| Alcoholic cardiomyopathy(ACM)is a kind of cardiomyopathy that is caused by long-term heavy drinking,mainly characterized by left ventricular dilatation,decreased ejection fraction and arrhythmia.Clinical and pathological data reveal:myocardial tissue in alcoholic cardiomyopathy shows swelling of cardiomyocytes,accumulation of glycogen and lipids,changing in sarcoplasmic reticulum and mitochondrial structure,reduction in the number of myocardial fiber streaks.Studies also show a focal collection of intramural thrombus and swollen cardiomyocytes.Although the specific pathogenesis of alcoholic cardiomyopathy is not well understood,recent studies have shown that as a key regulator of oxidative stress,the renin-angiotensin system(RAS)plays an important role in the development of ACM.Compared with the classical circulating RAS,myocardial tissue local RAS is more likely to cause people's enthusiasm.As a new member of local tissue RAS,the(pro)renin receptor(PRR)has attracted much attention due to its many biological functions.(Pro)renin receptor can bind to renin or prorenin(collectively referred to as:(pro)renin).On the one hand,this binding can significantly increase the biological activity of(pro)renin and increase the conversion efficiency of angiotensinogen(Angiotensinogen,AGT)to angiotensin I(Ang I),further strengthen the Ang II dependent effects;on the other hand,this binding can cause changes in the spatial conformation of PRR,by recruiting transcription factors promyelocytic leukemia zinc finger protein(PLZF)and transfer extracellular signals into the intracellular,which is also known as the Ang II independent pathways.Studies have reported that in the endomyocardial biopsy samples of patients with dilated cardiomyopathy,the expression level of PRR gene is significantly increased,and correlated with the severity of the disease.However,the role and mechanism of PRR in alcoholic cardiomyopathy has not been reported.In the present study,we mainly used animal experiments and in vitro cell experiments to study the expression of PRR in myocardial tissue of rats with alcoholic cardiomyopathy and to study the role of PRR in the pathological process of alcoholic cardiomyopathy,furthermore,to clarify the mechanisms of PRR in the pathogenesis of alcoholic cardiomyopathy.Our research may provide new insights into the mechanisms by which PRR involves in ACM and provide the possibility that PRR may be a potential target for the treatment of ACM.Our experiments are mainly divided into the following three parts:Part One:Establish An Animal Model of Alcoholic Cardiomyopathy in RatsBackgroundWith the development of social economy and the richness of material life,the use of alcoholic beverages is also increasing.The accumulation of toxic substances such as acetaldehyde,a metabolite of alcohol,has damaging effects on multiple organs within the body and the related health problems have attracted increasing attention.The most prominent disease is alcoholic cardiomyopathy(ACM),which causes clinical symptoms such as dilatation,arrhythmia and cardiac insufficiency due to long-term heavy drinking.A large number of clinical and pathological data reveal:myocardial tissue in alcoholic cardiomyopathy shows swelling of cardiomyocytes,accumulation of glycogen and lipids,changing in sarcoplasmic reticulum and mitochondrial structure,reduction in the number of myocardial fiber streaks.Studies also show a focal collection of intramural thrombus and swollen cardiomyocytes.Previous studies have showed that the pathophysiological mechanisms of alcoholic cardiomyopathy involve:oxidative stress,cell death,mitochondrial dysfunction and abnormal activation of neuroendocrine system,but the specific mechanism remains unclear.Therefore,the clinical diagnosis and treatment of alcoholic cardiomyopathy is mainly based on epidemiological observation.The treatment strategy is more empirical treatment or symptomatic treatment.However,the treatment effects are not significant.In the present study,rats received alcohol gavage to establish an animal model of alcoholic cardiomyopathy.The main pathological damage of myocardial tissue was observed.The potential pathological mechanisms of alcoholic cardiomyopathy were studied.We hope to provide new targets for the clinical treatment of alcoholic cardiomyopathy through our study.Objective1.To establish a rat model of myocardial injury by alcohol gavage and observe the effects of alcohol gavage on rats.2.To evaluate the feasibility of alcohol gavage for establishment of alcoholic cardiomyopathy model through the observation of the general state,the detection of cardiac function and the pathological