AIM:To investigate the protective effect of Yishen-Tongluo prescription(YTP)against oxidative stress-induced senescence of renal tubular epithelial cells via phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/AKT)/mammalian target of rapamycin(mTOR)pathway in vitro,and to explore its potential mechanism.METHODS:Human renal tubular epithelial HK-2 cells were cultured in vitro and exposed to 400 μmol/L H2O2 to establish a cellular se-nescence model.The cells were treated with 1 g/L YTP and/or 1 μmol/L 740 Y-P(PI3K activator)for 48 h,and were di-vided into control group,H2O2 group,YTP group,YTP+H2O2 group,740 Y-P+H2O2 group and 740 Y-P+YTP+H2O2 group.Senescent cells were analyzed by senescence-associated β-galactosidase(SA-β-Gal)staining.The protein levels of P21,P16,γ-H2AX,microtubule-associated protein 1 light chain 3B(LC3B),P62,PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,P70S6K and p-P70S6K were determined by Western blot.The proliferation of the cells was detected by EdU staining.Intracellular level of reactive oxygen species(ROS)was assessed by DCFH-DA probe,and laser confocal microscopy was used to observe autophagic flux in HK-2 cells.Each experiment was repeated 3 times.RESULTS:(1)Compared with control group,the SA-β-Gal-positive cells in H2O2 group were significantly increased,the EdU-positive cells were significantly reduced,and the protein levels of P21,P16 and γ-H2AX were significantly elevated(P<0.01).The ratios of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR and p-P70S6K/P70S6K were significantly increased(P<0.05).The protein level of LC3B-Ⅱ was incresed,the P62 expression was reduced(P<0.01),and the ratio of autolyso-somes to autophagosomes increased(P<0.05).(2)Compared with H2O2 group,the intracellular ROS level in YTP+H2O2 group significantly decreased(P<0.01).The SA-β-Gal-positive cells were significantly reduced,the EdU-positive cells were significantly increased,and the protein levels of P21,P16 and γ-H2AX were decreased(P<0.05).The ratios of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR and p-P70S6K/P70S6K were significantly reduced(P<0.05).The protein lev-el of LC3B-Ⅱ was increased,the P62 expression was reduced(P<0.05),and the ratio of autolysosomes to autophago-somes decreased(P<0.05).(3)Compared with YTP+H2O2 group,the ratios of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR and p-P70S6K/P70S6K,and the protein level of LC3B-Ⅱ were decreased in 740 Y-P+YTP+H2O2 group(P<0.01),while the P62 expression increased(P<0.05).CONCLUSION:The YTP can clear accumulated intracellular ROS,res-cue autophagic flux by inhibiting the PI3K/AKT/mTOR signaling pathway,thus alleviating oxidative stress-induced HK-2 cell senescence.

AIM:To investigate the protective effect of Yishen-Tongluo prescription(YTP)against oxidative stress-induced senescence of renal tubular epithelial cells via phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/AKT)/mammalian target of rapamycin(mTOR)pathway in vitro,and to explore its potential mechanism.METHODS:Human renal tubular epithelial HK-2 cells were cultured in vitro and exposed to 400 μmol/L H2O2 to establish a cellular se-nescence model.The cells were treated with 1 g/L YTP and/or 1 μmol/L 740 Y-P(PI3K activator)for 48 h,and were di-vided into control group,H2O2 group,YTP group,YTP+H2O2 group,740 Y-P+H2O2 group and 740 Y-P+YTP+H2O2 group.Senescent cells were analyzed by senescence-associated β-galactosidase(SA-β-Gal)staining.The protein levels of P21,P16,γ-H2AX,microtubule-associated protein 1 light chain 3B(LC3B),P62,PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,P70S6K and p-P70S6K were determined by Western blot.The proliferation of the cells was detected by EdU staining.Intracellular level of reactive oxygen species(ROS)was assessed by DCFH-DA probe,and laser confocal microscopy was used to observe autophagic flux in HK-2 cells.Each experiment was repeated 3 times.RESULTS:(1)Compared with control group,the SA-β-Gal-positive cells in H2O2 group were significantly increased,the EdU-positive cells were significantly reduced,and the protein levels of P21,P16 and γ-H2AX were significantly elevated(P<0.01).The ratios of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR and p-P70S6K/P70S6K were significantly increased(P<0.05).The protein level of LC3B-Ⅱ was incresed,the P62 expression was reduced(P<0.01),and the ratio of autolyso-somes to autophagosomes increased(P<0.05).(2)Compared with H2O2 group,the intracellular ROS level in YTP+H2O2 group significantly decreased(P<0.01).The SA-β-Gal-positive cells were significantly reduced,the EdU-positive cells were significantly increased,and the protein levels of P21,P16 and γ-H2AX were decreased(P<0.05).The ratios of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR and p-P70S6K/P70S6K were significantly reduced(P<0.05).The protein lev-el of LC3B-Ⅱ was increased,the P62 expression was reduced(P<0.05),and the ratio of autolysosomes to autophago-somes decreased(P<0.05).(3)Compared with YTP+H2O2 group,the ratios of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR and p-P70S6K/P70S6K,and the protein level of LC3B-Ⅱ were decreased in 740 Y-P+YTP+H2O2 group(P<0.01),while the P62 expression increased(P<0.05).CONCLUSION:The YTP can clear accumulated intracellular ROS,res-cue autophagic flux by inhibiting the PI3K/AKT/mTOR signaling pathway,thus alleviating oxidative stress-induced HK-2 cell senescence.