Pharmacodynamic Study Of Chikusetsu Saponin Ⅳa Against Diabetic Cardiomyopathy And Preparation And Evaluation Of Its Nanoparticles

Backgrounds:Diabetes mellitus（DM）has become one of the most common chronic diseases worldwide,and its incidence is rising year by year.As one of the serious complications related to DM,diabetic cardiomyopathy（DCM）refers to left ventricular hypertrophy and diastolic abnormalities in the absence of hypertension,coronary heart disease or valvular heart disease,which increases the risk of death such as arrhythmia and sudden cardiac death.DCM is caused by complex pathophysiological factors,including metabolic abnormalities,oxidative stress,inflammation,or cardiovascular autonomic neuropathy and so on.A large number of studies have shown that abnormal myocardial energy metabolism and mitochondrial dysfunction are crucial in the occurrence and development of DCM.Currently,lifestyle modifications,antidiabetic medications,lipid lowering medications and heart failure management are the main treatment strategies for DCM.Since the clinical efficacy of these interventions is not satisfactory,it is of great clinical significance to find new targets and drugs for DCM prevention and treatment.Chikusetsu saponinⅣa（C-Ⅳa）has a variety of pharmacological activities including cardio-protection,brain protection,hypoglycemic,lipid-lowering,anti-inflammatory,anti-coagulant,and anti-tumor.Our previous research has shown that the cardioprotective effect of C-Ⅳa is definite.C-Ⅳa could reduce the myocardial damage induced by diabetes,but the underlying mechanism is still unclear.Therefore,this study was to reveal the myocardial protection of C-Ⅳa against DCM by regulating energy metabolism on the animal,cellular and molecular levels.In addition,because C-Ⅳa is not easy to pass through biomembrane,the oral drug absorption is quick but elimination is slow,and aslo it takes a high dose orally to be effective,all these factors greatly limit the effect of the medicine,we conducted a preliminary exploration of C-Ⅳa.Based on nanotechnology,the lipid-polymer hybrid nanoparticles as a carrier of Chikusetsu saponinⅣa（C-Ⅳa/LPNs）was prepared to ameliorate anti-DCM efficacy.This research would contribute to theoretical basis for the application of C-Ⅳa.Objectives:1.To confirm that C-Ⅳa improves DCM by regulating energy metabolism;2.To clarify that C-Ⅳa regulates energy metabolism and improves DCM by activating Sirtuin 3（SIRT3）and inhibiting the opening of Mitochondrial permeability transition pore（m PTP）;3.To prepare C-Ⅳa/LPNs for improving the efficacy of anti-DCM myocardial injury.Methods:1.Methods of C-Ⅳa anti-DCM efficacy research:the wild type（WT）C57BL/6 mice were randomly divided into five groups:control group,DCM model group,and DCM treated C-Ⅳa（30,60,120mg/kg）groups.The DCM model of mice were induced by intraperitoneal injection of Streptozotocin（STZ）.Changes in fasting blood glucose and body weight were monitored every two weeks.The indicators of cardiac function indicators were monitored through multi-channel physiological recorders;the ratio of heart weight to body weight（HW/BW）was calculated;myocardial tissue pathological changes were observed by Hematoxylin-eosin（HE）staining;the contents of hydroxyproline,collagen I,and ATP in the left ventricular myocardial tissue were detected by the corresponding kits;the opening of m PTP was monitored by continuously observing changes in mitochondrial absorbance on a multi-functional microplate reader;western blot was used to detect the expression levels of SIRT3,Bax,Bcl-2,Caspase 3,etc.H9c2 cells model induced by high glucose（HG）was used to observe the protective effect of C-Ⅳa at different doses（10,20,40μM）on H9c2 cells.Cell growth was detected by MTT.The changes of lactate dehydrogenase（LDH）,ATP content,and mitochondrial membrane potential（ΔΨm）of cardiomyocytes were tested;the apoptosis rates of cells were measured by flow cytometry;the expression levels of SIRT3 were detected by western blot.2.Methods of research mechanism about C-Ⅳa regulating energy metabolism by activating SIRT3:H9c2 cells with SIRT3 overexpressed were constructed by plasmid transfection,and H9c2 cell model was induced by HG.To compare the effects of C-Ⅳa pretreatment in SIRT3-overexpressed H9c2 cells and normal H9c2 cells,MTT method was used to observe cell growth;LDH activity was