Study Of Liraglutide On Diabetic Myocardial Fibrosis And The Mechanism Of P4h?1 Expression

Chapter lEffects of liraglutide on myocardial fibrosis and myocardial function indiabetic rats1 BackgroundDiabetic cardiomyopathy refers to occur in people with diabetes,cannot use heart disease,coronary atherosclerosis,hypertensive heart disease,heart valve disease to definite myocardial disease.The disease on the basis of metabolic disorders and microvascular lesions caused by myocardial widely focal necrosis,subclinical cardiac anomalies,eventually progress for cardiogenic shock,myocardial infarction,heart failure,arrhythmia,severe patients even sudden death.Therefore,the search for effective drugs to treat diabetic cardiomyopathy has important scientific research value and clinical significance.GLP-1 is the expression product of glucagon genein intestinal L cells and neurons,mainly containing two bioactive forms,GLP-1(7-37)and GLP-1(7-36)amide.Studies showed that GLP-1,as a new diabetes drug,hasglucose-dependent insulin secretion function,can promote ?cell proliferation and differentiation,make glucagon secretion inhibited,loss of appetite and ingestion,and gastric emptying delayed,and recover the "enterotrophin effect" in patients with type 2 diabetes mellitus.The mechanism is that GLP-1 combined withGLP-1 receptors on the surface of P cells in the pancreas cells in the pancreas,stimulate G protein and activate adenosine cyclase(AC),and then form cyclic phosphate(cAMP).The increased cAMP activat protein kinase A(PKA)and guanine nucleotide exchange factor ?(cAMP-GEF ?),were eventually involved in the process of insulin secretion by downstream molecules.Research has also proved that GLP-1,serves as a kind of incretin,has a protective effect on multiple organ and cardiovascular system,and studies have found that GLP-1 can affect ? type and ? type collagen expressions,but its specific mechanism for diabetic cardiomyopathy is not yet clear.Lilalutin is the latest analogue of GLP-1,which can reduce blood sugar by promoting insulin synthesis and secretion,inhibiting glucagon secretion,and then suppress appetite and delay gastric emptying.In this study,we will further explore the effects of lilalutin on myocardial fibrosis and myocardial function in diabetic rats.2 PurposeTo investigate the effects of liraglutide on diabetic cardiomyopathy rat weight,blood pressure,heart rate,blood sugar.electrocardiogram and lipid metabolism related indexes,and the influences of liraglutide on diabetic cardiomyopathy rat COL-1,COL-3,MMP-1,MMP-9 and P4Hal expressions.3 MethodsThe Wistar rats were randomly divided into three groups:the control group(n=10);Diabetes group(n=20);Diabetes+liraglutide group(n=20).In the process of experiments,we measured the rat weight.blood pressure,heart rate,and blood sugar.We also extracted 1.5 ml blood,and separated serum.Automatic biochemical analyzer was used to determine the levels of blood total cholesterol,triglyceride,low-density lipoprotein cholesterol,and high-density lipoprotein cholesterol.After the experiment,the weight of the heart was measured,and the ratio of heart weight/weight[HW/BW(mg/g)]was calculated.Serum NEFA,hydroxyproline and serum insulin levels were measured by enzyme-linked immunoassay(ELISA)assay.Electrocardiogram(ECG)was used to observe the electrical activity of rats in each group.Echocardiography was used to detect the left ventricular systolic inner diameter(LVIDs),left ventricular diastolic(LVIDd),ejection fraction(LVEF),and left ventricular shortening(FS).Catheter method to detect and analyze the cardiovascular parameters of left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP)and left ventricular pressure maximum rising rate(+dp/dt),left ventricular pressure(dp/dt),the largest decline rate and calculate the left ventricular relaxation time constant Tau = P/(dp/dt).The collagen content of myocardial tissue in diabetic cardiomyopathy was determined by Masson staining and scarlet red staining.The cardiac lipid deposition in diabetic cardiomyopathy rats was detected by Oil Red 0 