ABSTRACT
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.
ABSTRACT
@#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.
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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.
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Objective To investigate the effect on differentiation of denervated skeletal muscle-derived stem cells (MDSCs) induced by TGF-β1 in vitro.Methods MDSCs were obtained from the rat denervated skeletal muscle by preplate technique,with TGF-β1 adding on medium.Cultured cells were divided into two groups.A,control group; B,10 ng/ml TGF-β1 group.Cell growth was observed with phase contrast microscope.lmmunocytochemistry,quantitative RT-PCR and Western blot was used to detect the expression of Sca-1,COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin in denervated MDSCs.Results The synthesis of COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin by denervated MDSCs was extremely low at protein level in vitro,while Sca-1 level was really high.Belong to the treatment with TGF-β1,COL-Ⅰ,COL-Ⅲ,oα-SMA and vimentin in the denervated MDSCs had strong expression,but Sca-1 in which had a weak expression.Under the stimulation of TGF-β1,COL-Ⅰ expression reached peak at the 2nd day (12.5591 ± 0.3389),which was about 3 times as control group.COL-Ⅲ reached highest value at the 5th day (0.8956 ± 0.0438),which was about 23 times as control group.α-SMA topped out to 18 times at the 5th day (1.1090 ± 0.0018).Vimentin expression rose by 8.5 times and peaked at the 5th day (0.1794 ± 0.0019).The expression of Sca-1 began to decline at the 2nd day,with a remarkable reduction at the 5th day (0.0636 ± 0.0015).Conclusion TGF-β1 could induce differentiation of the denervated MDSCs to myofibroblasts in vitro,and promote the synthesis and excretion of extracellular matrix.