The Effect And Mechanism Of Galectin-3 In Diabetic Retinopathy

Diabetic retinopathy(DR),as one of the serious microvascular complications of diabetes,has become an important cause of visual impairment and blindness in adults,seriously affecting patients’ quality of life and increasing medical burden.It is believed that multiple factors are involved in the occurrence and progression of diabetic retinopathy,among which oxidative stress and chronic inflammation play important roles.In view of this characteristic,antioxidant stress and inhibition of inflammatory response have become the key to prevent and treat DR.Galectin-3(Gal-3),an important member of the mammalianβ-galactoside-binding lectins family,has attracted much attention in recent years.It has been confirmed to be involved in a variety of pathophysiological processes in human body,such as inflammation,atherosclerosis,coronary heart disease and diabetes,and so on.Recent studies have shown that Gal-3 is associated with diabetic retinopathy,diabetic nephropathy,diabetic macrovasculopathy and other diabetic complications.However,the role of Gal-3 in the pathogenesis of diabetic retinopathy,especially in RPE cells,has been little investigated at home and abroad.In this study,the difference of Gal-3 expression in the plasma of NDR patients and DR patients was first investigated.Then,the adult retinal pigment epithelial cells(ARPE-19)was taken as the research object and cultured in groups in vitro.The high glucose induced cell model was constructed to detect the expression of related indexes.Expression of Gal-3 were inhibited by Si-Gal-3 transfection to verify its effects on cell apoptosis,localization of junctional proteins,intracellular oxidative stress and expression of inflammatory factors such as ICAM-1,VCAM-1,IL-1β and integrin-β1 in ARPE-19 cells under high glucose culture conditions.Furthermore,the possible role and related mechanism of Gal-3 in diabetic retinopathy was investigate.Part One Expression and significance of Gal-3 in plasm of pat-ients with diabetic retinopathyObjective: To investigate the expression of Gal-3 in plasma of NDR patients and DR patients,furthermore to explore its possible role in the development of DR.Methods:1.Clinical data: 62 patients without diabetic retinopathy and 54 patients with diabetic retinopathy were selected for the study.Baseline data such as age and gender of patients were recorded,routine ophthalmic examination was performed for all patients,and blood samples were collected from enrolled patients.2.Plasma Gal-3 concentration in each group were determined by enzyme-linked immunosorbent assay(ELISA).3.The expression concentration difference in each group was compared by the statistical analysis.Results:1.The concentration of Gal-3(ng/ml)in plasm of NDR group and DR group were(4.94±1.29)and(5.70±0.88)respectively.The concentration of Gal-3 in plasm of DR group was significantly higher than that of the NDR group.Therefore,there was significant statistical difference(P<0.05).Conclusions:1.Gal-3 is highly expressed in the plasm of diabetic retinopathy patients.2.Gal-3 may be involved in the pathogenesis of DR.Part Two Inhibition of Gal-3 ameliorates high-glucose-induced oxidative stress and inflammation in ARPE-19 cellsObjective: To establish the ARPE-19 cell model in high glucose culture environment in vitro,and then to detect the cell apoptosis and the localization of junctional proteins under high concentration of glucose.To targeted silencing Gal-3 gene expression in ARPE-19 cells,and investigate its effects on the expression of ICAM-1,VCAM-1,IL-1β and integrin-β1 and the intracellular ROS,MDA and SOD levels under high glucose environment.To investigate the effect of Gal-3 on cell apoptosis and tight junction protein expression of ARPE-19 cells,and to explore the possible role and related mechanism of Gal-3 in diabetic retinopathy.Methods:1.Adult retinal pigment epithelial cell line(ARPE-19 cell line)was used as the research object,and the expression of Gal-3 was inhibited by si RNA transfection technique.Meanwhile,the negative control group(NC group)was transfected with meaningless si RNA fragment.Real-time PCR and Western Blot were used to detect the silencing efficiency of Gal-3 si RNA on m RNA and protein levels.2.ARPE-19 cells of 3-5 generations with good growth status were selected in experiments and then divided into four groups: control group(C),high glucose group(HG),high glucose +NC group(HG+NC)and high glucose + Gal-3 si RNA group(HG+ Si-Gal-3).The cells in each group were cultured at 5.5mmol/L normal glucose concentration(control group)and25mmol/L high glucose concentration(experimental group)for 48 h.3.ARPE-19 cells apoptosis was detected by flow cytometry.Different oxidative stress kits(reactive oxygen species(ROS),malondialdehyde(MDA)and superoxide dismutase(SOD))were used to detect the oxidative stress levels in the grouping cells mentioned above.The expression,location and intensity of occludin and ZO-1 in ARPE-19 cells were observed by immunofluorescence chemical staining.The m RNA and protein expression levels of Gal-3,ICAM-1,VCAM-1,IL-1β and integrin-β1 in ARPE-19 cells were quantified by real-time PCR and Western Blot respectively.Results:1.Data from Real-time PCR and Western Blot indicated that Gal-3 can be expressed in ARPE-19 cells.2.Compared with the control group,high glucose treatment caused significantly increased Gal-3 expression in ARPE-19 cells(P<0.05).High glucose treatment also caused significantly increased apoptosis ratio of ARPE-19 cells(P<0.05),increased cellular oxidative stress,and decreased protein expression of occludin and ZO-1(P<0.05).High glucose treatment also caused elevated gene expression of ICAM-1,VCAM-1 and integrin-β1 at m RNA and protein levels(P<0.05).High glucose could induce IL-1βexpression at m RNA level(P<0.05).3.Compared with controls,Gal-3 knockdown might ameliorate high glucose-induced apoptosis and oxidative stress,increased occludin,ZO-1protein expression,and partly reversed increased inflammatory factors ICAM-1,VCAM-1 and integrin-β1caused by high glucose treatment at the m RNA and protein levels(all P<0.05).Gal-3 knockdown ameliorated IL-1βexpression induced by high glucose at m RNA level(P<0.05).Conclusions:1.High glucose treatment can induce Gal-3 expression in ARPE-19 cells.And high glucose also may increase apoptosis ratio,oxidative stress,reduce the expression of tight junction proteins,and increase the expression of inflammatory factors in ARPE-19 cells.2.Gal-3 knockdown may ameliorate apoptosis,improve tight junction protein expression,and improve cellular oxidative stress and inflammatory responses in ARPE-19 cells caused by high glucose.3.Gal-3 play key role in ARPE-19 cells damage caused by high glucose.