damage of the heart.3.To study the possible pathophysiological mechanisms of alcoholic cardiomyopathy.Methods1.Animal model and groupsThirty Wistar rats(200±15g)were randomly divided into the control group(n=15)and alcohol group(n=15).The rats in the alcohol group received alcohol gavage,the dose of alcohol accounted for 9%of total diet(v/v).The rats in the control group received normal saline gavage.After 8 weeks,the serum alcohol concentration of the model group reached 237±42 mg/100mL.Echocardiography showed that the heart chamber was enlarged and the systolic function was reduced as a successful model.At the end of 12 week,all rats were anesthetized and killed.2.Cardiac function assessmentRats in all groups were anesthetized by intraperitoneal injection of pentobarbital(60mg/kg)and cardiac function was measured using a VEVO770 imaging system.Two-dimensional ultrasound and M-mode ultrasound were used to evaluate cardiac function.The parameters including left ventricular ejection fraction(LVEF),shortened fraction(FS),left ventricular end-diastolic diameter(LVEDD),and left ventricle end-systolic diameter(LVESD)were recorded.The E/A values were measured to assess diastolic function.3.Histology and immunohistochemistryAfter myocardial tissues were immersed in 4%paraformaldehyde,all tissues were embedded in paraffin,and cut into 4.5?m sections.Myocardial injury was assessed by haematoxylin and eosin(HE)staining.Myocardial fibrosis was assessed by Masson's trichrome staining and Sirius red staining.Immunohistochemical staining was used to evaluate the protein expression of collagen I,NADPH oxidase 4,IL-1?,IL-6 and TNF-a in myocardial tissue.TUNEL staining was used to evaluate the apoptosis of cardiomyocyte.Results1.General status of rats in two groups after 12 weeks of alcohol gavageThe rats in the control group were lively and active,the hair color was bright and smooth,the diet was good,the mental state was good and the resistance was obvious when grasping.Compared with the control group,the hair of the alcohol group rats was dull,the diet was reduced.The mental state is not good and it was in a state of lethargy.Even some rats had coma.When they were grasped,the body was soft and had no obvious resistance and there was no obvious reaction to external stimulation.Two rats died in the alcohol group.At the end of the experiment,the body weight(g)and the heart weight(mg)of each group of rats was weighed,the value of heart weight/body weight(mg/g)was calculated.The results showed that compared with the control group,the body weight of the alcohol group significantly decreased(P<0.01).The value of heart weight/body weight(mg/g)in the alcohol group was significantly higher(P<0.01).The heart rates of rats in the alcohol group also increased(P<0.05).2.12 weeks of alcohol gavage deteriorates cardiac functionCompared with the control group,rats in the alcohol group showed different degrees of cardiac insufficiency.LVEDD and LVESD were significantly higher than those in the control group(P<0.01).Compared with the control group,LVEF and FS in the alcohol group were reduced(P<0.01),which indicated that the left ventricular dilatation and the systolic function were impaired in the alcohol group.The E/A value of rats in the alcohol group also decreased(P<0.01),indicating that the diastolic function of rats was also affected,and the myocardial compliance was decreased.3.12 weeks of alcohol gavage aggravates myocardial fiber injuryThe cardiomyocytes of control group were normal in size and uniform in morphology.The myocardial fibers were arranged neatly,the distribution was even and dense,the myocardial fiber transverse striation mechanism was clear and no interstitial infiltration of other inflammatory cells was observed.In contrast,the cardiomyocytes of alcohol group were different in size and uneven in morphology.Cardiomyocytes edema,lysis and necrosis were observed and the arrangement was disordered.Some of the cardiomyocytes showed vacuolization changes accompanied by infiltration of a few interstitial inflammatory cells.4.12 weeks of alcohol gavage increases myocardial fibrosisA small amount of collagen was scattered in the heart of the control group.Compared with the control group,the myocardial interstitial collagen content in the alcohol group increased(P<0.01),the distribution was more diffuse and the collagen deposition around the vessel wall was more obvious.Immunohistochemical staining of collagen I showed that compared with the control group,the myocardial interstitial collagen I content in the alcohol group increased(P<0.01)and the whole myocardial interstitial collagen I diffused.5.12 