The Role And Mechanism Of NEUraminidase 1 In Diabetic Cardiomyopathy

BackgroundIt is estimated that by 2040,diabetes will affect 10% of the world’s population.One of the main complications of diabetes is diabetic cardiomyopathy(DCM),which can progress to heart failure.DCM is an important cause of global heart failure and an important burden of national medical expenses.In DM patients,cardiovascular disease is the main cause of death.The pathophysiology of DCM is complex,including exposure of the heart to a hyperglycemic diabetic environment and increased fatty acids and cytokines.Hyperglycemia enhances the glucamide modification of cardiomyocyte proteins,and this change is an undesirable change.The increase in advanced glycation end products can also have deleterious effects.DM autonomic neuropathy is closely related to hyperglycemia.Exposure to increased lipid levels(including FA and triglycerides)can lead to increased accumulation of fat droplets in cardiomyocytes,thereby mediating cardiac lipotoxicity.Decreased insulin signal is a sign of T1 DM and T2 DM.Other signal cascades are altered,including decreased AMPK(AMP activated protein kinase)signal and increased PKC(protein kinase C)and MAPK(mitogen activated protein kinase)signals,leading to heart structure and the function has changed.DCM initially manifests as an isolated diastolic dysfunction,but it develops into a systolic dysfunction characterized by various neurohumoral pathway and metabolic disorders over time.In T2DM-related diabetic cardiomyopathy,coronary microvascular inflammation and its paracrine effect on cardiomyocytes and endothelial cells mediate left ventricular centripetal remodeling and hypertrophy,increase ventricular stiffness,and lead to diastolic dysfunction.Neuraminidase(NEUs),are a family of enzymes that can cleave sialic acid on the cell surface.Neuraminidase,as a key enzyme that regulates the state of sialylation,not only plays an important role in infectious diseases,but also plays a key role in other types of diseases.NEU1 is involved in regulating the metabolic behavior and signal transmission of various cells in the body,and affects the development of related diseases,such as respiratory system disease,cardiovascular disease,blood system disease,nervous system disease,and cancer.High expression of NEU1 in monocytes and macrophages contributes to the development of atherosclerosis and exacerbates plaque inflammation.Down-regulation of NEU1 reduces very low density lipoprotein and alleviates atherosclerosis.There are few studies on the role of NEU1 in cardiomyocytes.In addition,compared with healthy controls,neuraminidase activity in the plasma of patients with myocardial infarction increased.For cardiomyocytes,it has been reported that abnormal sialylation or desialylation of the cell membrane can cause cardiac ion channel dysfunction,leading to arrhythmia.NEU1 may be acts as an emerging target for the treatment of cardiovascular diseases.This study aims to establish a mouse diabetic cardiomyopathy model and evaluation criteria,explore the effect of NEU1 on DCM,explore the mechanism of NEU1 in in vivo and in vitro experiments,and design reversal experiments to verify the specific mechanism of NEU1 on DCM.Methods:Part OneAnimal experiment: select 8-10 weeks old(weight 20-25g)sterile male C57BL/6J mice,the pre-experimental mice are divided into control group and streptozocin(STZ)group,STZ group received intraperitoneal injection of STZ at a dose of 50mg/kg/d for 5 days.The control group was injected with citric acid buffer of the same volume for the same days.In the 16 th week,the samples will be collected.Western blotting,enzyme linked immunosorbent assay(ELISA)and immunofluorescence staining + wheat germ agglutinin(WGA)were used to co-localize and detect the expression and localization of NEU1 in the heart tissues of the two groups of mice.The second animal experiment grouping: Sterile male C57BL/6J mice aged 8-10weeks(body weight 20-25g)were injected with adeno-associated virus(AAV9-sh RNA or AAV9-sh NEU1)in situ in the myocardium.Four weeks later,STZ or citrate buffer was injected Construct the T1 DM model.Cardiac ultrasound and hemodynamics were used to monitor and analyze the changes in the heart function of each group of mice at specific time points,and then pathology and molecular biology tissue materials were taken.The transfection efficiency of AAV-sh NEU1 was assessed by Western blot.Cardiac tissue PSR staining,Western