| BackgroudsCardiac hypertrophy of mammalian is an adaptive response to high pressure,volume overload,and neurohormonal activation,and is associated with many heart diseases,including hypertension,coronary artery disease,valvular heart disease.Ventricular hypertrophy is closely related to an increased risk of heart failure.Therefore,alleviating hypertrophy is an important step for the prevention of heart failure.It is generally accepted that angiotensin ?(Ang ?),a crucial molecular component of the renin-angiotensin-aldosterone system,plays a significant role in cardiovascular homeostasis.Additionally,multiple studies have demonstrated that Ang ?-induced cardiac hypertrophy depends primarily on reactive oxygen species(ROS)and inflammatory factor overproduction.Excessive ROS levels can cause cardiomyocyte apoptosis,extracellular matrix remodelling,cardiac hypertrophy,and,ultimately,impaired cardiac contractile function.Cyclophilin A(CyPA)is a primarily intracellular protein,which is expressed abundantly in all mammalian cell types.As a housekeeping protein,it accounts for 0.4%of the complete intracellular proteome.As a pro-inflammatory cytokine,CyPA is secreted by several cell types,including monocytes/macrophages,vascular smooth muscle cells,activated platelets,and endothelial cells.Under pathological conditions,these cells secrete CyPA into the extracellular space.For example,CyPA is released by macrophages upon stimulation with LPS or high levels of glucose or during foam cell formation in vitro.Moreover,the release of CyPA by vascular smooth muscle cells appears to highly promote the pathophysiology of vascular inflammation in abdominal aortic aneurysm,vascular restenosis,and atherosclerosis.In addition,both activated platelets and hypoxic cardiomyoctes have been shown to secrete CyPA into the extracellular space.The exact mechanism of the CyPA release in these cells has not been described.However,a recent report describes that the acetylation of CyPA is required for its secretion into the extracellular space in vascular smooth muscle cells.Moreover,acetylated CyPA seems to exert a more pronounced inflammatory activity than unmodified CyPA.In patients,enhanced levels of soluble extracellular CyPA can be detected under classical inflammatory conditions,e.g.in synovial fluids of patients with rheumatoid arthritis,in serum of patients with sepsis,in nasal fluids of patients with asthma,and in plasma of patients with Type 2 diabetes.It has been found that enhanced serum CyPA has been characterized as a biomarker of coronary artery disease as well.Cyclosporine A(CsA),an immunosuppressant,inhibits CyPA peptidyl-prolyl cis-trans isomerase(PPIase)activity.It was recently reported that CyPA possibly plays a crucial role in cardiac hypertrophy pathophysiology and may be a key determinant of ROS production in cardiomyocytes.CD 147,an extracellular matrix metalloproteinase inducer(EMMPRIN),is a ubiquitously distributed integral transmembrane glycoprotein belonging to the immunoglobulin(Ig)superfamily.And it has been implicated in a number of physiological and pathological effects through interacting with different binding partners such as cyclophilins(CyPs),caveolin-1,integrins,and E-selectin.To date,lots of studies have demonstrated that CD 147 has taken part in the regulation of lymphocyte responsiveness,carcinoma metastasis,inflammatory responsive,and spermatogenesis.Among these partners,cyclophilins,especially cyclophilin A(CyPA),might be investigated most frequently in the recent years.Recent studies have found that it is thought to be the main cell surface receptor mediating CyPA signal transduction.Nowadays,a growing body of research suggested CyPA and CD 147 involvement in key processes of cardiovascular diseases pathologies.Nevertheless,the effects of CyPA and CD147 on cardiomyocyte hypertrophy and the underlying mechanism of its actions remain to be explored.Melatonin is an important chronobiological regulatory molecule that was discovered when Lerner isolated and characterized this indolamine from the bovine pineal gland.The production of melatonin is controlled by the suprachiasmatic nucleus(SCN),the central circadian pacemaker that triggers the pineal gland to produce melatonin at night.Melatonin