枸杞多糖对脂多糖诱导人视网膜色素上皮细胞凋亡的影响

The Effect of Lycium Barbarum Polysaccharide on the Apoptosis of Human Retinal Pigment Epithelial Cells Induced by Lipopolysaccharide

目的探讨枸杞多糖(lycium barbarum polysaccharide,LBP)预处理对脂多糖(lipopolysaccharide,LPS)诱导的人视网膜色素上皮细胞(adult retinal pigment epithelial cells-19,ARPE-19)炎性反应相关细胞凋亡的影响。方法将体外培养的ARPE-19随机分为空白组,模型组,0.1、0.5、1 mg·mL^(-1)LBP组,不同浓度LBP预处理24 h后与模型组同时给予10μg·mL^(-1)LPS诱导24 h。采用倒置荧光显微镜观察各组细胞形态,CCK-8检测细胞增殖情况,流式细胞仪检测细胞凋亡率,RT-PCR及Western blot观察凋亡相关因子Bcl-2、Bax的mRNA及蛋白表达量,ELISA检测细胞上清液中炎症因子白细胞介素(interleukin,IL)-6、IL^(-1)β及单核细胞趋化蛋白-1(MCP-1)的浓度。结果与空白组相比,模型组细胞活力下降,细胞凋亡率增加(P均<0.05),Bcl-2 mRNA及蛋白表达量下降,Bax升高(P均<0.05),细胞上清液中炎症因子浓度上调(P<0.05)。各LBP处理组CCK-8结果示细胞活力增加(P<0.05);流式细胞仪检测细胞凋亡率降低(P<0.05);RT-PCR显示Bcl-2的mRNA表达量增加、Bax表达量减少(P均<0.05);Western blot观察到Bcl-2的蛋白相对表达量上调、Bax的表达量下调(P均<0.05);ELISA结果示细胞上清液中炎症因子IL-6、IL^(-1)β及MCP-1的蛋白浓度降低(P均<0.05)。结论LBP可减少LPS诱导ARPE-19细胞外分泌炎症因子IL-6、IL^(-1)β及MCP-1,并通过逆转细胞内Bcl-2、Bax的转录和翻译过程,抑制炎性反应相关的RPE细胞凋亡。

Objective The effect of lycium barbarum polysaccharides(LBP)was explored on apoptosis associated with inflammatory response of human retinal pigment epithelial cells(ARPE-19)induced by lipopolysaccharide(LPS).Methods ARPE-19 cells cultured in vitro were randomly divided into blank group,model group(LPS group),0.1,0.5,1 mg·mL^(-1)LBP group.The cells were pretreated with LBP of different concentrations,then all groups were induced with10μg·mL^(-1)LPS for 24 hours.After that,cell morphology was viewed by an inverted fluorescence microscope.Flow cytometry assay was used to detect the apoptotic status,andthe cell proliferation was detected by the CCK-8 method.The mRNA and protein levels of Bcl-2 and Bax were measured by real-time fluorescent quantitative PCR(RT-PCR)and Western blot.The concentrations of IL-6,IL^(-1)βand MCP-1 in the supernatant were detected by ELISA.Results Compared with the blank group,the survival rate of the model group cells was decreased(P all<0.05).Besides,the apoptosis rate was increased(P all<0.05).The mRNA and protein expression of Bcl-2 was reduced,while Bax was opposite(P all<0.05).The concentration of inflammatory factors in the supernatant was up-regulated(P<0.05).On the contrary,in each LBP group,CCK-8 results showed that the cell viability went up(P<0.05).Also,flow cytometry exhibited that the apoptosis rate was decreased(P<0.05).The mRNA expression of Bcl-2 was promoted and Bax was cut down in RT-PCR(P all<0.05).Western blot displayed that Bcl-2 was up-regulated and Bax was down(P all<0.05).The protein levels of IL-6,IL^(-1)βand MCP-1 in the supernatant were decreased in ELISA test(P all<0.05).Conclusion LBP can reduce the secretion of IL-6,IL^(-1)βand MCP-1 in LPS-induced ARPE-19 cells,and inhibit inflammatory apoptosis through reversing the transcription and translation of Bcl-2 and Bax in RPE cells.