为学变化;HE染色观察大鼠骨骼肌形态组织变化;透射电镜观察骨骼肌线粒体结构及自噬体;Western blot法检测大鼠骨骼肌AMPK、磷酸化(p)-AMPK、ULK1、p-ULK1、微管相关蛋白1轻链3(LC3)-Ⅰ及LC3-Ⅱ蛋白表达水平并计算p-AMPK/AMPK、p-ULK1/ULK1、LC3-Ⅱ/LC3-Ⅰ比值;PCR法检测大鼠骨骼肌AMPK、ULK1和LC3 mRNA表达水平.结果:与空白组比较,造模组大鼠体质量下降(P<0.05),力竭游泳时间缩短、旷场实验跨格数与直立次数显著减少(P<0.05);与模型组比较,温针灸组与辅酶组大鼠体质量上升(P<0.05),力竭游泳时间延长、旷场实验跨格数与直立次数增加(P<0.05).与空白组比较,模型组p-ULK1/ULK1比值升高(P<0.05);与模型组比较,温针灸与辅酶组骨骼肌中p-AMPK/AMPK、p-ULK1/ULK1、LC3-Ⅱ/LC3-Ⅰ蛋白表达比值显著升高(P<0.05).与空白组比较,模型组大鼠骨骼肌ULK1、LC3 mRNA表达升高(P<0.05),骨骼肌中线粒体呈空泡样,肿胀明显;与模型组比较,温针灸组与辅酶组骨骼肌中AMPK、ULK1、LC3 mRNA表达升高(P<0.05),辅酶组大鼠骨骼肌中线粒体肿胀稍减轻,温针灸组大鼠骨骼肌中线粒体空泡样进一步减少,肿胀明显减轻.结论:温针灸干预CFS大鼠可通过激活AMPK/ULK1信号通路,提高AMPK、ULK1、LC3相关因子表达,促进骨骼肌线粒体自噬,改善线粒体形态,从而达到治疗慢性疲劳综合征的目的.
Objective To observe the effect of warming needle moxibustion(WNM)on the expression of AMP-activated protein kinase(AMPK)/Unc-51 like autophagy activating kinase 1(ULK1)signaling pathway in chronic fatigue syndrome(CFS)rats,so as to explore its mechanism underlying improvement of CFS.Methods Male SD rats were randomly divided into control,model,WNM and coenzyme groups,with 10 rats in each group.The CFS model was established by multi-factor compound stress stimulation method(exhaustive swimming+chronic restraint+alternate-day fasting).The rats of the WNM group received warming needle moxibustion stimulation at bilateral"Zusanli"(ST36),"Guanyuan"(CV4)and"Zhongwan"(CV12)for 15 min,once daily for 14 days.The rats of the coenzyme group were administered coenzyme Q10(1 mg/kg)by gavage once daily for 14 days.Body weight and swimming time of the rats were recorded before and after modeling and after intervention.Behavioral changes were assessed using the open-field test.Histological changes in skeletal muscle were observed via HE staining.The structure of mitochondria and autophagosomes in skeletal muscle were observed using transmission electron microscopy.The protein expression levels of AMPK,phosphorylated(p)-AMPK,ULK1,p-ULK1,microtubule-associated protein 1 light chain 3(LC3)-Ⅰ and LC3-Ⅱ in skeletal muscle were detected by Western blot,and the ratios of p-AMPK/AMPK,p-ULK1/ULK1 and LC3-Ⅱ/LC3-Ⅰ were calculated.The mRNA expression levels of AMPK,ULK1 and LC3 in skeletal muscle were detected by real-time PCR.Results Compared with the control group,the body weight,swimming time,and the duration of standing and the number of grid crossing were significantly reduced(P<0.05),while the mRNA expression levels of ULK1 and LC3,and the ratio of p-ULK1/ULK1 were significantly increased in the model group(P<0.05).Compared with the model group,the body weight,swimming time,and the duration of standing and the number of grid crossing were significantly increased(P<0.05),and the mRNA expression levels of AMPK,ULK1 and LC3,as well as the ratios of p-ULK1/ULK1 and LC3-Ⅱ/LC3-Ⅰ,were significantly up-regulated in both the WNM and coenzyme groups(P<0.05).Conclusion Warming needle moxibustion can alleviate chronic fatigue syndrome by activating the AMPK/ULK1 pathway,upregulating the expression of AMPK/ULK1/LC3,and promoting mitochondrial autophagy,thereby enhancing mitochondrial morphology.
原艺娜;刘娣;刘君伟;李花园;李龙;武永利
宁夏医科大学中医学院,银川 750004宁夏医科大学总医院中医骨伤科,银川 750004宁夏医科大学总医院中医骨伤科,银川 750004宁夏医科大学中医学院,银川 750004宁夏医科大学总医院中医骨伤科,银川 750004宁夏医科大学总医院中医骨伤科,银川 750004||宁夏少数民族医药现代化教育部重点实验室,银川 750004
慢性疲劳综合征腺苷酸活化蛋白激酶/Unc-51样激酶信号通路温针灸线粒体自噬骨骼肌
Chronic fatigue syndromeAMPK/ULK1 signaling pathwayWarming needle moxibustionMitochondrial autophagySkeletal muscle
《针刺研究》 2026 (2)
189-198,10
宁夏自然科学基金项目(No.2022AAC03532)
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