represents the biological signal of darkness and the duration of its release is proportional to the length of the night.Since melatonin was discovered,melatonin has been widely tested in numerous experimental and clinical studies and has been shown to have a variety of benefits.Studies have revealed that melatonin helps prevent many diseases,including inflammatory diseases,ischemia/reperfusion injury,hypertension,and various cardiovascular diseases.Moreover,recent studies indicate that the role of melatonin in cardiovascular disease is dependent on its activity as a powerful antioxidant.However,the effects of melatonin on cardiomyocyte hypertrophy and the underlying mechanism of its actions remain to be explored.In this study,we explored the role and molecular mechanisms of action of melatonin in Ang ?-induced cardiomyocyte hypertrophy,which could provide scientific evidence for new opportunities and therapeutic targets in prevention and treatment of cardiac hypertrophy.Objectives1.This study used the H9C2 cells induced by Ang 11 or CyPA as cardiac hypertrophy model to observe the influence of H9C2 cell surface area,the[3H]leucine incorporation at different concentrations of melatonin stimulus.2.To investigate the mechanism of the signal-transduction pathway about CyPA/CD147 in hypertrophic cardiomyocytes induced by Ang ?.3.To explore the role and mechanism of action of melatonin in cardiomyocyte hypertrophy.Methods1.Cell cultureCulture H9C2 cells until the desired cell density was reached.First,with or without 60 min of melatonin pretreatment at different concentrations,the cells were treated with Ang ? for 24 h.Second,the CsA group was pretreated with CsA for 60 min,and then the Ang ? and CsA groups were treated with Ang ? for 24 h.Third,H9C2 cells were pretreated with different melatonin doses for 60 min before being treated with CyPA for 24 h,as described above.2.[3H]Leucine incorporation To detect the level of protein synthesis in H9C2 cardiomyocytes.3.Immunofluorescence analysis Immunofluorescence was used to detect the protein expression and location of?-SMA,CyPA and CD 147.To detect the surface area of H9C2 cells using software.4.Western blot analysisTo detect the expression of ?-SMA,CyPA,CD147,NOx2 in protein levels.5.RT-PCRTo detect the expression of ?-SMA,?-MHC,ANP in gene levels.6.Intracellular ROS level evaluationIntracellular ROS levels in H9C2 cells were detected with the fluorescent indicator 2',7'-dichlorodihydrofluorescein triacetate diacetate(DCFH-DA).7.Accessment of cell prolifertationCell Counting kit-8(CCK-8)was used to examine cell proliferation.8.Statistical analysisDifferences between groups were analyzed with t-tests or ANOVAs using SPSS software version 18.0.All data are presented as the means ± SD of at least 3 three independent experiments.p<0.05 was considered statistically significant.Results1.Melatonin inhibits Ang ?-induced H9C2 cell hypertrophyTo determine whether melatonin has an effect on Ang ?-induced hypertrophy in H9C2 cells,we pretreated H9C2 cells with different concentrations of melatonin and then exposed them to 100 nmol/L of Ang ? for 24 h.We found that pretreatment with melatonin attenuated Ang ?-induced increases in[3H]leucine incorporation,reduced the surface area of these cells,downregulated the mRNA expression levels of ?-SMA,ANP and ?-MHC in a concentration-dependent manner(p<0.05).And western blot analyses and immunofluorescence revealed that ?-SMA protein was strongly expressed in H9C2 cells under Ang ? stimulation.However,melatonin pretreatment reduced it in a concentration-dependent manner(p<0.05).2.Melatonin decreases Ang ?-induced oxidation reaction in H9C2 cellsTo determine whether melatonin pretreatment inhibits ROS generation in H9C2 cells,we observed DCFH-DA fluorescence and western blot analyses in these cells in response to different treatments,and noted that pretreatment with melatonin significantly and concentration-dependently blocked ROS generation and the protein level of NOx2(p<0.05).3.Ang ?