blotting,RT-PCR(Reverse Transcription-Polymerase Chain Reaction)technology and immunofluorescence staining for α-SMA to detect the level of fibrosis;Western blot,RT-PCR and immunohistochemical staining of heart tissue to detect the level of inflammatory factors and inflammatory cell infiltration in heart tissue;Terminal deoxynucleotide transferase-mediated d UTP nick end labeling(TUNEL)staining,immunohistochemical staining and western blotting were used to detect the apoptosis levels of mouse heart tissues in each group;The expression levels of p67 phox and superoxide dismutase(SOD)protein was detected by Western blotting,and the corresponding ELISA kit detects the content of malondialdehyde(MDA),reduced glutathione(GSH),oxidized glutathione(GSSG),SOD activity,and4-hydroxynonenal(4-HNE)in myocardial tissue,RT-PCR detection of m RNA levels of NADPH oxidase subunit in myocardial tissue.Cell experiment: Isolate SD rat neonatal rat heart and extract Neonatal rat ventricular myocytes(NRVMs),culture NRVMs in a medium containing 15% FBS for 24 hours,and then switch to serum-free low-sugar medium;Inoculate in 6-well plate or 24-well plate according to experimental requirements.The 24-well plate was divided into two groups: the PBS group was cultured with a medium with a glucose concentration of 5.5 m M for 24 hours,and the HG group was cultured with a medium with a glucose concentration of 33 m M for the same time as the PBS group.Subsequently,cell immunofluorescence staining for NEU1 was performed.The6-well plate is divided into 4 groups: the PBS group is the same as described above;with the different culture time of the high-sugar medium,it is divided into the HG 6h group;the HG 12 h group;the HG 24 h group.Subsequently,cell protein was extracted and immunoblotting method was used to detect the expression of NEU1 protein in cells at different time points.Three types of AAV9-shNEU1(namely AAV9-shNEU1 #1,#2,#3)were used to transfect NRVMs,and RT-PCR was used to detect cell NEU1 m RNA levels.Part TwoCell experiment: According to experimental needs,the grouping method is as follows.The first grouping was used to silence SIRT3 with small interfering RNA to verify the effect of SIRT3: PBS group;sh NEU1 group;HG group;HG+sh NEU1group;si SIRT3 group;si SIRT3+sh NEU1 group;si SIRT3+HG group;si SIRT3+sh NEU1 +HG group.Western blotting was used to detect the transfection efficiency of si SIRT3.ROS levels were detected in each group,and RNA was extracted for RT-PCR to detect m RNA levels of gp91,p67,p22,BAX,Bcl-2,IL-6,TNF-α and MCP-1 genes.Cell Count analysis(CCK-8)was used to detect the Cell viability of each group.The ELISA kit detects cell MDA level and SOD activity.The second grouping was applied to H2O2 to induce cell death: PBS group;sh NEU1 group;H2O2 group;H2O2+sh NEU1 group;si SIRT3 group;si SIRT3+sh NEU1 group;si SIRT3+ H2O2 group;si SIRT3+sh NEU1+ H2O2 group.CCK-8 detects the cell viability level of each group.The third grouping was applied to silencing AMPKα with small interfering RNA to verify the role of AMPKα: HG group: HG+sh NEU1 group;HG+si AMPKα group;HG+sh NEU1+si AMPKα group.Western blotting was used to detect the transfection efficiency of si AMPKα and the influence of AMPKα on the expression of SIRT3 and SOD2 proteins.ROS level was detected in each group,RNA was extracted for RT-PCR to detect m RNA level of IL-6 TNF-α gene,SOD activity was detected by ELISA kit,and cell viability was detected by CCK-8 kit.The fourth group is applied to the inhibitors of the three agonists upstream of AMPKα(namely Takinib(a potent and selective transforming growth-factor-β-activated kinase-1(TAK1)inhibitor),STO-609(a selective Ca2+/calmodulin dependent protein kinases(Ca MKKβ)inhibitor)and Liver kinase B1(LKB1)small interfering RNA(si LKB1)): PBS group;HG group;HG+sh NEU1group;HG+sh NEU1+si LKB1 group;HG+sh NEU1+STO-609 group;HG+sh NEU1+Takinib group;Western blotting was used to detect the effects of various inhibitors on the activation level of AMPKα and the expression of SIRT3SOD2 protein.CCK-8 was used to detect the cell viability level of each group.Animal experiment: select SPF C57BL/6J male mice and AMPKα-KO male mice aged 8W and weighing between 23.5-27.5,grouped as follows: WT+Saline+sh RNA group;WT+Saline+sh NEU1 group;WT+STZ+sh RNA group;WT+STZ+sh NEU1group;KO+Saline+sh RNA group;KO+Saline+sh NEU1 group;KO+STZ+sh RNA group;KO+STZ+sh NEU1 group.Ultrasound and hemodynamics were used to detect the heart function of