| Aim:Heart failure,as the last stage of the development of various heart diseases,has the characteristics of poor prognosis,high hospitalization rate,and high mortality.China has entered an aging society,and the prevalence of heart failure has reached 1.3%.In the next few decades,the number of patients with heart failure will increase rapidly,and it has become a worldwide concern.The heart is the largest energy-consuming organ in the body,and mitochondria are the main source of energy.The stability of mitochondrial function is particularly important for maintaining the normal physiological function of the heart.During heart failure,the mitochondrial structure of the cell changes,the activity of respiratory chain enzymes increases,and the function decreases.The dysfunction of the mitochondria leads to impaired mitochondrial energy and increased oxidative stress.At the same time,the opening of mitochondrial membrane transition holes promotes cell death and myocardial weight.This study establishes a model of chronic heart failure induced by myocardial infarction through coronary artery ligation in rats,and observes the effect of Liguzinediol(LZDO)on the heart function of chronic heart failure rats,and to determine whether it can improve heart function by improving the function of cardiomyocytes’ mitochondria.The effect is to provide a theoretical basis for the clinical treatment of heart failure in LZDO.Methods1.Chronic heart failure rat model replication and LZDO interventionCoronary artery ligation was used to reproduce the model of congestive heart failure induced by myocardial infarction.Sixty male SD rats were randomly divided into a sham operation group,a model group,a positive drug TMZ(10 mg/kg)group,and a low,medium,and high dose of LZDO 5,10,20 mg/kg group,each group of 10 only.Eight weeks after the administration,the echocardiogram of each group of rats was measured,and the hemodynamics were recorded.The left ventricular systolic pressure(LVSP)and left ventricular end-diastolic pressure(LVEDP)of LZDO,Maximum left ventricular pressure rise rate(+dp/dtmax),maximum left ventricular pressure fall rate(-dp/dtmax),systolic blood pressure(SBP),diastole blood pressure(DBP)and mean arterial pressure(MAP)were observed.Weigh the total heart mass(HM)and the left ventricular mass(including ventricular septum)(LVM),and calculate the heart mass index(HMI)and left ventricular mass index(LVMI).The effect of LZDO on myocardial tissue morphology in rats with heart failure was observed by HE staining and Masson staining.LZDO was used to detect myocardial damage markers such as brain natriuretic peptide(BNP),Rat creatine kinase(CK)and troponin(cTn I).2.Effect of LZDO on mitochondria in cardiomyocytes of rats with chronic heart failureThe kit was used to detect the expression of superoxide dismutase(SOD)and malondialdehyde(MDA)in myocardial tissue,and to observe the effect of LZDO on the mitochondrial antioxidant capacity of heart failure rats;Death,Western blot detection of myocardial tissue apoptosis-associated protein B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cysteine Protease 3(Cysteineprotease 3,Caspase 3)to observe the effect of LDZO on cardiomyocyte apoptosis;Western blot detection of myocardial tissue mitochondrial fusion mitochondrial fusion 1(Mitofusin 1,MFN1),mitochondrial fusion factor 2(Mitofusin 2,MFN2),mitochondrial division Protein 1(Fissionl,Fis1),Dynamin-related protein 1(Dynamin-related proteinl,Drp1)expression to observe the effect of LDZO on myocardial mitochondrial dynamics in heart failure rats;kit to detect Adenosine triphosphate(ATP)1.The level of free fatty acid(FFA)was used to observe the effect of LDZO on the energy metabolism of myocardial tissue in rats with heart failure;Western blot was used to detect the phosphorylated AMP-activated protein kinase(p-AMPK),AMP-activated protein kinase(AMPK),CD36,peroxisome proliferator-activated receptor coactivator 1α(PGC1α),Silent mating-type information regulation 2 homolog 3(SIRT3),carnitine palmitoyl transferase 1(CPT1)expression