-induced H9C2 cell hypertrophy through CyPA/CD147 signaling pathwayWe subjected H9C2 cells to 24 h of Ang ? treatment in either the absence or presence of 60 min of CsA pretreatment.Subsequent Western blot analyses revealed that Ang ? treatment significantly increased the levels of inflammatory factors including CyPA and CD 147(p<0.05).Immunofluorescence analyses revealed that CyPA and CD147 were also strongly expressed in H9C2 cells during Ang ?stimulation(p<0.05).However,these increases were markedly suppressed by CsA pretreatment(p<0.05).Meanwhile,data revealed by CCK-8 assays indicated that CsA had no influence in the proliferation of H9C2 cells(p>0.05).4.Melatonin at least partially blocks CyPA/CD147 signaling pathway-related Ang?-induced H9C2 cell hypertrophyWe detected the expression levels of CyPA and CD 147 in response to Ang ?stimulation via immunofluorescence and Western blot analyses,which demonstrated that Ang ? treatment significantly increased CyPA and CD 147 levels,which were markedly and concentration-dependently suppressed by melatonin pretreatment(p<0.05).5.Melatonin inhibits CyPA-induced cardiac hypertrophyWe tested whether 100 nmol/L of CyPA stimulation induces H9C2 cell hypertrophy and investigated the effects of melatonin pretreatment on CyPA-induced cardiac hypertrophy.Our results show that pretreatment with melatonin attenuated CyPA-induced increases in[3H]leucine incorporation,reduced the surface area of these cells,downregulated the mRNA expression levels of ?-SMA,ANP and ?-MHC in a concentration-dependent manner(p<0.05).And western blot analyses and immunofluorescence revealed that a-SMA protein was strongly expressed in H9C2 cells under CyPA stimulation.Further,melatonin pretreatment reduced it in a concentration-dependent manner(p<0.05).However,to compared Ang ? and CyPA groups,they have no statistical significant in the above-mentioned experimental methods(p>0.05).6.Melatonin decreases oxidation reaction in CyPA-induced H9C2 cell hypertrophyThe results indicate that CyPA stimulation alone increases ROS generation in H9C2 cells and that melatonin blocks ROS generation in a concentration-dependent manner in CyPA-induced cardiac hypertrophy(P<0.05).We simultaneously observed the effects of melatonin on NOx2 expression in CyPA-induced cardiac hypertrophy.Western blot results suggest that melatonin significantly decreased the levels of NOx2 in a concentration-dependent manner(p<0.05).Meanwhile,data revealed by CCK-8 assays indicated that CyPA had no influence in the proliferation of H9C2 cells(p>0.05).Conclusions1.Melatonin markedly inhibits Ang ? or CyPA-induced H9C2 cell hypertrophy.2.The CyPA/CD147 signaling pathway is at least partially activated in Ang?-induced cardiac hypertrophy and that melatonin attenuates Ang ?-induced cardiac hypertrophy via its potential antioxidant property.BackgroudsHypertension is a widespread and multifaceted public health disease throughout the world,and a major cause of cardiac damage.Left ventricular hypertrophy(LVH)resulting from systemic hypertension is characterized by structural remodelling of the heart.In hypertension,LVH is initially a transitional phase and a compensatory process,then can gradually lead to dysfunction of cardiac electric system and ventricular arrhythmia,rise of myocardial stiffness,damage of cardiac function,which can cause various kinds of cardiovascular events,for example,myocardial infarction,congestive heart failure,sudden cardiac death.Individuals with LVH are at two to four times higher risk of having adverse cardiovascular events compared to patients without this condition.Hypertension due to pressure load is a clinical indicator of progressive LVH in hypertensive heart disease,LVH induced by hypertension is a multi-factor pathological process,and identifying the key determinants involved is critically important for developing strategies to prevent heart failure.Now many studies have confirmed that acknowledged LVH risk factors include blood pressure,duration of hypertension,age,obesity,diet,and pharmacologic treatment.The mechanisms of LVH are intricate,LVH can be caused by many pathways,the primary of them are inflammation and oxidative stress.Recent clinical and fundamental researches have shown that many cytokines and chemokines have participated in the process of LVH morbidity.Among them,brian natriuretic peptide(BNP)or N-terminal pro-brian natriuretic peptide(NT-proBNP)and endothelin-1(ET-1)have been confirmed to having an intimate