weeks of alcohol gavage increases myocardial oxidative stressImmunohistochemical staining showed that NOX4 protein was slightly expressed in the control group.Compared with the control group,the expression of NOX4 in the myocardial tissue of the alcohol group was significantly increased(P<0.01).Meanwhile,the expressions of protein nitration products(3-Nitrotyrosine,3-NT)and lipid peroxidation products(4-Hydroxynonenal,4-HNE)in the myocardial tissue of alcohol group were increased(P<0.01).6.12 weeks of alcohol gavage exacerbates myocardial inflammation responsesIn general,the expression levels of inflammatory factors in myocardial tissue are very low,and are only secreted by a few interstitial infiltrating inflammatory cells.The results of immunohistochemical staining showed that the expression levels of inflammatory cytokines(IL-1?,IL-6,TNF-?)in the alcohol group were significantly increased compared with the control group(P<0.01).7.12 weeks of alcohol gavage increases the apoptosis of cardiomyocytesAt the end of the experiment,TUNEL staining was used as apoptotic cell detection.TUNEL staining was positive and combined with nucleus shrinkage and other morphological features were identified as apoptotic cells.The results showed that compared with the control group,the number of cardiomyocyte apoptosis in the alcohol group increased(P<0.01).Conclusion1.A rat model of alcoholic cardiomyopathy can be successfully established by alcohol gavage.2.Alcoholic cardiomyopathy in rats mainly involves myocardial cell injury,apoptosis,oxidative stress,inflammatory response and increased interstitial fibrosis in myocardial tissue.Part Two:The Effects of(Pro)renin Receptor on Myocardial Remodeling and Cardiac Dysfunction in Alcoholic CardiomyopathyBackgroundAlcoholic cardiomyopathy(ACM)is a type of cardiomyopathy that is caused by long-term heavy drinking.Long-term alcohol abuse causes the accumulation of myocardial toxic substances such as alcohol and acetaldehyde,and a series of clinical symptoms characterized by left ventricular dilatation,decreased ejection fraction,and arrhythmia.Numerous studies have shown that long-term heavy drinking causes accumulation of acetylated fatty acids in cardiomyocytes,mitochondrial dysfunction,oxidative stress,apoptosis,and cardiac remodeling eventually evolve into refractory heart failure,which brings a heavy burden to our health care system.Although the specific pathogenesis of alcoholic cardiomyopathy is not well understood,recent studies have shown that as a key regulator of oxidative stress,the renin-angiotensin system(RAS)plays an important role in the development of ACM.Compared with the classic circulating RAS,myocardial tissue local RAS is more likely to cause people's enthusiasm.As a new member of local RAS,the(pro)renin receptor(PRR)has attracted much attention due to its biological functions.(Pro)renin receptor can bind to renin or prorenin(collectively referred to as:(pro)renin).On the one hand,this binding can significantly increase the biological activity of(pro)renin and increase the conversion efficiency of angiotensinogen(Angiotensinogen,AGT)to angiotensin I(Ang ?),further strengthen the Ang ?-dependent pathways,on the other hand,this binding can cause changes in the spatial conformation of PRR,by recruiting transcription factors promyelocytic leukemia zinc finger protein(PLZF)or self-phosphorylation,transducting extracellular signals into the intracellular,which is known as the Ang ? independent pathways.Studies have reported that in the endomyocardial biopsy samples of patients with dilated cardiomyopathy,the expression level of PRR gene is significantly increased,and correlated with the severity of the disease.However,the role and mechanism of PRR in alcoholic cardiomyopathy has not been reported.Therefore,this part of the experiment was based on the animal model of alcoholic cardiomyopathy established in the first part,to observe the expression of PRR in myocardial tissue of alcoholic cardiomyopathy,and to study its effects on myocardial injury and remodeling.Objective1.To establish a rat model of alcoholic cardiomyopathy and observe the expression of PRR in myocardial tissue of ACM.2.To investigate the effects of PRR overexpression and PRR silencing on myocardial remodeling and cardiac function in ACM.3.To explore the mechanism of PRR gene on myocardial remodeling and cardiac function in alcoholic cardiomyopathyMethods1.Construction of rat PRR gene overexpression and PRR gene silencing adenoviral vectorThe recombinant adenovirus overexpression vector containing rat PRR gene was constructed according to the rat PRR gene sequence in GeneBank.Following