枸杞多糖通过miR-29b/MAPK1通路发挥视网膜血管内皮细胞的抗凋亡作用

Lycium barbarum polysaccharide exerts anti⁃apoptotic effects on retinal vascular endothelial cells through the miR⁃29b/MAPK1 pathway

目的 探究枸杞多糖(LBP)通过调控miRNA-29b/丝裂原活化蛋白激酶1(miR-29b/MAPK1)对高糖诱导人视网膜血管内皮细胞(HREC)凋亡的影响。方法 加入(100、200、400、800)mg/L LBP以及使用LipofectamineTM2000转染inhibitor NC和miR-29b inhibitor至高糖处理的HREC损伤模型中进行干预处理。随后,采用CCK-8检测细胞活力;实时荧光定量PCR(qRT-PCR)检测细胞中miR-29b、MAPK1 m RNA水平;Hoechst 33258染色法检测细胞凋亡情况;2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)法检测活性氧(ROS)水平;蛋白质免疫印迹法检测细胞中MAPK1、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白表达变化。双荧光素酶实验验证miR-29b与MAPK1的靶向关系。结果 与对照组相比,高糖组细胞A450值、miR-29b水平降低(P<0.05),MAPK1 mRNA水平升高(P<0.05);在高糖基础上添加LBP,随着LBP浓度增加,A450升高(分别为0.42±0.003,0.52±0.023,0.67±0.042和0.78±0.051)(P<0.05)、miR-29b水平逐渐升高(分别为0.42±0.29,0.57±0.27,0.63±0.29,0.78±0.51,P<0.05),MAPK1 mRNA水平则逐渐降低(1.67±0.61,1.53±0.29,1.41±0.27,1.32±0.73)(P<0.05)。与对照组相比,高糖组细胞活力、miR-29b水平,Bcl-2蛋白水平降低(P<0.05),MAPK1 mRNA和蛋白水平,ROS水平,Bax、Caspase-3蛋白水平升高(P<0.05);与高糖组相比,在加入LBP干预处理的细胞中上述现象得到逆转;进一步发现,在LBP基础上联合miR-29b inhibitor,发现细胞活力、miR-29b水平,Bcl-2蛋白水平降低(P<0.05),MAPK1 m RNA和蛋白水平,凋亡率,ROS水平,Bax、Caspase-3蛋白水平升高(P<0.05)。此外,MAPK1 m RNA的3’UTR含有miR-29b序列保守碱基,且经双荧光素酶实验得到证实。结论 LBP能够上调miR-29b的表达,来降低MAPK1表达,进而抑制高糖导致的HREC凋亡发生。

Objective To investigate the effect of Lycium barbarum polysaccharide(LBP)on the apoptosis of human retinal vascular endothelial cells(HRECs)induced by high glucose through regulation of miRNA⁃29b/mito⁃gen activated protein kinase 1(miR⁃29b/MAPK1).Methods Intervention was performed by adding(100,200,400,800)mg/L LBP and transfection with Lipofectamine TM 2000 inhibitor of NC and miR⁃29b inhibitor to HREC in⁃jury model treated with high glucose.Subsequently,CCK⁃8 was used to detect cell viability.Quantitative real⁃time PCR(qRT⁃PCR)was used to detect the mRNA levels of miR⁃29b and MAPK1.Hoechst 33258 staining was used to detect cell apoptosis.Reactive oxygen species(ROS)were detected by 2',7'⁃dichlorofluorescence yellow diacetate(DCFH⁃DA)method.The expression changes of MAPK1,Bcl⁃2 associated X protein(Bax),B lymphoblastoma⁃2 gene(Bcl⁃2)and cysteine aspartic protease 3(Caspase⁃3)were detected by western blotting.Double luciferase as⁃say verified the targeting relationship between miR⁃29b and MAPK1.Results Compared with the control group,the levels of OD 450 and miR⁃29b in high glucose group were decreased(P<0.05),while the level of MAPK1 mRNA was increased(P<0.05).With the increase of LBP concentration,the levels of A 450(0.42±0.003,0.52±0.023,0.67±0.04,0.78±0.051,respectively)(P<0.05)and miR⁃29b were gradually increased(0.42±0.29,0.57±0.27,0.63±0.29,0.78±0.51,respectively)(P<0.05),while the level of MAPK1 mRNA was gradually decreased(1.67±0.61,1.53±0.29,1.41±0.27,1.32±0.73,respectively)(P<0.05).Compared with the control group,the cell viability,miR⁃29b level and Bcl⁃2 protein level in high glucose group were decreased(P<0.05),while the mRNA and protein levels of MAPK1,ROS level,Bax and Caspase⁃3 protein levels were increased(P<0.05).Compared with the high glucose group,the above phenomenon was reversed in the cells treated with the intervention of LBP.It was further found that cell viability,miR⁃29b level,Bcl⁃2 protein level,MAPK1 mRNA and protein level,apoptosis rate,ROS level,and miR⁃29b inhibitor were decreased based on LBP.Bax and Caspase⁃3 protein levels were increased(P<0.05).In addition,the 3’UTR of MAPK1 mRNA contained the conserved base of miR⁃29b sequence,which was confirmed by double luciferase assay.Conclusion LBP can up⁃regulate the expression of miR⁃29b to reduce the ex⁃pression of MAPK1,and thus inhibit the occurrence of HREC apoptosis caused by high glucose.