摘要
目的探讨益肾养肝明目方(YYM)对过氧化氢(H_(2)O_(2))诱导的人视网膜色素上皮(RPE)细胞凋亡的保护作用及药理机制。方法常规培养ARPE-19细胞,筛选H_(2)O_(2)、康柏西普和YYM的最佳浓度,之后将细胞分为空白组(BG)、模型组(MG)、益肾养肝明目方组(YYM)、阳性对照药康柏西普组(CB)、益肾养肝明目方+康柏西普组(YYM+CB)、益肾养肝明目方对照组(YYM-CG),以400μM H_(2)O_(2)、2μg/mL康柏西普和40μg/mL YYM的最佳浓度处理。使用相差显微镜观察各组ARPE-19细胞形态,流式细胞仪检测细胞凋亡率,化学荧光法检测细胞活性氧(ROS)表达,Western Blot检测血管内皮生长因子/丝裂原活化蛋白激酶(VEGF/MAPKs)通路相关蛋白外源性调节蛋白激酶1/2(eERK1/2)、B细胞淋巴瘤蛋白-2(Bcl-2)、Bcl-2相关蛋白X(Bax)、细胞色素c(Cyc)表达。结果(1)细胞形态:MG组细胞出现皱缩,部分脱落;而YYM、CB组的细胞损伤明显减少,凋亡细胞数量下降。(2)细胞凋亡:与BG组比较,MG组细胞凋亡率较高(t=12.408,P=0.000);与MG组比较,YYM、CB、YYM+CB组细胞凋亡率均降低(t_(YYM)=6.023,t_(CB)=5.967,t_(YYM+CB)=9.804,均P=0.000)。(3)ROS:与BG组比较,MG组ROS表达较高(t=14.388,P=0.000);与MG组比较,YYM、CB、YYM+CB组ROS表达较低(t_(YYM)=8.621,t_(CB)=8.774,t_(YYM+CB)=12.620,均P=0.000)。(4)VEGF/MAPKs:与BG组比较,MG组VEGF、p-p38 MAPKs、p-SAPK/JNK、p-ERK1/2、Bax、Bcl-2、Cyc蛋白表达均较高(t_(VEGF)=18.569,t_(p-p38 MAPK)=22.097,t_(p-SAPK/JNK)=17.548,t_(p-ERK1/2)=18.567,t_(Bax)=20.575,t_(Bcl-2)=18.879,t_(Cyc)=28.196,均P=0.000);与MG组比较,YYM、CB、YYM+CB组VEGF、p-p38 MAPK、p-SAPK/JNK、p-ERK1/2、Bax、Bcl-2、Cyc蛋白表达均较低(YYM组:t_(VEGF)=9.493,t_(p-p38 MAPK)=8.942,t_(p-SAPK/JNK)=8.683,t_(p-ERK1/2)=8.929,t_(Bax)=8.849,t_(Bcl-2)=4.863,t_(Cyc)=11.832,均P=0.000;CB组:t_(VEGF)=16.065,t_(p-p38 MAPK)=16.033,t_(p-SAPK/JNK)=15.558,t_(p-ERK1/2)=13.890,t_(Bax)=16.043,t_(Bcl-2)=6.579,t_(Cyc)=22.028,均P=0.000;YYM+CB组:t_(VEGF)=18.569,t_(p-p38 MAPK)=21.378,t_(p-SAPK/JNK)=17.005,t_(p-ERK1/2)=16.583,t_(Bax)=20.071,t_(Bcl-2)=11.442,t_(Cyc)=26.560,均P=0.000),差异均有统计学意义。结论YYM能通过调控VEGF/MAPKs通路相关蛋白的表达,降低H_(2)O_(2)诱导RPE细胞凋亡率和ROS表达,减轻氧化应激损伤,且联合康柏西普使用效果更佳。
OBJECTIVE To investigate the protective effect and pharmacological mechanism of Yishen Yanggan Mingmu Formula(YYM)on H_(2)O_(2)-induced apoptosis in human retinal pigment epithelial(RPE)cells.METHODS ARPE-19 cells were cultured and treated with various concentrations of H_(2)O_(2),Conbercept,and YYM to determine the optimal concentrations.The cells were divided into blank group(BG),model group(MG),YYM group(YYM),positive control drug Conbercept group(CB),YYM+Conbercept group(YYM+CB),and YYM control group(YYM-CG).The optimal concentrations of 400μM H_(2)O_(2),2μg/mL Conbercept,and 40μg/mL YYM were selected for further experiments.Cellular morphology was observed using phase-contrast microscopy.Flow cytometry was used to measure apoptosis rate,chemical fluorescence was used to detect