the principle of gene silencing,three pairs of shRNAs sequences targeting rat PRR gene were designed and synthesized.The sequence with the highest silencing efficiency was measured by RT-PCR and Western blot,and the adenovirus silencing vector was constructed for subsequent experiments.2.Animal model and groupsNinety Wistar rats(200±15g)were randomly divided into the control group(Control,n=15)and the alcohol group(Alcohol,n=75).The rats in the alcohol group were given alcohol accounting for 9%of total diet.The rats in the control group were given normal saline.After 8 weeks,the serum alcohol concentration in the alcohol group reached 237±42 mg/100mL.Echocardiography showed that the heart chamber was enlarged and the systolic function was reduced.Rtas in the alcohol group was further divided into:alcoholic cardiomyopathy group(ACM,n=15),EGFP group(EGFP,n=15),Scramble-shRNA group(Scr-shRNA,n=15),PRR group(PRR group,n=15)and PRR-shRNA group(PRR-shRNA,n=15).Rats of each group were injected with adenovirus 1×109 v.g(dissolved in 100 ?l normal saline)via the tail vein to express EGFP,Scr-shRNA,PRR and PRR-shRNA for subsequent experiments.3.Cardiac function measurementCardiac function was measured using the VEVO770 imaging system 4 weeks after virus injection.The left ventricular function parameters such as:left ventricular ejection fraction(LVEF),fractional shortening(FS),left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD)and E/A values were measured.4.Histology and immunohistochemistryAfter myocardial tissues were immersed in 4%paraformaldehyde,all tissues were embedded in paraffin,and cut into 4.5?m sections.Myocardial injury was assessed by haematoxylin and eosin(HE)staining.Myocardial fibrosis was assessed by Masson's trichrome.Immunohistochemical staining was used to evaluate the protein expression of collagen ?,collagen ?,connective tissue growth factor(CTGF),NADPH oxidase 4,IL-1? and IL-6 in myocardial tissue.Toluidine blue staining was used to measure the degranulation of mast cells5.Western blotTotal protein from the myocardium was extracted.Protein was separated by SDS-PAGE gel,and transferred to polyvinylidene difluoride membranes.The membranes were incubated with anti-rat PRR?ERK1/2?p-ERK1/2?NOX4?TGF-?.Protein levels were normalized to GAPDH.6.Measurement of oxidative stress in myocardiumFresh myocardium was embedded with OCT.Frozen sections were cut and incubated with DHE(10?M)at 37 C for 30 min in the dark.The fluorescent images of tissue sections were observed by fluorescence microscope.7.Real time PCRTotal RNA was extracted from myocardial tissue.The mRNA expression level of PRR was measured by RT-PCR.Results1.The protein expression of PRR in myocardial tissue gradually increases with the development of alcoholic cardiomyopathyWe observed the protein expression of PRR in myocardial tissue of alcoholic cardiomyopathy at different time points.Immunohistochemical staining showed that compared with the control group,protein expression of PRR in myocardial tissue was significantly increased at 4 weeks after alcohol gavage(P<0.05).With the development of alcoholic cardiomyopathy,the protein expression of PRR in myocardial tissue could be maintained up to 12 weeks(P<0.05).2.PRR gene mediates the NOX4 protein expression and the ERK1/2 protein phosphorylation in myocardial tissue of alcoholic cardiomyopathyRT-PCR and Western blot confirmed that the expression levels of PRR mRNA and protein in rat myocardium of PRR group increased significantly(P<0.05).However,the mRNA expression and protein expression of PRR were decreased in the PRR-shRNA group(P<0.05).Meanwhile,HE staining showed that the cross-sectional area of rat cardiomyocytes in the PRR group was higher than that in the ACM group(P<0.05),but there was no statistical difference between the ACM group and the EGFP group(P=0.987);the cross-sectional area of cardiomyocytes in the PRR-shRNA group was decreased(P<0.05).Western blot showed that compared with the ACM group,the NOX4 protein expression and phosphorylation of ERK1/2 protein in the myocardial tissue homogenate of the PRR group increased significantly(P<0.05),while in the PRR-shRNA group,the NOX4 protein expression and phosphorylation of ERK1/2 protein in rat myocardium homogenate were decreased(P<0.05)3.PRR gene mediates cardiac dysfunction in alcoholic cardiomyopathyCompared with the control group,the rats in the ACM group showed heart dilatation with cardiac dysfunction.The left ventricular end-systolic diameter(LVESD)and left ventricular end-diastolic diameter(LVEDD)were significantly increased(P<0.05);left ventricular ejection fraction(LVEF)and shortening