| Objective:The study clarified the correlation between renin-angiotensin system（RAS）abnormality and skeletal muscle atrophy,and explored the effect mechanism of vitamin D（Vit.D）and RAS in regulating glucocorticoid-induced skeletal muscle atrophy（GM）.To investigate the efficacy and mechanism of Shengyu Decoction（SYD）in the prevention and treatment of skeletal muscle atrophy.Methods:1.The murine myoblast cell line C2C12 was induced to differentiate into myotubes with 2%horse serum,and then treated with angiotensin II（Ang II）and its type 1 receptor（AT1R）blocker（ARB）Olmesartan.Myosin heavy chain（MHC）-stained fluorescent images were analyzed to quantifying the average area of the myotubule.The protein expression of ubiquitin ligase-related proteins（MURF1 and MAFbx）,myogenic regulatory factors（MRFs）-related proteins（Myo D and MHC）and PI3K/Akt/FOXO1signaling pathway-related proteins were detected by Western blot（WB）.2.The wild type and VDR（-/-）mice were treated with vehicle and dexamethasone（Dex,i.p.,25 mg/kg once daily）for 10 consecutive days to induce GM.Evaluation for skeletal muscle mass using body weight and skeletal muscle wet weight coefficient and skeletal muscle function measuring with the four-limb grip test and weight-loaded swimming test.Dystrophin-immunofluorescence-stained and haematoxylin–eosin（H&E）-stained sections were used to define the cross section area of muscle fibers.Myostatin（MSTN）and Myo D expression were assessed by PCR,while the protein levels of ubiquitin ligase,MRFs and PI3K/Akt/FOXO1 signaling pathway were assessed by WB.Expression levels of genes and proteins in RAS were identified by PCR and western blot.3.C57BL/6J mice were induced GM and treated with low dose（1μg/kg）and high dose（3μg/kg）of active Vit.D calcitriol administered by gavage for 10 consecutive days.The changes of skeletal muscle mass and function were calculated as well as skeletal muscle morphology was measured by immunofluorescence and H&E staining.PCR and WB were used to detect the expression of genes and proteins related to skeletal muscle atrophy and formation in mice,as well as the local RAS components of skeletal muscle.4.Sprague-Dawley（SD）rats were given normal saline and low（6.75 g/kg）,medium（20.25 g/kg）,and high doses（60.75 g/kg）of SYD for 7 days continuously.Blood was taken from the abdominal aorta,and test the serum 1,25-dihydroxyvitamin D3（1,25D）levels with ELISA.In addition,Stable transgenic strain with VDR gene knockout（VDR-sh）and its control strain（VDR-con）were constructed by sh RNA and interfering lentiviral vectors in the myoblast cell line C2C12,and treated with medicated serum of SYD low,medium,and high doses（10%）and active Vit.D paricalcitol（Pari）(10-7M).MHC immunofluorescent staining was used to detect the changes in the mean area of the myotubule.WB was used to detect the protein expression of ubiquitin ligase and MRFs.5.C57BL/6J mice were induced GM and treated with low dose（4.875 g/kg）,medium dose（9.75 g/kg）and high dose（19.5 g/kg）of SYD and calcitriol（0.1μg/kg）,The changes of skeletal muscle mass and function in mice were detected,and the changes of skeletal muscle morphology were detected by dystrophin immunefluorescent staining and H&E staining.Blood was taken from the abdominal aorta,and test the serum 1,25D levels with ELISA.The genes of MSTN,Myo D,AGT and renin and the proteins expression of ubiquitin ligase-related,MRFs,VDR,CYP27B1,CYP24A1 and RAS components were detected by PCR.and WB.Results:1.Compared with the Control group,after Ang II intervention,the IF staining with MHC showed that the average area of the myotube was significantly reduced（P<0.001）,and the expression of myotube MURF1（P<0.05）and MAFbx（P<0.01）protein were significantly increased,while the expression of MHC（P<0.05）and Myo D（P<0.05）protein were significantly reduced（P<0.05）.The ratio of p-PI3K/PI3K（P<0.01）,p-Akt/Akt（P<0.05）0.001)and p-FOXO1/FOXO1（P<0.05）were decreased.Compared with the Ang II group,after ARB pretreatment,the average area of myotubes was significantly increased（P<0.001）,myotubes MURF1（P<0.01）and MAFbx（P<0.05）protein expression decreased,and MHC（P<0.05）)And Myo D（P<0.05）protein expression increased,and the ratios of p-PI3K/PI3K（P<0.01）,p-Akt/Akt（P<0.001）and p-FOXO1/FOXO1（P<0.01）increased.2.The decline of body weight was obviously found in mice with Dex treatment,and more profound in Dex-treated VDR（-/-）mice as compared to Dex-treated WT mice.Muscle index showed that Dex induced decrease in tibialis anterior weight（P<0.01）and gastrocnemius weight（P<0.01）,and affected muscle function demonstrated by the drop in weight-loaded swimming time（P<0.05）and grip strength（P<0.05）.There was severe decrease in muscle weight（P<0.01）,weight-loaded swimming time（P<0.05）and grip strength（P<0.001）in the Dex-treated VDR（-/-）mice as compared to the WT counterparts.H&E staining and IF staining with anti-dystrophin antibody showed the reduction in cross-sectional area of tibialis anterior in the Dex-induced atrophy model groups compared with vehicle-treated