AG3340对高糖诱导人视网膜色素上皮细胞迁移和侵袭的影响

Effects of AG3340 on the migration and invasion of human retinal pigment epithelial cells induced by high glucose

目的:探讨基质金属蛋白酶(MMPs)抑制剂AG3340对高糖(HG)培养状态下人视网膜色素上皮细胞(ARPE-19细胞)迁移和侵袭能力的影响及作用机制。方法:将体外培养的ARPE-19细胞分为4组,其中对照组(Control组)用含5.6mmol/L葡萄糖的DMEM/F12培养基培养;HG组用含30mmol/L葡萄糖的DMEM/F12培养基培养;HG+AG3340组用AG3340预处理细胞12h后,再用含30mmol/L葡萄糖的DMEM/F12培养基继续培养;甘露醇(MA)组用含5.6mmol/L葡萄糖和24.4mmol/L甘露醇的DMEM/F12培养基培养作为渗透压对照组。采用划痕实验检测细胞的迁移能力,Transwell小室实验检测细胞的侵袭能力,Western blot法检测MMP-9、MMP-2、纤连蛋白(Fibronectin)及胶原蛋白(Collagen)Ⅳ的相对表达水平。结果:划痕实验结果显示,划痕24、48h后HG组细胞迁移率均较Control组明显增加(均P<0.001),采用AG3340预处理后细胞迁移率均较HG组降低(均P<0.01)。Transwell小室实验结果显示,HG组细胞侵袭数目较Control组明显增加(P<0.001),采用AG3340预处理后细胞侵袭数目较HG组减少(P<0.01)。Western blot检测结果显示,HG组细胞中MMP-9和MMP-2相对表达量均较Control组增加,Fibronectin和CollagenⅣ相对表达量均较Control组减少(均P<0.001),采用AG3340预处理后细胞中MMP-9和MMP-2蛋白相对表达量均较HG组降低,Fibronectin和CollagenⅣ相对表达量均较HG组增加(均P<0.05)。结论:高糖环境诱导ARPE-19细胞迁移和侵袭能力增强,AG3340则可以部分逆转该作用,该过程可能与AG3340抑制细胞内MMP-9和MMP-2的表达,稳定细胞外基质组分有关。

AIM: To investigate the effect and mechanism of matrix metalloproteinase(MMPs) inhibitor AG3340 on the migration and invasion ability of retinal pigment epithelial cells-19(ARPE-19) cultured in high glucose(CHG). METHODS: ARPE-19 cells cultured in vitro were divided into four groups: Control group, the glucose at the concentration of 5.6 mmol/L in DMEM/F12 medium;HG group, the glucose at the concentration of 30 mmol/L was cultured with DMEM/F12 medium;HG+AG3340 group, the cells were pretreated with AG3340 for 12 h, and then cultured in DMEM/F12 medium containing 30 mmol/L glucose;The mannitol(MA) group, cultured with DMEM/F12 medium of 5.6 mmol/L glucose and 24.4 mmol/L mannitol, which used as hypertonic control group. The migration ability of cells was detected by wound healing assay, the invasion ability of cells was detected by Transwell assay, and the relative expression levels of MMP-9, MMP-2, fibronectin and collagen Ⅳ were detected by Western blot.RESULTS: The results of wound healing assay showed that compared with the Control group, the cell migration rate of scratching after 24 h and 48 h in the HG group was significantly increased(all P<0.001).After pretreated by AG3340, the cell migration rate was significantly lower than that in the HG group(all P<0.01).Transwell assay showed that compared with the Control group, the number of cell invasion in the HG group was significantly higher than that in the Control group(all P<0.001). After pretreated by AG3340, the number of cell invasion was decreased than the HG group(all P<0.01). Western blot results showed that compared with the Control group, the relative expression levels of MMP-9 and MMP-2 of the cells in the HG group were increased, and the relative expression levels of Fibronectin and Collagen Ⅳ were decreased(all P<0.001). Compared with the HG group, the relative expression levels of MMP-9 and MMP-2 protein in AG3340 pretreatment group were decreased, and the relative expression levels of Fibronectin and Collagen Ⅳ were increased(all P<0.05). CONCLUSION: High glucose induced ARPE-19 cells with enhanced migration and invasion ability, and AG3340 partially reversed this effect, which was related to the inhibition of MMP-9 and MMP-2 expression and the stability of extra-cellular matrix components.