The Role Of Epithelial Sodium Channel In Glucocorticoid Induced Skeletal Muscular Atrophy

Ami: To establish the model of glucocorticoid induced skeletal muscle atrophy in Wistar inbred line and closed group rats and compare the difference of GIMA model in two strains of rats.To observe mRNA content changes of glucocorticoid receptor,alpha ENaC and muscle atrophy F-box gene of GIMA models gastrocnemius.To explore the role of ENaC in GIMA from the level of whole animal.To observe the changes of proliferation and differentiation of myoblasts C2C12 in vitro under ENaC specificity intervention agent amiloride and dexamethasone,explore the possible role of ENaC in the effect of GC on skeletal muscle regeneration at the cellular level.Method: Inbred Wistar and outbred rats each 32 were randomly divided into 4 groups:the control group administered(group C)intramuscular injection of 1 mg/kg normal saline,4 times per week,for 90 days;low frequency group(L group),medium group(M group)and High group(H group)were 1,3,5 a week intramuscular injection of Dex 1 mg/kg for90 days.The right gastrocnemius of rats was examined by paraffin section and HE staining to observe the morphology and cross-sectional area of muscle fibers;Take the left gastrocnemius muscle for frozen section and muscle fiber type staining,observe the changes of type two fibers,and extract the total RNA for RT-PCR and q-PCR,and detect the mRNA content of GR,-ENaC,MAFbx.Different concentrations Dex(5 * 10-7M~1.5 *10-4M)and Ami(10-5M)alone and combination act on cultured mouse myoblasts C2C12 in vitro,cell proliferation and differentiation was measured by CCK-8 and myotube diameter methods.Results:(1)Compared with C group and L group,the weight of M and H in two strains rats were significantly reduced(P<0.01).(2)Compared with C group(P<0.01)and L group,the gastrocnemius muscle coefficient of the two strains rats in H group was significantly lower(P<0.05).(3)The cross-sectional area of muscle fiber in M group and H group of inbred strain rats was significantly lower than that of C group and L group(P<0.01).The cross sectional area of muscle fiber was significantly decreased with the increase of administration frequency(P<0.01).(4)With the increase of the frequency ofdrug use,the cross-sectional area of type II fibers in the two strains rats was significantly smaller than that in the C group(P<0.01),and the decrease of cross-sectional area of inbred strain rats was frequency dependent.(5)The mRNA content of GR,-ENaC and MAFbx gene in inbred rats were higher than those in C group,alpha-ENaC gene mRNA content in H group was significantly higher than C group and L group(P<0.01).MAFbx gene mRNA cotent in each medication group of closed group rats was significantly higher than C group(P< 0.05);Compared with C group(P<0.05)and L group(P<0.01),alpha-ENaC mRNA in M group and H group increased significantly.Compared with C group(P<0.05),L group(P<0.01),GR mRNA content in M group and H group was significantly reduced.(6)The effect of Dex on C2C12 cells for 24 h,could significantly promote cell proliferation under 5* 10-4M(P<0.05),and up to 1.5 * 10-4M there was a trend of inhibition cell proliferation,but When the time is extended to 48 h,Dex significantly inhibited cell proliferation(P<0.05).The effect of 10-5M Ami on C2C12 cell proliferation was significantly promoted for 24h(P<0.05),and no significant change was observed for 48 h.10-5M Ami and Dex combination acting on C2C12 cells for 24 h,under 5*10-4M there was a trend of promote cell proliferation,but increased to 1.5*10-4M Dex can inhibit cell proliferation;Dex have an inhibited cell proliferation trend for 48 h,and the combination acting with Ami more significantly inhibited(P<0.05).Ami and Dex used alone or in combination on C2C12 cells in 72 h differentiation stage,muscle diameter were significantly reduced(P<0.05),when Dex concentration increased to 1.0~1.5 * 10-4M,Dex combined with Ami had a more inhibitory effect on cell differentiation(P<0.05).Conclusion: The GIMA model was successfully induced in two strains of rats with Dex of 1 mg/kg per week for intramuscular injection of 3 or 5 times.The inbred strain model was more typical than the closed group model,and the dose effect of GC was more significant.GC can make the alpha-ENaC and MAFbx gene mRNA in two strains GIMA rats skeletal muscle tissue increased.At the same time,with the increase of Dex administration frequency,the expression of GR gene in inbred line model had a dependent trend,and expression of alpha-ENaC?MAFbx gene and GR gene were increased,showing the skeletal muscle fibers of the inbred line model have a consistent population response toGC as a signal molecule,it shows the advantages of inbred strains in the study of genetic background.It is suggested that skeletal muscle fiber GR mediated exogenous GC can induce the high expression of ENaC gene and atrophy gene in skeletal muscle,which may be related to GIMA.In addition,The Dex and Ami intervented myoblast C2C12 proliferation and differentiation in different drug concentration and time.The results show that GC has a dual regulating effect on myoblast proliferation,short term use of low concentrationto promoted cell proliferation and long-term use of high concentration inhibited cell proliferation.And Dex can inhibit cell differentiation.When the ENaC function by short-term inhibition can promote myoblast proliferation,but on myoblast differentiation were inhibited,and with the role of GC,the inhibition of Ami on cell proliferation and differentiation is more significant,suggesting that ENaC of the skeletal muscle fibers may be involved in the regulation of myoblast proliferation and differentiation,normal ENaC function can help myoblasts differentiation,may have a positive impact on the skeletal muscle fiber regeneration of GIMA.