control,moreover,the reduction was more robust in VDR（-/-）mice than that in WT mice.Furthermore,Dex treatment for 10 days induced MSTN m RNA expression in both WT（P<0.001）and KO mice（P<0.001）,and the induction on MSTN was much more robust in VDR（-/-）mice（P<0.001）as compared to VDR（+/+）group.The expressions of Myo D at both m RNA level and protein level as well as the protein expression of MHC were significantly down-regulated in tibialis anterior of mice after treatment with Dex as compared to respective vehicle control,and the down-regulation was more dramatic in VDR（-/-）mice.Treatment with Dex highly stimulated the protein expression of MURF1 and MAFbx,and this up-regulation was much higher in VDR（-/-）mice than that in VDR（+/+）mice..Dex treatment caused a dramatic down-regulation in the ratio of p-PI3K/PI3K（P<0.001）,p-Akt/Akt（P<0.05）and p-FOXO1/FOXO1（P<0.01）,and immunoblotting clearly showed that the ratios of these proteins were much lower in VDR（-/-）mice than those in VDR（+/+）mice after Dex treatment.RAS components including angiotensinogen（AGT）,renin and Ang II were measured in mice tibialis anterior.Gene expression of AGT（P<0.001）and renin（P<0.001）and protein expression of renin（P<0.01）and Ang II（P<0.001）were markedly induced in Dex-treated VDR（+/+）and VDR（-/-）mice.Furthermore,the up-regulation in AGT and renin m RNA expression and renin and Ang II protein expression was much more significant in KO mice as compared with VDR（+/+）mice.3.Calcitriol did not affect Dex-induced decrease in body weight.However,treatment with calcitriol significantly increased skeletal muscle mass shown by the elevation in muscle index of tibialis anterior and gastrocnemius,and suppressed Dex-induced reduction in weight-loaded swimming time and grip strength.Moreover,the fiber area of tibialis anterior as shown by both H&E staining and dystrophin-positive IF staining was dramatically enhanced in calcitriol-treated groups as compared with those in vehicle-treated model group.The genes expression of MSTN and Myo D were increased and decreased,respectively,in tibialis anterior of mice with Dex-induced skeletal muscle atrophy.Calcitriol played a regulation on MSTN m RNA expression and Myo D m RNA expression with an decrease and increase.Treatments of mice with muscle atrophy by calcitriol at low dose and high dose significantly down-regulated protein expression of MURF1 and MAFbx as compared to those treated by vehicle.Furthermore,the substrate proteins for myogenesis,including MHC and Myo D,of ubiquitin ligases were profoundly up-regulated by calcitriol.Gene expression of AGT and renin was significantly lower in calcitriol-treated group than those in vehicle-treated atrophy group.Similarly,calcitriol dose-dependently down-regulated protein level of renin and Ang II in tibialis anterior of mice with muscle atrophy induced by Dex.4.Target virus（PLVE3005）of VDR infecting C2C12 cell showed that the expression of VDR gene and protein decreased significantly.Compared with the control group,after medium and high dose of SYD intervention,the serum 1,25D concentration of rats increased significantly.Challenging the VDR-con and VDR-sh with different concentrations of SYD containing serum and Pari,the average area of myotube was significantly increased in the VDR-con,but no significant changing in the VDR-sh.Furthermore,the expression of MURF1 and MAFbx protein were significantly reduced,and the expression of MHC and Myo D protein were significantly higher in the low,medium and high dose of SYD and Pari groups as compared with the control group.However,there were no significant changes in the expression of MURF1,MAFbx,MHC and Myo D protein in the VDR-sh.5.Compared with the model group,after the intervention of medium and high doses of SYD and calcitriol,the wet weight coefficient of tibialis anterior and gastrocnemius,the grip strength,and the exhaustion time of the weight-loaded swimming experiment increased significantly.The average cross-sectional area of the tibialis anterior was significant increase in the SYD and calcitriol groups.Compared with the control group,after SYD and calcitriol intervention,the serum 1,25D concentration of mice increased significantly.The MSTN gene expression decreased,while Myo D gene expression increased after treating with SYD and calcitriol.The expression of MURF1,MAFbx and CYP24A1 proteins and RAS component genes and proteins were decreased challenging with medium and high doses of SYD and calcitriol,but the expression of MHC,Myo D,VDR and CYP27B1 protein were increased.Conclusions:Ang II combined with AT1R induces skeletal muscle atrophy through PI3K/Akt/FOXO1 signaling pathway.Activating the Vit.D system can inhibit skeletal muscle local RAS and improve skeletal muscle atrophy.Furthermore,SYD can activate the Vit.D system in vivo to effectively improve skeletal muscle atrophy confirmed that the Vit.D system may be one of the targets of SYD to improve muscle atrophy. |
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