fraction(FS)were decreased in the ACM group(P<0,05);The E/A ratio was also reduced,indicating that the diastolic function was also impaired and myocardial compliance was abnormal.Compared with the ACM group,PRR overexpression further aggravated cardiac dysfunction in the PRR group(P<0.05);PRR gene silencing increased LVEF and FS(P<0.05),reduced LVESD and improved systolic function(P<0.05).But in terms of E/A ratio,however,there was no statistical differences between the PRR-shRNA group and the ACM group(P=0.460).4.PRR gene mediates myocardial fibrosis in alcoholic cardiomyopathyMasson staining showed that compared with the control group,the deposition of interstitial collagen in the ACM group was significantly increased(P<0.05);compared with the ACM group,PRR gene overexpression further increased the deposition of interstitial collagen(P<0.05).However,PRR gene silencing alleviated the deposition of collagen fibers in ACM myocardial tissue(P<0.05)Immunohistochemical staining showed that PRR gene overexpression increased the expression of myocardial interstitial collagen I,collagen III,connective tissue growth factor and fibronectin(P<0.05),However,compared with ACM group,PRR gene silencing reduced the deposition of these fibrotic factors in ACM myocardial tissues(P<0.05)5.PRR gene mediates myocardial oxidative stress in alcoholic cardiomyopathyCompared with the control group,the DHE staining-positive fluorescent spots in the ACM group were dense and the fluorescence intensity was enhanced(P<0.05)The DHE staining-positive fluorescent spots of the PRR group were the most dense and the fluorescence intensity was the strongest(P<0.05),while PRR gene silencing attenuated the density and intensity of DHE staining-positive fluorescent spots in ACM myocardial tissue(P<0.05).Immunohistochemical staining showed that the expression levels of 3-NT,4-HNE and NOX4 in the ACM group were significantly higher than those in the control group(P<0.05).PRR gene overexpression further increased the expression levels of 3-NT,4-HNE and NOX4 in rat myocardium(P<0.05).However,PRR gene silencing weakened the expression levels of 3-NT,4-HNE and NOX4 in ACM myocardial tissue(P<0.05)6.PRR gene mediates cardiac inflammatory injury in alcoholic cardiomyopathyToluidine blue staining showed that compared with the control group,the number of degranulation mast cells in the ACM group was significantly increased(P<0.05)Meanwhile,the expression levels of IL-1? and IL-6 were also increased in the ACM group as indicated by immunohistochemical staining(P<0.05).PRR gene overexpression further increased the number of degranulation mast cells(P<0.05)and inreased the expression levels of IL-1? and IL-6(P<0.05).Compared with the ACM group,the number of degranulation mast cells and the expression levels of inflammatory factors such as IL-1? and IL-6 were ameliorated by PRR gene silencing(P<0.05).Conclusion1.The expression of PRR gene is significantly increased in myocardial tissue of alcoholic cardiomyopathy.2.PRR gene mediates myocardial oxidative stress,increases myocardial fibrosis,inflammatory responses,deteriorates cardiac function and cardiac remodeling in alcoholic cardiomyopathyPart Three:The Mechanism of(Pro)renin Receptor in Alcohol-induced Injury in Primary Cardiac FibroblastsBackgroundAlcoholic cardiomyopathy(ACM)is a kind of myocardial specific disease caused by long-term heavy drinking.Ethanol and its metabolite acetaldehyde accumulated in the body induce hypertrophy,apoptosis and myocardial fibrosis.Myocardial fibrosis is an important pathological basis for myocardial remodeling and heart failure.One of the main functions of cardiac fibroblasts is responsible for the production of extracellular matrix(ECM)and its metabolic homeostasis,providing a stable three-dimensional network for cardiac function.However,under the stimulation of a variety of virulence factors,a large number of cardiac fibroblasts accumulate and activate into myofibroblasts,resulting in excessive deposition of ECM,involving in myocardial remodeling and damage to cardiac function.It has been going on for more than a hundred years to study the renin-angiotensin system(RAS)since the renin was named in 1898.As the research progresses,new problems will continue to emerge,and new members will continue to join.Gradually,we divide RAS into two relatively independent but mutually influential systems:circulating RAS and local RAS.The former plays a major role in body fluid metabolism,water and electrolyte balance and blood pressure homeostasis,while the latter plays an important role in local tissue effects such as cell growth,proliferation,proteins synthesis and organ damage.It is called relatively independent because the local tissue RAS can perform its function independently of the circulating RAS,even more,the content of local tissue RAS in the interstitial fluid is much higher than the circulating RAS.So what is the reason of the difference between the concentration of RAS components in the interstitial fluid and the concentration of RAS components in the circulating system?This problem has been confusing researchers.Until 2002,Nguyen et al first reported and cloned human renin/prorenin receptor(PRR)to provide a new target for us to explain this problem.Accumulating studies have reported that PRR is widely distributed in various cells of the body.In the mouse kidney collecting duct cells,PRR increased fibrotic proteins such as CTGF and PAI-1 through the MAPK-ROS pathway and increased renal interstitial fibrosis damage.In rat mesangial cells,high glucose stimulation can autophosphorylate PRR,further activate TGF-?/CTGF signaling pathway and participated in the damage of diabetic renal fibrosis.In H9C2 cell line,PRR binds to renin,recruites more promyelocytic leukemia zinc finger protein and promotes nuclear translocation,which increases downstream target gene expression,promotes proliferation and reduces apoptosis.So is there any PRR expression in cardiac fibroblasts?Does it have important pathophysiological functions?Therefore,in this part of the experiment,we observed the expression of PRR in primary cardiac fibroblasts,and then study the mechanism of PRR in alcohol-induced injury in primary cardiac fibroblasts through a series of "Gain-of-function","Loss-of-function"and "rescue experiment".Objective 1.To isolate,culture and identificate rat primary cardiac fibroblasts.2.To observe the expression and distribution of PRR in primary cardiac fibroblasts stimulated by alcohol in vitro.3.To study the mechanisms of PRR in alcohol-induced injury in primary cardiac fibroblasts through a series of "Gain-of-function","Loss-of-function" and "rescue experiment".Methods 1.Construction of rat PRR gene overexpression and PRR gene silencing recombinant adenoviral vector The recombinant adenovirus overexpression vector containing rat PRR gene was constructed according to the rat PRR gene sequence in GeneBank.Following the principle of gene silencing,three pairs of shRNA against rat PRR gene were designed and synthesized.After RT-PCR and Western blot,the sequence with the highest silencing efficiency was selected to construct adenovirus silencing vector.2.Isolation,culture and identification of primary cardiac fibroblasts Primary cardiac fibroblasts were isolated from 1-3 days old rats and cultured with DMEM containing 10%FBS.Immunocytochemical staining of Vimentin was used to identification.3.Cell culture and groups Cardiac fibroblasts were seeded on 6-well plates and divided into nine groups as follows:?control group,?alcohol group,?alcohol+scramble-shRNA(scramble-shRNA)group,? alcohol+PRR-shRNA(PRR-shRNA)group,?alcohol+PRR(PRR)group,?alcohol+PRR+Telmisartan(PRR+Tel)group,?alcohol+PRR+PD98059(PRR+PD98059)group,?alcohol+PRR+Telmisartan+PD98059(PRR+Tel+PD98059)group,?alcohol+PD98059 group4.Western blotTotal protein was extracted and SDS-PAGE was performed to measure the expression levels of PRR,ERK1/2,p-ERK1/2,NOX4 and TGF-?.GAPDH was used as an internal reference5.ELISAELISA was performed to measure the content of collagen ?,collagen ? and TGF-?in the cell culture medium.6.Measurement of oxidative stressAll cells were incubated with DHE(10?M)at 37? for 30 min in the dark.The fluorescent images of cells were observed by fluorescence microscope7.NADPH oxidase activityAll cells were collected and the NADPH oxidase activity was measured according to the manufacturer instructionsResults1.Identification of primary cardiac fibroblasts and expression of PRR in cardiac fibroblasts stimulated by alcoholPrimary cardiac fibroblasts grew well,irregular shapes were common,long spindles were rare,no autonomous beat,no clusters but overlap occurred when growth was too dense.Immunocytochemical staining showed that Vimentin staining was positive and the middle silk skeleton protein was arranged densely and orderly in the cytoplasm.Western blot and RT-PCR showed that alcohol stimulation increased the expression of PRR in fibroblasts in a concentration-and time-dependent manner,and the expression was highest at 200 mM for 8 h(P<0.01).The fluorescent staining showed that PRR was mainly expressed in the cytoplasm,especially in the area around the nucleus,no obvious expression was observed in the nucleus.PRR expression was also observed in the cell membrane.Alcohol stimulation significantly