枸杞多糖通过miRNA-21-5p靶向PTEN调控PI3K/Akt/mTOR通路抗人视网膜色素上皮细胞光损伤机制

Mechanism of LBP targeting PTEN through miRNA-21-5p to regulate PI3K/Akt/mTOR pathway against photodamage in human retinal pigment epithelial cells

目的:研究枸杞多糖(LBP)对光诱导人视网膜色素上皮(RPE)细胞凋亡作用的影响及其保护机制。方法:将体外培养的人视网膜色素上皮细胞(ARPE-19)随机分为空白对照组,模型组,枸杞多糖低、中、高剂量组,建立人RPE细胞光损伤模型,于光照24h后进行相应指标检测。采用CCK-8法检测细胞存活率;流式细胞术检测细胞凋亡率;qRT-PCR法检测miRNA-21-5p、PTENmRNA表达水平;WesternBlot法检测PTEN、磷脂酰肌醇3-激酶(PI3K)、p-PI3K、蛋白激酶B(Akt)、p-Akt、哺乳动物雷帕霉素靶蛋白(mTOR)、P-mTOR蛋白表达量。结果:与空白对照组比较,模型组细胞存活率显著降低,细胞凋亡率显著升高,miRNA-21-5p表达显著下降,PTENmRNA表达显著升高,PI3K、Akt、mTOR磷酸化水平显著降低,PTEN蛋白表达量显著升高(P<0.01);与模型组比较,枸杞多糖低、中、高剂量组细胞存活率显著升高,细胞凋亡率显著下降,miRNA-21-5p表达显著升高,PTENmRNA表达均显著降低,PI3K、Akt、mTOR磷酸化水平均显著升高,PTEN蛋白表达量显著降低(P<0.05,P<0.01);与LBP低剂量组比较,中、高剂量组细胞存活率显著升高,细胞凋亡率显著降低,miRNA-21-5p表达显著升高,PTENmRNA表达显著降低,PI3K、Akt、mTOR磷酸化水平均显著增高,PTEN蛋白表达量显著降低(P<0.05,P<0.01);LBP高剂量组与LBP中剂量组相比细胞存活率显著上升,细胞凋亡率显著降低,miRNA-21-5p表达显著上升,PTENmRNA表达显著下降,mTOR、PI3K、Akt磷酸化水平均显著升高,PTEN蛋白表达量显著降低(P<0.05,P<0.01)。结论:枸杞多糖可抑制光诱导下ARPE-19细胞凋亡,其机制可能是通过上调miRNA-21-5p、下调PTENmRNA表达,从而激活PI3K/Akt/mTOR信号通路来增强保护RPE细胞对抗光损伤作用,且呈浓度依赖性。

Objective:To study the effect of Lycium barbarum polysaccharide(LBP)on light-induced apoptosis of human retinal pigment epithelium(RPE)cells and its protective mechanism.Methods:Human retinal pigment epithelial cells(ARPE-19)cultured in vitro were randomly divided into blank control group,model group and low,medium and high dose groups of Lycium barbarum polysaccharide,and the light injury model of hRPE cells was established,and the corresponding indexes were detected after 24 h of light exposure.The cell survival rate was detected by CCK-8 method.Apoptosis rate was detected by flow cytometry.The expression levels of miRNA-21-5p and PTEN mRNA were detected by qRT-PCR.The expressions of PTEN,p-PI3K,PI3K,p-Akt,Akt,p-mTOR and mTOR were detected by Western Blot.Results:Compared with the blank control group,the cell survival rate,the apoptosis rate,the expression of miRNA-21-5p,the expression of PTEN mRNA,the phosphorylation levels of PI3K,Akt and mTOR in the model group were significantly decreased,and the expression of PTEN protein was significantly increased(P<0.01).Compared with the model group,the low,middle and high doses of LBP significantly increased the cell survival rate,significantly decreased the apoptosis rate,significantly increased the expression of miRNA-21-5p,significantly decreased the expression of PTEN mRNA,significantly increased the phosphorylation levels of PI3K,Akt and mTOR,and significantly decreased the expression of PTEN protein(P<0.05,P<0.01).Compared with the LBP low-dose group,the cell survival rate,the apoptosis rate,the expression of miRNA-21-5p and the expression of PTEN mRNA in the middle and high-dose groups were significantly increased,and the phosphorylation levels of PI3K,Akt and mTOR were significantly decreased.The expression of PTEN protein was significantly increased and decreased(P<0.05,P<0.01).Compared with the LBP middle dose group,the LBP high dose group significantly increased the cell survival rate,significantly decreased the apoptosis rate,significantly increased the expression of miRNA-21-5p,significantly decreased the expression of PTEN mRNA,significantly increased the phosphorylation levels of mTOR,PI3K and Akt,and significantly decreased the expression of PTEN protein(P<0.05,P<0.01).Conclusion:LBP can inhibit the apoptosis of ARPE-19 cells induced by light,and its mechanism may be through upregulating miRNA-21-5p and down-regulating PTEN mRNA expression,thus activating PI3K/Akt/mTOR signaling pathway to enhance protection.RPE cells have anti-photodamage effect in a concentration-dependent manner.