[Effect of electroacupuncture on amyotrophia and expression of myogenic differentiation-related genes of gastrocnemius in rats with chronic constriction injury of sciatic nerve].

OBJECTIVE: To explore the effect of electroacupuncture(EA) on amyotrophia and expression of paired box7(Pax7), myogenic differentiation antigen-1 (Myod1), myogenin (Myog), myosin heavy chain- Ⅱa (Myh2), myosin heavy chain-Ⅱx (Myh1) and myosin heavy chain-Ⅰ (Myh7) of denervated gastrocnemius in rats with chronic constriction injury (CCI) of the right sciatic nerve, so as to explore its mechanisms underlying postponing development of amyotrophia. METHODS: Sixty-six SD adult male rats were randomly divided into sham operation (sham) group (n＝24), model group (n＝24) and EA group (n＝18). The denervated muscle (gastrocnemius) atrophy model was established by CCI of the right sciatic nerve. EA (2 Hz，1.0 mA) was applied to the right "Zusanli"(ST36) and "Huantiao"(GB30) for 10 min, once a day, six times a week and for 1, 2 and 4 weeks, respectively. After complete dissection of bilateral gastrocnemius muscles, their wet weight levels were measured and the ratio of wet weight (＝that of the operation side/that of the non-operation side) was calculated, and the cross-sectional area (CSA) and diameter of the gastronemius were detected after fixation in 4% paraformaldehyde, sectioning, and H．E. staining. The expression levels of Pax7, Myod1, Myog, Myh2, Myh1 and Myh7 mRNAs in the gastrocnemius tissue after 3 weeks of modeling were detected with quantitative real time-PCR (qPCR). RESULTS: After 1 week of modeling, the ratios of wet weight of gastrocnemius and the CSA and fiber diameter at the 2nd, 3rd and 5th week were significantly smaller in the model group than in the sham group (P<0.05). The expression levels of Myod1 and Myog mRNAs were significantly up-regulated (P<0.01), and those of Myh2, Myh1 and Myh7 considerably down-regulated in the model group compared with the sham group (P<0.05, P<0.01). No significant changes were found in the expression levels of Pax7 mRNA after modeling and EA intervention (P>0.05)．Following EA intervention, the CSA and diameterof the gastronemius were obviously increased (P<0.05), and the expression levels of Myod1, Myog and Myh7 further markedly or remarkably up-regulated in comparison with the model group (P<0.05, P<0.01). No significant changes were found in the ratio of wet weight of gastrocnemius at the 3 time-points, and the expression levels of Myh2 mRNA and Myh1 mRNA in the EA group relevant to the model group after 3 weeks of modeling (P>0.05). CONCLUSION: EA treatment may delay gastrocnemius atrophy in CCI rats, which is possibly associated with its effects in up-regulating the expression of Myod1, Myog and Myh7 mRNAs to control the differentiation of the satellite cells and the muscle fiber type transformation.

Effects of Electroacupuncture on Mammalian Target of Rapamycin/70-KD Ribosomal Protein S6 Kinase Pathway in Gastrocnemius Muscle in Denervated Rats / 中国康复理论与实践

@#Objective To explore the effects and mechanism of electroacupuncture (EA) on denervation-induced atrophy in rats. Methods A total of 18 male Sprague-Dawley rats were divided into sham group (n=6), model group (n=6) and EA group (n=6). The latter two groups were clamped right sciatic nerve to establish atrophy model of skeletal muscle. On the second day after modeling, EA group accepted electroacupuncture on right Zusanli (ST36) and Huantiao (GB30) for two weeks. Their gastrocnemius muscles were obtained after intervention, and the wet weight ratio of the gastrocnemius muscles was calculated. The cross-sectional area (CSA) and diameter of muscle fibers were measured after HE staining. The protein expression of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), 70-KD ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6k (p-p70S6k) was tested with Western blotting. The gene expression of mTOR and p70S6K was detected with real-time quantitative polymerase chain reaction. Results Compared with the sham group, the wet weight ratio of the gastrocnemius muscle, CSA and diameter of the muscle fibers decreased in the model group and EA group (P<0.001), which were more in EA group than in the model group (P<0.01); the protein expression of mTOR, p-mTOR, p70S6K and p-p70S6K increased in the model group (P<0.01), and increased more in EA group (P<0.05); the gene expression of mTOR and p70S6K increased in the model group (P<0.05) , and increased more in EA group (P<0.05).Conclusion Electroacupuncture delays the atrophy of denervated skeletal muscles, which may relate to activation of mTOR/p70S6K signal pathway to impact synthesis of skeletal muscle proteins.