increased the fluorescence intensity of PRR2.Effects of PRR gene silencing on NOX4,TGF-P protein expression and phosphorylation of ERK1/2 in cardiac fibroblastsWestern blot showed that compared with the control group,the expression of PRR in the alcohol group was significantly increased(P<0.01);compared with the alcohol group,the expression of PRR in the PRR-shRNA group was significantly decreased(P<0.05).Alcohol stimulation increased the expression levels of NOX4,TGF-? and phosphorylation of ERK1/2 in fibroblasts while PRR gene silencing reversed these changes.In addition,there was no significant difference in the protein expression of NOX1 in all groups(P=0.208).No significant expression of NOX2 was detected in all groups regardless of mRNA level or protein level3.PRR gene silencing reduces oxidative stress levels in cardiac fibroblasts stimulated by alcoholDHE staining showed that compared with the control group,the fluorescence intensity of the alcohol group was significantly enhanced and the activity of NADPH oxidase in the cell homogenate was enhanced(P<0.01).Compared with the alcohol group,PRR gene silencing reduced the fluorescence intensity of DHE and decreased the activity of NADPH oxidase in cell homogenate(P<0.05).4.PRR gene silencing reduces the content of collagen ?,collagen ? and TGF-?in cardiac fibroblasts culture medium stimulated by alcoholAfter alcohol stimulation was completed,cell culture medium was collected for ELISA.The results showed that the content of collagen ?,collagen ? and TGF-? in the culture medium of the alcohol group was significantly higher than those of the control group.Compared with the alcohol group,the content of collagen ?,collagen? and TGF-? in the culture medium of the PRR-shRNA group was significant reduced(P<0.05).5.Effects of telmisartan on NOX4,TGF-? protein expression and phosphorylation of ERK1/2 in cardiac fibroblasts overexpressing PRRWestern blot showed that compared with the alcohol group,PRR overexpression significantly increased the PRR protein expression(P<0.05)and PRR overexpression further increased NOX4,TGF-? protein expression and phosphorylation of ERK1/2 in fibroblasts stimulated by alcohol(P<0.05).However,telmisartan attenuated the increased expression of NOX4 protein induced by PRR overexpression(P<0.05).There was no statistical difference between the PRR+Tel group and the PRR group in terms of the expression of TGF-? and phosphorylation of ERK1/2 protein(P>0.05).6.Effects of PD98059 on NOX4,TGF-? protein expression and phosphorylation of ERK1/2 in fibroblasts overexpressing PRRWestern blot showed that compared with the alcohol group,PD98059 significantly decreased the phosphorylation of ERK1/2 with or without PRR overexpression(P<0.05).Compared with the alcohol group,PD98059(Alcohol+PD98059 group)reduced the increased expression of NOX4 and TGF-? in fibroblasts induced by alcohol(P<0.05).Telmisartan decreased NOX4 protein expression(P<0.05),however,the effect of PD98059 was better than that of telmisartan in terms of reducing NOX4 protein expression(P<0.05).In terms of TGF-? protein expression and ERK1/2 protein phosphorylation,the effect of PD98059 was significantly better than that of telmisartan(P<0.01).The combination of the two drugs significantly reduced NOX4,TGF-? protein expression and phosphorylation of ERK1/2(P<0.01).7.Effects of telmisartan and PD98059 on oxidative stress in cardiac fibroblasts overexpressing PRRDHE staining and cell homogenate NADPH oxidase activity were measured after the experiment.The results showed that compared with the control group,the DHE fluorescence intensity of alcohol group was increased(P<0.05)and PRR overexpression further increased the fluorescence intensity(P<0.01).Compared with the PRR group,the fluorescence intensity of the PRR+Tel group and the PRR+PD98059 group were decreased(P<0.05),however,the decrease in fluorescence intensity of the PRR+PD98059 group was more prominent.The combination of two drugs has the most significant reduction in fluorescence intensity.There was a similar trend in NADPH oxidase activity in cell homogenate.Compared with the alcohol group,the activity of NADPH oxidase in the PRR group was significantly increased(P<0.01).While the activity of NADPH oxidase in cell homogenate of the PRR+Tel group and the PRR+PD98059 significantly decreased(P<0.05)and the decline was most obvious in the PRR+Tel+PD98059 group.8.Effects of telmisartan and PD98059 on the contents of collagen ?,collagen ?and TGF-? in the culture medium of cardiac fibroblasts overexpressing PRRThe cell culture medium was collected f... |
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