staining.The ultrastructure of diabetic cardiomyopathy was detected by transmission electron microscopy.Morphological changes of diabetic cardiomyopathy rats were detected by HE staining;The mRNA expression level of P4Hal was detected by qRT-PCR assay,the expression levels of COL-1,COL-3,mmp-1,mmp-9 and P4Hal were measured by immunohistochemistry and Western blot assays.4 ResultsLiraglutide reduced HW/BW ratio,blood glucose and heart rate in diabetic rats.Liraglutide reduced the left ventricular LVEDP and Tau in diabetic cardiomyopathy rats,and increased the left ventricle +dp/dt in diabetic cardiomyopathy,indicating that liraglutide improved left ventricular function in diabetic cardiomyopathy.Liraglutide reduced the total cholesterol,triglycerides,LDL cholesterol and HDL cholesterol in diabetic cardiomyopathy.Liraglutide increased cardiac ultrasonography(LVPWd,LVEF and FS)in diabetic cardiomyopathy,indicating that liraglutide improved cardiac ultrasound function in diabetic cardiomyopathy.In addition,we showed that liraglutide decreased serum NEFA and hydroxyproline content in diabetic cardiomyopathy and elevated fasting serum insulin levels.Liraglutide can improve the left ventricular structure of rat heart and reduce the collagen content of myocardial tissue and heart lipid deposition.Liraglutide reduced myocardial hydroxyproline content,and the expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4H?1 in diabetic cardiomyopathy rats.It is suggested that liraglutide may improve the myocardial function by inhibiting the expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4H?1.5 ConclusionLiraglutide reduced HW/BW ratio,blood glucose and heart rate in diabetic rats,and improved left ventricular function in diabetic cardiomyopathy,lipid metabolism,the left ventricular structure and cardiac ultrasonography in diabetic cardiomyopathy rats.In addition,liraglutide inhibited COL-1,COL-3,MMP-1,MMP-9 and P4H?1 expressions.It is suggested that liraglutide can improve myocardial function of diabetic cardiomyopathy.In chapter 2Liraglutide suppresses P4H?1 expression inhyperglycemic-stimulated fibroblast via CD36-JNK-MAPK-AP1 pathway1 BackgroundIn patients with ype 1 and type 2diabetes,cardiovascular complications are the main cause of morbidity and mortality.Although the primary reasonesof morbidity and mortality are an increased incidence of atherosclerosis and coronary artery disease,many patients suffer from clinically significant ventricular dysfunction even in the absence of these conditions.This ventricular dysfunction has been termed diabetic cardiomyopathy and is defined as myocardial left ventricular(LV)dysfunction independent of atherosclerosis and coronary artery disease.Diabetic cardiomyopathy is a major cause of heart failure in people with diabetes.Despite the lack of coronary artery disease,a strong association exists between diabetic cardiomyopathy and the presence of microvascular complications.For example,an impaired angiogenic response,abnormal vascular remodeling,endothelial dysfunction and inflammation are related to the myocardial apoptosis,hypertrophy and fibrosis in diabetic cardiomyopathy.However.the mechanisms of diabetic cardiomyopathy have not been clearly defined.P4Hal is a key enzyme in the collagen synthesis process,and can makethe proline on collagenhydroxylated.It plays an important role in the synthesis of all known types of collagen.Studies have shown that P4H 1 can slow the pathological process of atherosclerotic plaque.However,the mechanism of P4Hal for diabetic cardiomyopathy is not clear.Our preliminary results showed that the promoter region of P4Ha1 contained the binding site of AP-1,and JNK could participate in the regulation of P4Ha1.In addition,studies have found that liraglutide can regulate CD36.Therefore,we hypothesized that liraglutide may regulate P4Ha1 to reduce myocardial fibrosis by CD36-JNK-AP1 pathway,thereby plays a protective role in the heart.2 PurposeTo explore the Effect of liraglutide on P4Ha1 expression,and the influences of liraglutide on cell