基于自噬途径探讨补肾益精方对RPE细胞氧化应激损伤的保护机制研究

Study on the Protective Mechanism of Bushen Yijing Formula on Oxidative Stress Induced Damage in RPE Cells Based on the Autophagy Pathway

目的基于自噬途径探讨补肾益精方(BSYJ)对氧化应激状态下的视网膜色素上皮(RPE)细胞的保护机制。方法制备补肾益精方含药血清和空白血清,将体外培养的RPE细胞分为6组,对照组(CG)常规培养,模型组(MG)以90μmol/L氢醌处理24 h,空白血清组(BSG)以空白血清+氢醌处理,低剂量补肾益精方组(L-BSYJ)以2.5%含药血清+氢醌处理,高剂量补肾益精方组(H-BSYJ)以10%含药血清+氢醌处理,自噬抑制剂3-甲基腺嘌呤组(3-MA)以10 mmol/L 3-MA+10%含药血清+氢醌处理。细胞计数试剂-8(CCK-8)法检测各组RPE细胞活力,透射电镜观察RPE细胞自噬小体形态及数量,实时荧光定量PCR(qRT-PCR)检测RPE细胞Kelch样环氧氯丙烷相关蛋白-1(Keap1)、核因子E2相关因子2(Nrf2)、肌球蛋白样卷曲螺旋蛋白1(Beclin1)和自噬相关基因5(Atg5)mRNA表达,Western Blot法检测Keap1、微管相关蛋白1轻链3(LC3)、Nrf2、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、Beclin1蛋白表达。结果(1)细胞活力:MG组细胞活力低于CG组(t=19.277,P=0.000),LBSYJ组、H-BSYJ组细胞活力均高于MG组(tL-BSYJ=7.688、tH-BSYJ=10.433,均P=0.000),3-MA组细胞活力低于H-BSYJ组(t=2.426,P=0.023),差异均有统计学意义。(2)自噬:CG组RPE细胞自噬小体极少,MG组较多,L-BSYJ组自噬小体数量少于MG组,3-MA组较L-BSYJ组进一步减少。(3)相关因子mRNA:MG组细胞中Atg5、Beclin1、Keap1表达高于CG组(t_(Atg5)=18.191、t_(Beclin1)=16.766、t_(Keap1)=13.361,均P=0.000),Nrf2表达低于CG组(t=5.494,P=0.000);L-BSYJ组的Atg5、Beclin1、Keap1表达低于MG组(t_(Atg5)=4.958、t_(Beclin1)=7.831、t_(Keap1)=7.488,均P=0.000),Nrf2表达高于MG组(t=16.141,P=0.000);H-BSYJ组的Atg5、Beclin1、Keap1表达低于MG组(t_(Atg5)=12.791、t_(Beclin1)=12.683、t_(Keap1)=14.920,均P=0.000),Nrf2表达高于MG组(t=32.582,P=0.000);3-MA组的Atg5、Beclin1、Keap1表达低于H-BSYJ组(t_(Atg5)=5.243、t_(Beclin1)=9.320、t_(Keap1)=8.879,均P=0.000),Nrf2表达高于H-BSYJ组(t=8.060,P=0.000),差异均有统计学意义。(4)相关蛋白:MG组的Beclin1、LC3、Keap1表达高于CG组(t_(Beclin1)=18.328、t_(LC3)=13.930、t_(Keap1)=20.720,均P=0.000);p-mTOR、Nrf2表达低于MG组(t_(p-mTOR)=21.515、t_(Nrf2)=21.244,均P=0.000);L-BSYJ组的Beclin1、LC3、Keap1表达低于MG组(t_(Beclin1)=6.987,P=0.000;t_(LC3)=4.767,P=0.001;t_(Keap1)=22.070,P=0.000),p-mTOR、Nrf2表达高于MG组(t_(p-mTOR)=11.351、t_(Nrf2)=23.551,均P=0.000);H-BSYJ组的Beclin1、LC3、Keap1表达低于MG组(t_(Beclin1)=14.266、t_(LC3)=9.116、t_(Keap1)=40.284,均P=0.000),pmTOR、Nrf2表达高于L-BSYJ组(t_(p-mTOR)=28.164、t_(Nrf2)=43.930,均P=0.000);3-MA组Beclin1、LC3、Keap1表达低于H-BSYJ组(t_(Beclin1)=5.362、t_(LC3)=5.000、t_(Keap1)=20.817,均P=0.000),p-mTOR、Nrf2表达高于H-BSYJ组(t_(p-mTOR)=10.449、t_(Nrf2)=20.571,均P=0.000),差异均有统计学意义。结论补肾益精方可降低RPE细胞由于氧化应激而导致的自噬水平提高,减轻氧化应激对RPE细胞的损伤。

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 90μmol/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 HBSYJ 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(t_(Atg5)=18.191,t_(Beclin1)=16.766,t_(Keap1)=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(t_(Atg5)=4.958,t_(Beclin1)=7.831,t_(Keap1)=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(t_(Atg5)=12.791,t_(Beclin1)=12.683,t_(Keap1)=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(t_(Atg5)=5.243,t_(Beclin1)=9.320,t_(Keap1)=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(t_(Beclin1)=18.328,t_(LC3)=13.930,t_(Keap1)=20.720,all P=0.000)and lower p-mTOR and Nrf2 expression(t_(p-mTOR)=21.515,t_(Nrf2)=21.244,both P=0.000)than CG group.L-BSYJ group had lower Beclin1,LC3,and Keap1 expression(t_(Beclin1)=6.987,P=0.000;t_(LC3)=4.767,P=0.001;t_(Keap1)=22.070,P=0.000)and higher pmTOR and Nrf2 expression(t_(p-mTOR)=11.351,t_(Nrf2)=23.551,both P=0.000)than MG group.H-BSYJ group had lower Beclin1,LC3,and Keap1 expression(t_(Beclin1)=14.266,t_(LC3)=9.116,t_(Keap1)=40.284,all P=0.000)and higher p-mTOR and Nrf2 expression(t_(p-mTOR)=28.164,t_(Nrf2)=43.930,both P=0.000)than MG group.3-MA group had lower Beclin1,LC3,and Keap1 expression(t_(Beclin1)=5.362,t_(LC3)=5.000,t_(Keap1)=20.817,all P=0.000)and higher p-mTOR and Nrf2 expression(t_(p-mTOR)=10.449,t_(Nrf2)=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.