reactive oxygen species(ROS)expression,and Western Blot was used to detect the expression of vascular endothelial growth factor/mitogen-activated protein kinases(VEGF/MAPKs)pathway-related proteins extracellular signal-regulated kinase 1/2(eERK1/2),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and Cytochrome c(Cyc).RESULTS(1)Cellular morphology:Microscopy revealed cell shrinkage and partial detachment in the MG,whereas cell damage were significantly reduced in the YYM and CB.(2)Cell apoptosis:Compared to the BG,the MG showed increased apoptosis rate(t=12.408,P=0.000);Compared to the MG,the YYM,CB,and YYM+CB exhibited decreased apoptosis rates(t_(YYM)=6.023,t_(CB)=5.967,t_(YYM+CB)=9.804,all P=0.000).(3)ROS:ROS expression was elevated in the MG compared to the BG(t=14.388,P=0.000);ROS expression was reduced in the YYM,CB,and YYM+CB compared to the MG(t_(YYM)=8.621,t_(CB)=8.774,t_(YYM+CB)=12.620,all P=0.000).(4)VEGF/MAPKs:Compared to the BG,protein expression of VEGF,p-p38 MAPK,p-SAPK/JNK,p-ERK1/2,Bax,Bcl-2,and Cyc were elevated in the MG(t_(VEGF)=18.569,t_(p-p38 MAPK)=22.097,t_(p-SAPK/JNK)=17.548,t_(p-ERK1/2)=18.567,t_(Bax)=20.575,t_(Bcl-2)=18.879,t_(Cyc)=28.196,all P=0.000);Compared to the MG,the YYM,CB,and YYM+CB showed decreased expression of these proteins(YYM:t_(VEGF)=9.493,t_(p-p38 MAPK)=8.942,t_(p-SAPK/JNK)=8.683,t_(p-ERK1/2)=8.929,t_(Bax)=8.849,t_(Bcl-2)=4.863,t_(Cyc)=11.832,all P=0.000;CB:t_(VEGF)=16.065,t_(p-p38 MAPK)=16.033,t_(p-SAPK/JNK)=15.558,t_(p-ERK1/2)=13.890,t_(Bax)=16.043,t_(Bcl-2)=6.579,t_(Cyc)=22.028,all P=0.000;YYM+CB:t_(VEGF)=18.569,t_(p-p38 MAPK)=21.378,t_(p-SAPK/JNK)=17.005,t_(p-ERK1/2)=16.583,t_(Bax)=20.071,t_(Bcl-2)=11.442,t_(Cyc)=26.560,all P=0.000),and all differences were statistically significant.CONCLUSIONS YYM can regulate the expression of VEGF/MAPKs pathway-related proteins,decrease H_(2)O_(2)-induced RPE cell apoptosis rate and ROS expression,alleviate oxidative stress damage,and achieve better effects when used in combination with Conbercept.
作者
龚雁
李萌
廖燕红
高健
章晓林
王桑桑
GONG Yan;LI Meng;LIAO Yanhong;GAO Jian;ZHANG Xiaolin;WANG Sangsang(Ningbo Eye Hospital,Ningbo 315042,China)
出处
《中国中医眼科杂志》
2023年第11期1007-1013,共7页
China Journal of Chinese Ophthalmology
基金
浙江省卫生科技计划(2021PY073)
浙江省医药卫生科技计划(2023KY1141)
浙江省中医药科技计划(2021ZB268)
宁波市鄞州区农业与社会发展领域科技项目(2021AS0058)
