Mechanism Of Capsaicin Ameliorating Diabetic Retinopathy By Inhibiting Poldip2-mediated Oxidative Stress

Backround and objectiveDiabetes retinopathy（DR）is a common microvascular complication of diabetes（DM）.Oxidative stress caused by long-term hyperglycemia is an important cause of DR.Studies have shown that Poldip2 is a key regulator of endothelial barrier function and inflammation.Poldip2 can increase the production of H₂O₂by enhancing the activity of Nox4.But at present,the role of Poldip2 in DR is unclear.Capsaicin（CAP）plays an important role in anti-inflammatory,antioxidant and lipid-lowering.Recent studies have found that capsaicin can improve diabetes,but there is little research on whether it has a protective effect on DR.Our previous work found that Poldip2,Nox4 and H₂O₂were highly expressed in the retina of diabetic rats and in HRMECs which cultured by high glucose,and capsaicin treatment could reverse these changes.Therefore,the purpose of this study was to investigate:1.whether Poldip2 is involved in the pathogenesis of diabetic retinopathy.2.Whether capsaicin can exert a protective effect on DR by inhibiting the oxidative stress induced by Poldip2.MethodsIn this study,male Sprague-Dawley（SD）rats and human retinal microvascular endothelial cells（HRMECs）were used as research objects,the diabetic model were established in vitro and in vivo by intraperitoneal injection of STZ in rats and HRMECs treated with high glucose respectively.The experiments consisted of two parts in vivo:1.The protective effect of CAP on DR.The rats were randomly categorized into control group（NC）,diabetic group（DM）,diabetic group with capsaicin treatment（DM+CAP）.4 weeks after successful modeling,the rats in DM+CAP group were given capsaicin solution（0.5 mg/kg/d）by gavage,and the other two groups was intragastric with dimethyl sulfoxide（DMSO）.The samples were collected after 8 weeks of capsaicin treatment.2.Poldip2 participated in DR by regulating H₂O₂derived from Nox4.The rats were randomly divided into control group（NC）,diabetic group treated with scrambled-AAV9-sh RNA（DM+NCsh RNA,n=10）,diabetic group treated with AAV9-poldip2-sh RNA（DM+poldip2sh RNA,n=10）.4 weeks after successful modeling,the rats in the DM+NCsh RNA and DM+poldip2sh RNA group were injected with 5μl scrambled-AAV₉-sh RNA or AAV₉-poldip2-sh RNA in vitreous cavity while the rats in the NC group were left untreated.All sample were collected after 4weeks.The experiments in vitro were divided into two parts:1.Protective effect of CAP on HRMECs cultured with high glucose.The HRMECs were divided into normal glucose group（NG）,high glucose group（HG）and high glucose with capsaicin group（HG+CAP）.The cells were respectively cultured in medium containing 5.5 m M glucose,30 m M glucose,or 30 m M glucose and 1μM capsaicin for 48 h.2.Cells were divided into normal control group（si-NC）and Poldip2 si RNA transfection group（si-poldip2）.Cells were cultured with negative control si RNA or Poldip2 si RNA respectively.The body weight and fasting blood glucose of rats were recorded weekly.Total cholesterol（TC）,triglycerides（TG）,low density lipoprotein（LDL）,high density lipoprotein（HDL）concentrations in serum were detected by assay kit（Nanjing Jiancheng,Nanjing）.HE staining was used to observe the changes of retinal pathological.Evans Blue angiography was used to observe the changes of retinal vascular permeability.The immunofluorescence was used to detect the distribution and expression levels of TRPV1,Poldip2 and Nox4 in rat retina.Western blot was used to detect the expression of TRPV1,Poldip2,Nox4,HIF-1α,VCAM-1,VEGF in rat retina and HRMECs.The content of ROS in rat retina and HRMECs were detected by DHE staining and DCFH-DA staining respectively.The H₂O₂concentrations in retina,serum and cells were detected by xylenol orange method.The permeability of retinal vascular endothelial cells was observed by transwell assay.Results1.Compared with the NC group,the concentration of blood glucose,TC,TG and HDL of the DM group were increased,the body weight and LDL levels decreased. Capsaicin treatment could reduce the concentrations of TC,TG and LDL in serum,had no significant effect on blood glucose,body weight and the concentration of HDL.2.Compared with the NC group,the expression of Poldip2,Nox4,VCAM-1,HIF-1α,VEGF,ROS and H₂O₂was increased in rat retinas with DM.Besides,the overt hyperpermeability of retinal microvessels and the increasd number of retinal neovascularization in diabetic rat were observed.However,capsaicin treatment could activate TRPV1 and inhibit those increases.3.Compared with the NG group,the levels of Poldip2,Nox4,VCAM-1,HIF-1α,VEGF,ROS,and H₂O₂were increased in HRMECs treated with high glucose,while the paracellular permeability of HRMECs was significantly increased.Capsaicin intervention could activate TRPV1and reverse these changes.4.After Poldip2 knockdown in HRMECs,the levels of Nox4,VCAM-1,HIF-1α,VEGF,ROS and H₂O₂were decreased concomitantly,while the paracellualr permeability of HRMECs was decreased.5.After Poldip2 silence in the retina of DM rats,the levels of Nox4,VCAM-1,HIF-1α,VEGF,ROS and H₂O₂were decreased,and the hyperpermeabilirty of retinal microvessel in diabetic rat retinas was decreased.Conclusions1.Hyperglycemia-induced oxidative stress and endothelial dysfunction through Poldip2-Nox4-H₂O₂pathway.2.Capsaicin treatment may attenuate the progression of DR by activating TRPV1 to exert antioxidant effects through the Poldip2-Nox4-H₂O₂signaling pathway.