| BackgroundDiabetic cardiomyopathy(DCM)is a kind of serious complication of diabetes mellitus and damages to the life quality of diabetic patients.The principal manifestations of DCM are left ventricular(LV)hypertrophy,LV systolic and diastolic dysfunction in the absence of hypertension,coronary heart disease as well as heart valve disease.The pathogenesis of diabetic cardiomyopathy is complicated,including oxidative stress,interstitial fibrosis and myocardial apoptosis.Above all,chronic hyperglycemia is the most prominent pathophysiological stimuli involved in the development of DCM.However,the molecular mechanisms of chronic hyperglycemia induced cardiomyocyte apoptosis remain to be elucidated.The heart is composed mostly of cardiomyocytes,non-cardiomyocytes(including cardiac fibroblasts,endothelial cells and vascular smooth muscle cells)and extracellular matrix(ECM).Chronic hyperglycemia leads to increased apoptosis of cardiomyocytes and collagen synthesis of cardiac fibroblasts,resulting in accumulation of ECM,ventricular remodeling,and eventually heart failure.Although there have been a lot of studies about DCM,it is still necessary to further explore the specific pathogenesis and the molecular therapeutic targets of DCM.Pleckstrin homology(PH)domain leucine-rich repeat protein phosphatase 1(PHLPP1),a newfound serine/threonine phosphatases,is a member of the type 2C phosphatase(PP2C)family.It is well known that PHLPP1 plays a significant role in suppressing cell survival.And mounting evidences support the fact that PHLPP1 could directly dephosphorylate the hydrophobic motif of Akt(Ser473)which would lead to the inhibition of kinase activity and suppress the occurrence and progression in several cancers,such as colon cancer,prostate cancer,pancreatic cancer and lymphoma.Moreover,a previous study stated that impaired insulin action could increase PHLPP1 level in the adipose and muscle tissues of obese patients.While many studies have studied the function of PHLPP1,the role of PHLPP1 in DCM has not been reported yet.The PI3K/Akt/mTOR signaling pathway is central to regulating cell transcription,metabolism,survival and inflammation.It is well established that during diabetes,defective insulin signaling down-regulates the activity of PI3K,leading to Akt and mTOR inactivation.Previous studies have demonstrated that Akt is closely related to PHLPP1 in cell survival and inflammation.However,the role of the PHLPP1 in PI3K/Akt/mTOR signal pathway in the process of DCM has not been investigated.Here,we hypothesized that PHLPP1 inhibition is likely to have a protective effect in DCM.To make clear the role of PHLPP1 in the development of DCM,we investigated in primary rat cardiomyocytes as well as H9c2 cells stimulated by high glucose in vitro and in a DCM rat model in vivo.ObjectiveTo investigate the expression level of PHLPP1 in DCM and to explore the function and mechanism of PHLPP1 in DCM through inhibition of PHLPP1 by shPHLPPl lentivirus.To investigate the effect and mechanism of PHLPP1 expression in primary cardiomyocytes and H9c2 cells under HG stimuli.To study whether inhibition of PHLPP1 in the HG-treated cardiomyocytes can alleviate apoptosis induced by HG stimuliMethods1.AnimalsAfter adaptive feeding with standard diet for one week,sixty male 4-week old Sprague-Dawley(SD)rats were randomly allocated into the following four groups(n=15 for each group):control,diabetes mellitus(DM),DM+shRNA-negative control(shN.C),and DM+shPHLPP1.All of the model rats were intraperitoneally injected with a high-dose of streptozotocin(60mg/kg,STZ,Solarbio,China)dissolved in 0.5 mL citrate buffer(pH 4.5)in order to inducing type I diabetes.While the control rats were administered with 0.5mL citrate buffer.12 weeks after diabetes successful built,an amount of 1×108 UT/50?L of lentivector with PHLPP1 shRNAor 50?L lentivehicle was injected into the jugular vein.4 weeks after PHLPP1 shRNA injection,the left ventricular function of rats were detected through echocardiography before sacrifice.All experiments were in compliance with animal protocols approved by the Shandong University Animal Care Committee.2.Real time RT-PCRTotal RNA samples of rat hearts were extracted with the help of TRIzol reagent(Ambion).qRT-PCR were conducted making use of the PrimescrioptTM RT reagent kit with gDNA Eraser(TakaRa).We used the 2-??CT method in order to calculate the fold changes of BNP and ?