摘要
目的探讨黄连素对高糖培养大鼠离体视网膜Müller细胞的影响及其作用机制。方法将体外培养的Sprague Dawley(SD)大鼠视网膜Müller细胞分为正常对照组、高糖组、高糖+10μmol/L黄连素组和高糖+25μmol/L黄连素组,正常对照组和高糖组细胞分别采用5 mmol/L葡萄糖和25 mmol/L葡萄糖培养,后2个组分别采用25 mmol/L葡萄糖+相应浓度黄连素处理,培养后72 h采用流式细胞仪检测细胞凋亡情况,采用酶联免疫吸附测定(ELISA)法及实时荧光定量PCR法分别检测细胞培养液上清中炎性因子肿瘤坏死因子(TNF-α)、白细胞介素-8(IL-8)和环氧合酶2(COX-2),L-谷氨酸-L-天冬氨酸转运体(GLAST)及相关蛋白的表达情况,采用Western blot法检测细胞质中GLAST、镁离子依赖的蛋白磷酸酶1A(PPM1A)、核因子-κB(NF-κB)和cleaved caspase-3及细胞核中NF-κB蛋白的表达情况。结果正常对照组、高糖组、高糖+10μmol/L黄连素组和高糖+25μmol/L黄连素组细胞凋亡率分别为(1.37±0.21)%、(17.67±1.17)%、(10.60±0.17)%和(5.57±0.35)%,总体比较差异有统计学意义(F=375.97,P<0.01),其中高糖组细胞凋亡率较正常对照组明显升高,高糖+10μmol/L黄连素组和高糖+25μmol/L黄连素组细胞凋亡率较高糖组明显降低,高糖+25μmol/L黄连素组较高糖+10μmol/L黄连素组细胞凋亡率明显降低,差异均有统计学意义(均P<0.01)。ELISA检测结果显示,各组细胞中TNF-α、IL-8、COX-2质量浓度总体比较差异均有统计学意义(F=28.36、35.88、41.59,均P<0.01),其中高糖组细胞中TNF-α、IL-8和COX-2质量浓度均明显高于正常对照组,高糖+10μmol/L黄连素组和高糖+25μmol/L黄连素组细胞中TNF-α和COX-2质量浓度明显低于高糖组,高糖+25μmol/L黄连素组细胞中TNF-α和IL-8浓度明显低于高糖+10μmol/L黄连素组,差异均有统计学意义(均P<0.05)。实时荧光定量PCR结果显示,各组间Müller细胞中GLAST mRNA的相对表达量总体比较差异有
Objective To investigate the effects of berberine on Sprague Dawley(SD)rat retinal Müller cells cultured by high concentration glucose.Methods The cultured SD rat retinal Müller cells were divided into normal-glucose group,high-glucose group,high-glucose+10μmol/L berberine group and high-glucose+25μmol/L berberine group,and the cells were cultured in 5 mmol/L glucose,25 mmol/L glucose,25 mmol/L glucose+10μmol/L berberine,and 25 mmol/L glucose+25μmol/L berberine,respectively.After 72 hours cultured,cell apoptosis rate was detected by flow cytometry;the expressions levels of tumor necrosis factor-α(TNF-α),interleukin-8(IL-8)and cyclooxygenase-2(COX-2)in the culture supernatant were detected by enzyme linked immunosorbent assay(ELISA);the L-glutamate-L-aspartate transporter(GLAST)and the related protein expression levels were detected by real-time fluorescence quantitative PCR;the expressions levels of GLAST,protein phosphatase magnesium-dependent 1A(PPM1A),nuclear factor-κB(NF-κB)and cleaved caspase-3 in the cytoplasm,and the expression level of NF-κB protein in the nucleus were detected by Western blot.Results The cell apoptosis rate was(1.37±0.21)%,(17.67±1.17)%,(10.60±0.17)%and(5.57±0.35)%in the normal-glucose group,high-glucose group,high-glucose+10μmol/L berberine group and high-glucose+25μmol/L berberine group,respectively,and the overall comparative difference was statistically significant(F=375.97,P<0.01).The cell apoptosis rates in the high-glucose group was increased in comparison with those in the normal-glucose group(P<0.01).The cell apoptosis rates in the high-glucose+10μmol/L berberine group and high-glucose+25μmol/L berberine group were significantly reduced in comparison with that in the high-glucose group(both at P<0.01).And the cell apoptosis rate in the high-glucose+25μmol/L berberine group was lower than that in the high-glucose+10μmol/L berberine group(P<0.01).ELISA results revealed that the overall comparative differences of the concentrations of TNF-α,IL-8 and COX-2 amon
作者
金轶平
朱皓皓
廖宇洁
于晓彦
Jin Yiping;Zhu Haohao;Liao Yujie;Yu Xiaoyan(Department of Ophthalmology,Shanghai Fifth People's Hospital,Fudan University,Shanghai 200240,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2020年第12期1016-1024,共9页
Chinese Journal Of Experimental Ophthalmology
基金
上海市闵行区自然科学研究课题项目(2013MHZ010)。
关键词
MÜLLER细胞
高糖
黄连素
细胞凋亡
炎症
细胞培养
Müller cell
Glucose,high concentration
Berberine
Cell apoptosis
Inflammation
Cell culture
