Effect Of Neferine On Diabetic Myocardial Fibrosis And Its Signal Transduction Mechanism

BackgroundDiabetes mellitus(DM)is a global health concern.The burden of diabetes as a major cause of premature illness and death is mostly due to the associated increased risk of cardiovascular disease,cardiac remodeling and heart failure.Cardiac fibrosis is reported to be a key pathogenic component of cardiovascular diseases.Specifically,cardiac fibrosis contributes to cardiac remodeling,increases myocardial stiffness,reduces the pumping capacity of the heart,and eventually leads to heart failure.Nevertheless,no curative treatment for cardiac fibrosis has been developed so far.Cardiac fibroblasts(CFs)are the predominant cell type in the heart,and are responsible for the basal deposition and degradation of the extracellular matrix(ECM)in the normal heart.As the main matrix-producing cells,CFs are critically involved in all cardiac fibrotic conditions.In response to various stimuli,CFs may proliferate,migrate,differentiate into myofibroblasts,generate or degrade the ECM,secrete cytokines and growth factors,and so on.High glucose(HG)in the blood(hyperglycemia),the main feature of diabetes mellitus,can stimulate collagen deposition by inducing CF proliferation and activation in vitro.On the basis of these concepts,inhibiting the activation of CFs could be a viable strategy for treating cardiac fibrosis."Lianzixin," the seed embryo of Nelumbo nucifera(Gaertn.),has been commonly used in traditional Chinese medicine as a sedative,antipyretic and hemostatic agent.Neferine is a major bisbenzylisoquinline alkaloid derived from this plant,along with liensinine and isoliensinine.Neferine has been reported to have a variety of biological and pharmacological effects,such as anti-hypertensive,anti-arrhythmic,anti-agglutinating,anti-thrombotic,antioxidant,anti-inflammatory,neuroprotective,anticancer,negative inotropic and vascularsmooth-muscle-relaxing effects.In addition,neferine exerts antifibrotic effects.Zhao et al.found that neferine attenuated bleomycin-induced pulmonary fibrosis in vitro and in vivo.Niu et al.demonstrated that neferine significantly inhibited amiodarone-induced pulmonary fibrosis.A recent study revealed that neferine had an antifibrotic effect on CCl4-induced hepatic fibrosis in mice,which may have been partly due to the reduced expression of transforming growth factor-?1(TGF-?1)in the liver.Hence,the current study was designed to determine whether and by what molecular pathways neferine could attenuate cardiac fibrosis induced by HG in CFs,and whether neferine could thus serve as an alternative and safe drug for clinical applications.Objective1.To study the effect and mechanism of neferine on myocardium fibrosis induced by diabetes mellitus.2.To study the effect of neferine on the proliferation,migration and collagen synthesis of cardiac fibroblasts induced by high glucose.MethodsPart ?1.Establishment of animal model and administration of neferine in vivoEight-week-old male C57BL/6J mice were randomly divided into STZ group(n=45)and normal control group(n = 15).The rats in the model group were induced by streptozotocin(STZ)solution for 5 consecutive days,and the dose was 60 mg/kg/d.Control mice were injected with the same volume of sterile citrate buffer(pH 4.5)at a concentration of 0.1 mmol/l.One week after the injection of STZ solution,blood glucose was measured in the tail vein,and mice whose blood glucose concentration in the tail vein was greater than 18 mmol/1 were included in the diabetic model group,and were randomly divided into 3 groups:model group,low dose of neferine group(60mg/kg/d)and high dose of neferine(120mg/kg/d).Neferine was administered by intragastric administration twice a day from the end of the fourth week.The control group was given the same amount of normal saline.2.Blood pressure and heart rate testingThe tail-cuff method was used to measure systolic blood pressure,diastolic blood pressure,mean arterial blood pressure and heart rate.3.Echocardiographic examination of cardiac functionThe cardiac function of the left ventricle was assessed by two-dimensional,M-mode ultrasonography,pulsed Doppler and tissue Doppler imaging.4.The ultrastructure of myocardium was observed by transmission electron microscopeThe fresh heart was quickly removed and the left ventricle was cut into 1 mm3 blocks for observation by a transmission electron microscope.5.Histological stainingFormaldehyde was fixed in the heart of mice,prepared paraffin sections,HE,Masson,Sirius red staining,showing the general shape of the heart and collagen content.Immunohistochemical staining was used to observe the distribution and expression of intracellular factor.Part ?1.Cell cultureIn the present study,cardiac fibroblasts were isolated and cultured in the heart of C57 