and glucose transport pathway related protein phosphatidylinositol 3(phosphatidylinositol 3-kinase,PI3K),phosphorylated phosphatidylinositol 3-kinase(p-PI3K),protein phosphatase 2(PP2),protein phosphatase 1(PP1),protein kinase B(Akt),phosphorylated protein kinase B(Phosphorylated protein kinase B,p-Akt)3.Effects of Liguzinediol on Mitochondria in Hypoxic and Hypoglycemic CardiomyocytesFirst,the co-culture of cobalt chloride(CoCl2)and sugar-free medium was used to replicate the cardiomyocyte hypoxia and hypoglycemia model,and MTT was used to determine the concentration of injured cardiomyocytes by 50%.Then use different concentrations of LZDO on H9C2 cells for 24 hours to determine whether LZDO is toxic to normal cardiomyocytes.Based on this,LZDO is screened for high,medium and low concentrations that protect against hypoxia and hypoglycemia H9C2 cell damage.Cells were divided into:blank group,hypoxic and hypoglycemic group,LZDO high,medium and low dose groups to observe the protective effect of LZDO on hypoxic and hypoglycemic H9C2 cell damage,collagen contraction experiment to observe the contraction of LZDO on hypoxic and hypoglycemic H9C2 cells The effect of function was verified by hypoxia inducible factor-1α(HIF-1α)protein expression.Observe the effect of LZDO on the mitochondrial function of hypoxic and hypoglycemic cardiomyocytes,and detect the superoxide dismutase(SOD),malondialdehyde(MDA),mitochondrial membrane potential and reactive oxygen species(ROS),in different groups of cells apoptosis-related proteins Bcl2,Bax,Caspase3,mitochondrial fusion MFN1,MFN2,fission protein Fis1,Drp1 expression,observe the effect of LZDO on mitochondrial function of heart failure rats;detect myocardial tissue FFA,ATP levels,energy Fatty acid transport pathway related proteins p-AMPK,AMPK,CD36,PGC1α,SIRT3,CPT1 expression and glucose transport pathway related proteins p-PI3K,PI3K,PP2,PP1,P-Akt,Akt,GLUT4 expression in the metabolic pathway,observed The effect of LZDO on the energy metabolism pathway of myocardial tissue.4.Pathway of LZDO to improve energy metabolism in hypoxia and hypoglycemia cardiomyocytes AMPK inhibitor Compound C and AMPK agonist AICAR were used to inhibit or stimulate cardiomyocyteAMPK expression,while PI3K/AKT inhibitor wortmannin and PI3K/AKT agonist IGF-I inhibited or stimulated PI3K/AKT expression in cardiomyocytes,and then passed WB To observe the effect of LZDO on the mitochondrial fusion and division of cardiomyocytes and the related protein levels of AMPK-CD36-fatty acid pathway and PI3K-GLUT4-glucose pathway to determine the way LDZO improves energy metabolism.Results1.LZDO can improve cardiac function in rats with chronic heart failure caused by myocardial infarctionWhen the coronary artery ligation method was used to prepare a rat model of chronic heart failure caused by myocardial infarction,the electrocardiogram showed abnormal S-T node elevation.After 4 and 8 weeks of modeling,the body weight of the rats in each group was significantly different.The body weight of the rats in the TMZ and LZDO groups increased significantly compared to the model group.The results of echocardiography showed that after 8 weeks of intragastric administration,the heart pumping function of the rats in the model group decreased,indicating that the model was successfully replicated.After giving different concentrations of LZDO,the heart ejection fraction,left ventricular short axis shortening rate,stroke volume,and stroke volume of heart failure rats increased significantly,and the left ventricular systolic volume decreased.The hemodynamic results were the same as echocardiograms.The three dose groups of LZDO could significantly increase LVSP,±dp/dtmax,SBP,DBP,and MAP,while LVEDP decreased.HE staining results showed that the model group’s cardiac myocytes were severely degenerated and necrotic,myofibrils were dissolved,the arrangement was disordered,the matrix density was reduced,and the intercellular space was enlarged.After treatment with LZDO,it can reduce myocardial cell necrosis,interstitial