4-苯基丁酸对高糖诱导的人晶状体上皮细胞发生上皮间质转化的影响

目的　探讨4-苯基丁酸（4-PBA）对高糖诱导的人晶状体上皮细胞发生上皮间质转化（EMT）的影响。方法　体外培养人晶状体上皮细胞系HLE-B3细胞，将培养的HLE-B3细胞分为正常对照组、高糖组和4-PBA预处理+高糖组。利用流式细胞术检测各组细胞内活性氧（ROS）的含量；利用Western blot检测各组细胞中内质网应激（ERS）相关通路分子的蛋白表达；采用免疫荧光染色观察各组细胞中上皮因子E-cadherin和间质因子α-SMA的表达；分别采用实时荧光定量PCR和Western blot检测各组细胞中EMT标志分子E-cadherin、α-SMA、Snail的 mRNA及蛋白表达。结果　高糖组细胞呈长梭形改变，丢失上皮细胞特性，类似纤维细胞的表型；4-PBA预处理+高糖组细胞形态不规则，相对于高糖组细胞，长梭形细胞数量较少。流式细胞术检测结果显示，高糖组细胞内ROS含量高于正常对照组，4-PBA预处理后ROS含量降低，差异均有统计学意义（均为P<0.01）。Western blot检测结果显示，高糖组细胞中ERS相关通路分子GRP78、CHOP、p-eIF2α的蛋白相对表达水平均高于正常对照组（均为P<0.05）；4-PBA预处理+高糖组细胞中GRP78、CHOP、p-eIF2α的蛋白相对表达水平均明显低于高糖组（均为P<0.05）。实时荧光定量PCR和Western blot检测结果显示，与正常对照组相比，高糖组细胞中E-cadherin mRNA及蛋白表达下调，α-SMA和Snail的mRNA及蛋白表达均上调（均为P<0.05）；与高糖组相比，4-PBA预处理+高糖组细胞中E-cadherin mRNA及蛋白的相对表达量均明显上调，α-SMA和Snail的mRNA及蛋白的相对表达量则与之相反（均为 P<0.05）。免疫荧光染色结果显示，高糖组细胞中E-cadherin荧光表达明显弱于正常对照组，4-PBA 预处理后荧光增强；α-SMA荧光表达则与之相反。结论　4-PBA通过抑制ERS相关通路分子的表达、减少ROS产生来抑制高糖诱导的晶状体上皮细胞发生EMT，从而对晶状体上皮细胞起到一定的保护作用。

Effects of 4-phenylbutyric acid on the high glucose-induced epithelial-mesenchymal transition of human lens epithelial cells

Objective　To explore the effect of 4-phenylbutyric acid (4-PBA) on the epithelial-mesenchymal transition (EMT) of human lens epithelial (HLE) cells induced by high glucose.Methods　HLE-B3 cells cultured in vitro were divided into the normal control (CON) group, high-glucose (HG) group, and 4-PBA pretreatment combined with high glucose (4-PBA+HG) group. The content of reactive oxygen species (ROS) in each group was detected by the flow cytometry. The expression of endoplasmic reticulum stress (ERS) related proteins was determined by the Western blot assay. The levels of epithelial E-cadherin and mesenchymal α-SMA were observed by immunofluorescence staining. The mRNA and protein levels of EMT markers, such as E-cadherin, α-SMA, and Snail were detected by the real-time quantitative polymerase chain reaction (Q-PCR) and Western blot assay.Results　In the HG group, the HLE-B3 cells showed a long spindle shape and lost the characteristics of epithelial cells, similar to the phenotype of fibroblasts. In the 4-PBA+HG group, the HLE-B3 cells had an irregular phenotype, and the long spindle cells were fewer than the HG group. The flow cytometry showed that the ROS content in the HG group was higher than that in the CON group, and decreased when the cells were pretreated with 4-PBA (both P<0.01). The Western blot assay showed that the expression levels of ERS related proteins such as GRP78, CHOP and p-eIF2α in the HG group were higher than those in the CON group (all P<0.05), while after 4-PBA pretreatment, they were significantly reduced (all P<0.05). The real-time Q-PCR and Western blot assay showed that on both mRNA and protein levels, the expression of E-cadherin in the HG group was down-regulated, while the expression of α-SMA and Snail were up-regulated compared with the CON group (all P<0.05). In the 4-PBA+HG group, on both mRNA and protein levels, the expression of E-cadherin was significantly increased, while the expression of α-SMA and Snail were reduced compared with the HG group (all P<0.05). The immunofluorescence staining showed that the fluorescence expression of E-cadherin in the HG group was significantly lower than that in the CON group, but increased after 4-PBA pretreatment. On the contrary, the fluorescence expression of α-SMA was significantly higher than that in the CON group, but reduced after 4-PBA pretreatment.Conclusion　4-PBA can inhibit the expression of ERS related proteins and reduce the ROS production to prevent the EMT of lens epithelial cells induced by high glucose; thus, it has a protective effect on the lens epithelial cells.