Background: Functional disorders of mitochondria are closely related to muscle diseases. Many studies have also shown that oxidative stress can stimulate the production of a large number of reactive oxygen species (ROS), which have various adverse effects on mitochondria and can damage muscle cells. Purpose: In this study, based on our previous research, we focused on the PINK1/Parkin pathway to explore the mechanism by which AS-IV alleviates muscle injury by inhibiting excessive mitophagy. Methods: L6 myoblasts were treated with AS-IV after stimulation with hydrogen peroxide (H2O2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Then, we detected the related indices of oxidative stress and mitophagy by different methods. A PINK1 knockdown cell line was established by lentiviral infection to obtain further evidence that AS-IV reduces mitochondrial damage through PINK1/Parkin. Results: After mitochondrial damage, the expression of malondialdehyde (MDA) and intracellular ROS in L6 myoblasts significantly increased, while the expression of superoxide dismutase (SOD) and ATP decreased. The mRNA and protein expression levels of Tom20 and Tim23 were decreased, while those of VDAC1 were increased. PINK1, Parkin, and LC3 II mRNA and protein expression increased, and P62 mRNA and protein expression decreased·H2O2 combined with CCCP strongly activated the mitophagy pathway and impaired mitochondrial function. However, abnormal expression of these factors could be reversed after treatment with AS-IV, and excessive mitochondrial autophagy could also be reversed, thus restoring the regulatory function of mitochondria. However, AS-IV-adjusted function was resisted after PINK1 knockdown. Conclusion: AS-IV is a potential drug for myasthenia gravis (MG), and its treatment mechanism is related to mediating mitophagy and restoring mitochondrial function through the PINK1/Parkin pathway.

线粒体自噬是通过有选择性地将功能障碍或受损的线粒体清除,PTEN诱导激酶1(PINK1)/E3泛素连接酶(Parkin)信号通路是线粒体自噬发生的经典信号通路,其与神经肌肉病发生机制的研究是目前研究的热点.通过线粒体自噬保持细胞内线粒体的质量和数量的平衡,对维持骨骼肌生理功能具有重要意义.笔者基于中医"脾藏肌肉之气"理论,从营卫角度阐发脾与肌肉之间的联系,结合现代医学对线粒体自噬的研究,分析线粒体自噬与重症肌无力的相关性,以期阐明健脾益气法治疗重症肌无力强肌健力作用的科学内涵,为临床治疗神经肌肉相关疾病提供理论支撑.

Due to challenges in diagnosing myasthenia gravis (MG), identifying novel diagnostic biomarkers for this disease is essential. Mitochondria are key organelles that regulate multiple physiological functions, such as energy production, cell proliferation and cell death. In the present study, Mfn1/2, Opa1, Drp1, Fis1, AMPK, PGC-1α, NRF-1 and TFAM were compared between patients with MG and healthy subjects to identify potential diagnostic biomarkers for MG. Blood samples were collected from 50 patients with MG and 50 healthy subjects. The participants' demographic information and routine blood test results were recorded. Mitochondrial dynamics were evaluated and levels of Mfn1/2, Opa1, Drp1, Fis1, AMPK, PGC-1α, NRF-1 and TFAM were determined in peripheral blood mononuclear cells using western blotting and reverse transcription-quantitative PCR, respectively. Receiver operating characteristic curve analysis was used to evaluate the diagnostic accuracy of these indicators. The areas under the curve values of Mfn1/2, Opa1, Drp1, Fis1,AMPK, PGC-1α, NRF-1 and TFAM were 0.5408-0.8696. Compared with control subjects, mRNA expression levels of Mfn1/2, Opa1, AMPK, PGC-1α, NRF-1 and TFAM were lower, while those of Drp1 and Fis1 were higher in patients with MG. The protein expression levels of all these molecules were lower in patients with MG than in control subjects. These results suggested that mitochondrial dynamics and biogenesis indicators may be diagnostic biomarkers for MG.

