Study On Anti-inflammatory Mechanism Of Netrin-1 In Diabetic Retinopathy

ObjectiveTo investigate the anti-inflammatory effect of Netrin-1,and the effects of its receptor UNC5 B and downstream molecule PPARγ on the inflammation of retinal Müller cells in diabetic mice.MethodsC57 BL / 6 mice with high-fat and high-sugar diet,were induced diabetes mellitus(DM)model by intraperitoneal injection of streptozotocin.In order to investigate the effect of endogenous Netrin-1 on diabetic retinopathy(DR),four groups were set up for experiments.Normal mice / DM mice were injected with si-Netrin-1 in right eyes to inhibit the expression of Netrin-1(si-Netrin-1 group / si-Netrin-1 +DM group)and negative control inhibitor in left eyes(si-NC group / si-NC +DM group).To investigate the effect of exogenous Netrin-1 on DR,30 mice were randomly divided into three groups: the control group,the DM group and the Netrin-1 group(intravitreal injection of Netrin-1 after successful DM modeling 1 month).The m RNA expressions of inflammatory factors(IL-1β,IL-6,TNF-α)in the retinal tissues of mice in each group were measured by real-time quantitative PCR.Müller cells and IL-6 proteins were detected by immunofluorescence staining in the control group,the DM group and the Netrin-1 group.In order to investigate the effect of Netrin-1 on the inflammatory response of Müller cells induced by hyperglycemia,5 groups were set up for experiments: the high-glucose group simulated hyperglycemia in vivo(DMEM complete medium with 30 mmol/L glucose added),the control group(DMEM complete medium with 30 mmol/L manneol added)to exclude the influence of hyperosmolar pressure of glucose,the Netrin-1 group(exogenous Netrin-1 was added to the medium of high-glucose group),the si-Netrin-1 group(Netrin-1 si RNA was added to the medium of high-glucose group to silence endogenous Netrin-1)and the Netrin-1 negative inhibition group(negative nucleotide inhibitor was added to the medium of high-glucose group).Netrin-1 has a variety of receptors.In order to investigate whether Netrin-1 plays a role through UNC5 B receptor and regulating downstream PPARγ,three groups were divided for experiments: the si-UNC5 B group(the medium of Netrin-1 group with UNC5 B si RNA added to silence receptor UNC5B),UNC5 B negative suppression group(the medium of Netrin-1 group with negative nucleotide inhibitor added),BADGE group(the medium of Netrin-1 group with BADGE,PPARγ specific antagonist,added).The expression levels of Netrin-1,PPARγ,NF-B and IL-6 were detected by Western blot and immunofluorescence after 48 hours.ResultsThe expression levels of IL-1β,IL-6 and TNF-α m RNA increased after retinal tissue silencing Netrin-1(P<0.05).The protein levels of IL-1β,IL-6 and TNF-α decreased after injection of Netrin-1 solution(P<0.05).The fluorescence intensity of IL-6 in the DM group was greater than that in the control group,and the increase of IL-6 in the Netrin-1 group was reduced.Compared with the control group,the expression of Netrin-1 protein in Müller cell increased slightly in the high-glucose group,while the expressions of PPARγ,NF-B and IL-6 protein increased significantly(P<0.05).Compared with the high-glucose group,the expression of PPARγ protein in the Netrin-1 group increased,and the expression of NF-B and IL-6 protein decreased.The expression of PPARγ protein in si-Netrin-1 group decreased,and the expression of NF-B and IL-6 protein increased(P<0.05).Compared with the Netrin-1 group,the si-UNC5 B group had lower PPARγ protein expression and increased NF-B and IL-6 protein expression(P<0.05).NF-B and IL-6 protein expression decreased in the BADGE group compared with the Netrin-1 group(P<0.05).The negative control group was established.ConclusionsNetrin-1 can reduce the inflammatory response in DR,which may be involved in the PPARγ / NF-B signaling pathway in Müller cells in combination with its receptor UNC5 B to reduce the hyperglycemic-induced inflammatory response.