mice,and RT-PCR was used to detect the gene expression levels of TGF-β,COL 1,IL-6,TNF-α,BAX and BCL-2 in the heart tissue of each group of mice.Results:Part oneThe pre-experimental immunoblotting results showed that NEU1 was significantly increased in the hearts of diabetic mice.The ELISA results showed that STZ treatment activated the expression of NEU1 in the mouse hearts.The co-localization of immunofluorescence staining and WGA staining showed that NEU1 was mainly located in cardiomyocytes.The immunoblotting results of HG-stimulated NRVMs showed that the expression of NEU1 was up-regulated with the time of HG stimulation,and immunofluorescence staining also proved that the expression of NEU1 was significantly up-regulated in the HG environment.Three kinds of AAV-sh NEU1 were used to transfect NRVMs,and it was found that #2 AAV9-sh NEU1 inhibited the NEU1 m RNA level most significantly,so the subsequent studies all used #2 AAV9-sh NEU1.Detection of the expression of NEU1 in myocardial tissue found that the transfection of sh NEU1 significantly reduced the expression of NEU1 protein in the heart.After STZ induced diabetes,the blood glucose level,insulin content,Hb A1 c and body weight of the mice showed different degrees of adverse changes,but inhibiting the expression of NEU1 did not significantly improve the above indicators.The left ventricular ejection fraction(LVEF)and short axis shortening rate(FS)of the diabetic mice were significantly reduced,and the left ventricular end diastolic diameter(LVEDD)was significantly increased,but the inhibition of NEU1 significantly reversed the adverse changes in the above indicators.In addition,NEU1 inhibits and improves the ±dp/dt and E/A values of the heart in diabetic conditions.The results of PSR staining,immunofluorescence staining and RT-PCR found that the level of cardiac tissue fibrosis in diabetic mice was significantly increased,and the treatment of sh NEU1 could significantly reduce the level of cardiac fibrosis in mice caused by STZ.STZ treatment significantly increased the phosphorylation levels of p65 and IκBα,and increased the degradation of IκBα,but sh NEU1 treatment could significantly down-regulate the phosphorylation levels of p65 and IκBα.RT-PCR results showed that the m RNA levels of various inflammatory factor genes in myocardial tissue were significantly increased in the diabetic state,but the inhibition of NEU1 could down-regulate the m RNA levels of these genes.The results of immunohistochemical staining showed that the infiltration of inflammatory cells in diabetic cardiomyopathy increased significantly,and sh NEU1 can significantly reduce the migration and infiltration of inflammatory cells.Tunel staining results showed that the Tunel positive signal of diabetic cardiomyopathy myocardial tissue was significantly increased,and sh NEU1 could significantly reduce the Tunel positive signal.Immunohistochemical staining showed that NEU1 knockdown significantly inhibited the expression of C-caspase3 in myocardium under the condition of DM.Western blot results further showed that NEU1 knockdown significantly increased the expression of BCL-2 protein and down-regulated the expression of BAX and C-Caspase3 protein in myocardium induced by STZ.The results of western blotting showed that the expression level of p67 phox protein in the diabetic group was significantly increased,and the protein expression of corresponding SOD2 was significantly reduced,and sh NEU1 could reverse the expression of the two proteins.The level of GSH in DM was significantly decreased,and the GSSG in myocardial tissue was significantly increased.Sh NEU1 could increase the decrease of GSH induced by STZ and decrease the increase of GSSG induced by STZ.The SOD activity of myocardial tissue in the diabetic group decreased significantly,and sh NEU1 can increase the SOD activity in DCM.In addition,sh NEU1 can also significantly reduce lipid peroxide levels.PCR results showed that sh NEU1 reduced the up-regulation of p67 phox,p22phox and gp91 phox m RNA in the myocardial tissue of diabetic mice induced by STZ injection.Part TwoThe results of western blot and PCR showed that the m RNA and protein expression levels of SIRT3 in diabetic myocardial tissues were significantly reduced,while sh NEU1 could increase the m RNA and protein expression levels of SIRT3 in myocardial tissues in diabetic conditions.In vitro experiments,high glucose stimulation increased NRVMs ROS levels,as well as p67 