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

Abstract

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-associatedβ-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 activation of AMPK/ULK1 signaling and increase autophagy.On the whole,for the renal tubular epithelial cells aging models induced by D-gal,MCs not only has the in vitro actions of anti-aging,but also intervenes aging process by inhibiting autophagy-related AMPK/ULK1 signaling activation,which may be the novel molecular mechanisms of MCs protecting against aging of the renal tubular epithelial cells.