Study On The Effect Of Chinese Herbal Compound Shuangshen Ningxin On Myocardial Ischemia / Reperfusion Injury And Mitochondrial Protection

Coronary heart disease (CHD) is the most common cause of death for both men and women and remains a major public health threat worldwide. Myocardial ischemia and hypoxia play a fundamental role in the pathophysiology of common causes of CHD, so restoring normal epicardial blood flow promptly can save the lives of patients with myocardial infarction, but the consequent myocardial ischemia-reperfusion (I/R) injury can easily cause myocardial stunning, arrhythmias or sudden cardiac death, which seriously influence prognosis. Currently, studies have shown that the pathogenesis of myocardial I/R injury mainly include cellular energy metabolism disturbance, free radical damage, calcium overload and apoptosis, etc. The mitochondrion is a vital component in cellular energy metabolism and intracellular signaling cascades. Myocardial I/R have been shown to result in mitochondrial dysfunction, and the importance of mitochondria as both targets and mediators of Myocardial I/R injury is well recognized. Thus, the preservation of mitochondrial integrity and function is a potential therapeutic strategy to limit MI/R injury and attenuate the disease process.In traditional Chinese medicine, CHD is characterized as "Xiong Bi, Xin Tong" meaning stagnant blood of heart will lead to malnourishment of heart and attacks of sharp and intense pain. Deficiency of Heart-Qi has been implicated as a major determinant of pathogenesis, which is indicated by essence Qi deficiency and superficiality blood stasis excess. Thus, the treatment for "Xiong Bi, Xin Tong" usually depends on tonifying Qi, activating blood circulation, removing blood stasis and relieving pain. Shuang Shen Ning Xin (SSNX), a traditional Chinese medicine for invigorating Qi, activating blood circulation and relieving pain, has been developed for the treatment of coronary heart diseases. The SSNX is composed of three kinds of effective fractions in certain proportion including total ginsenosides, total salvianolic acids and total alkaloids which are isolated from Renshen (Panax ginseng C.A. Mey.), Danshen (Salvia miltiorrhiza Bge.) and Yanhusuo{Corydalis yanhusuo W. T. Wang), respectively. Previous experimental studies have shown that SSNX can effectively limit myocardial infarction, improve cardiac hemodynamic parameters and preserve left ventricular structure and function. Thus, due to the compatibility with unique features and good therapeutic effect, SSNX will have good prospects in application and deserve further research and development. Moreover, we have found that the beneficial effects of SSNX may be partly attributed to improving cardiac energy metabolism and alleviating cellular irreversible injury during ischemia or reperfusion, so to systemically clarify this protective effect and mechanism of SSNX will provide a strong basis for its clinical application.Normally, mitochondria provide approximately97%ATP for cardiac myocytes to maintaining systolic and diastolic function and ion pumps function, and as a result, these cells are particularly vulnerable to mitochondrial defects. During myocardial ischaemia and reperfusion, mitochondria can quickly turn into death-promoting organelles by depressing respiratory chain complex activity, disrupting adenosine triphosphate synthesis, releasing pro-death proteins to active apoptosis. In addition, a key aspect of mitochondrial involvement in cell demise is control of the opening of the mitochondrial permeability transition pore (mPTP). It is proposed that myocardial I/R promotes mPTP opening, especially in reperfusion conditions such as increased matrix Ca2+, Pi, and ROS which favor long-lasting pore opening, resulting in swelling and subsequent rupture of mitochondria. Currently, inhibiting mPTP opening by intervening its component or reducing induction factors has been recognized as critical therapeutic interventions for myocardial I/R injury.Accordingly, in the present study, we investigated the effects of SSNX on cardiac energy metabolism and cardiomyocyte apoptosis in in vitro primary neonatal rat cardiomyocyte model of hypoxia/reoxygenation and in vivo rat model of myocardial ischemia/reperfusion, and explored the possible mechanisms involved in mitochondria protection. The main contents of animal experiments are as follows:1. The rat model of myocardial I/R injury was made in order to evaluate the effects of SSNX on myocardial infarct size by NBT staining, histopathological changes by HE staining, leakage of LDH and CK-MB by UV spectrophotometry, as well as the levels of MDA and the activities of GSH-Px.2. Based on the above research, the effects of SSNX on cardiac energy metabolism and cardiomyocyte apoptosis are investigated further. The levels of ATP, ADP and AMP in myocardial tissue were measured by HPLC, and the TAN and EC were calculated. TUNEL assay was performed to determine myocardial apoptosis, and western blot was