The Mechanism Of Fasudil Inhibits ROCK1 Induced Mitochondrial Fission Mediated Muscle Wasting In Chronic Kidney Disease

ObjectiveTo confirm whether ROCK1 stimulates mitochondrial fission,leading to the progression of muscle wasting in chronic kidney disease;MethodIn vitro:Male,C57BL/6J mice,40,were divided into 4groups according to the different treatment: sham operation group(WT group),CKD group,sham +Fasudil group(WF group),CKD+Fasudil group(CF group);The mice in WT group with renal capsule stripping,the mice in CKD group with 5/6 nephrectomy,after one month,take tail venous blood,test serum urea nitrogen(BUN)and serum creatinine(Cr)to select the succeeed CKD model mice;The mice in WT and CKD groups were randomly divided into the above four groups.The WT and CKD group mice were injected intraperitoneally with normal saline(3 times per week for 8 weeks).WF and CF groups mice were intraperitoneal injection of Fasudil(30mg/kg/time,3 times/week,sustained 8 weeks),high protein feed.Measure the weight of mice every week.After the end of the experiment,the lower limb muscles such as gastrocnemius and tibialis anterior muscle were collected according to the purpose and procedure of the experiment.The samples were treated with immunofluorescence staining and Western Blot or Real time-PCR to detect the target molecules such as ROCK1 and MuRF1 Expression to clarify whether Fasudil can reverse the increase in CKD-induced muscle atrophy and mitochondrial fissure-related proteins.In vitro cell culture: C2C12 myoblasts were cultured in vitro,2% horse serum induced cell differentiation and maturation.Adenovirus vector ROCK1 stimulated cell overexpression of ROCK1.Adenovirus vector GFP(green fluorescent protein)was administered in the control group.According to the different stimuli divided into four groups:1)Ad-GFP group,with AAV-GFP,5nmol/;2)Ad-ROCK group with AAV-ROCK1,5nmol/;3)Ad-GFPF groups,with AAV-GFP 5nmoland Fasudil 10umol/l);4)Ad-ROCK1 F group,with AAV-ROCK1 5nmoland Fasudil 10umol/l.Each group of virus stimulated for 48 hours.The changes of oxygen consumption and acid production rate of C2C12 myoblasts under different stimulation conditions were evaluated by the cellular energy metabolism analyzer(Seahorse).The expression of ROCK1 overexpression on C2C12 myoblast mitochondrial morphology was confirmed by staining.Evaluate the mitochondrial fission-related gene expression in each group.ResultsCompared with WT group,CKD group increased the expression of ROCK1,the muscle cross-sectional area decreased muscle fiber distribution,and electron microscopy showed that the mitochondrial fission increased in CKD group.Fasudil intraperitoneal injection of mice,CKD-induced muscle cross-sectional area decreased significantly,muscle fiber distribution right shift,muscle atrophy-related protein MuRF1 and mitochondrial fission-related protein p-Drp1 expression decreased.In vitro experiments,Seahorse test mitochondrial stress changes,Ad-ROCK1 group than Ad-GFP group of cells basal breathing,maximum breathing,extract breathing increased,after given FCCP stimulation,cell respiration oxygen consumption and acidity also increased;Cells mitochondrial staining showed that the mitochondrial fission increased in Ad-ROCK group,and the expression of mitochondrial fission protein p-Drp1 increased,and the release of reactive oxygen species increased.After Fasudil stimulation,the expression of ROCK1 overexpression was significantly improved P-Drp1,MuRF1,Atrogin1 decreased correct cell respiration abnormalities.ConclusionCKD can activate ROCK1,stimulate mitochondrial fission,increased cellular oxygen consumption,which mediates the occurrence of muscle atrophy;Fasudil as an inhibitor of ROCK1,can block ROCK1 to some extent through the activation of p-Drp1 induced mitochondrial fission,and eventually reach the purpose of improving muscle atrophy.