| Objective:Liqi Buxue Decoction was used to intervene the rat model of denervation gastrocnemius atrophy,to investigate its effect on TGF-β1/Smad pathway of denervated gastrocnemius muscle and MyoD1 and myogenin expression closely related to the proliferation and differentiation of muscle satellite cells,so as to provide a theoretical basis for its clinical application and follow-up research.Methods:54 SD rats were divided into sham operation group,model group and Liqi Buxue Decoction group by random number table method.Denervated gastrocnemius atrophy model l was made by cutting right tibial nerve.The wet weight ratio of gastrocnemius muscle in each group was measured on the 7th,28 th and 56 th day of intervention;HE staining was used to observe muscle atrophy and measure the cross-sectional area and diameter of muscle cells;Masson staining was used to observe the proliferation of collagen fibers and determine the volume fraction of collagen;The TGF-β1,Smad2,Smad3,MyoD1,myogenin mRNA expression of gastrocnemius muscle in each group was detected by RT-qPCR;The TGF-β1,p-Smad2,p-Smad3,MyoD1,myogenin protein expression of gastrocnemius muscle in each group was detected by Western blot;The expression of myosin in gastrocnemius muscle was detected by immunofluorescence.Results:1.Effect of Liqi Buxue Decoction on wet weight of rat denervation gastrocnemius muscleCompared with the sham operation group,the model group and Liqi Buxue Decoction group had significantly lower plumpness,weaker elasticity and progressive muscle atrophy,The gastrocnemius muscle on the operative side of Liqi Buxue Decoction group was fuller and more elastic than that of the model group at the same time point.Compared with the sham operation group in the same period,the wet weight ratio of gastrocnemius muscle on the operation side in the model group and Liqi Buxue Decoction group decreased significantly at each time point(P<0.01);The wet weight ratio of gastrocnemius muscle at each time point in Liqi Buxue Decoction group was higher than that in model group at the same time point(P<0.05,P<0.01).2.Effect of Liqi Buxue Decoction on cross sectional area and diameter of rat denervation gastrocnemius cellsOn the 7th,28 th and 56 th day of intervention,the size of muscle cells in the sham operation group was normal,arranged neatly and compactly,and the gap was small;Compared with the sham operation group,the muscle cells in the model group and Liqi Buxue Decoction group were significantly reduced,with different sizes,irregular shapes and gradually disordered arrangement.Compared with the model group at the same time point,the muscle cells in Liqi Buxue Decoction group were significantly larger and the cell size was relatively uniform.Compared with the sham operation group in the same period,the cross-sectional area and diameter of gastrocnemius muscle cells at each time point in the model group and Liqi Buxue Decoction group were significantly lower(P<0.01).At the same time,the cross-sectional area and diameter of gastrocnemius muscle cells at each time point in Liqi Buxue Decoction group were significantly higher than those in the model group in the same period(P<0.05,P<0.01).3.Effect of Liqi Buxue Decoction on fibrosis degree and collagen volume fraction of denervation gastrocnemius muscle in ratsThe sham operation group contained a small amount of collagen fibers.The collagen fibers in the model group were gradually increased compared with those in the Liqi Buxue Decoction group on the 7th,28 th and 56 th days,and the distance between muscle fibers widened due to the proliferation of collagen fibers.At the same time,the collagen fibers of gastrocnemius in the operation side of Liqi Buxue Decoction group were less than those in the model group.Compared with the sham operation group,the collagen volume fraction of sural muscle on the operation side in the model group and Liqi Buxue Decoction group increased significantly at each time point(P<0.01),However,the collagen volume fraction of sural muscle at each time point in Liqi Buxue Decoction group was significantly lower than that in model group at the same time(P<0.01).4.Effect of Liqi Buxue Decoction on mRNA expression of TGF-β1,Smad2,Smad3,MyoD1 and Myogenin in rat denervation gastrocnemiusCompared with the sham operation group in the same period,there was no difference in Smad2 mRNA expression between the model group and the Liqi Buxue Decoction group at 7 days(P>0.05).The mRNA expressions of TGF-β1,Smad2,Smad3,MyoD1 and myogenin were all increased(P<0.05,P<0.01),in model group and Liqi Buxue Decoction group at each time point.The mRNA expressions of TGF-β1,Smad2 and Smad3 in the model group were lower than those in the model group(P<0.05,P<0.01);The mRNA expressions of MyoD1 and myogenin in the model group were higher than those in the model group at the same time(P<0.05,P<0.01).5.Effect of Liqi Buxue Decoction on protein expression of TGF-β1,Smad2,Smad3,MyoD1 and Myogenin in rat denervation gastrocnemiusCompared with the sham operation group in the same period,the expression of TGF-β1,p-Smad2,p-smad3,MyoD1 and myogenin protein in the model group and Liqi Buxue Decoction group increased at each time point(P<0.05,P<0.01),and the expression of TGF-β1,p-Smad2 and p-smad3 protein in Liqi Buxue Decoction group was lower than that in the model group in the same period(P<0.05,P<0.01);The expressions of MyoD1 and myogenin protein in the model group were higher than those in the model group at the same time(P<0.05,P<0.01).Conclusion:1.Liqi Buxue Decoction can delay the atrophy of denervated skeletal muscle by slowing down the decline of skeletal muscle wet weight,muscle cell cross-sectional area and diameter,and reducing the expression of collagen fibers.2.Liqi Buxue Decoction can effectively delay denervated skeletal muscle atrophy and inhibit TGF-β1/Smad signaling down regulates the increase of MyoD1 and myogenin,promotes the proliferation and differentiation of muscle satellite cells,and reduces fibrosis. |
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