-MHC.3.Histology and immunohistochemistryThe heart sections of all rats were stained with hematoxylin and eosin(H&E)to evaluate the width of cardiomyocyte.We perfomed the masson saining as well as sirius red staining to observe the collagen deposition in heart.Immunohistochemistry was conducted to detect of expression of PHLPP1,collagen ? and collagen ?.4.Acquisition of cellsPrimary neonatal rat cardiomyocytes(NRCMs)were obtained from neonate SD rats(2 to 3 days old,Shandong University).NRCMS were isolated by differential adhesion and purified by chemical inhibition.The H9c2 cell line was purchased from ATCC.5.Cell cultureNRCMs were cultured in DMEM ?g/L glucose)with 8%horse serum,5%calf and 0.1?mol/L Brdu.H9c2 cells were cultured in DMEM(lg/L glucose)with 10%fetal bovine serum(FBS).After starvation,cells were incubated in normal glucose(NG,5.5 mM glucose)or high glucose(HG,33.3 mM glucose)and harvested at 6,12,24 and 48 h of HG stimulation.In order to investigate the function of PHLPP1 on cardiomyocytes apoptosis,we transfected PHLPP1-siRNA plasmids into H9c2 cells 24 h prior to HG stimulation.Moreover,we used LY294002(a specific PI3K inhibitor,25?mol/L)to explore the potential role of PHLPP1 on PI3K/Akt/mTOR pathway in HG stimulated H9c2 cells.6.Immunofluorescence microscopyAfter fixed in 4%paraformaldehyde for 20min at room temperature,NRCMs and H9c2 cardiomyoblasts were permeabilized,blocked and then incubated with rabbit anti-PHLPP1 overnight at 4?.In the next day,after rewarming,we treated cells with a secondary antibody 30 min at 37? and used the Prolong Gold Anti-Fade Reagent with DAPI to seal cells.Finally,the distribution and expression of PHLPP1 in NRCMs and H9c2 cells could be observed under an immuno-fluorescence microscopy.7.Measurement of ROSDCFH-DA was used to observe ROS production.HG+NAC group was exposed to NAC(3mmol/L)2h before HG stimuli.8.TUNEL assayTUNEL assay were performed to detecte the apoptotis rate of cells in myocardium and H9c2 cells.The apoptotic cells were observed via an immunofluorescence microscopy.9.Western blot analysisWe extracted total protein from freshly dissected rat hearts,NRCMs and H9c2 cells.The BCA Protein Assay Kit was used to measure the concentration of extracted protein.After gel electrophoresis,transmembrane and antibody incubation,we detected the expression level of the target protein.10.Statistical analysisEach experiment was performed at least 3 times.All statistical analysis were carried out using SPSS.Data were reported as means±standard deviation Differences between two groups were performed by unpaired t test,and multiple groups involved one-way ANOVA.p<0.05 was considered as statistically significant.Result1.The expression of PHLPP1 was upregulated in diabetic rat hearts,and PHLPP1 inhibition alleviated cardiac remodeling in diabetes.As demonstrated by Western blotting,the protein level of PHLPP1 was much higher in the diabetic rat hearts than that in controls.While treatment with shPHLPP1 reduced the expression of PHLPP1 in DM+shPHLPP1 group.The pictures of rat hearts showed that the diabetic rats had enlarged heart with spherical apex of the left ventricular.And the myocardial cell diameter of DM group was obviously increased compared with the normal rats.PHLPP1 inhibition could effectively restored the increment of cardiac morphology and the diameter of myocardial cell.What's more,qRT-PCR suggested that PHLPP1 ihbition could down regulate the increment of ?-MHC and BNP in diabetic rat hearts.To sum up,diabetic rat had remodeled heart and upregulated myocardial PHLPP1 expression.While PHLPP1 inhibition could effectively alleviate the remodeling of diabetic rat hearts.2.PHLPP1 inhibition improved left ventricular function of diabetic rats.To explore the effect of PHLPP1 inhibiton on left ventricular function,we conducted echocardiographic measurement.It was found that the FS and LVEF was significantly decreased in DM group,and E/A of mitral valve as well as e'/a'was also fall compared with the control group.However,PHLPP1 inhibition reversed those reduction to a certain extent.What's more,the diameter of LVED was increased in the DM group,and PHLPP1 inhibition attenuated the wall thickening in diabetic rats.3.PHLPP1 downregulation prevented diabetes-induced myocardial fibrosis.Masson staining and sirius red staining showed serious collagen deposition in the hearts of diabetes.Western blot analysis showed that with the severity of cardiac