proliferation,cycle,apoptosis,migration and invasion ability of high-glucose induced fibroblastsby regulating the AP-1/P4Ha1 signal transduction pathway.3 MethodsDifferent concentration of glucose(0,5,10,25,50mmol/L)were used to treat myocardial fibroblasts(CFs)for 24 hrs,CCK8 assay was used to detect cell proliferation activity.qRT-PCR assay was performed to measure the mRNA expression level of P4Ha1;Western blot assay was used to analyzed the protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4Hal.Different concentration of liraglutide(0,10,50,100nmol/L)were used to treat myocardial fibroblasts(CFs)for 1h,25 mmol/L glucose stimulated myocardial fibroblast 48hrs,CCK8 assay was used to detect cell proliferation activity.qRT-PCR assay was performed to measure the mRNA expression level of P4H?1;Western blot assay was used to analyzed the protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4H?1.25 mmol/L glucose and 50 nmol/L liraglutide were used to treat human myocardium fibroblasts.Cell proliferation ability was measured by EDU assay;The cell cycle and cell apoptosis rate were detected by flow cytometry;The cell migration ability was accessed by Wound Healing assay;The cell invasion ability was detected by Transwell assay.siP4H?1 sequence was obtained and transfected into myocardial fibroblasts,qRT-PCR and Western blot assays were used to analyzed the expression level of P4Hal and screenthe effective sequence of siP4H?1.And then myocardial fibroblasts were treated with siP4H?1 andliraglutide,the expression level of P4H?1 was detected by qRT-PCR and Western blot assays;Cell proliferation ability was measured by EDU assay;The cell cycle and cell apoptosis rate were detected by flow cytometry;The cell migration ability was accessed by Wound Healing assay;The cell invasion ability was detected by Transwell assay.Myocardial fibroblasts were treated with liraglutide and AP-1 inhibitor(20 mmol/L curcumin),and then were stimulated by high glucose.EMSA assay was used to detect the changes in the DNA binding activity of AP-1 and P4H?1.Immunofluorescence assay was performed to detect the subcellular distribution of AP-1;qRT-PCR assay was performed to measure the mRNA expression level of P4H?1;Western blot assay was used to analyzed the protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4H?1.Myocardial fibroblasts were treated with liraglutide and JNK inhibitor(50 mumol/L SP600125),and then were stimulated by high glucose.qRT-PCR assay was performed to measure the mRNA expression level of P4H?1;Western blot assay was used to analyzed the protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4H?1.Myocardial fibroblasts were treated with liraglutide and CD36 inhibitors(1,mol/L SSO),and then were stimulated by high glucose.EMSA assay was used to detect the changes in the DNA binding activity of AP-1 and P4H?1;qRT-PCR assay was performed to measure the mRNA expression level of P4Ha1;Western blot assay was used to analyzed the protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4Hal.4 ResultsDifferent concentration of glucose(0,5,10,25,50mmol/L)promoted myocardial fibroblasts proliferation ability,increasedthe mRNA expression level of P4Hal,upregulatedthe protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4Ha1,and the impact on myocardial fibroblasts was greatest when the concentration of glucose was 25mmol/L.25mmol/L glucose significantly improved myocardial fibroblasts proliferation ability,increased the mRNA expression level of P4Hal,upregulatedthe protein expression levels of COL-1,COL-3,MMP-1.MMP-9 and P4Ha1,and the impact on myocardial fibroblasts was greatest when myocardial fibroblasts were treated with 25mmol/L glucose for 48 hrs.Different concentration of liraglutide(0,10,50,100nmol/L)inhibited myocardial fibroblasts proliferation ability,decreased the mRNA expression level of P4Hal,downregulatedthe protein expression levels of COL-1,COL-3,MMP-1,MMP-9 and P4Hal,and the impact on myocardial fibroblasts was greatest when the concentration of liraglutide was 50nmol/L.Myocardial fibroblasts were treated with 25mmol/L