柚皮素基于TGF-β2介导对白内障晶状体病理性上皮间充质转化的作用研究

A Study on the Effect of Naringenin on Pathological Epithelial-mesenchymal Transi⁃tion in Cataract Lenses Mediated by TGF-β2

目的探讨白内障的发病机制是否与人晶状体上皮细胞系B3(HLE-B3)的病理性上皮间充质转化(EMT)有关,并评价柚皮素基于转化生长因子-β2(TGF-β2)介导对白内障晶状体病理性EMT的作用及机制。方法常规培养HLE-B3细胞,筛选TGF-β2和柚皮素的最佳浓度后,TGF-β2处理HLE-B3细胞诱导EMT,建立白内障体外模型。随机将细胞分为对照组(CG)、TGF-β2组(TGF-β2)、柚皮素组(Nar)、柚皮素+TGF-β2组(Nar+TGF-β2)培养48 h,采用跨孔迁移实验检测细胞侵袭能力,伤口愈合实验测定HLE-B3的迁移能力,细胞计数法检测细胞活性,流式细胞术检测细胞凋亡情况,5-乙炔基-2'-脱氧尿苷(EdU)法测定细胞增殖能力。Western Blot和实时荧光定量PCR(RT-qPCR)法检测EMT相关标志物α-平滑肌肌动蛋白(α-SMA)、E-钙粘蛋白(E-cadherin)和胞质紧密粘连蛋白1(ZO-1)的表达水平。结果(1)细胞活性与增殖:与CG组比较,Nar组和Nar+TGF-β2组细胞活性较低(t_(Nar)=16.404,t_(Nar)+TGF-β2=16.119,均P=0.000),EdU阳性细胞数较少(t_(Nar)=13.918,t_(Nar)+TGF-β2=13.473,均P=0.000),差异有统计学意义。(2)细胞侵袭和迁移:与CG组比较,TGF-β2组细胞侵袭个数较多(t=26.198,P=0.000),细胞迁移率较高(t=11.703,P=0.000);而Nar组细胞侵袭个数较少(t=23.975,P=0.000),细胞迁移率较低(t=7.198,P=0.000),差异均有统计学意义。(3)细胞凋亡:与CG组比较,TGF-β2组细胞凋亡率较高(t=5.806,P=0.000),Nar组细胞凋亡率较低(t=3.838,P=0.005),差异均有统计学意义。(4)EMT相关蛋白和mRNA:与CG组比较,TGF-β2组HLE-B3细胞α-SMA蛋白表达较高(t=4.847,P=0.001)、mRNA表达较高(t=8.684,P=0.000),E-cadherin和ZO-1蛋白表达较低(t_(E-cadherin)=5.584,t_(ZO-1)=5.251,均P=0.001)、mRNA表达较低(t_(E-cadherin)=5.379,P=0.001;t_(ZO-1)=2.365,P=0.045);Nar组α-SMA蛋白表达较低(t=5.144,P=0.001)、mRNA表达较低(t=3.961,P=0.004),E-cadherin和ZO-1蛋白表达较高(t_(E-cadherin)=6.175,t_(ZO-1)=8.383,均P=0.000)、mRNA表达较高(t_(E-cadherin)=5.379,t_(ZO-1)=5.125,均P=0.001),差异均有统计学意义。结论柚皮素能抑制TGF-β2诱导的HLE-B3细胞的增殖、侵袭、迁移、凋亡能力及EMT。

OBJECTIVE To investigate whether the pathogenesis of cataracts is related to pathologic epithelial-mesenchymal transition(EMT)in human lens epithelial-B3(HLE-B3)cells and to evaluate the effect and mechanism of naringenin mediated by transforming growth factor-β2(TGF-β2)in pathological EMT of cataract lenses.METHODS HLE-B3 cells were cultured conventionally,and the optimal concentrations of TGF-β2 and naringenin were screened.TGF-β2 was used to induce EMT in HLE-B3 cells,establishing an in vitro model of cataracts.The cells were randomly divided into the control group(CG),the TGF-β2 group(TGF-β2),the naringenin group(Nar),and the naringenin+TGF-β2 group(Nar+TGF-β2).The cells were cultured for 48 h.Cell invasion was determined by the transwell migration assay,wound healing experiments were performed to assess cell migration,cell viability was measured using a cell counting kit,apoptosis was evaluated by flow cytometry,and cell proliferation was assessed by the 5-ethynyl-2'-deoxyuridine(EdU)assay.The expression levels of epithelial-mesenchymal transition(EMT)related markersα-smooth muscle actin(α-SMA),E-cadherin,and zonula occludens-1(ZO-1)were detected by Western Blot and real-time quantitative PCR(RT-qPCR).RESULTS(1)Cell viability and proliferation:Compared to the CG,the cell viability was lower in the Nar group and the Nar+TGF-β2 group(t_(Nar)=16.404,t_(Nar)+TGF-β2=16.119,both P=0.000),and the number of EdU-positive cells was lower(t_(Nar)=13.918,t_(Nar)+TGF-β2=13.473,both P=0.000),with statistically significant differences.(2)Cell invasion and migration:Compared to the CG,the TGF-β2 group had a higher number of invading cells(t=26.198,P=0.000)and a higher cell migration rate(t=11.703,P=0.000).In contrast,the Nar group had fewer invading cells(t=23.975,P=0.000)and a lower cell migration rate(t=7.198,P=0.000),with statistically significant differences.(3)Cell apoptosis:Compared to the CG,the TGF-β2 group had a higher cell apoptosis rate(t=5.806,P=0.000),whereas the Nar group had a lower cell apoptosis rate(t=3.838,P=0.005),with statistically significant differences.(4)EMT-related proteins and mRNA:Compared to the CG,the TGF-β2 group had higher protein(t=4.847,P=0.001)and mRNA expression ofα-SMA(t=8.684,P=0.000),lower protein(t_(E-cadherin)=5.584,t_(ZO-1)=5.251,both P=0.001)and mRNA expression of E-cadherin(t_(E-cadherin)=5.379,P=0.001;t_(ZO-1)=2.365,P=0.045).In contrast,the Nar group had lower protein(t=5.144,P=0.001)and mRNA expression ofα-SMA(t=3.961,P=0.004),higher protein expression of E-cadherin(t_(E-cadherin)=6.175,t_(ZO-1)=8.383,both P=0.000)and mRNA expression of E-cadherin(t_(E-cadherin)=5.379,t_(ZO-1)=5.125,both P=0.001),with statistically significant differences.CONCLUSIONS Naringenin can inhibit the proliferation,invasion,migration,and apoptosis of HLE-B3 cells induced by TGF-β2 and the EMT process.