| Objective:In recent years,research has shown that diabetes(especially type 2 diabetes)has become the third largest killer of human health after cancer cardiovascular disease.According to the 2017 China Diabetes Association in the Chinese population incidence survey report shows that China has become the world's largest diabetic patients.Diabetes is often accompanied by abnormal microcirculation of diabetes,especially in the fundus,glomeruli,nerves,myocardium,muscle and other microvascular mainly caused by fundus lesions,kidney disease,neuropathy and myocardium and other diseases,become the main factor in determining the patient's life span The Diabetic retinopathy(DR),one of the most common complications of diabetes,is the leading cause of blindness in diabetic patients.However,for a long time,diabetic retinopathy screening methods and targeted treatment of drug scarcity.In order to achieve the purpose of prolonging the life span of diabetic patients and improving the quality of life of patients,it is of great theoretical and practical significance to provide more simple clinical screening methods and new intervention targets.CTRP3 as a very promising means of diagnosis and treatment of metabolic syndrome has become a recent research focus.It has been reported that CTRP3 can activate Akt and other signaling pathways to reduce the apoptosis of diabetic macrovascular endothelial cells and protect vascular endothelial cell function.A large number of researchers believe that further reveal the mechanism of CTRP3 on diabetic vascular endothelial dysfunction in the early prevention and treatment of diabetic vascular disease is important,but its role in microcirculation vascular endothelium and its mechanism has not been reported.Purposes:In this study,CTRP3 was applied to the microcirculation level,and the protective effect and mechanism of CTRP3 on DR blood retinal barrier(iBRB)were analyzed.Which provides an important theoretical basis for the new ideas and new targets of clinical screening and intervention of CTRP3 as diabetic retinopathy(DR).Methods:The research program of this subject is mainly from four aspects(1)Analysis of normal people,patients with diabetes without complications,the severity of different DR patients related to test data,to explore the correlation between CTRP3 and the severity of DR from the perspective of population,and to provide an important theoretical basis for the early diagnosis and screening of diabetic retinopathy(DR)by CTRP3.(2)To establish the iBRB model in vitro,using X-CELLigence system and Transwell chamber to detect the effect of CTRP3 on the injury of iBRB barrier induced by high glucose and high lipid barrier function injury protection;(3)High glucose and high lipid(HGHL)were used to simulate DR injury in vitro.Microcapsule microvascular endothelial cells(HRMECs)CTRP3 were used to activate AMPK,and siRNA was used to block AMPK and western blot to analyze the intracellular signal transduction mechanism of CTRP3 in HRMECs.(4)HFD/STZ method was used to establish a type 2 diabetic induced DR mouse model,FITC-Dextran combined with FRITC-ConcanavalinA mouse retinal microvascular staining and western blot in vivo to verify the inhibitory effect of CTRP3 on diabetic iBRB injury and its mechanism.Results:(1)In this study,we found that serum CTRP3 and CTRP5 concentrations in patients with T2 DM were significantly lower than those in the control group,and the serum CTRP3 and CTRP5 concentrations in the PDR group were even lower(P <0.001).Subsequently,we established a single factor and multivariate regression model and found that only the CTRP3 concentration had a significant independent relationship with DR(P <0.05).After that,ROC and AUC analysis showed that CTRP3 was a biomarker of DR [AUC(area under the ROC curve)was 0.900,with a 95% confidence interval of 0.838 to 0.962(P <0.001)],and the CTRP3 was negatively correlated with DR severity [AUC(area under the ROC curve)was 0.919,with a 95% confidence interval of 0.850 to 0.989(P <0.001)].This discovery has important theoretical and practical significance.Finally,by analyzing the correlation between CTRP3 and covariates,it was found that sVCAM-1 was negatively correlated with CTRP3 in normal subjects,T2 DM patients and DR patients(P <0.001).(2)Establishment of iBRB in vitro model,high glucose and high lipid(HGHL)in vitro simulated DR injury.The in vitro model function of iBRB was detected by X-CELLigence system and Transwell chamber on tracer detection.The results were stable and consistent.HGHL resulted in a decrease in Cell Index of single-layer HRMECs(P <0.05)and an increase in the permeability of tracer FITC-Dextran(P <0.05),that HGHL caused iBRB in vitro model dysfunction The In the whole experiment,the effect of CTRP3 on the effect of HGHL on single-layer HRMECs was significantly reduced(P<0.05),indicating that CTRP3 had protective effect on DR,and its mechanism was to maintain the barrier function of iBRB.(3)When HRMECs were incubated with CTRP3(3 ?g / ml)for 30 min,CTRP3 activated AMPK signaling molecules in HRMECs(P <0.05)and had no significant effect on AKT signaling molecules.The expression of ZO-1,Claudin1,Claudin1,Claudin5 and Occludin was significantly decreased by HGHL in vitro(P <0.05),while the intervention of CTRP3 significantly inhibited the expression of Occludin and Claudin5,and the results were dose-dependent(P < 0.05).The inhibition of the expression and release of endogenous AMPK in HRMECs cells was blocked by siRNA-AMPK.siRNA-AMPK intervention in X-CELLigence system to detect the permeability of i BRB in vitro model also confirmed that CTRP3 through the AMPK / Occludin,Claudin5 pathway inhibition of HGHL induced inflammation of the iBRB dysfunction(P <0.05).(4)HFD / STZ binding method,can establish a stable type 2 diabetes induced DR mouse model.In vivo experiments with this model confirmed that CTRP3 could inhibit the iBRB barrier function in DR mice,and the inhibitory target was Occludin and Claudin5(P <0.05).Conclusion:(1)There was a negative correlation between CTRP3 and DR severity in serum,CTRP3 could be used as a simple clinical screening method for DR.(2)The establishment of iBRB in vitro model,the use of X-CELLigence system and Transwell chamber on the tracer permeability testing both methods found that CTRP3 can inhibit HGHL-induced iBRB in vitro model of barrier function damage.(3)In vitro and in vivo experiments can be confirmed,CTRP3 AMPK / Occludin,Claudin5 pathway to inhibit DR-induced iBRB dysfunction.Therefore,this study provides an important theoretical basis for CTRP3 as a new idea and new target for clinical screening and intervention of diabetic retinopathy(DR),which is an important microcirculation disease of diabetes mellitus. |
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