fibrosis,the expression levels of collagens I,collagen III,MMP2 and MMP9 were significantly elevated in the DM group.Whereas PHLPP1 inhibition reduced the collagen deposition as well as the expression of each protein in diabetic rat hearts4.PHLPP1 downregulation alleviated diabetes-induced cardiomyocytes apoptosis.The results of TUNEL assay showed that the rate of TUNEL-positive cells was remarkably increased in diabetic rat hearts compared with the control group.Meanwhile,the expression level of cleaved caspase-3 and Bax/Bcl-2 were also elevated in the DM group.Inhibition of PHLPP1 could reverse those increment.5.HG increased the expression of PHLPP1in NRCMs and H9c2 cells.Immunofluorescence microscopy and western blot analysis revealed that the expression of PHLPP1 was significantly increased in both NRCMs and H9c2 cells exposed to HG stimulation.6.ROS mediated HG-induced PHLPP1 overexpressionAs shown by immunofluorescence microscopy,the production of ROS was significantly increased under HG treatment.What's more,the expression level of PHLPP1 in H9c2 cells was also significantly increased under HG treatment.However,when H9c2 cells were pretreated by NAC before HG stimuli,the expression level of PHLPP1 was declined.7.PHLPP1 inhibition ameliorated HG-induced apoptosis of H9c2 cells.In HG-treated H9c2 cells,with the increased expression of PHLPP1,the expression of cleaved caspase-3 as well as the ratio of Bax to Bcl-2 were also elevated.And the results of TUNEL assay showed that the percentage of TUNEL-positive cells in HG group was remarkably increased.To explore the role of PHLPP1 inhibition in HG-induced apoptosis of H9c2 cells,H9c2 cells were transfected with PHLPP1-siRNA plasmids 24h prior to HG stimulation.As a result,PHLPP1 inhibition revesed all the changes.8.PHLPP1 inhibition attenuated HG-induced cardiomyocyte apoptosis via activating the PI3K/Akt/mTOR signaling pathway.Compared with the normal glucose group,HG treatment significantly reduced the phosphorylation of PI3K,AKT and mTOR.However,PHLPP1 inhibition revesed all the decrement in HG treated H9c2 cells.So we hypothesized that inhibition of PHLPP1 may allleviae HG-induced cardiomyocyte apoptosis via activating the PI3K/Akt/mTOR signaling pathway.To prove our hypothesis,we pretreated cells with a specific PI3K inhibitor,LY294002,to block the activation of the PI3K/Akt/mTOR signaling pathway.Results showed that the apoptosis indexes were higher in the HG+siPHLPP1+LY294002 group than the HG+siPHLPP1 group.The above results indicated that inhibition of PHLPP1 attenuated HG-induced cardiomyocyte apoptosis via activating the PI3K/Akt/mTOR signaling pathway.Conclusion(1)The expression level of PHLPP1 was significantly increased in the myocardial tissue of diabetic rats.(2)Inhibition of PHLPP1 expression could significantly ameliorate the cardiac structural changes and dysfunction caused by diabetes.(3)Diabetic rats showed changes of pathological structure.Silencing the expression of PHLPP1 could inhibit diabetes induced myocardial fibrosis and alleviate cardiomyocyte apoptosis induced by diabetes.(4)High glucose stimulation significantly increased the expression of PHLPP1 in both NRCMs and H9c2 cells.And high glucose stimulation increased the expression of PHLPP1 through promoting ROS production in cardiomyocytes.(5)Inhibition of PHLPP1 attenuated HG stimulation induced cardiomyocyte apoptosis via activating the PI3K/Akt/mTOR signaling pathway.BackgroundDiabetes mellitus(DM),a metabolic disease,can damage multiple organs and systems and lead to their dysfunction.Studies have shown that the risk of cardiovascular death in diabetic patients is significantly higher than that in non-diabetic patients.According to the "guidelines for the prevention and treatment of type 2 diabetes in China" in 2020,the prevalence of diabetes in China has reached 11.2%,and diabetes has become a major threat to national health.Since the incidence rate of diabetes has been increasing year by year,prevention and treatment of diabetes and its complications have gained more and more attention.Diabetic cardiomyopathy(DCM)refers to primary cardiac dysfunction without potential coronary heart disease,hypertension,valvular heart disease,etc.In the early stage,the main manifestations of DCM are asymptomatic myocardial fibrosis and increased left ventricular end diastolic pressure.The further the disease progresses,the more severe the myocardial fibrosis.Over