Mechanism Of Action Of MiR-21 In Diabetic Myocardial Fibrosis

Diabetes leads to microvascular,macrovascular diseases and multiple target organ damage.In the absence of hypertension and coronary artery disease,diabetic patients also have myocardial damage,that is,diabetic cardiomyopathy.At first,there are slight structural and functional(diastolic relaxation)abnormalities,which develop into serious diastolic heart and/or systolic dysfunction heart failure.Diabetic cardiomyopathy involves major pathological changes such as myocardial fibrosis,cardiac vascular disease and cardiac cell death.Cardiac interstitial fibrosis and perivascular fibrosis are important factors for the progressive development of diabetic cardiomyopathy.Myocardial fibroblasts(CFs)transdifferentiation and Endothelial-toMesenchymal Transition(EndMT)are the main sources of myocardial fibrosis;miRNAs,especially miR-21,play important roles in fibrosis of lung,kidney,heart and other organs,and are related to diabetic renal fibrosis.The objective of this study is to study the mechanism of miR-21 in diabetic myocardial fibrosis.The model of Type 1 Diabetes Mellitus(T1DM)was constructed by intraperitoneal injection of Streptozocin(STZ)(50 mg/kg/d)into male C57BL/6 mice for 8-12 weeks.The model was injected 5 days,once a day.Fasting for 12 hours before injection,only water was given.One week later,the fasting blood glucose was measured,and the model was constructed successfully when the content was ≥ 12 mmol/L.The successful T1 DM mice were randomly divided into two groups(n = 8/group): STZ group and STZ + inhibitor group.They were fed with normal diet for 4 weeks,and injected with inhibitor NC and miR-21 inhibitor respectively.The blank control group(C57BL/6,n = 8)were injected with inhibitor NC at 4 weeks.At 12 weeks,echocardiography was used to detect the changes of cardiac function in each group;qRT-PCR was used to detect the mRNA level of miR-21;Masson and Sirius red staining were used to trace the process of cardiac interstitial fibrosis and peripheral vascular fibrosis;immunohistochemistry was used to detect SMAD7 and p-p65;tissue immunofluorescence co-staining was used to detect the fluorescence changes of α-SMA and CD31;Western blotting was used to detect the changes of SMAD7,CD31,α-SMA,Collagen I,Fibronectin,Collagen III and other proteins at translation level.At the cell level,Human Umbilical Vein Endothelial Cells(HUVECs)and Human Embryonic Kidney 293(HEK293)were used as tool cells.HUVECs were stimulated by high concentration of D-glucose to simulate hyperglycemia in T1 DM mice,and L-glucose was used in the control group;HEK293 was used in luciferase experiment to verify the regulation of p65 on miR-21 upstream or the targeting of miR-21 on SMAD7 downstream.The main experimental techniques involved in cell level include qRT-PCR,Western Blotting,cell immunofluorescence,luciferase,etc.In vivo studies showed that,(1)the transcription level of miR-21 was up-regulated by 2.24 times(P<0.01);T1DM mice have disordered heart function and the Left Ventricular Ejection Fraction(LVEF)were decreased significantly(P<0.01);the collagen deposition was increased(interstitial fibrosis,P<0.01)and the perivascular fibrosis was enhanced(P<0.01)in T1 DM mice.The results indicated that injection of miR-21 inhibitor improved the cardiac function,partially recovered the LVEF(P<0.01),reduced collagen deposition(P<0.01),and alleviated interstitial fibrosis(P<0.01);(2)the expression of CD31 and α-SMA in the cardiac tissues of T1 DM mice decreased(P<0.01),suggesting that EndMT may be involved in the process of diabetic myocardial fibrosis;injection of miR-21 inhibitor partially inhibited the conversion process;the mechanism of miR-21 promoting diabetic myocardial fibrosis may be through down-regulating SMAD7 and activating p-SMAD2 and p-SMAD3.In vitro cytological studies confirmed that,(1)NF-κB(p65)regulated the expression of miR-21,and high glucose(HG)upregulated the mRNA level of miR-21 in HUVECs by activating NF-κB;(2)SMAD7 is a direct target of miR-21 in HUVECs,and miR-21 inhibitor inhibited the EndMT process induced by HG via regulating SMADs signal pathway.This thesis pointed out that inhibition of miR-21 prevented and treated myocardial fibrosis and cardiac function change caused by diabetes via inhibiting EndMT,slowing down the process of cardiac interstitial and perivascular fibrosis caused by T1 DM,and the regulation of NF-κB/miR-21/SMAD7 signaling pathway may be the main mechanism involved.At the same time,inhibiting miR-21 improved the fibrosis process of many organs including heart,lung and kidney,suggesting that miR-21 may be an important target for the prevention and treatment of T1 DM mediated multiple organ fibrosis.