天冬酰胺合成酶对移植黑色素瘤APP/PS1小鼠骨骼肌减少的影响

Effect of asparagine synthetase on reduction of skeletal muscle in APP/PS1 mice transplanted with melanoma

目的:探讨天冬酰胺合成酶(ASNS)在移植黑色素瘤模型淀粉样蛋白前体蛋白/早老素1(APP/PS1)小鼠骨骼肌减少中的作用,并阐明其作用机制。方法:C57BL/6J(C57)小鼠和APP/PS1小鼠分别移植黑色素瘤B16细胞(C57移植瘤组和APP/PS1移植瘤组),同时设C57对照组和APP/PS1对照组,每组7只。取小鼠腓肠肌组织,分析天平称量小鼠腓肠肌质量,计算小鼠腓肠肌质量与体质量比值即肌肉减少症指数(SI),采用HE染色观察小鼠腓肠肌组织形态表现,实时荧光定量PCR(RT-qPCR)法检测小鼠腓肠肌组织中肌肉组织特异萎缩相关基因E3泛素连接酶1(Atrogin-1)、肌肉环指蛋白1(MuRF1)和ASNS mRNA表达水平,Western blotting法检测小鼠腓肠肌组织中ASNS及内质网应激途径相关蛋白葡萄糖调节蛋白78(GRP78)、活化转录因子4(ATF4)、真核细胞起始因子2α(eIF2α)和磷酸化eIF2α(p-eIF2α)蛋白表达水平。结果:与C57对照组比较,C57移植瘤组小鼠腓肠肌SI降低(P<0.05),腓肠肌细胞呈玻璃样变性,肌浆蓝染,部分细胞核可见核内移现象,小鼠腓肠肌组织中Atrogin-1、MuRF1和ASNS mRNA表达水平升高(P<0.05),ASNS蛋白表达水平升高(P<0.05),GRP78、ATF4蛋白表达水平和p-eIF2α/eIF2α蛋白表达水平比值明显升高(P<0.05);与APP/PS1对照组比较,APP/PS1移植瘤组小鼠腓肠肌SI降低(P<0.05),腓肠肌部分肌纤维肿胀,部分肌纤维变细,细胞核内移现象多见,肌浆颜色以红色为主,腓肠肌组织中Atrogin-1、MuRF1和ASNS mRNA表达水平升高(P<0.05),ASNS蛋白表达水平升高(P<0.05),GRP78、ATF4蛋白表达水平和p-eIF2α/eIF2α蛋白表达水平比值明显升高(P<0.05);与C57移植瘤组比较,APP/PS1移植瘤组小鼠腓肠肌SI降低(P<0.05),腓肠肌组织中Atrogin-1 mRNA表达水平升高(P<0.05),ASNS mRNA水平降低(P<0.05),ASNS蛋白表达水平差异无统计学意义(P>0.05),GRP78和ATF4蛋白表达水平及p-eIF2α/eIF2α蛋白表达水平比值明显降低(P<0.05)。结论:APP/PS1移植黑色素瘤模型小鼠腓肠肌更易发生肌肉减少,其机制可能与适应性反应ASNS转录水平降低有关,内质网应激PERK-eIF2a-ATF4信号途径可能参与调控ASNS转录活性。

Objective:To investigate the role of asparagine synthetase(ASNS)on reduction of skeletal muscle in the amyloid precursor protein/presenilin 1(APP/PS1)mice transplanted with melanoma,and to clarify its mechanism.Methods:The C57BL/6J(C57)and APP/PS1 mice were transplarted with melanoma cells(C57 transplanted tumor group and APP/PS1 transplanted tumor group),at the same time,C57 control group and APP/PS1 control group were set up.The mass of gastrocnemius was measured and the sarcopenia index(SI)was calculated with the gastrocnemius mass to body weight ratio.The morphology of gastrocnemius tissue of the mice was determined by HE staining.The mRNA expression levels of muscle specific atrophy gene E3 ubiquitin protein ligase 1(Atrogin-1),muscle ring finger protein 1(MuRF1)and ASNS in gastrocnemius tissue of the mice were measured by Real-time fluorescence quantitative PCR(RT-qPCR).The expression levels of ASNS and endoplasmic stress related glucose regulated protein 78(GRP78),activating transcription factor-4(ATF4),eukaryotic cell initiation factor 2α(eIF2α),phosphorylated eIF2α(p-eIF2α)in gastrocnemius tissue of the mice were measured by Western blotting method.Results:Compared with C57 control group,the SI of the mice in C57 transplanted tumor group was decreased(P<0.05),gastrocnemius cells had hyaline degeneration,the sarcoplasm was stained blue,and some muscle fiber showed intranuclear migration;the expression levels of Atrogin-1,MuRF1 and ASNS mRNA in gastrocnemius tissue of the mice were increased(P<0.05),and the expression level of ASNS protein was increased(P<0.05);the expression levels of GRP78 and ATF4 and the ratio of p-eIF2α/eIF2αwere increased(P<0.05).Compared with APP/PS1 control group,the SI of the mice in APP/PS1 transplanted tumor group was decreased(P<0.05),some muscle fibers of gastrocnemius were swelling,and some were thin;intranuclear migration was easily seen,and the sarcoplasm was red;the expression levels of Atrogin-1,MuRF1 and ASNS mRNA in gastrocnemius tissue were increased(P<0.05),and the expression level of ASNS protein was increased(P<0.05);the expression levels of GPR78 and ATF4 proteins and the ratio of p-eIF2α/eIF2αwere increased(P<0.05).Compared with C57 transplanted tumor group,the SI of the mice in APP/PS1 transplanted tumor group was decreased(P<0.05),the level of Atrogin-1 mRNA was increased(P<0.05),and the level of ASNS mRNA in gastrocnemius tissue was decreased(P<0.05);there was no difference in the expression level of ASNS protein(P>0.05);the expression levels of GRP78 and ATF4 proteins and the ratio of p-eIF2α/eIF2αwere decreased(P<0.05).Conclusion:Gastrocnemius in the APP/PS1 transplanted melanoma mice is more prone to reduction of skeletal muscle,and its mechanism may be associated with the reduced levels of adaptive response in ASNS transcription,and endoplasmic reticulum stress in PERK-eIF2a-ATF4 signaling pathway may be involved in the regulation of ASNS transcriptional activity.