fibrous connective tissue hyperplasia and inflammatory cell infiltration,and the symptoms have been significantly improved compared with the model group.Masson staining results showed that in the model group,blue collagen deposition increased in the rat heart,extracellular matrix remodeling was obvious,collagen deposition increased,and the degree of fibrosis was significant.After the administration of LZDO,each dose group can improve this phenomenon to varying degrees,especially in the high-dose group.2.LZDO improves mitochondrial function of cardiomyocytes in rats with chronic heart failureThe mitochondrial function of myocardial cells in heart failure rats is decreased,which is manifested by a significant decrease in SOD content and a significant increase in MDA.Looking for the cause,it is found that myocardial cell apoptosis is increased,the ratio of apoptosis-related protein Bcl-2/Bax is significantly reduced,and the expression of Caspase3 is significantly increased Because the expression of mitochondrial fusion proteins MFN1 and MFN2 is significantly reduced,the significant increase in fission-related proteins Fisl and Drpl leads to a decrease in the energy produced by mitochondria,a decrease in ATP levels and a significant increase in FFA content,and fatty acid transport-related proteins p-AMPK/AMPK,CD36,PGC1α,SIRT3,CPT1 protein levels were significantly reduced,glucose transport-related protein PDH expression was significantly reduced,p-PI3K/PI3K,p-Akt/Akt,GLUT4,PP2,PP1 significantly increased leading to decreased energy metabolism.After administration of LZDO,the mitochondrial function of myocardial cells in heart failure rats was significantly improved,the MDA content was reduced,the SOD content was increased,and the mitochondrial antioxidant capacity was enhanced.As the ratio of Bcl-2/Bax increased significantly,the expression of Caspase3 decreased significantly and the apoptosis decreased.Myocardial mitochondrial fusion protein MFN1,MFN2 expression increased significantly,fission protein Fisl,Drpl decreased significantly resulting in increased mitochondrial productivity,increased ATP content,increased use of FFA,in addition to fatty acid transport related proteins p-AMPK/AMPK,CD36,PGC1α,SIRT3,The level of CPT1 protein was significantly increased,the level of glucose transport-related protein PDH protein was significantly increased,and p-PI3K/PI3K,p-Akt/Akt,GLUT4,and PP1 were significantly reduced,which significantly improved energy metabolism.3.Liguzinediol significantly improves mitochondrial function in hypoxic and hypoglycemic cardiomyocytesFirstly,the concentration of CoCl2 replication hypoxia and hypoglycemia cardiomyocyte model is 600μM,and then the modeling time is 12 hours.Under this condition,the expression of HIF-1αprotein in cardiomyocytes increases.It is clear that the cytotoxicity of LZDO has no significant effect on the viability of H9C2 cells below 1 mM,indicating that the safety range of LZDO is large.On this basis,it was found that LZDO has a clear protective effect on hypoxia and hypoglycemia myocardial cell damage,and there is a concentration-dependent relationship between the concentration of 1,10,100 μM.Collagen contraction test results showed that compared with the model group,LZDO concentration can reduce the area of collagen ring,suggesting that it can enhance the contractility of myocardial cells.The myocardial cells in the hypoxic and glucose deficient group were damaged,so the LDH in the cell supernatant increased significantly,the intracellular MDA content increased,the SOD content decreased,the mitochondrial membrane potential decreased,and the expression of ROS increased.The reason is related to the increase in myocardial cell apoptosis.Death-related protein Bcl-2/Bax ratio decreased significantly,Caspase3 expression increased significantly,mitochondrial fusion proteins MFN1,MFN2 expression decreased significantly,and fission-related proteins Fisl,Drpl increased significantly,indicating decreased mitochondrial function,decreased ATP production,and