relationship of LVH induced by hypertension,and they can evaluate extent of LVH.However,the serum inflammatory factors including c-reactive protein(CRP)and interleukin-6(IL-6)have been elevated in LVH patients;then we can conclude that inflammatory factors have an important role in the process of LVH.Overproduction of reactive oxygen species(ROS)is regarded as an important driver that lies upstream of the inflammatory cascade.Some mediators,which are induced in response to ROS,were named secreted oxidative stress-induced factors(SOXFs).Cyclophilin A(CyPA)is not only a new pro-inflammatory cytokine,but also highlighted as a major SOXF.CyPA is a ubiquitously distributed protein belonging to the cyclophilin family,and is mainly secreted by monocytes/macrophages,vascular smooth muscle cells,endothelial cells under the stimulation of inflammation and ROS.Recently,elevated levels of CyPA have been reported in advanced atherosclerosis lesions,hypertension,abdominal aortic aneurysm(AAA),ischaemia/reperfusion of myocardium and the myocardiac remodelling.Therefore,we inferred that CyPA may participate in the earlier period of hypertension-induced LVH.Melatonin,an indole produced in several organs but most notably in the pineal gland,has a variety of effects that influence cardiac pathophysiology.Several studies show that humans with cardiovascular disease have noticeably lower circulating melatonin levels than age-matched subjects without significant cardiovascular deterioration.Recent investigations confirmed a relationship between melatonin concentrations and many cardiovascular diseases,for example hypertension,acute myocardial infarction(AMI),atherosclerosis and ischaemia/reperfusion of myocardium because of its activity as a potent powerful antioxidant and antiinflammatory agent.However,the research about association between serum melatonin concentration and the earlier LVH in essential hypertension patients is fewer.Moreover,the investigation of melatonin and cardiomyocyte hypertrophy is primarily on some clinical manifestation and the underlying mechanism of its actions remain to be explored.Our present clinic experiment was performed to elucidate the relationships between serum CyPA,melatonin and LVH in essential hypertension patients;moreover,we wanted to find the role of CyPA and melatonin in the complexly physiopathologic mechanisms with hypertension-induced LVH.Then we would find a convenient and simple method to predict the earlier damage of hypertension-induced LVH and a new target for the treatment of it.Objectives1.To study the expression of melatonin and CyPA in the serum of hypertension-induced LVH.2.To investigate the correlations between melatonin,CyPA and hypertension induced LVH,then statistically analyze other independent risk factors for hypertension-induced LVH.3.To explore the correlations between melatonin and CyPA in hypertension-induced LVH patients.MethodsOne hundred and thirty five essential hypertensive patients who were treated with hypertensive drugs in Shandong Provincial Chest Hospital were enrolled in this study between October 2015 and October 2016.The enrolled hypertensive patients were divided into two groups according to Doppler echocardiography as following:? the group of LVH(+)patients was seventy;?the group of LVH(-)patients was sixty five.All the subjects were taken medical examinations for blood pressure,heart rate,height,weight,et al.Determine serum CyPA,Melatonin,ET-1,CRP,IL-6,NOx2,NT-proBNP and TnI concentrations by enzyme-linked immunosorbent assay(ELISA).Ultrasonography for measuring left ventricular internal diameter at end-diastole(LVIDd),left ventricular internal diameter at end-systole(LVIDs),left ventricular posterior wall thickness(LVPWd),interventricular septal thickness(IVST)and left ventricular ejection fraction(LVEF),then calculate left ventricular mass(LVM),relative wall thickness(RWT)and left ventricular mass index(LVMI)by equations.The data were analyzed using SPSS version 18.0(SPSS,Inc.,Chicago,IL,USA).Continuous variables are presented as the mean±standard deviation,whereas categorical variables are presented as frequencies and percentages.The significance of the differences in continuous variables between the LVH(+)and LVH(-)groups was determined