Role Of Sestrin2 In The Anti-cardiac Remodeling Effect Of Pentamethylquercetin

Cardiac remodeling is a response of heart to a series of physiologic and pathologic stimuli,and is an independent risk factor for heart failure.Ascertaining the pathophysiology process of cardiac remodeling in order to slow or reverse it,has been a new target of heart failure treatment.Heart failure is divided into HFr EF and HFp EF,and the latter accounts for more than 50%.Traditional treatment for reversal of cardiac remodeling such as RAAS inhibitors,beta-blockers,only had effect on partial patients with HFr EF,with poor efficacy on HFp EF patients.Obesity,diabetes,hypertension,valvular disease,usually develop as the concentric hypertrophy,which has more possibility of developing into HFp EF.It is extremely urgent to find out the regulatory mechanism of cardiac remodeling,and search for effective drugs to inhibit cardiac remodeling in HFp EF.Pentamethylquercetin(PMQ)is one of the members of natural polymethoxyflavonoids,mainly exists in the seabuckthorn fruit and rhizome of the genus galanga resurrectionlily plants.PMQ have multiple pharmacological effects,such as resisting diabetes and improving the body’s metabolism;Resisting renal fibrosis;Improving cardiac remodeling induced by pressure overload and Ang Ⅱ in rat,while the mechanism remains to be further studied.This topic was proposed by establishing the monosodium glutamate induced obesity(MSG-IO)model and thoracic aorta constriction(TAC)induced pressure overload model to establish cardiac remodeling in mice,and ISO induced neonatal rat cardiomyocyte hypertrophy model,to evaluate the effect and mechanism of PMQ on cardiac remodeling in vivo and in vitro,and to provide theoretical support for searching new drugs and targets to restrain cardiac remodeling.Part Ⅰ Effects of PMQ on metabolic disorders and cardiac remodeling in MSG-IO micePurpose: To observe the effects of PMQ on metabolic disorders and cardiac remodeling in obese type 2 diabetic mice.Methods: Injecting newborn CD-1 rats of monosodium glutamate(MSG,3 mg/g)subcutaneously for continuous 7d from the 2nd day after born,once a day.After 21 d of birth,weaning male MSG-IO mice were divided into different groups as follows randomly(n=10): MSG-IO model group,PMQ groups(5,10,20 mg/kg/d),and metformin(Met)300 mg/kg/d group as positive control.All these groups were given conventional feed and water until 8wk of ages.PMQ groups were given fodder containing the corresponding concentration of PMQ and conventional water,and Met group were given water containing 300 mg/kg/d of Met and standard chow,while the Control group(n = 10)and MSG-IO model mice were still given conventional feed and water as before.Oral glucose tolerance test(OGTT)was performed at 18 th week.Cardiac echocardiography was measured at the age of 23 wk.Body weight,body length,waist circumference,Lee index were measured and calculated.Serum glucose,insulin level was detected,and the indexes of insulin resistance(HOMA-IR)were calculated.After that,picking the heart and fixing part of myocardial tissue for Masson staining to detect myocardial fibrosis degree.Results:(1)PMQ significantly improved the metabolic abnormality of MSG-IO mice.Compared with control group,MSG-IO mice showed obvious obese characteristics,accompanied with type 2 diabetes,such as body weight,waist circumference and Lee index increased significantly.Fasting plasma glucose,plasma insulin and HOMA-IR elevated markedly,and glucose tolerance impaired.PMQ(5、10、20 mg/kg)significantly improved the above indicators dose dependently.PMQ(20 mg/kg)had equivalent effects with Met group.(2)PMQ significantly improved cardiac remodeling in MSG-IO mice.The hearts of MSG-IO mice showed typical characteristic of concentric hypertrophy compared with those of control mice,and the thickness of left ventricular anterior wall(LVAW)and interventricular septum(IVS)were increased obvious,at the same time left ventricular internal diameter(LVID)was smaller.The hearts were stiffness,and myocardial fibrosis increased significantly in MSG-IO mice.PMQ was able to improve the ventricular wall stiffness and attenuate the increase of ventricular wall thickness including LVAW and IVS in systolic and diastolic,at the same time PMQ restored the size of LVID,which showing that PMQ could effectively suppressed concentric hypertrophy of the heart in obese type 2 diabetes model.Furthermore,PMQ reduced the cardiac weight index obviously,and decreased the degree of myocardial interstitial fibrosis significantly.PMQ(20 mg/kg)improving cardiac remodeling in MSG-IO mice was better than Met(300 mg/kg).Conclusion: PMQ could improve the metabolic disorders and concentric hypertrophy significantly in MSG-IO mice.Part Ⅱ Effect of PMQ on cardiac remodeling in TAC induced pressure overload mice modelPurpose: To