富含半胱氨酸蛋白61对慢性肾脏病小鼠骨骼肌萎缩的影响

Effects of cysteine-rich protein 61 on skeletal muscle atrophy in mice with chronic kidney disease

目的探讨富含半胱氨酸蛋白61(CCN1)对慢性肾脏病(CKD)小鼠骨骼肌萎缩的影响。方法构建骨骼肌条件性敲除CCN1小鼠品系,将得到的29只SPF级雄性野生型小鼠和敲除型小鼠随机分为野生型假手术组(WOS组)6只、野生型造模组(WOC组)9只、敲除型假手术组(COS组)8只、敲除型造模组(COC组)6只。WOC组、COC组小鼠经5/6肾切除术建立CKD模型,WOS组、COS组小鼠进行假手术。饲养21个月后处死小鼠,检测各组小鼠血清CCN1、肌酐、尿素氮、尿白蛋白肌酐比值及体重、胫骨前肌重量、腓肠肌重量;HE染色观察肾脏病理学改变,laminin免疫荧光观察胫骨前肌肌纤维横截面积;real-time PCR检测腓肠肌CCN1、肌肉特异性环指蛋白1(MuRF1)、肌肉萎缩盒F基因(MAFbx)mRNA表达水平;Western blot法检测腓肠肌CCN1、p53蛋白表达水平。结果WOC组较WOS组、COC组较COS组小鼠血清CCN1、肌酐、尿素氮、尿白蛋白肌酐比值均升高(均P<0.05),胫骨前肌体重比值、腓肠肌体重比值和胫骨前肌肌纤维横截面积均下降(均P<0.05);腓肠肌组织CCN1、MuRF1、MAFbx mRNA表达水平均升高(均P<0.05),CCN1表达水平升高(P<0.05)。而COC组较WOC组小鼠胫骨前肌肌纤维横截面积、胫骨前肌体重比值、腓肠肌体重比值均升高(均P<0.05),腓肠肌CCN1、MuRF1、MAFbx mRNA表达水平均下降(均P<0.05),CCN1、p53蛋白表达水平均下降(均P<0.05)。结论CCN1可能通过p53途径促进CKD小鼠骨骼肌衰老和蛋白降解途径参与骨骼肌萎缩的发生。

Objective To investigated the effects of cysteine-rich protein 61(CCN1)on skeletal muscle atrophy in mice with chronic kidney disease(CKD).Methods The skeletal muscle conditional knockout CCN1 mouse strain was constructed.A total of 29 SPF male wild-type mice and knockout mice were randomly divided into wild-type sham operation group(WOS,n=6),wild-type model group(WOC,n=9),knockout sham operation group(COS,n=8),and knockout model group(COC,n=6).CKD model was established with 5/6 nephrectomy in mice of WOC group and COC group,while sham operation was performed in WOS group and COS group.The mice were sacrificed 21 months after modeling,and the serum CCN1,serum creatinine,blood urea nitrogen,urinary albumin creatinine ratio and body weight,tibialis anterior muscle weight and gastrocnemius muscle weight were measured.Renal pathological changes were observed with HE staining,and the cross-sectional area of tibialis anterior muscle was observed by Laminin immunofluorescence.Real-time PCR was used to detect CCN1,MuRF1 and MAFbx mRNA expressions in gastrocnemius muscle.The expression levels of CCN1 and p53 protein in gastrocnemius were detected by Western blot.Results Compared with WOS and COS groups,serum CCN1,creatinine,urea nitrogen levels and urinary albu-min creatinine ratio in WOC and COC groups were increased(all P<0.05),and tibialis anterior body weight ratio,gastrocnemius muscle weight ratio and tibialis anterior muscle fiber cross-sectional area were decreased(all P<0.05).The mRNA expressions of CCN1,MuRF1 and MAFbx in gastrocnemius muscle were increased(all P<0.05),and the protein expressions of CCN1 was increased(P<0.05).Compared with WOC group,tibialis anterior muscle fiber cross-sectional area,tibialis anterior body weight ratio and gastrocnemius muscle weight ratio were increased(all P<0.05),and the mRNA expression levels of CCN1,MuRF1 and MAFbx in gastrocnemius muscle were decreased(all P<0.05).The protein expression levels of CCN1 and p53 were decreased(all P<0.05).Conclusion CCN1 may promote skeletal muscle senescence and participate in skeletal muscle a-trophy through p53 pathway in CKD mice.