Study on the Protective Mechanism of Bushen Yijing Formula on Oxidative Stress-Induced Damage in RPE Cells Based on the Autophagy Pathway
OBJECTIVE To explore the protective mechanism of Bushen Yijing Formula(BSYJ)on retinal pigment epithelium(RPE)cells under oxidative stress based on the autophagy pathway.METHODS The medicated serum containing BSYJ and blank serum was prepared.Cultured RPE cells were divided into six groups:Control group(CG),model group(MG)treated with 90 μmol/L hydroquinone for 24 h,blank serum group(BSG)treated with blank serum and hydroquinone,low-dose BSYJ group(L-BSYJ)treated with 2.5%medicated serum and hydroquinone,high-dose BSYJ group(H-BSYJ)treated with 10%medicated serum and hydroquinone,and autophagy inhibitor 3-methyladenine(3-MA)group treated with 10 mmol/L 3-MA,10%medicated serum,and hydroquinone.Cell viability was measured using the cell counting kit-8(CCK-8)assay.Autophagosomes in RPE cells were observed via transmission electron microscopy.Quantitative real-time PCR(qRT-PCR)was used to detect mRNA expression of Keap1,Nrf2,Beclin1,and Atg5.Protein expression of Keap1,LC3,Nrf2,p-mTOR,and Beclin1 was detected by Western Blot.RESULTS(1)Cell viability:MG group had lower cell viability than CG group(t=19.277,P=0.000),while L-BSYJ and H-BSYJ groups had higher viability than MG group(tL-BSYJ=7.688,tH-BSYJ=10.433,both P=0.000).3-MA group had lower viability than H-BSYJ group(t=2.426,P=0.023),all differences were statistically significant.(2)Autophagy:RPE cells in CG group had few autophagosomes,MG group had many,L-BSYJ group had fewer than MG group,and 3-MA group had even fewer than L-BSYJ group.(3)Related factor mRNA:MG group had higher Atg5,Beclin1,and Keap1 expression(tAtg5=18.191,tBeclin1=16.766,tKeap1=13.361,all P=0.000)and lower Nrf2 expression(t=5.494,P=0.000)compared to CG group.L-BSYJ group had lower Atg5,Beclin1,and Keap1 expression(tAtg5=4.958,tBeclin1=7.831,tKeap1=7.488,all P=0.000)and higher Nrf2 expression(t=16.141,P=0.000)than MG group.H-BSYJ group had lower Atg5,Beclin1,and Keap1 expression(tAtg5=12.791,tBeclin1=12.683,tKeap1=14.920,all P=0.000)and higher Nrf2 expression(t=32.582,P=0.000)than MG group.3-MA group had lower Atg5,Beclin1,and Keap1 expression(tAtg5=5.243,tBeclin1=9.320,tKeap1=8.879,all P=0.000)and higher Nrf2 expression(t=8.060,P=0.000)than H-BSYJ group,all differences were statistically significant.(4)Related protein:MG group had higher Beclin1,LC3,and Keap1 expression(tBeclin1=18.328,tLC3=13.930,tKeap1=20.720,all P=0.000)and lower p-mTOR and Nrf2 expression(tp-mTOR=21.515,tNrf2=21.244,both P=0.000)than CG group.L-BSYJ group had lower Beclin1,LC3,and Keap1 expression(tBeclin1=6.987,P=0.000;tLC3=4.767,P=0.001;tKeap1=22.070,P=0.000)and higher p-mTOR and Nrf2 expression(tp-mTOR=11.351,tNrf2=23.551,both P=0.000)than MG group.H-BSYJ group had lower Beclin1,LC3,and Keap1 expression(tBeclin1=14.266,tLC3=9.116,tKeap1=40.284,all P=0.000)and higher p-mTOR and Nrf2 expression(tp-mTOR=28.164,tNrf2=43.930,both P=0.000)than MG group.3-MA group had lower Beclin1,LC3,and Keap1 expression(tBeclin1=5.362,tLC3=5.000,tKeap1=20.817,all P=0.000)and higher p-mTOR and Nrf2 expression(tp-mTOR=10.449,tNrf2=20.571,both P=0.000)than H-BSYJ group,all differences were statistically significant.CONCLUSIONS Bushen Yijing Formula can reduce the increase in autophagy levels in RPE cells caused by oxidative stress and mitigate oxidative stress-induced damage to RPE cells.
万方数据
维普