investigate the effect of PMQ on cardiac remodeling in TAC induced pressure overload mice model.Methods: Male C57BL/6 mice were adaptive fed for 1wk,then were randomly divided into 6 groups: sham,TAC,TAC+Vehicle,TAC+PMQ 5,TAC+PMQ 10 and TAC+PMQ 20 mg/kg.Transverse aortic constriction(TAC)was created to induce pressure overload model.Cardiac echocardiography was performed at the end of 2wk after operation to detect LVAW,LVPW and IVS which reflecting ventricular wall thickness,to confirm the heart appeared typical concentric hypertrophy.The mice were gavage given corresponding doses of PMQ(5,10,20 mg/kg)or Vehicle(0.1% DMSO)continuous for 8wk after finishing echocardiography detection.Echocardiography was performed again at 10 wk after operation to measure the thickness of the ventricular wall and cardiac function.Weighing viscera organ weight and calculating viscera organ weight index after slaughter.H-E and Masson staining to observe the myocardial structure abnormal changes.Measuring the expression level of ANP,BNP and TGF-β1 in myocardial tissue using Real-Time PCR method.Results:(1)At 10 wk after TAC operation,ventricular wall thickness increased significantly,just the same trend as postoperative 2wk,showing the typical characteristic of cardiac concentric hypertrophy at 10 wk after TAC.Moreover,ventricular wall was stiffness,and cardiac weight index increased significantly.Myocardial structure disordered with inflammatory cells infiltration,and myocardial fiber cross sectional area and the degree of fibrosis increased significantly.Myocardial remodeling markers of ANP,BNP and TGF-β1 gene expression were upregulated significantly.There were no differences about these parameters between TAC+Vehicle and TAC group.(2)Compared with TAC+Vehicle group,PMQ inhibited ventricular wall thickening,and reduced the cardiac weight index and left ventricular mass index significantly,and improved the structure of the myocardial disorder.Myocardial fiber cross sectional area and the degree of fibrosis were also reduced.PMQ significantly downregulated the m RNA expression levels of ANP,BNP and TGF-β1 in myocardial tissue.Conclusion: PMQ treatment could have a protective effect against cardiac remodeling in TAC induced pressure overload mice model.Part Ⅲ Mechanism of PMQ on cardiac remodelingPurpose: to explore the mechanisms of PMQ protecting against cardiac remodeling.Methods:(1)MSG-IO model and TAC model: Detecting oxidative stress markers of 4-HNE and 8-OHd G by using immunohistochemical method,and testing Nrf2 nuclear transfer by using immunofluorescence staining.Extracting total protein or plasma/nucleoprotein from myocardial tissue to detect protein expression of antioxidants Sestrins and Nrf2/ keap1 pathway by Western blot.(2)Neonatal rat cardiomyocytes hypertrophy model: ISO(10 μM,24 h)induce cardiomyocytes hypertrophy,pretreatment with different concentrations of PMQ(0,1,3,10 μM)for 1h.Cytoskeleton protein α-Actinin were stained to show cell morphology by immunofluorescence staining.Hypertrophy markers ANP,BNP and β-MHC expression were detected by Real-time PCR.The expression of antioxidants Sestrin2 and Nrf2 / keap1 pathway protein were determined by Western blot.After silencing Sestrin2 by using Si RNA,detecting the same indexes as above with the same method,to determine Sestrin2 is the key target mediating the effect of PMQ on cardiac remodeling.Results:(1)MSG-IO model: Compared with MSG-IO group,PMQ significantly increased the protein expression of Sestrin 1/2,HO-1,and reduced keap1 protein expression,and at the same time facilitated the Nrf2 nuclear translocation.(2)TAC model:(1)Compared with sham group,4-HNE and 8-OHd G expression were significantly increased in TAC model group,and protein expression of Sestrin2,Nrf2 and HO-1 decreased,while keap1 protein expression increased significantly.There were no statistic differences between TAC and TAC+Vehicle group;(2)PMQ rectified the abnormal expression of Sestrin2,Nrf2 / keap1 and HO-1,yet it failed to stimulate Sestrin1 protein expression.(3)Neonatal rat cardiomyocyte hypertrophy model: ISO treatment for 24 h to stimulate neonatal rat cardiomyocyte hypertrophy,it showed that cell area increased,and m RNA expression of ANP,BNP and β-MHC were higher,and the protein levels of Sestrin2,Nrf2 and HO-1 decreased obviously,while keap1 protein expression increased significantly.PMQ(1,3,10 μM)intervention could reverse the ISO-induced changes significantly,which coincide with the animal experiment results in vivo.Silencing Sestrin2 with Si RNA weakened the effect of PMQ on cardiomyocyte hypertrophy and its regulating signaling pathways of Nrf2 / keap1.Conclusion: The protecting effect of PMQ on cardiac remodeling was Sestrin2 dependent,at least partially by upregulating Sestrin2 and enhancing the function of Nrf2 / keap1 endogenous antioxidant system.