[目的]探讨脾虚证中线粒体能量代谢障碍机制及健脾益气方药的干预作用.[方法]采用皮下注射利血平法构建脾虚大鼠模型,将造模成功后的大鼠分为脾虚模型组和四君子汤组,每组各7只,另取正常组7只.四君子汤组给予四君子汤1.0 g·mL-1·kg-1灌胃,脾虚模型组和正常组灌胃等体积生理盐水,每日1次,持续21 d.给药期间记录大鼠一般情况.给药结束后,采用对溴苯胺法测定尿D-木糖排泄率,采用苏木素-伊红(HE)染色法观察骨骼肌病理改变,采用聚合酶链反应(PCR)法检测大鼠骨骼肌细胞色素c氧化酶(COX)Ⅰ、COXⅣmRNA表达,采用免疫组织化学法、蛋白免疫印迹法检测大鼠骨骼肌COXⅠ、COXⅣ蛋白表达.[结果]与正常组比较,脾虚模型组大鼠骨骼肌肌纤维排列紊乱、间隙增大,细胞核分布不均,胞质染色不匀;与脾虚模型组比较,四君子汤组骨骼肌纤维排列较整齐.脾虚模型组大鼠骨骼肌COXⅠ、COXⅣmRNA和蛋白表达水平较正常组降低(P<0.05或P<0.01);四君子汤组大鼠骨骼肌COXⅠ、COXⅣmRNA和蛋白表达水平较脾虚模型组升高(P<0.05或P<0.01).[结论]脾虚大鼠存在骨骼肌病理形态损伤,骨骼肌COXⅠ、COXⅣmRNA和蛋白表达均降低.四君子汤改善脾虚大鼠骨骼肌病理结构,提高COXⅠ、COXⅣmRNA和蛋白表达可能是其修复脾虚证能量代谢障碍的机制之一.

Oxidative stress can cause the excessive generation of reactive oxygen species (ROS) and has various adverse effects on muscular mitochondria. Qiangji Jianli decoction (QJJLD) is an effective traditional Chinese medicine (TCM) that is widely applied to improve muscle weakness, and it has active constituents that prevent mitochondrial dysfunction. To investigate the protective mechanism of QJJLD against hydrogen peroxide- (H2O2-) mediated mitochondrial dysfunction in L6 myoblasts. Cell viability was determined with MTT assay. Mitochondrial ultrastructure was detected by transmission electron microscope (TEM). ROS and mitochondrial membrane potential (MMP) were analyzed by fluorescence microscope and flow cytometry. The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) level were determined by WST-1, TBA, and DTNB methods, respectively. The mRNA and protein levels were measured by quantitative real-time PCR (qRT-PCR) and Western blot. The cell viability was decreased, and the cellular ROS level was increased when L6 myoblasts were exposed to H2O2. After treatment with QJJLD-containing serum, the SOD and GSH-Px activities were increased. MDA level was decreased concurrently. ROS level was decreased while respiratory chain complex activity and ATP content were increased in L6 myoblasts. MMP loss was attenuated. Mitochondrial ultrastructure was also improved. Simultaneously, the protein expressions of p-AMPK, PGC-1α, NRF1, and TFAM were upregulated. The mRNA and protein expressions of Mfn1/2 and Opa1 were also upregulated while Drp1 and Fis1 were downregulated. These results suggest that QJJLD may alleviate mitochondrial dysfunction through the regulation of mitochondrial dynamics and biogenesis, the inhibition of ROS generation, and the promotion of mitochondrial energy metabolism.

The Qiangji Jianli Decoction (QJJLD) is an effective Chinese medicine formula for treating Myasthenia gravis (MG) in the clinic. QJJLD has been proven to regulate mitochondrial fusion and fission of skeletal muscle in myasthenia gravis. In this study, we investigated whether QJJLD plays a therapeutic role in regulating mitochondrial biogenesis in MG and explored the underlying mechanism. Rats were experimentally induced to establish autoimmune myasthenia gravis (EAMG) by subcutaneous immunization with R97–116 peptides. The treatment groups were administered three different dosages of QJJLD respectively. After the intervention of QJJLD, the pathological changes of gastrocnemius muscle in MG rats were significantly improved; SOD, GSH-Px, Na+-K+ ATPase and Ca2+-Mg2+ ATPase activities were increased; and MDA content was decreased in the gastrocnemius muscle. Moreover, AMPK, p38MAPK, PGC-1α, NRF-1, Tfam and COX IV mRNA and protein expression levels were also reversed by QJJLD. These results implied that QJJLD may provide a potential therapeutic strategy through promoting mitochondrial biogenesis to alleviate MG via activating the AMPK/PGC-1α signaling pathway.