phox,p22phox and gp91 phox m RNA levels.Sh NEU1 treatment significantly reduced oxidative stress levels induced by high glucose stimulation,but in si SIRT3 transfected cells,sh NEU1 inhibited ROS production induced by high glucose,and did not significantly reduce the m RNA levels of p67 phox,p22phox and gp91 phox.In addition,high glucose stimulation can lead to increased expression of inflammatory factors in NRVMs cells,such as IL-6,TNF-α and MCP-1 m RNA levels in HG group significantly increased,but after treatment with si SIRT3,sh NEU1 treatment did not decrease the m RNA levels of IL-6,TNF-α and MCP-1 induced by HG.After 24 h HG stimulation,NRVMs cell viability was significantly decreased,while NEU1 inhibition could significantly improve cell viability under HG environment.However,sh NEU1 lost the effect of improving cell viability after application of si SIRT3.HG stimulation down-regulated BCL-2 m RNA level and up-regulated BAX m RNA level in NRVMs cells.However,NEU1 inhibition can reverse these unfavorable changes,but the protective effect of NEU1 knockdown on cells from HG-induced apoptosis is eliminated by SIRT3 silencing.At the same time,SIRT3 silencing completely eliminated the protection of sh NEU1 against H2O2-induced apoptosis.Sh NEU1 treatment significantly down-regulated the high level of MDA induced by HG and the decreased activity of SOD induced by HG up-regulation,but silencing SIRT3 made sh NEU1 lose its beneficial effect on NRVMs lipid peroxidation and oxidative damage stimulated by HG.The results of western blotting showed that shNEU1 treatment increased the phosphorylation level of AMPKα in cardiomyocytes caused by HG,and activated AMPKα.After sh AMPKα was used,the expression level of AMPKα was about 8times lower.Western blot results showed that when sh AMPKα was not used,sh NEU1 treatment could up-regulate the protein expression levels of SIRT3 and SOD2,but when sh AMPKα was used,sh NEU1 treatment could not increase the down-regulation of SIRT3 and SOD2 protein expression levels caused by HG.Consistent with the down-regulation of SIRT3,after sh AMPKα was used,NEU inhibition could not significantly reduce the up-regulation in ROS level caused by HG,and could not increase the downregulation in SOD activity caused by HG.Without sh AMPKα,NEU1 inhibition can significantly reduce the high expression levels of IL-6 and TNF-α m RNA caused by HG,and improve cell viability,but sh NEU1 loses these protective effects after sh AMPKα is used.There was no statistical difference in LVEF and LVFS between the STZ+Vehicle group and STZ+sh NEU1 group of AMPKα-KO mice.In AMPKα-KO mice,sh NEU1 could not significantly down-regulate the levels of TGF-β and col I m RNA in the myocardial tissue of diabetic cardiomyopathy.In AMPKα-KO mice,sh NEU1 treatment could not significantly down-regulate the levels of inflammatory factors such as TNF-α and IL-6 in DCM myocardial tissue.In AMPKα-KO mice,NEU1 inhibition did not significantly down-regulate the expression level of BAX m RNA and up-regulate the expression level of BCL-2 m RNA.The results of immunoblotting showed that the phosphorylation level of AMPKαin the si LKB1 group was significantly reduced,while the phosphorylation level of AMPKα in the Takinib group and the STO-609 group was not significantly changed compared to the HG+sh NEU1 group,but compared with the AMPKα in the si LKB1 group.There was a significant difference in phosphorylation levels;after knocking down LKB1,the protein expression levels of SIRT3 and SOD2 did not increase in the sh NEU1 treatment group.In addition,the proteins of SIRT3 and SOD2 in the Takinb group and the STO-609 group were not significantly different from the HG+sh NEU1 group,but they were significantly different from the si LKB1 group.In addition,the cell viability test found that after the addition of si LKB1,sh NEU1 could not improve the cell viability induced by HG,while after the addition of Takinb and STO-609,sh NEU1 treatment could still improve the cell viability in the HG environment.Conclusion1.The expression of NEU1 is significantly increased in diabetic cardiomyopathy,and it is mainly located in cardiomyocytes.2.NEU1 deletion can significantly improve the levels of cardiac dysfunction and myocardial fibrosis,inflammation,apoptosis and oxidative stress in diabetic conditions.3.NEU1 deletion may activate AMPK-SIRT3 signaling pathway through LKB1,thereby improving the pathological state of myocardial tissue or myocardial cells.