The Effect Of IL-37 On Apoptosis Of Human Retinal Microvascular Endothelial Cells Induced By High Glucose Culture

Objective: To establish a high glucose model of human retinal microvascular endothelial cells(HRMECs)in vitro and observe the apoptosis of HRMECs under high glucose environment.To detect the expression of interleukin-37(IL-37)in HRMECs under normal and high glucose conditions and to investigate the role of interleukin-37(IL-37) in high glucose-induced apoptosis of HRMECs.Methods: 1.Identification of HRMECs by immunofluorescence staining.2.CCK-8method was used to detect the activity of HRMECs at different glucose concentrations(5.5 mmol/L,20 mmol/L,35 mmol/L,50 mmol/L)and different time(24 h,48 h,72 h).3.Flow cytometry(Annexin V-FITC/PI staining)was used to detect the apoptosis rate of HRMECs treated with 5.5 mmol/L,20 mmol/L,35 mmol/L,50 mmol/L mannitol(35 mmol/L)for 48 h.Hoechst33342 staining was used to observe the apoptosis of HRMECs in normal controls group(5.5 mmol/L),high glucose group(35 mmol/L)and mannitol group(35 mmol/L).4.qRT-PCR and Western blot were used to detect the m RNA and protein expressions of Bax,Caspase 3,Bcl-2 and IL-1β in HRMECs of the normal controls group(5.5 mmol/L)and high glucose group(35 mmol/L).5.qRT-PCR was used to detect the expression of IL-37 mRNA in HRMECs in the normal controls group(5.5 mmol/L)and high glucose group(35 mmol/L).6.ELISA method was used to detect the expression of IL-37 in the cell supernatant of the normal controls group and the high glucose group(35 mmol/L).Results: 1.The cell-specific marker antibodies CD31 and Factor Ⅷ used in this experiment were positive,and the cells HRMECs needed for this experiment were positive.2.CCK-8 results showed that the cell viability of HRMECs decreased with the increase of concentration and time.The cell viability of HRMECs cultured in 35mmol/L glucose for 48 h was significantly lower than that of normal group(P < 0.05).3.The results of flow cytometry showed that the apoptosis rates of HRMECs at 35mmol/L and 50 mmol/L were higher than those in the normal controls group(5.5mmol/L),and there was no significant difference between the mannitol group,20mmol/L group and the normal group.Hoechst33342 results showed that compared with the normal control group,the apoptosis of HRMECs in high glucose environment increased(P < 0.05),and the number of cells decreased.The results of q RT-PCR and WB showed that compared with the normal group,the m RNA and protein expressions of apoptotic proteins(Bax,Caspase 3)and IL-1β in the high glucose group(35 mmol/L)were increased,and the m RNA and protein expressions of anti-apoptotic protein Bcl-2were decreased.4.qRT-PCR results showed that compared with the normal control group,the expression of IL-37 m RNA in HRMECs induced by high glucose was increased(P < 0.05).5.ELISA results showed that IL-37 in the cell culture supernatant of the high glucose group(35 mmol/L)was higher than that of the normal controls group(P < 0.05).Conclusion:(1)With the increase of glucose concentration,the viability of HRMECs in vitro decreased and the apoptosis rate increased.35 mmol/L glucose for 48 h was the best concentration and time of high glucose cultured HRMECs in vitro.(2)Increased expression of IL-37 in HRMECs under high glucose environment may be involved in apoptosis of HRMECs.