reduced FFA utilization,Fatty acid transport related proteins p-AMPK/AMPK,CD36,PGC1 α,SIRT3,CPT1 protein levels were significantly reduced,glucose transport related protein PDH expression was significantly reduced,p-PI3K/PI3K,p-Akt/Akt,GLUT4,PP2,PP1 were significantly Increased mitochondrial capacity decreased.After administration of LZDO,the LDH content in the cell supernatant decreased,the intracellular MDA content decreased,the SOD content increased,the mitochondrial membrane potential was restored,the ROS table decreased,the Bcl-2/Bax ratio increased significantly,and the Caspase3 expression decreased significantly,indicating decreased apoptosis,The function of mitochondria is obviously improved.Myocardial mitochondrial fusion protein MFN1,MFN2 expression increased significantly,mitotic protein Fis1,Drpl decreased significantly increased mitochondrial productivity,increased ATP content,increased FFA utilization,fatty acid transport related proteins p-AMPK/AMPK,CD36,PGC1αSIRT3,CPT1 The protein level was significantly increased,the glucose transport-related protein PDH protein level was significantly increased,and p-PI3K/PI3K,p-Akt/Akt,GLUT4,and PP1 were significantly reduced.4.LZDO Improves Energy Metabolism in Hypoxic and Hypoglycemia CardiomyocytesAfter using the agonist AICAR,the results showed that compared with the model group,the protein levels of P-AMPK/AMPK,CD36,PGC1α,SIRT3,and CPT1 increased significantly;the expression of MFN1 and MFN2 increased significantly,and Fisl and Drpl decreased significantly;In comparison,P-AMPKAMPK,CD36,PGC1α,SIRT3,and CPT1 increased significantly,and the combined effect of AICAR and LZDO showed a tendency to superimpose,the expressions of MFN1 and MFN2 increased significantly,and Fisl and Drpl decreased significantly.After using Compound C,the AMPK inhibitor,it was found that compared with the model group,P-AMPK/AMPK,CD36,PGC1α,SIRT3,and CPT1 protein levels were significantly reduced,MFN1 and MFN2 expressions were significantly reduced,and Fisl and Drpl were significantly increased.The results of LZDO given on the basis of Compound C showed that compared with the inhibitor group,P-AMPK/AMPK,CD36,PGC1α,SIRT3,CPT1 increased significantly;MFN1,MFN2 expression increased significantly,Fis1,Drpl decreased significantly,suggesting that LZDO can pass AMPK/CD36 pathway to regulate mitochondrial fusion and division to improve energy metabolism.After using PI3K agonist IGF-I,the results showed that compared with the model group,P-PI3K,PP2A,PP1,P-Akt,GLUT4 increased significantly,and the PDH protein level did not increase significantly;the results of LZDO showed that compared with the model group,P-PI3K/PI3K,PP2A,PP1,P-Akt/Akt,GLUT4 were significantly reduced,and PDH protein levels were significantly increased;compared with the IGF-I group,PP1 protein was significantly reduced.After using PI3K inhibitor wortmannin,the results showed that compared with the model group,P-PI3K/PI3K,PP2A,PP1,P-Akt/Akt,GLUT4 increased significantly,and the level of PDH protein decreased significantly;the results of LZDO showed that compared with the inhibitor group,P-PI3K/PI3K,PP2A,PP1,P-Akt/Akt,GLUT4 were significantly reduced,and PDH protein levels were significantly increased;suggesting that LZDO can also improve energy metabolism through the PI3K/GLUT4 pathway.Conclusion1.LZDO can significantly improve the heart function of myocardial infarction-induced chronic heart failure rats,improve myocardial tissue morphology and fibrosis,and reduce myocardial cell apoptosis.2.LZDO can significantly improve mitochondrial function of cardiomyocytes,promote fatty acid transport and promote fatty acid metabolism in cardiomyocytes,relatively reduce glucose transport and increase glycolysis to increase the production of cardiomyocytes,and promote energy metabolism by promoting cardiac mitochondria fusion and reducing division.Myocardial contractility,which can achieve its effects through two pathways:AMPK/CD36 pathway and PI3K/GLUT4 pathway. |
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