| Part ? miR? 223? 3p reduces high glucose and high fat? induced endothelial cell injury in diabetic mice by regulating NLRP3 expression?Research background and objective?Cardiovascular disease in patients with diabetes begins with vascular endothelial cells.hyperglycemia and hyperlipidemia in body fluid are the main causes of vascular endothelial cell injury.There is evidence that vascular endothelial cell injury has occurred in diabetic patients in the early stage,so the intervention of vascular endothelial cell injury in diabetic patients is of great significance.Mi R-223-3p is also the focus of recent cardiovascular disease research.The activation of inflammatory body NLRP3 is associated with vascular dysfunction and proinflammatory phenotype associated with diabetes.According to the prediction of Target Scan targeting,miR-223-3p and NLRP3 have targeted binding sites.In order to confirm the conjecture that miR-223-3p can inhibit the injury of cardiac microvascular endothelial cells in diabetic patients by regulating the expression of NLRP3,we studied the diabetic mouse model and cardiac microvascular endothelial cells.To explore the expression of miR-223-3p and NLRP3 in diabetic mice induced by high glucose and high fat and the mechanism of cardiac endothelial cell injury.?Methods?1.Experimental group: 30 4-week-old C57BL/6J mice were randomly divided into three groups: control group(control group(10),model group(model group(10)and miR-223-3p-mimics group(10).The model group was fed with high-glucose and high-fat diet to establish diabetic model,and the mouse heart microvascular endothelial cell line(MCMECs)was purchased to construct vector by transient transfection.They were divided into normal group normal group(cultured in normal environment),model group model group(without transfection),blank vector group blank carrier group(transfected miR-NC),miR-223-3p-mimics group and miR-223-3p-inhibitor group.2.The targeting relationship between miR-223-3p and NLRP3 was predicted by targetscan,and the relationship between miR-223-3p and NLRP3 was detected by double luciferase reporter gene assay.3.RT-q PCR method was used to detect the expression of miR-223-3p and NLRP3 in cardiac tissue and cardiac microvascular endothelial cells.Western blot method was used to detect the expression of NLRP3 protein and apoptosis-related proteins Bax,caspase-3 and Bcl2 in cardiac tissue and cardiac microvascular endothelial cells.Annexin V-FITC/PI staining flow cytometry was used to detect the apoptosis level of MCMECs cells.4.Statistical analysis of data:RT-qPCR,Annexin V-FITC/PI staining,flow cytometry and Western blot were repeated for 3 times.SPSS 23.0 [Bizinsight(Beijing)Information Technology Co.,Ltd.] was used to statistically analyze the data.Graph Pad Prism 6(Graph Pad Software,Inc.)was used to plot the figures.Measurement data were expressed as the mean ± standard deviation(?x±s),and independent samples t-test was used for comparisons between two groups,whereas analysis of variance with LSD post hoc test were used for comparisons between multiple groups.P<0.05 was considered to indicate a statistically significant difference.?Results?1.The target relationship between miR-223-3p and NLRP3.According to the prediction of Target Scan,the base 406-412 of NLRP3 3 'UTR is the binding site of miR-223-3p.The direct interaction between miR-223-3p and NLRP3 was confirmed by double luciferase reporter gene detection.Co-transfection with miR-223-3p decreased the luciferase activity of the plasmid containing NLRP33'UTR-WT fragment(Fig.1A).The results of RT-q PCR after transfection showed that the expression of miR-223-3p in miR-223-3p mimics group was significantly higher than that in miR-NC group,while the expression of miR-223-3p in miR-223-3p inhibitor group was significantly lower than that in miR-NC group(Fig.1B).Western blot analysis showed that the expression of NLRP3 in MCMECs transfected with miR-223-3p-mimics was significantly decreased,while the expression of NLRP3 in MCMECs transfected with miR-223-3p-inhibitor was significantly increased(P < 0.05)(Fig.1C).2.The expression level of miR-223-3p,NLRP3 and apoptosis-related protein in heart tissue.Mice in miR-223-3p-mimics group were injected with neutral fat emulsion and miR-223-3p-mimics synthetic drug delivery system through tail vein.The results showed that the expression of miR-223-3p in control group and miR-223-3p mimic group was significantly higher than that in model group.However,the expression levels of m RNA and protein in NLRP3 were significantly decreased.The expression of Bax and caspase-3 protein decreased significantly,while the expression level of Bcl-2 protein increased significantly(P < 0.05)(Fig.2).3.Effect of miR-233-3p on NLRP3 expression and endothelial cell apoptosis.After cell modeling and HGHF culture,the expression of miR-223-3p in model group and blank vector group was significantly lower than that in normal group,while the m RNA and protein expression level of NLRP3 in model group