mice,1-3 days after birth.2.Real-time quantitative RT-PCRTissue and cellular RNA were extracted for reverse transcription,and real-time quantitative PR-PCR was used to determine mRNA levels in tissues and cells.3.Western blottingTissue and cell proteins were collected for extraction and tested according to standard procedures.4.Cell proliferation and migration experimentsThe cell proliferation rate was measured by CCK-8,and the migration of the cells was observed by Transwell chamber.5.Statistical analysisSPSS 18.0 software was used to analyze the experimental data.Continuous variables were expressed as the mean ± standard deviation.Variables between groups were examined using one-way ANOVA.P less than 0.05 was considered statistically significant.ResultsPart?1.The general properties of miceAfter induction of STZ,the random blood glucose of diabetic mice was significantly higher than that of normal control mice.At the same time,the systolic blood pressure,diastolic blood pressure,mean arterial pressure were significantly higher in diabetic mice than in normal control mice,while heart rate was lower in diabetic mice.However,low-dose or high-dose neferine drug intervention have no significant effect on above indicators,indicating that neferine improving diabetic cardiomyopathy is not achieved by lowering blood glucose.2.Neferine reduced diabetes-induced cardiac remodeling and cardiac dysfunctionIn vivo,high and low doses of neferine were administered for 12 weeks.The left ventricular remodeling was observed in all groups.Compared with the normal mice,the heart of the diabetic mice became larger and the apex was blunt.The cardiomyocyte size and morphology of the mice were significantly improved,and the weight ratio of the tibia to the tibia(LW/TL)and the ratio of HW/TL to LW/TL were also decreased.Compared with the normal mice,the diabetic mice showed mitochondrial morphological changes,Cristae disruption and disordered Z-line structure.Low-dose or high-dose neferine treatment reduced mitochondrial swelling and cristae fragmentation and Z-line structural damage.Echocardiography showed a decrease in cardiac function,left ventricular ejection fraction(LVEF),short axis shortening(FS)and early and late mitral inflow velocity(E/A)in diabetic mice,neferine can improve the above cardiac function parameters,and improve heart function in diabetic mice.3.Neferine attenuated diabetes-induced cardiac fibrosisImmunohistochemistry and immunoblot showed that the expressions of type ?,type ? collagen and TGF-?1 were increased in the left ventricular heart tissue of diabetic mice;Neferine decreased the expressions of type ?,type ? collagen and TGF-?1 in myocardium.4.Neferine attenuated diabetes-induced cardiac inflammationThe results of Western blot showed that the expression of TNF-?,ICAM-1 and VCAM-1 in diabetic mice were significantly higher than that in normal mice.The expression of TNF-?,ICAM-1 and VCAM-1 in myocardium of diabetic mice was significantly decreased after low or high dose of neferine treatments.Part ?1.Neferine inhibited the proliferation of cardiac fibroblasts stimulated by hyperglycemiaThe proliferation of fibroblasts was stimulated by high glucose(30mmol/l)for different time(24h,48h,72h),compared with the normal control group.The proliferation of fibroblasts was induced by different concentrations of neferine(1?M,2?M,5?M)could inhibit the proliferation of fibroblasts stimulated by high glucose,and the concentrations of 2?M and 5?M were statistically significant.2.Neferine inhibited the proliferation of cardiac fibroblasts in G1 phaseNeferine(2?M,5?M)for 48h could increase the percentage of G1 phase cells and decrease the proportion of S phase and G2 phase cells.3.Neferine reduced the collagen secretion of cardiac fibroblasts,reduced the expression of TGF-?1 and inhibited the migration of cardiac fibroblasts.Neferine inhibited the secretion of collagen ?,collagen ? and TGF-?1 in cardiac fibroblasts stimulated by high glucose,and inhibited the migration of cardiac fibroblasts stimulated by high glucose.4.Cardiac fibroblast-associated signaling pathways were mediated by neferineImmunofluorescence showed that Smad2/3?ERK1/2 and p38 phosphorylation increased in cardiac fibroblasts stimulated by hyperglycemia.Neferine treatments reduced the phosphorylation of Smad2/3?ERK1/2 and p38.Conclusion1.Neferine attenuated diabetes-induced myocardial remodeling,dysfunction and fibrosis in diabetic mice;2.Neferine attenuated diabetes-induced cardiac inflammation;3.Neferine inhibited the proliferation,migration and collagen secretion of cardiac fibroblasts induced by high glucose and inhibited the activation of cardiac fibroblasts.4.The mechanism of neferine alleviating diabetic cardiomyopathy may be related to the inhibition of TGF-?1/Smad2/3,ERK1/2 and p38 phosphorylation.