time,the heart shows cardiac remodeling,left ventricular hypertrophy and HFpEF.Finally,HFpEF turns into HFrEF.Up to now,there is little effective laboratory marker of DCM,and the therapies for DCM are still mainly to control blood glucose.Dapagliflozin,the first kind of SGLT2 inhibitor listed in China,plays a hypoglycemic role through inhibiting glucose reabsorption.Its unique cardiovascular protective effect put dapagliflozin into a sharper focus.Numerous clinical studies and meta-analysis results have shown that dapagliflozin can lower blood pressure and reduce the risk of thrombosis.Although multiple researchers have demonstrated that dapagliflozin can inhibit the development of diabetic cardiomyopathy through decreasing blood glucose,anti-inflammatory,antioxidant,reducing apoptosis and inhibiting fibrosis,its specific molecular mechanism is still unclear.Studies have shown that Wnt/?-catenin pathway plays an important role in tissue fibrosis.Fibrogenic stimulation makes Wnt3 combine with frizzled receptor to form a complex,which leads to GSK-3? phosphorylation.After GSK-3?inactivation by phosphorylation,it lost the ability to degrade ?-catenin.And the excessive accumulated ?-catenin in the cytoplasm would transferred into the nucleus,binding to the transcription region of downstream factors and increasing their expression.It is reported that dapagliflozin treatment can alleviate the development of DCM through inhibiting the activation of NLRP3/ACS inflammasome.Other researchers also shown that dapagliflozin can inhibit oxidative stress by regulating the expression level of Zn2+transporter.In addition,empagliflozin can inhibit renal fibrosis through reducing the expression of Wnt.And canagliflozin can suppress hepatocellular carcinoma by down-regulating the expression of ?-catenin.Therefore,we speculate that dapagliflozin can also act on Wnt/?-catenin pathway to inhibit the fibrosis process of DCM.In this study,we established a rat model of DCM by 6-week high-sugar-fat diet(HSFD)combined with intraperitoneal injection of streptozotocin(STZ)to explore the protective function of dapagliflozin in the fibrotic process of DCM.ObjectiveTo establish a rat model of type 2 diabetes mellitus,and to explore the effect and mechanism of dapagliflozin on myocardial interstitial fibrosis in diabetic rats.To explore the effect and molecular mechanism of dapagliflozin on collagen synthesis of cardiac fibroblasts stimulated by high glucose in vitro.Method1.Animal modelForty-five SD rats(100-120g,4 weeks old)were randomly divided into three groups:control group,DM(diabetes mellitus)group and DM+DAPA(diabetes with dapagliflozin treatment)group.The control rats were given normal diet,and the diabetes rats were given high-sugar-fat diet(HSFD).Six weeks later,intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed.Rats with insulin resistance(IR)were intra-peritoneally injected with 35 mg/kg STZ buffer.One week later,the rats with FBG?11.1mmol/L were enrolled.8 weeks after STZ injection,the DM+DAPA rats were administrated with lmg/(kg·d)dapagliflozin through drinking water.IPITT,IPGTT and echocardiography were performed after 8 weeks of dapagliflozin intervention.Then,all rats were euthanized,and the hearts were taken for subsequent molecular biology experiments.Animal experiments were conducted in accordance with the requirements of animal ethics committee of Shandong University.2.EchocardiographyThe fractional shortening(FS),left ventricular ejection fraction(LVEF),peak E,peak A,early(e'),late(a')and the left ventricular end-diastolic dimension(LVEDd)were measured using the Vevo 770 imaging system with the RMB710 transducer(VisualSonics,Toronto,Canada).3.Serological testThe fasting blood glucose(FBG)was measured after fasting for 8 hours.After euthanasia,the blood was drawn from cardiac apex to detect the serum triglyceride(TG)and the total cholesterol(TC).4.Heart specimenThe body weight and heart weight of rats were weighed,and the heart size was measured.After fixation,2-3 mm heart tissue was transected at the level of mitral valve for paraffin embedding.The slicing procedure is the same as the first part.5.Histological experimentThe heart sections were stained with H&E to evaluate the thickness of ventricular wall and the diameter of myocardial cells.Masson and Sirius red staining were used to evaluate the level of fibrosis.Collagen ?,collagen ? and other indicators were went on