| Objective To investigate the effects and relevant mechanism of ethanol on the human corneal keratocytes(HCKs),and the protect effect and mechanism of thymosin ?4(T?4)in the above process.Methods HCKs and BALB/c mice were chosen as our study subject.(1)In vitro experiment: HCKs were cultured to exponential phase and divided into four groups according to the experimental design: control group(Ctrl),T?4 group(T),ethanol group(E),ethanol+T?4 group(E+T).Cell viability was measured by Cell Counting Kit-8(CCK-8).2',7'-Dichlorodihydrofluorescein diacetate(DCFH-DA)was used to detect the level of reactive oxygen species(ROS).Real-time quantitive polymerase chain reaction(RT-q PCR)was used to detect the expression of oxidative stress related genes,including Catalase and Cu Zn SOD,in cells.The cell apoptotic rate was detected by TUNEL.RT-q PCR was used to detect the expression level of apoptosis-related proteins which including Caspase-3 and Bcl-2.ELISA was used to detect the expression level of Caspase-3.The ability of cell migration and proliferation was tested by wound healing assay.RT-q PCR was used to detect the expression of mi R-21 in cells.The expression of PDCD4 was detected by Western blot(WB).(2)In vivo experiment: BALB/c mice of 6 to 8 weeks were also divided into four groups according to the experimental design: Ctrl,T,E,and E+T group.The pathological changes of corneal stroma of mice were observed by HE staining.The expression of Ki67 was detected by immunofluorescence staining.RT-q PCR was used to detect the relative expression of apoptosis-related proteins,Caspase-3 and Bcl-2.RT-q PCR was used to detect the expression of mi R-21.The expression of PDCD4 was detected by immunofluorescence staining.Results(1)The results of cell level were as follows.The activity of HCKs in E group was significantly lower than it of Ctrl group,and the cell viability in E+T group was significantly higher than it of E group(P<0.01).The ROS level of E group was significantly higher than it of Ctrl,while in E+T group,it was significantly lower than it of E group(P<0.01).The expression of Catalase and Cu Zn SOD in HCKs of E group was lower than it of Ctrl group,while the expression of the above two genes of group E+T was significantly higher than it of group E(P<0.01).The apoptotic rate of E group cells was significantly higher than it of Ctrl group,and the apoptotic rate of E+T group was significantly lower than that of E group(P<0.01).The expression of Caspase-3 and Bcl-2 increased significantly in E group,while the expression of the above two m RNAs decreased in E+T group(P<0.01).The expression of Caspase-3 in protein level was consistent with the RT-q PCR results.The cell migration rate of E group was significantly slower than it of Ctrl,and the cell migration rate of E+T group was significantly higher than that of group E(P<0.01).The expression of intracellular mi R-21 in E group was higher than it in Ctrl group(P<0.05).As the target gene of mi R-21,the expression of PDCD4 met the expression pattern of mic R-21.(2)The corneal healing speed of E+T group was significantly higher than that of E group(P<0.01).The expression of Ki67 of group E decreased,while the expression of Ki67 of group E+T increased(P<0.01).The expression of Caspase-3 and Bcl-2 in cornea tissue were consistent with those of cells in each group,and the difference was significant(P<0.01).The expression of mi R-21 in cornea was also consistent with that in HCKs,and the difference was statistically significant(P<0.01).Immunofluorescence staining results also confirmed that the expression PDCD4 match it of the cell level.Conclusions Ethanol can induce oxidative stress injury and secondary cell apoptosis on HCKs,and T?4 can effectively inhibit oxidative stress injury and cell apoptosis.Ethanol can lead to the damage and edema in cornea stroma of mice,while T?4 can promote corneal stroma repair of mice.In addition,The expression of mi R-21 was up regulated by ethanol in HCKs and corneal stroma of mice. |
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