Advance in Model Establishment for Denervated Skeletal Muscle Atrophy (review) / 中国康复理论与实践

The skeletal muscle atrophy could be induced by the injury of nerve. According to the source of denervated skeletal mus-cle atrophy, it could be divided into exogenous muscle atrophy and endogenous muscle atrophy. In recent years, the ex-ogenous muscle atrophy models are mainly established by operating, physically injuring or chemically injuring, while the endogenous muscle atrophy models are mainly established by the transgenic animals of amyotrophic lateral sclero-sis. The selection and optimazation of animal models are crucial for the basic studies of denervated skeletal muscle atro-phy.

The evolution of the denervated gastrocnemii and it's miR-206 and myoD expression during skeletal muscle atrophy in rats / 中华显微外科杂志

Objective To investigate the expression pattern of skeletal muscle specific miR-206,myogenesis related myoD which change with time in dcnervated muscle atrophy rats.Methods From June,2015 to January,2016,40 SPF sprague-dawley rats were equally classified into 5 groups randomly according to standard settled before,5 groups were separately defined as denervated 0d group,denervated 1d group,denervated 7d group,denervated 14d group,and denervated 28d group.Each group contained 8 rats.The rats atrophy models were established by cutting sciatic never on left side.According to the different denervated time,the gastrocnemii on both sides were obtained under anesthesia,respectively.The wet weight ratio of two compared gastrocnemii were measured,and the gastrocnemii transection was observed by HE stain,measured the expression of myoD protein by western blot,obtained the expression of miR-206,myoD mRNA by qPCR.Results According to our study on rats denervated atrophy models,the wet ratio of compared gastrocnemius would decrease rapidly,by HE stain,decease of cross sectional area in muscle fiber was observed as well as degeneration.Collagen fibers hyperplasia appeared and increased with time change.Wet ratio and transaction aera ratio of group Od,1d,7d,14d,28d were 0.99±0.04,0.92±0.07,0.68±0.11,0.39±0.06,0.27±0.07 and 0.99±0.02,0.96±0.04,0.51±0.09,0.34±0.08,0.23±０.03 respectively,difference between experimental groups and control group were statistically significant (P＜ 0.05),the differences between each experimental groups were also statistically significant (P＜ 0.05).After qPCR test of miR-206,myoD mRNA expression,it was found that their expression patterns were similar,miR-206,myoD mRNA increased at first and would reach the expression peak at the 7 th day,after that their contents decreased but still higher at the 14th day when compared with that at the 1 st day.Their expression of group 0d,1 d,7d,14d,28d were 0.24±0.06,0.34±0.04,0.68±0.04,0.49± 0.07,0.25±0.03 and 0.41 ±0.06,0.49±0.09,0.93±0.06,0.66±0.03,0.39±0.04,respectively.All experimental groups were statistically significant different when compared with 0d group except 1d group (P＜ 0.05),the differences between each experimental groups were also statistically significant(P＜ 0.05).The protein expression of myoD was also measured by western blot test,which showed nearly the same expression pattern as the mRNA expression pattern.After injury,the protein expression increased and reached the expression peak at the 7th day.The relative expression of myoD of group 0d,1d,7d,14d,28d measured by grey ratio were 1.03±0.05,1.06±0.06,1.42±0.10,0.66±0.13,0.24±0.07,respectively.The difference between experimental groups and control group were statistically significant (P ＜ 0.05),the differences between each experimental groups were statistically significant (P ＜ 0.05) as well.Conclusion The degree of muscle denervation atrophy was related to the denervated duration in rats.The expression regulation of miR-206 and myoD in gastrocnemius was similar during the muscle denervation atrophy,which suggesting having internal relationship between miR-206 and myoD.