using the paired t-test and one-way analysis of variance(ANOVA).Categorical variables were compared with the ?2 test.The relationships between variables were assessed by Pearson and Partial correlation analyses,and the related factors were assessed by multivariate logistic regression and multiple linear regression analyses for their contribution to LVM and the LVMI in the population.P<0.05 was considered statistically significant.Results1.The comparison of groups common characteristics and laboratory resultsAs shown in Table 1,both LVH(-)and LVH(+)subjects have no statistical significance in age,diabetes,smoking index,drinking index,use of anti-hypertensivedrugs,DBP,WBC,TC,TG,HDL,LDL,FPG,Scr,UA,ALT,AST,CK,CK-MB(p>0.05).However,compared with LVH(-)group,LVH(+)subjects have significantly increased in the percentage of female subjects,morbidity of coronary artery disease,SBP,duration of hypertension,body mass index(BMI)(p<0.05).2.The comparison of groups echocardiographic characteristicsCompared with the LVH(-)group,LVH(+)subjects have significantly increased in LVM,LVMI,LVIDd,LVPWT,IVST,RWT(p=0.000).But they have no statistical significant in LVEF and LVIDs(p>0.05).The results are presented in Table 1.3.The comparison of groups CyPA,melatonin,ET-1,CRP,IL-6,NOx2,cTnI,NT-proBNP concentrations in serumCompared with the LVH(-)group,LVH(+)group has significantly increased in serum CyPA,ET-1,IL-6 concentrations,and decreased in serum melatonin and NOx2(p<0.05).However,they have no statistical significance in serum CRP,cTnl and NT-proBNP concentrations(p>0.05).The results are also presented in Table 1.4.Multivariate logistic regression analysis Multivariate logistic regression analysis was applied to assess the significance of the factors contributing to LVH shown in Table 1,and the results are presented inTable 2.The percentage of female subjects,morbidity of coronary artery disease,duration of hypertension,serum ET-1,IL-6 and NOx2 levels were not significantly correlated with LVH(p>0.05).However,serum CyPA concentration(p=0.008),serum melatonin level(p=0.005),BMI(p=0.000)and SBP(p=0.036)exhibited a significant association with LVH.Thus,high CyPA level and low melatonin level are independent risk factors for LVH.In addition,BMI and SBP are also correlated with the risk of developing LVH.5.Pearson correlation analysis between serum CyPA,melatonin,BMI,SBP and LVM,LVMI(1).Table 3 and figure 1 showed that BMI was positively correlated with LVM,LVMI(p=0.000).(2).Table 3 and figure 1 showed that SBP was positively correlated with LVM,LVMI(p=0.000).(3).Table 3 and figure 1 showed that serum CyPA concentration was positively correlated with LVM,LVMI(p<0.01).(4).Table 3 and figure 1 showed that serum melatonin concentration was negatively correlated with LVM,LVMI(p=0.000).6.Pearson correlation analysis between serum CyPA and melatoninThe Pearson correlation analysis showed that serum CyPA concentration was negatively correlated with melatonin(p<0.01)in LVH.The results is presented in Table 4 and figure 2.7.Multiple linear regression analysisTo determine the factors independently associated with LVH,multiple linear regression analysis was performed.LVM and LVMI were employed as dependent variables,and BMI,SBP,CyPA,and melatonin were employed as independent variables.As Table 5 shows,LVM and LVMI were positively associated with BMI,SBP,and CyPA(p<0.05),whereas they were negatively associated with melatonin(p=0.000).8.Partial correlation analysisTo further distinguish the relationship among CyPA,SBP,the morbidity of coronary artery disease,and LVH,Table 6 shows the results of the Partial correlation analysis.When two of those factors were employed as controlled variables,CyPA was positively correlated with the LVMI(p<0.05),but it was not associated with SBP or the morbidity of coronary artery disease(p>0.05).Conclusions1.The serum CyPA and melatonin concentrations are correlated with LVH,and serum CyPA concentration was negatively correlated with melatonin;suggesting that CyPA and melatonin are independent risk factors for LVH.2.BMI and SBP are increased and positively correlated with LVH,suggesting that BMI and SBP are also independent risk factors for LVH. |
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