吲哚菁绿对人晶状体上皮细胞生物学行为和转分化的抑制作用及其机制

Inhibitory effect of indocyanine green on biological behavior and transdifferentiation of human lens epithelial cells and its mechanism

目的研究吲哚菁绿(ICG)对人晶状体上皮细胞(HLECs)生物学行为和转分化的抑制作用及其机制。方法将HLECs细胞系分为空白对照组、5%葡萄糖溶液(GS)组和0.5%、1.5%、2.5%ICG组,分别用平衡盐溶液、5%GS以及0.5%、1.5%和2.5%的ICG溶液处理3 min,然后在新鲜培养基中孵育24 h。采用流式细胞术检测HLECs凋亡水平;采用Western blot法检测HLECs凋亡相关蛋白Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤因子2(Bcl-2)、半胱氨酸蛋白酶3(caspase-3)和caspase-9表达水平;采用细胞计数试剂盒8(CCK-8)和5-溴-2-脱氧尿嘧啶(EdU)法检测HLECs增生过程;采用细胞划痕实验检测HLECs迁移能力;采用Transwell法检测HLECs迁移和侵袭过程。采用Western blot法检测HLECs转分化相关蛋白α-平滑肌肌动蛋白(α-SMA)、N-钙黏蛋白(N-cadherin)、纤维连接蛋白(FN)和波形纤维蛋白(vimentin)表达水平。结果空白对照组、5%GS组、0.5%ICG组、1.5%ICG组和2.5%ICG组细胞凋亡率分别为(4.35±0.60)%、(4.63±0.19)%、(8.17±0.69)%、(13.90±0.33)%和(23.08±1.12)%,总体比较差异有统计学意义(F=412.74,P<0.05),其中0.5%ICG组、1.5%ICG组和2.5%ICG组细胞凋亡率明显高于空白对照组和5%GS组,差异均有统计学意义(均P<0.05)。0.5%ICG组、1.5%ICG组、2.5%ICG组caspase-3、caspase-9和Bax蛋白相对表达量明显高于空白对照组和5%GS组,1.5%ICG组、2.5%ICG组Bcl-2蛋白相对表达量较空白对照组和5%GS组降低,差异均有统计学意义(均P<0.05)。0.5%ICG组、1.5%ICG组和2.5%ICG组EdU阳性细胞率明显低于空白对照组和5%GS组,差异均有统计学意义(均P<0.05)。0.5%ICG组、1.5%ICG组、2.5%ICG组细胞生存率明显低于空白对照组和5%GS组,差异均有统计学意义(均P<0.05)。细胞划痕实验结果显示,0.5%ICG组、1.5%ICG组、2.5%ICG组细胞迁移率明显低于空白对照组和5%GS组,差异均有统计学意义(均P<0.05)。Transwell实验结果显示,0.5%ICG组、1.5%ICG组、2.5%ICG组细胞迁移数和侵袭细胞数明显少于空白对照组和5%GS组,差异均有统计学意义(均P<0.05)。0.5%ICG组、1.5%ICG组和2.5%ICG组α-SMA、N-cadherin和FN蛋白相对表达量明显低于空白对照组和5%GS组,1.5%ICG组和2.5%ICG组vimentin蛋白相对表达量较空白对照组和5%GS组降低,差异均有统计学意义(均P<0.05)。结论ICG可促进HLECs凋亡,抑制其增生、迁移、侵袭和转分化,并呈浓度依赖性。

Objective To investigate the inhibitory effect of indocyanine green(ICG)on biological behavior and transdifferentiation of human lens epithelial cells(HLECs)and its mechanism.Methods HLECs were divided into blank control group,5%glucose solution(GS)group and 0.5%ICG group,1.5%ICG group and 2.5%ICG group,which were treated with balanced salt solution,5%GS and 0.5%,1.5%and 2.5%ICG solutions for 3 minutes,respectively,and then were incubated in fresh medium for 24 hours.The apoptosis level of HLECs was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2-associated X protein(Bax),B-cell lymphoma-2(Bcl-2),caspase-3 and caspase-9 were detected by Western blot.Cell proliferation was detected via the cell counting kit-8(CCK-8)assay and 5-ethynyl-2'-deoxyuridine(EdU)incorporation assay.The migration ability of HLECs was detected by cell scratch assay.Cell migration and invasion were determined by Transwell assays.The expression levels transdifferentiation-related proteins,α-smooth muscle actin(α-SMA),nerve calcium adhesion protein(N-cadherin),fibronectin(FN)and vimentin were assessed by Western blot.Results The apoptosis rates of blank control group,5%GS group,0.5%ICG group,1.5%ICG group and 2.5%ICG group were(4.35±0.60)%,(4.63±0.19)%,(8.17±0.69)%,(13.90±0.33)%and(23.08±1.12)%,with a statistically significant difference in the overall comparison(F=412.74,P<0.05).The apoptosis rate was significantly higher in 0.5%ICG group,1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group(all at P<0.05).The relative expressions of caspase-3,caspase-9 and Bax proteins were significantly higher in 0.5%ICG group,1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group,and the relative expression of Bcl-2 protein was lower in 1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group,and the differences were statistically significant(all at P<0.05).The rate of EdU-positive cells was significantly lower in 0.5%ICG group,1.5%ICG group and 2.5%ICG groups than in blank control group and 5%GS group(all at P<0.05).The survival rate of cells was significantly lower in 0.5%ICG group,1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group(all at P<0.05).The migration rates of scratch cells were significantly lower in 0.5%ICG group,1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group,and the differences were statistically significant(all P<0.05).The number of migrating cells and the number of invading cells were significantly lower in 0.5%ICG group,1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group(all at P<0.05).The relative expressions ofα-SMA,N-cadherin and FN were significantly lower in 0.5%ICG group,1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group,and the relative expression of vimentin was lower in 1.5%ICG group and 2.5%ICG group than in blank control group and 5%GS group,and the differences were statistically significant(all at P<0.05).Conclusions ICG can promote HLECs apoptosis and inhibit HLECs proliferation,migration,invasion and transdifferentiation in a concentration-dependent manner.