detected in cells;flow cytometry was used to investigate cell apoptosis;changes ofΔΨm and m PTP opening were observed by laser confocal.In the DCM model mice,the protection of C-Ⅳa on cardiac energy metabolism and mitochondrial function in SIRT3^-/-and WT mice were further compared.Myocardial mitochondrial structures were observed by transmission electron microscope;ATP content in myocardial tissue was detected;m PTP opening was monitored;the expression levels of mitochondrial energy metabolism related protein were detected by western blot.3.Preparation and evaluation methods of C-Ⅳa/LPNs:the prescription of C-Ⅳa/LPNs was optimized by orthogonal test.C-Ⅳa/LPNs was prepared by nanoprecipitation method;the physical morphology and homogeneity of nanoparticles were observed by transmission electron microscope,the particle size and Zeta potential were detected;the encapsulation efficiency（EE）and drug loading（DL）of C-Ⅳa/LPNs were measure by ultrafiltration.The stability was investigated by simulating artificial gastric juice,intestinal juice,and high-speed centrifugation in vitro.The dialysis bag diffusion technology was used for drug release experiments in vitro.In addition,the efficacy of C-Ⅳa/LPNs was evaluated in the DCM mice.Changes in fasting blood glucose and body weight were monitored every two weeks.The level of HW/BW was calculated;the changes of LDH in serum and ATP content in myocardial tissue were tested.Results:1.In the DCM model of WT mice,C-Ⅳa significantly reduced the levels of fasting blood glucose and HW/BW,improved cardiac function indicators such as left ventricular diastolic pressure（LVDP）,left ventricular end diastolic pressure（LVEDP）,the maximum increase and decrease rate of left ventricle pressure(±dp/dt_max),reduced Hydroxyproline and Collagen I contents,ameliorated myocardial tissue lesions and necrosis,reduced the ratio of Bax/Bcl-2,Cleaved-caspase 3/Pro-caspase 3.C-Ⅳa increased the ATP content and SIRT3 expression in myocardial tissue,and inhibited the opening of m PTP.It might be seen that C-Ⅳa had a significant protective effect on myocardium damage caused by DCM,which was likely to be closely related to regulating SIRT3 and energy metabolism.In the H9c2 cells induced by HG,C-Ⅳa pretreatment improved cell survival and decreased the LDH activity of the supernatant.C-Ⅳa increased the ATP content and SIRT3 expression,which also increasedΔΨm in H9c2 cells.2.The pretreatment of C-Ⅳa in SIRT3-overexpressed H9c2 cells significantly increased viability and decreased LDH activity;flow cytometry results showed good anti-apoptotic activity;C-Ⅳa increasedΔΨm and inhibited m PTP opening.Therefore,SIRT3overexpression enhanced the protective effect of C-Ⅳa in HG-damaged H9c2 cells.In the DCM model of SIRT3^-/-mice,C-Ⅳa could neither significantly increase the ATP content of myocardial tissue;nor improve mitochondrial damage and inhibit m PTP opening.The expression levels of peroxisome proliferator-activated receptor-γcoactivator-1α（PGC-1α）,mitochondrial transcription factor A（TFAM）,carnitine palmitoyl transferase-1（CPT-1）and pyruvate dehydrogenase（PDH）protein expression levels were significantly reduced.Therefore,the knockout of SIRT3 severely affected myocardial energy metabolism and mitochondrial function in DCM mice.3.According to the optimized prescription,the prepared C-Ⅳa/LPNs presented relatively uniform round spheres with good dispersion;the particle size was（105.4±2.2）nm,the zeta potential was（-35.4±1.4）m V,the encapsulation efficiency was（90.4±1.4）%,and the drug loading was（12.6±1.0）%.C-Ⅳa/LPNs could maintain good stability in artificial gastric juice,intestinal juice and high-speed centrifugation test;had obvious sustained release characteristics in vitro.Under the circumstances of same C-Ⅳa dose,compared with the C-Ⅳa group,the fasting blood glucose,HW/BW and LDH activity of C-Ⅳa/LPNs group decreased significantly,while ATP contents in myocardial tissue increased significantly.Conclusions:1.It was clarified that C-Ⅳa improved DCM by regulating energy metabolism;2.It was confirmed that C-Ⅳa could regulate energy metabolism and ultimately improve DCM by activating SIRT3 and inhibiting m PTP opening;3.The prepared C-Ⅳa/LPNs was stable with high encapsulation efficiency,had good drug loading and long-term drug release in vivo,which significantly improved the anti-DCM effect of C-Ⅳa.