虫草菌丝抑制自噬相关AMPK/ULK1信号活性改善肾小管上皮细胞衰老的分子机制  

Molecular mechanisms of mycelium of Cordyceps sinensis ameliorating renal tubular epithelial cells aging induced by D-galactose via inhibiting autophagy-related AMPK/ULK1 signaling activation

作　　者：刘不悔 何伟明[1] 万毅刚[2] 高坤[1] 涂玥[3,4] 吴薇 陶佳音[1] 朱婧婧 陆映丹 孙伟 LIU Bu-hui;HE Wei-ming;WAN Yi-gang;GAO Kun;TU Yue;WU Wei;TAO Jia-yin;ZHU Jing-jing;LU Ying-dan;SUN Wei(Department of Nephrology,the Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;Department of Traditional Chinese Medicine,Nanjing Drum Tower Hospital Clinical College of Chinese Medicine and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210008,China;Department of Traditional Chinese Medicine Health Preservation,Second Clinic Medical School,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学附属医院肾内科,江苏南京210029 [2]南京大学医学院附属鼓楼医院中医科,江苏南京210008 [3]南京中医药大学第二临床医学院中医养生学教研室,江苏南京210023 [4]Section of Comparative Medicine,Yale University School of Medicine

出　　处：《中国中药杂志》2019年第6期1258-1265,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81774245;81673912;81573903);国家自然科学基金青年基金项目(81603675);江苏省自然科学基金青年基金项目(BK20161046);江苏省高校自然科学研究面上项目(16KJB360004);2017年江苏政府留学奖学金项目;江苏省研究生科研与实践创新计划项目(KYCX17_1324);南京市医学科技发展基金项目(QRX17042)

摘　　要：为了探讨虫草菌丝(mycelium of Cordyceps sinensis,MCs)改善D-半乳糖(D-galactose,D-gal)诱导的肾小管上皮细胞衰老的作用和分子机制,将体外培养的大鼠近端肾小管上皮细胞(NRK-52E)分为正常组(normal group,N),模型组(D-gal model group,D),低剂量MCs组(low dose of MCs,L-MCs),中剂量MCs组(medium dose of MCs,M-MCs),高剂量MCs组(high dose of MCs,H-MCs),分别进行不同的干预。具体而言,N组加入1%胎牛血清(fetal bovine ser-um,FBS)1 m L;D组加入100 mmol·L^(-1)D-gal;L-MCs组加入100 mmol·L^(-1)D-gal+20 mg·L^(-1)MCs;M-MCs组加入100 mmol·L^(-1)D-gal+40 mg·L^(-1)MCs;H-MCs组加入100mmol·L^(-1)D-gal+80 mg·L^(-1)MCs。在干预后的24或48 h,首先,观察D-gal对NRK-52E细胞klotho,P27,P16蛋白表达水平,β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)染色以及腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)/不协调的51类激酶1(uncoordinated 51-like kinase 1,ULK1)信号活性的影响;其次,观察MCs对NRK-52E细胞增殖活性的影响;最后,观察MCs对D-gal诱导的NRK-52E细胞klotho,P27,P16蛋白表达水平,SA-β-gal染色以及哺乳动物同族物微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)和AMPK/ULK1信号活性的影响。结果表明,对于NRK-52E细胞,D-gal能引起衰老,并诱导磷酸化AMPK(phosphorylated-AMPK,p-AMPK)和磷酸化ULK1(phosphorylated-ULK1,p-ULK1)蛋白高表达,激活AMPK/ULK1信号通路;中、高剂量MCs与D-gal联合干预能明显改善klotho,P27,P16蛋白表达水平和SA-β-gal染色程度,具有抗细胞衰老的作用;此外,中、高剂量MCs与D-gal联合干预能明显改善LC3,p-AMPK,p-ULK1蛋白表达水平,抑制AMPK/ULK1信号活性,提高自噬水平。总之,对于D-gal诱导的肾小管上皮细胞衰老模型,MCs在体外有抗衰老的作用,并且,通过抑制自噬相关AMPK/ULK1信号活性而干预其衰老进程。这可能是MCs抗肾小管上皮细胞衰老的新的分子机制。To explore the effects and molecular mechanisms of mycelium of Cordyceps sinensis(MCs)improving renal tubular epithelial cells aging induced by D-galactose,the renal proximal tubular epithelial cells(NRK-52E cells)of rats in vitro were divided into the normal group(N),the D-gal model group(D),the low dose of MCs group(L-MCs),the medium dose of MCs group(M-MCs)and the high dose of MCs group(H-MCs),and treated by the different measures,respectively.More specifically,the NRK-52E cells in each group were separately treated by 1%fetal bovine serum(FBS)or D-galactose(D-gal,100 mmol·L^-1)or D-gal(100 mmol·L^-1)+MCs(20 mg·L^-1)or D-gal(100 mmol·L^-1)+MCs(40 mg·L^-1)or D-gal(100 mmol·L^-1)+MCs(80 mg·L^-1).After the intervention for24 h or 48 h,firstly,the effects of D-gal on the protein expression levels of klotho,P27 and P16,the staining of senescence-associated β-galactosidase(SA-β-gal)and the activation of adenosine monophosphate activated protein kinase(AMPK)/uncoordinated 51-like kinase 1(ULK1)signaling in the NRK-52E cells were detected,respectively.Secondly,the effects of MCs on the activation of the NRK-52E cells proliferation were investigated,respectively.Finally,the effects of MCs on the protein expression levels of klotho,P27,P16and microtubule-associated protein 1 light chain 3(LC3),the staining of SA-β-gal and the activation of AMPK/ULK1 signaling in the NRK-52E cells exposed to D-gal were examined severally.The results indicated that,for the NRK-52E cells,D-gal could cause aging,induce the protein over-expression levels of the phosphorylated AMPK(p-AMPK)and the phosphorylated ULK1(p-ULK1)and activate AMPK/ULK1 signaling pathway.The co-treatment of MCs at the medium and high doses and D-gal could significantly ameliorate the protein expression levels of klotho,P27,P16 and the staining of SA-β-gal,suggesting the anti-cell aging actions.In addition,the cotreatment of MCs at the medium and high doses and D-gal could obviously improve the protein expression levels of LC3,p-AMPK,and p-ULK1,inhibit the

关 键 词：衰老 虫草菌丝 肾小管上皮细胞 自噬 AMPK/ULK1信号通路 

分 类 号：R285.5[医药卫生—中药学]