immunohistochemical staining to evaluate the level of collagen deposition in myocardial interstitium.6.Cell treatmentCardiac fibroblasts(CFs)were isolated from 2-3 days old SD rats and cultured in low glucose DMEM(5.5 mM)with 10%fetal bovine serum.The experiments were carried out when CFs were at the forth generation to the eighth.7.Western blotThe cells were treated as follows:(1)LG(low glucose,5.5mmol/L);(2)HG(high glucose,33.3mmol/L);(3)HG+low concentration dapagliflozin(DAPA1,3 ?mol/L dapagliflozin);(4)HG+high concentration dapagliflozin(DAPA2,10?mol/L dapagliflozin).The protein were extracted from rat myocardium or cardiac fibroblasts.And the expression levels of collagen ?,collagen ?,wnt3,?-Catenin,p-GSK-3? and GSK-3? were detected by Western blot.9.Data analysisThe data were processed by SPSS and Graphpad.Continuous variables were expressed as mean±SD.The comparison between two groups was performed by unpaired t test.One way ANOVA was used for the comparison among multiple groups.There was statistical difference when p<0.05.Result1.Dapagliflozin improves glucose metabolism in diabetic rats.After 6 weeks of high-sugar-fat diet(HSFD),the area under curve(AUC)of IPGTT and IPITT in the model group were significantly higher than those in the control group.At the end of the experiment,DM rats showed polydipsia,polydipsia and polyuria.The area under curve(AUC)of IPGTT and IPITT in DM group were significantly higher than those in the control group.Moreover,the FBG,TG and TC of DM rats were significantly higher than the normal rats.After 8 weeks treatment with 1 mg/(kg·d)of dapagliflozin,the FBG and TG of diabetic rats were significantly decreased,and the AUC of DM+DAPA group was significantly lower than that of the DM group,but the effect of dapagliflozin on TC was not significant.2.Dapagliflozin alleviates diabetes induced cardiac hypertrophy and dysfunction.HE staining showed that the diameter of myocardial cells in DM rats was significantly higher than that in the control group,which could be alleviated by dapagliflozin treatment.Compared with the control group,the FS,LVEF and E/A,e'/a' of DM rats were significantly decreased at the end of the experiment,while LVEDd was significantly increased;the changes of the above indexes were reversed to a certain extent after 8 weeks of treatment with dapagliflozin.3.Dapagliflozin alleviates diabetes-induced myocardial fibrosis.Masson and sirius red staining identified the area of interstitial fibrosis.The ratio of collagen deposition area to total tissue area was increased in the untreated DM group,and dapagliflozin treatment decreased the percentage of interstitial fibrosis in diabetic rat hearts.Both immunohistochemical staining and western blot analysis showed that the expression of collagen ? and collagen ? were increased in diabetic rat hearts,whereas dapagliflozin can reduce those abnormal expression to a certain extent.4.Dapagliflozin attenuates diabetes-induced myocardial fibrosis via blockage of the Wnt/?-catenin signaling pathway.The expression level of ?-catenin and p-GSK-3? were increased significantly in diabetic rat hearts.Treatment with dapagliflozin significantly decreased ?-catenin and p-GSK-3? in comparison with the untreated diabetic group5.Dapagliflozin attenuates HG-induced collagen production in primary cardiac fibroblasts.Western blotting showed that 48h HG treatment increased the expression of collagen ? and collagen ? in CFs.Nevertheless,Dapagliflozin at 3 ?mol/L and 10?mol/L can significantly alleviate HG-induced collagen production in primary cardiac fibroblasts.6.Dapagliflozin decreased the activity of the Wnt/?-catenin signaling pathway in primary cardiac fibroblasts exposed to HG.Western blotting analysis showed that HG stimuli could activate the Wnt3/?-catenin signaling pathway,resulting in up-regulated expression of ?-catenin and p-GSK-3? in CFs.Compared with HG group,the expression level of p-GSK-3?and ?-catenin were decreased when pretreated with dapagliflozin at both 3?mol/L and 10?mol/L.Conclusion(1)Dapagliflozin has a protective effect on diabetic cardiomyopathy through inhibiting fibrosis.(2)Dapagliflozin can protect diabetic rat hearts from fibrosis through decreasing the activity of the Wnt/?-catenin pathway.(3)Dapagliflozin can attenuate HG-induced collagen synthesis of CFs through decreasing the activity of the Wnt/?-catenin signaling pathway. |
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