glucose for 48 hrs and 50nmol/Lliraglutide foe 1 h,the results showed liraglutide inhibited the proliferation,migration and invasion abilities,promoted cell cycle arrest and apoptosis ability of myocardial fibroblasts induced by high glucose.Myocardial fibroblasts were transfected with P4Hal siRNAs,the results from qRT-PCR and Western blot assays indicated that theinterference effect was best of siP4Hal-523.P4Hal siRNAs and liraglutide were used to treat myocardial fibroblasts induced by high glucose,and the results revealed that silence of P4Hal or liraglutidedown-regulatedthe expression level of P4Ha1,and silence of P4Hal inhibited the expression level of P4Hal mediated by liraglutide.Silence of P4Hal or liraglutide inhibited cell proliferation,migration and invasion abilities,and silence of P4Ha1 inhibited cell proliferation,migration and invasion abilities mediated by liraglutide.Silence of P4Ha1 or liraglutidepromoted cell cycle arrest and apoptosis ability,and silence of P4Halpromoted cell cycle arrest and apoptosis ability mediated by liraglutide.Myocardial fibroblasts induced by high glucose were treated with liraglutide and 20 pmol/L AP-1 inhibitor(Curcumin).The results indicated that liraglutide enhancedthe DNA binding activity of AP-1 in the process of P4Hal expression,AP-1 inhibitors reduced the DNA binding activity of AP-1 in the process of P4Hal expression,liraglutide enhanced the DNA binding activity of AP-1 in the process of P4Hal expression mediated by AP-1 inhibitors.Liraglutide promotedAP-1 expression,AP-1 inhibitor inhibited AP-1 expression,and liraglutide promotedAP-1 expression mediated by AP-1 inhibitors.Liraglutide inhibited P4H?1 expression,promoted CD36 and p-JNK expressions,AP-1 inhibitors promoted P4H?1 expression,inhibited CD36 and p-JNK expressions,and liraglutide inhibited P4Hal expression mediated by AP-1 inhibitors,promoted CD36 and p-JNK expressions mediated by AP-1 inhibitors.Myocardial fibroblasts induced by high glucose were treated with liraglutide and 50 ?mol/L JNK inhibitor(SP600125).The results indicated that liraglutide inhibited P4Hal expression,promoted CD36 and p-JNK expressions,JNKinhibitors p.romoted P4Hal expression,inhibited CD36 and p-JNK expressions,and liraglutide inhibited P4Hal expression mediated by JNK inhibitors,promoted CD36 and p-JNK expressions mediated by JNK inhibitors.Myocardial fibroblasts induced by high glucose were treated with liraglutide and 1 ?mol/L CD36 inhibitor(SSO).The results indicated that liraglutide enhancedthe DNA binding activity of AP-1 in the process of P4Hal expression,CD36 inhibitors reduced the DNA binding activity of AP-1 in the process of P4Ha1 expression,liraglutide enhanced the DNA binding activity of AP-1 in the process of P4Hal expression mediated by CD361 inhibitors.Liraglutide inhibited P4Hal expression,promoted CD36 and p-JNK expressions,CD36 inhibitors promoted P4Hal expression,inhibited CD36 and p-JNK expressions,and liraglutide inhibited P4Hal expression mediated by CD36 inhibitors,promoted CD36 and p-JNK expressions mediated by CD36 inhibitors.5 ConclusionGlucose promoted myocardial fibroblast proliferation and P4H?1,collagen 1,collagen 3,MMP1 and MMP9 expressions.Liraglutide inhibited myocardial fibroblast proliferation and P4Ha1,collagen 1,collagen 3,MMP1 and MMP9 expressions.Silence of P4Ha1 inhibited cell proliferation,migration and invasion abilities,and promoted cell cycle arrest and apoptosis ability of myocardial fibroblasts mediatedliraglutide.Liraglutide enhanced the binding activity of P4H?1 and AP-1 DNA,liraglutide inhibited P4H?1 expression mediated by AP-1 inhibitor,and promotedAP-1,CD36 and p-JNK expressions mediated by AP-1 inhibitor.Liraglutide inhibited P4Ha1 expression mediated by JNK inhibitor,and promotedAP-1 CD36 and p-JNK expressions mediated by JNK inhibitor.Liraglutide inhibited P4Hal expression mediated by CD36 inhibitor,and promotedAP-1.CD36 and p-JNK expressions mediated by CD36 inhibitor.Therefore,liraglutideaffectshigh glucose-induced fibroblasts function by P4Hal mediated AP-1,JNK and CD36.