A Preliminary Investigation Of The Effect Of TGF-β1 Via LOX On Apoptosis In Human Retinal Microvascular Endothelial Cells Induced By High Glucose

Objective: In this study,we verified the involvement of transforming growth factorβ1(TGF-β1)in the regulation of lysyl oxidase(LOX)expression and observed the apoptosis of human retinal microvascular endothelial cells(HRMECs)under high glucose environment.To investigate whether TGF-β1 is involved in the apoptotic process of HRMECs induced by high glucose through the regulation of LOX.Methods: 1.5.5 mmol/L sugar concentration was set as the normal control,and the concentrations of 0.1 ng/m L,1 ng/m L,and 10 ng/m LTGF-β1 were set sequentially for24 h.The expression of LOX protein in each group was detected by Western Blot.2.ELISA was performed to detect the expression of TGF-β1 in the supernatants of the normal group(5.5 mmol/L),the high sugar group(30.0 mmol/L),and the mannitol group(30.0 mmol/L).3.HRMECs were divided into five groups: normal group(5.5mmol/L),high glucose group(30.0 mmol/L),high glucose + TGF-β1 receptor inhibitor(SB525334)group,high glucose + dimethylsulfoxide(DMSO)group and mannitol group(mannitol concentration was set at 30.0 mmol/L as a hypertonic control for the high glucose group);the apoptosis of HRMECs in each of the five groups was detected by TUNEL method;q RT-PCR and Western Blot were used to detect the apoptosis in the normal group,high glucose group,high glucose + TGF-β1receptor inhibitor group,and high glucose group.The expression of LOX,TGF-β1,Bax,Bcl-2,Caspase 3/Cleaved Caspase 3 m RNA and protein in HRMECs in normal group,high glucose group,high glucose + TGF-β1 receptor inhibitor group,high glucose + DMSO group and mannitol group were examined by q RT-PCR and Western Blot.Results: 1.Western Blot results showed that TGF-β1 upregulated LOX expression in HRMECs in a dose-dependent manner(P < 0.05).2.ELISA results showed an increase in TGF-β1 in the supernatant of the hyperglycemic group compared to the normal control group(P < 0.05).3.TUNEL(red TRITC-labeled fluorescence assay)The results showed that the apoptosis rate of HRMECs was significantly increased in the hyperglycemic environment compared with the normal group(P < 0.05);the apoptosis rate was significantly decreased in the hyperglycemic + TGF-β1 receptor inhibitor(SB525334)group compared with the hyperglycemic group(P < 0.05).q RT PCR and Western Blot results showed that,compared with the normal group,the hyperglycemic group had significantly increased LOX,TGF-β1 m RNA and its protein expression were significantly increased in the high glucose group compared with the normal group(P < 0.05);the ratio of apoptotic protein Caspase 3 m RNA,Cleaved Caspase 3 protein and Bax/Bcl 2 m RNA and its protein were increased(P < 0.05);LOX and TGF-β1 in the high glucose + TGF-β1 receptor inhibitor(SB525334)group m RNA and their protein expressions were significantly increased in the mannitol group compared with the normal control group,and the protein expressions of LOX,TGF-β1,apoptotic protein Bax,Cleaved Caspase 3 and anti-apoptotic protein Bcl 2were no significantly difference in the hyperglycemia group compared with the hyperglycemia + DMSO group.Conclusions:1.TGF-β1 upregulates the expression of LOX in HRMECs.2.LOX and TGF-β1 expression were increased in HRMECs under high glucose environment.3.TGF-β1 receptor inhibitor(SB525334)reduced LOX expression under high glucose conditions and played a protective role against high glucose-induced apoptosis in HRMECs.