The Mechanism Of Buyang Huanwu Decoction In Delaying Denervated Skeletal Muscle Atrophy Was Studied Via TGF-?1/Smad3 Pathway

Objective:To evaluate the effects of Buyang Huanwu Decoction(BHD)on skeletal muscle fibrosis and TGF-?1 / Smad3 pathway in denervated rats,so as to explain its mechanism of delaying skeletal muscle atrophy.Methods:Sciatic nerve was severed in 32 SD rats to establish a denervated muscle atrophy model,the rats was randomly divided into a sham-operated group,a model group,a BHD group,a methycobal group,with 8 in each group.After 21 days of intervention,bilateral gastrocnemius muscles were completely removed to calculate the wet-weight ratio.Skeletal muscle atrophy and fibrosis were observed by HE staining and the cross-sectional area of the muscle fiber was measured.The expression of TGF-?1?Smad3?MyoD1?Pax7 mRNA were tested by RT-PCR.The expression level of TGF-?1?p-Smad3 protein were detected by Western blot.Immunofluorescence was used to detect the positive expression of MyoD1 and Pax7,and the correlation between TGF-?1,Smad3,MyoD1 and Pax7 proteins was analyzed by bioinformatics.Results: 1.Muscle wet weight ratio and cross-sectional area results: Compared with the sham-operated group,the model group,methycobal group,and BHD group were significantly reduced,especially in the model group.The muscle wet weight ratio and cross-sectional area of the methycobal group and the BHD group were significantly higher than those of the model group(P <0.05);however,there was no significant difference between methycobal group and BHD group(P> 0.05).2.HE staining results: In the sham-operated group,the muscle cells were round and full with small space,and muscle cells atrophy and fibrosis were not obvious;In the model group,a large number of muscle cells atrophied,distorted,and degenerated,and the intercellular space significantly widened,the fiber bundles were significantly shrunk,the arrangement structure was disordered,the shape was irregular,the structure was not clear,the cross-sectional area was reduced,the intercellular space significantly widened,a large number of connective tissue hyperplasia,and fibrosis was obvious.Muscle cells in the BHD group and methycobal group were atrophic,but the fiber bundles were arranged in an orderly structure,regular morphology,uniform staining,clear structure,less connective tissue hyperplasia,lighter fibrosis,and less degeneration of muscle cells,and the intercellular space,cross-sectional area of the fiber bundles and degree of fibrosis were significantly better than those in the model group.3.RT-PCR results: TGF-?1,Smad3,MyoD1,and Pax7 mRNA expressions were lowest in the sham-operated group;compared with the model group,TGF-?1 and Smad3 mRNA expression levels were significantly reduced in the methycobal group and BHD group(P <0.05),while the expression levels of MyoD1 and Pax7 mRNA were significantly increased(P <0.05);Compared with the methycobal group,the expression levels of TGF-?1 and Smad3 mRNA were lower in the BHD group(P <0.05),and the expression levels of MyoD1 and Pax7 mRNA were higher,the differences were statistically significant(P <0.05).4.Western blot results: The expression levels of TGF-?1 and p-Smad3 protein were the lowest in the sham-operated group;Compared with the model group,the expression levels of TGF-?1 and p-Smad3 protein in the methycobal group and BHD group were significantly reduced(P <0.05);Compared with methycobal group,the expression level of TGF-?1 and p-Smad3 protein was lower in the BHD group,the difference was statistically significant(P <0.05).5.Immunofluorescence results: The MyoD1 and Pax7 positive expression rates were the lowest in the sham-operated group;Compared with the model group,the positive expression rates of MyoD1 and Pax7 in the methycobal group and BHD group were significantly increased(P <0.05).However,there was no significant difference in the positive expression rate of MyoD1 and Pax7 between the BHD group and the methycobal group(P> 0.05).6.Bioinformatics results: TGF-?1,Smad3,MyoD1,and Pax7 were closely related,and have a one-to-one correspondence relationship.TGF-?1 directly acts on Smad3,and Smad3 then sequentially on MyoD1 and Pax7.Conclusions: 1.BHD can effectively reduce the decrease of skeletal muscle wet weight ratio and cross-sectional area in denervated rats,reduce the degree of skeletal muscle fibrosis,and delay the process of denervated skeletal muscle atrophy.2.BHD can reduce the degree of skeletal muscle fibrosis by inhibiting the activation of the TGF-?1 / Smad3 pathway,and then promote the activation of Muscle Satellite Cells(MSC)to delay the progression of skeletal muscle atrophy.