缺氧对巩膜成纤维细胞内质网应激反应的激活作用及其对巩膜重塑的影响

Effects of hypoxia on the activation of endoplasmic reticulum stress response and on the scleral remodeling in human fetal scleral fibroblasts

目的探讨缺氧对巩膜成纤维细胞(HFSF)内质网应激反应的激活作用及其对巩膜重塑的影响。方法取HFSF随机分为缺氧0 h组、缺氧12 h组、缺氧48 h组。缺氧0 h组细胞正常培养,缺氧12 h组及48 h组细胞分别在含氧体积分数2%的三气培养箱中缺氧处理12 h及48 h。采用Western blot检测肌醇需求酶l(IRE1α)、P-IRE1α、COL1A1、基质金属蛋白酶-2(MMP-2)、α平滑肌肌动蛋白(α-SMA)、BAX及BCL-2蛋白表达情况,免疫荧光染色检测HFSF中α-SMA蛋白含量,流式细胞仪检测细胞凋亡情况,CCK-8检测细胞增殖情况。结果Western blot检测结果显示:与缺氧0 h组相比,缺氧12 h组IRE1α、α-SMA蛋白表达均升高,COL1A1蛋白表达降低,差异均有统计学意义(均为P<0.05),其余蛋白表达变化不明显,差异均无统计学意义(均为P>0.05);与缺氧0 h组相比,缺氧48 h组IRE1α、P-IRE1α、MMP-2、α-SMA、BAX蛋白表达均升高,COL1A1、BCL-2蛋白表达均降低,差异均有统计学意义(均为P<0.05);与缺氧12 h组相比,缺氧48 h组COL1A1、BCL-2蛋白表达降低,BAX蛋白表达升高,差异均有统计学意义(均为P<0.05),其余蛋白表达变化不明显,差异均无统计学意义(均为P>0.05)。免疫荧光染色结果显示:缺氧12 h组及缺氧48 h组α-SMA蛋白荧光强度均较缺氧0 h组增高,且缺氧48 h组较缺氧12 h组α-SMA蛋白荧光强度升高更明显。流式细胞仪检测结果显示:缺氧0 h组、缺氧12 h组、缺氧48 h组细胞凋亡率分别为(0.617±0.032)%、(2.187±0.212)%、(4.130±0.395)%;缺氧12 h组及缺氧48 h组细胞凋亡率均高于缺氧0 h组,差异均有统计学意义(均为P<0.05),且缺氧48 h组细胞凋亡率高于缺氧12 h组,差异有统计学意义(P<0.05)。CCK-8检测结果显示:缺氧12 h组及缺氧48 h组D 450均低于缺氧0 h组,差异均有统计学意义(均为P<0.05);缺氧48 h组与缺氧12 h组相比,差异无统计学意义(P>0.05)。结论缺氧激活HFSF内质网应激反应,并且可能通过调节胶原代谢、促进细胞转分化及凋亡来参与巩膜重塑。

Objective To investigate the effects of hypoxia on the activation of endoplasmic reticulum stress response and on the scleral remodeling in human fetal scleral fibroblasts(HFSFs).Methods HFSFs were randomly divided into the hypoxia 0 h group,hypoxia 12 h group,and hypoxia 48 h group.HFSFs in the hypoxia 0 h group were cultured normally,while HFSFs in the hypoxia 12 h and 48 h groups were treated under hypoxia for 12 h and 48 h respectively in a three-gas incubator containing 2%oxygen.The expression levels of inositol-requiring enzyme 1α(IRE1α),P-IRE1α,collagen type I alpha 1(COL1A1),matrix metallopeptidase 2(MMP-2),alpha-smooth muscle actin(α-SMA),BAX,and BCL-2 proteins were detected by Western blot.The content ofα-SMA protein in HFSFs was detected by immunofluorescence.Cell apoptosis was detected by flow cytometry,and cell proliferation was detected by CCK-8.Results Western blot results showed that compared with the hypoxia 0 h group,the expression of IRE1αandα-SMA proteins increased,while the expression of COL1A1 protein decreased in the hypoxia 12 h group,and the differences were statistically significant(all P<0.05),but there were no significant differences in other proteins between the two groups(all P>0.05).Compared with the hypoxia 0 h group,the expression of IRE1α,P-IRE1α,MMP-2,α-SMA,and BAX proteins increased,while the expression of COL1A1 and BCL-2 proteins decreased in the hypoxia 48 h group,and the differences were statistically significant(all P<0.05).Compared with the hypoxia 12 h group,the expression of COL1A1 and BCL-2 proteins decreased,while the expression of BAX protein increased in the hypoxia 48 h group,and the differences were statistically significant(all P<0.05),but there were no significant differences in other proteins between the two groups(all P>0.05).Immunofluorescence results showed that the fluorescence intensity ofα-SMA protein was higher in both the hypoxia 12 h group and the hypoxia 48 h group than in the hypoxia 0 h group,and the fluorescence intensity was more obvious in the hypoxia 48 h group than in the hypoxia 12 h group.Flow cytometry results showed that the cell apoptosis rate in the hypoxia 0 h group,hypoxia 12 h group,and hypoxia 48 h group was(0.617±0.032)%,(2.187±0.212)%,and(4.130±0.395)%,respectively.The cell apoptosis rate in the hypoxia 12 h and 48 h groups was higher than that in the hypoxia 0 h group(both P<0.05),and meanwhile the cell apoptosis rate in the hypoxia 48 h group was higher than that in the hypoxia 12 h group(P<0.05).CCK-8 results showed that the D 450 in the hypoxia 12 h and 48 h groups was lower than that in the hypoxia 0 h group(both P<0.05),but there was no significant difference between the hypoxia 48 h group and the hypoxia 12 h group(P>0.05).Conclusion Hypoxia activates the endoplasmic reticulum stress response in HFSFs and may participate in scleral remodeling by regulating collagen metabolism and promoting cell transdifferentiation and apoptosis.