and blank vector group was significantly higher than that in normal group.Compared with model group and blank vector group,the expression of miR-223-3p in miR-223-3p mimic group was significantly increased,while the expression of NLRP3 m RNA and protein was significantly decreased.The expression levels of NLRP3 m RNA and protein in miR-223-3p inhibitor group were significantly higher than those in model group and blank carrier group.The apoptosis rate in the model group and blank vector group was significantly higher than that in the normal group,and the expression level of Bax and caspase-3 protein was significantly higher than that in the normal group,while the expression level of Bcl-2 protein was significantly lower than that in the normal group.Compared with the model group and the blank vector group,the apoptosis rate and the expression of Bax and caspase-3 protein in the miR-223-3p mimic group were significantly lower than those in the model group and the blank vector group,the expression of Bcl-2 protein was significantly increased(P< 0.05).In addition,compared with the model group and blank vector group,the apoptosis rate,the expression of Bax and caspase-3 protein and the expression level of Bcl-2 protein in miR-223-3p inhibitor group were significantly higher than those in model group and blank vector group(P < 0.05)(Fig.3).?Conclusions?(1)double luciferase reporter gene assay verified the targeted prediction of Target Scan,that is,miR-223-3p targeted regulation of NLRP3 expression.(2)the expression of miR-223-3p was low in the heart of diabetic mice induced by high glucose and high fat(HGHF),while the expression of NLRP3 was high in the myocardium.MIR-223-3p can reduce the injury of mouse microvascular endothelial cells and inhibit endothelial cell apoptosis by regulating the expression of NLRP3.Part ? Association between serum thyroid hormone levels and diabetic peripheral neuropathy in euthyroid patients with type 2 diabetes mellitus?Background and objective? Diabetic peripheral neuropathy(DPN)is a common chronic complication of DM,with a prevalence rate of 50% in patients with T2 DM [1-2].Lu et al.[3] found that the proportion of DPN in hospitalized T2 DM patients is about 61.8%.It is of great significance to find DPN risk factors early and take effective prevention and treatment measures.Some studies [4-6] have shown that the level of thyroid hormone(TH)is related to the occurrence and development of microvascular complications in DM.This study retrospectively analyzed the correlation between serum thyroid hormone(TH)level and DPN in T2 DM patients with normal thyroid function,in order to provide a basis for exploring the effect of TH on DPN.?Methods? 1114 patients with T2 DM were divided into two groups: DPN group(DPN,n =415)and simple T2 DM group(T2DM).The indexes of the two groups were compared: age,course of diabetes,waist to hip ratio((WHR)),history of hypertension,fasting blood glucose(FPG),2 hours postprandial blood glucose(2h PG),fasting C peptide(FC-P),postprandial 2h C peptide(2h C-P),triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),free triiodothyronine(FT3),Free thyroxine(FT4),thyroid stimulating hormone(TSH),FT3/FT4,glycosylated hemoglobin(Hb A1c),glutamic pyruvic transaminase(ALT),glutamic oxaloacetic transaminase(AST),serum creatinine(Scr),smoking history(Smoking).The influencing factors of DPN were analyzed by Logistic regression,and the correlation between different serum free triiodothyronine(FT3)levels and the risk of DPN was analyzed.The value of FT3 in the diagnosis of DPN was analyzed by ROC.?Results? Proportion of men in the DPN group(69.4% vs 50.2%),smoking rate(38.3% vs28.5%),duration of diabetes [91(32-162)vs 69(15-133)months],WHR [0.94(0.90-0.98)vs0.93(0.89-0.97)],FPG[8.80(6.90-11.53)vs8.40(6.59-10.53)mmol/L],2h P G[19.06±5.08vs18.02±5.73mmol/L],Hb A1c[9.7(7.6-11.5)vs8.60(6.90-10.50)%]and Cr[69.1(54.8-78.6)vs64.9(54.7-75.5)umol/L] were higher than those in the non-DPN group(P<0.05).DPN group FC-P [1.37(0.82-2.02)vs 1.59(1.06-2.24)ng/ml],2h C-P [3.11(1.88-4.90)vs 4.37(2.70-6.50)ng/ml],ALT[ 19.8(14.2-30.0)vs 21.5(15.8-30.5)U/L],AST [18.4(15.1-23.7)vs 19.3(15.8-24.4)U/L],FT3 [4.52±0.64vs4.67±0.58 pmol/L] and FT3/FT4 [0.270(0.239-0.308)vs 0.277(0.247-0.307)] were lower than those in the non-DPN group(P<0.05).Logistic regression analysis showed that FT3 was independently negatively correlated with the risk of DPN after adjusting for gender,disease duration,Hb A1 c,and 2h C-P(OR=0.65,P<0.01).According to the serum FT3 level,patients were quartile grouped(Q1,Q2,Q3,Q4).Compared with Q1,the risk of DPN in Q2(OR=0.580),Q3(OR=0.560)and Q4(OR=0.473)patients Lower(P<0.01).FT3 receiver operating characteristic(ROC)area under the curve(AUC=0.568;95%CI 0.532~0.603;P<0.01).?Conclusion? There is an independent and negative association between serum FT3 and the prevalence of DPN in euthyroid patients with T2 DM.FT3 predicts that the risk of DPN is not high. |
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