Celastrol Alleviates Vascular Smooth Muscle Cell Senescence Via Induction Of Autophagy

Background and ObjectiveThe senescence of vascular smooth muscle cells(VSMCs)is closel y related to cardiovascular diseases.Senescent cells exhibit increased oxidative stress,telomere dysfunction and stable growth arrest as well as increased expression of senescence-associated ?-galactosidase(SA?-gal),p53,p21,and p16.Angiotensin II(Ang II)plays an important role in the pathogenesis of atherosclerosis.Ang II increases ROS production and reduces cellular antioxidant capacity,leading to vascular senescence through a p53/p21-dependent mechanism in VSMCs.Inhibition of Ang II activity has been shown to reduce the morbidity and mortality due to cardiovascular diseases.A growing body of evidence has shown that treatment with pharmaceuticals can reduce premature cellular senescence,providing a potential treatment for aging-related diseases.Celastrol(CeT)is a quinone methide triterpenoid isolated from the traditional Chinese medicinal plant Tripterygium wilford II Hook F.Ce T has attracted increasing attention due to its potential therapeutic effects of antioxidation and induction of autophagy.A recent study reported that a short time treatment using tripterine(i.e.celastrol)reduced ox-LDL-induced ROS generation and senescence in endothelial progenitor cells.We hypothesized that CeT plays an anti-senescence role by inducing autophagy to reduce ROS production.This hypothesis was investigated in this study using VSMCs.Methods1.Senescence ?-Galactosidase Staining Kit,Western blot assay and cell cycle analysis were used to investigate that if Ce T alleviates angiotensin II-mediated VSMC senescence.2.Flow cytometry and an ROS assay kit were used to investigate whether CeT attenuated the Ang II-induced ROS activity in VSMCs.3.Immunofluorescence assay was used to investigate the level of total LC3,Western blot was used to investigate the LC3 II level.Flow cytometry and ROS assay kit were used to find if the autophagy inhibitor influenced the anti-ROS effect of Ce T.To further confirm whether CeT-induced autophagy can reduce the Ang II-induced senescence of VSMCs,senescence ?-galactosidase staining and Western blot analysis were performed.4.Western blot assay was used to test whether CeT could suppress the PI3K/Akt/ mTOR signaling pathway.Results1.Ce T treatment counteracts Ang II-induced cellular senescence in VSMCs.Treatment with CeT alone had little effect on VSMC senescence compared with treatment with vehicle control(DMSO).A significant increase in VSMC senescence upon stimulation with Ang II was evidenced by the increase in the number of SA-?-gal-positive cells.In this setting,CeT showed a dose-dependent inhibition of VSMC senescence with its maximal effect achieved at 50 nmol/L.Expression levels of p53 and p21 were significantly increased in VSMCs after a 2-day period of stimulation with Ang II.Pretreatment with CeT significantly counteracted this effect of Ang II.Flow cytometry was also used to investigate the effect of CeT on the cell cycle of VSMCs.After stimulation with 100 nmol/L Ang II for 2 days,the percentage of G0/G1 phase cells was significantly increased compared with that of the DMSO control group.In contrast,in VSMCs treated with Ang II,the number of G0/G1-arrested cells was decreased while the number of cells in S/G2 phase was increased by CeT(50 nmol/L)-pretreatment,indicating that the proliferative capacity of senescent VSMCs induced by Ang II was restored by Ce T.2.CeT alleviates senescence through reducing ROS activity in VSMCs.Treatment with CeT alone had little effect on basal ROS generation similar to that of the DMSO control.ROS production was increased in the Ang II-treated group and this was significantly decreased in the Ce T-pretreated group,although such inhibitory efficacy was less potent compared to that cells treated with NAC.NAC did not affect the basal senescence of VSMCs,it obviously prevented the Ang II-induced increase in the SA-?-gal-positive cell counts.3.CeT-induced autophagy reduces Ang II-mediated ROS and senescence in VSMCs.Immunofluorescence assay showed that,compared with the control group,the total LC3 of CeT-pretreated group was significantly increased.Western blot assay was further demonstrated that compared with DMSO control,the protein level of LC3 II was increased in the Ce T-treated group,which could be further increased by bafilomycin A1,or decreased by 3Ma.Furthermore,the p62 protein level was decreased in the Ce T-treated group,and treatment with the two autophagy inhibitors reduced these effects.Ang II stimulation significantly increased cellular ROS levels in VSMCs,and CeT reduced the Ang II-stimulated ROS levels,similar effects of the positive control rapamycin;however,pretreating with the autophagy inhibitor 3Ma reversed the effect of Ce T.The results showed that,just like in the autophagy inducer rapamycin-pretreated group,the SA?-gal positive cell counts were significantly decreased in the CeT-pretreated group,which was reversed by the autophagy inhibitors 3Ma and Baf A1.Western blot assay further demonstrated that the autophagy inhibitor 3Ma largely abolished the effects of Ce T on p53 and p21 expression.4.CeT inhibited PI3K/Akt/m TOR signaling pathway.The results showed that,compared with the control group,the phosphorylation levels of PI3 K,Akt,mTOR and p70S6 K in the CeT group were down-regulated,indicating that Ce T treatment suppressed the PI3K/Akt/mTOR signaling pathway.ConclusionIn general,CeT up-regulates autophagy,which confers resistance to Ang II-induced ROS and the resultant senescence.Notably,the effects of Ce T on stimulating autophagy may be associated with its inhibition of m TOR activity.