used for analysis of cleaved-caspase3expression.3. To investigate the effect of SSNX on mitochondrial structure and function after myocardial I/R injury. Transmission electron microscopy was used to observe the changes of mitochondrial ultrastructure. Isolated Mitochondria were used for determination of the activities of metabolic complex I, Complex IV and citrate synthase (CS) and the opening of mPTP, respectively, while the cytolic fraction were used for determination of the Cyt c levels.According to the previous studies on pharmacokinetics in vivo and PK/PD data processing of SSNX, six representative indicated compounds, such as Rg1, Re, SAA, RA, THP and DHC were chosen for the further cell experiments study. The main contents are as follows:1. Using primary neonatal rat cardiomyocyte culture system, the effects of Rg1, Re, SAA, RA, THP and DHC on cardiomyocyte viability were investigated. On this basis, the primary neonatal rat cardiomyocyte model of hypoxia or hypoxia/reoxygenation (H/R) was made in order to evaluate the effects of SSNX on hypoxia or H/R injury by determining cell viability, LDH leakage and ROS production.2. To investigate the effect of Rg1, Re, SAA, RA, THP and DHC on energy metabolism of cardiomyocyte after hypoxia or H/R injury. In this experiment, the ATP levels, oxygen consumption and intracellular oxygen consumption were determined respectively.3. To investigate the effect of SAA, RA, THP and DHC on apoptosis of cardiomyocyte after hypoxia or H/R injury. The cell apoptosis and△Ψm changes were analysed by flow cytometry, and the protein expressions of cleaved-caspase3, Bax, Bcl-2and p-Akt(Ser473) were investigated by western blot.4. To investigate the effect of SAA on opening of cardiomyocyte mPTP after hypoxia or H/R injury. The opening of cardiomyocyte mPTP was analysed by flow cytometry, and the protein expressions of HKII, mitoHKII, GSK-3β and p-GSK-3β(Ser9) were investigated by western blot.The results are summarized as follows:1. SSNX at dose of22.5mg·kg-1and45mg·kg-1markedly reduced the infarct size, prevented the leakage of LDH and CK-MB from cardiomyocytes, reduced the production of MDA and increased the activity of GSH-Px. In addition, SSNX treated rats displayed a reduced degree of tissue edema, degenerative changes and disruption of myofibers, and inflammatory cell infiltration.2. Pretreatment with SSNX at doses of45mg·kg-1significantly preserved ATP and maintained energy charge of cardiomyocytes. In addition, SSNX at dose of22.5mg-kg-1and45mg·kg-1markedly reduced TUNEL-positive cells, and SSNX at doses of45mg·kg-1significantly decreased cleaved-caspase3expression levels.3. SSNX treated rats hearts displayed little change in mitochondrial structure which seemed to be highly dense with well organized cristae and a decreased mitochondrial swelling. Furthermore, pretreatment with SSNX markedly inhibited the release of Cyt c from the mitochondria, attenuated the decline in the complex I activity and decreased the swelling rate of mitochondria induced by Ca2+4. SAA, RA, THP and DHC had obvious effect in decreasing LDH leakage, increasing cell viability and reducing ROS production of cardiomyocytes induced by H/R injury. Moreover, SAA and RA but not THP and DHC markedly preserved ATP levels of cardiomyocytes after H/R injury.5. Rg1and Re had no obvious effect in decreasing LDH leakage, increasing ATP levels of cardiomyocytes induced by H/R injury, but Rg1and Re had obvious effect in increasing cell viability, reducing ROS production, increasing intracellular oxygen consumption and ATP levels of cardiomyocytes induced by hypoxia injury, in addition to significantly increased the activation of Akt signals and upregulated the expression of p-Akt (Ser473).6. SAA, RA and THP markedly inhibited cardiomyocytes apoptosis induced by H/R injury. SAA and RA but not THP also prevented the loss of△Ψm, downregulated the expression of cleaved-caspase3and upregulated the expression of p-Akt (Ser473), furthermore, SAA markedly downregulated the expression of Bax/Bcl-2induced by H/R injury.7. SAA markedly inhibited the opening of mPTP induced by H/R injury. The results of western blot experiments revealed that SAA had obvious effect in increasing p-GSK-3β(Ser9) expression but not mitoHKII expression. Furthermore, after inhibition of the Akt signalling pathway with LY294002, the effect of SAA in increasing p-GSK-3β(Ser9) expression decrease distinctly.In summary, we have shown that pretreatment with SSNX limited the myocardial I/R injury, and this protection was accompanied by a decrease in cardiomyocyte apoptosis and improvement in cardiac energy metabolism. Furthermore, SSNX improved structural mitochondrial changes, inhibited the mitochondrial apoptosis pathway by block the release of Cyt c, promoted the Complex I activities recovery and reduced the mPTP opening, so these results can also partly account for the preservation of mitochondrial function may be a new mechanism by which SSNX exerts its cardioprotective effects. On the other hand, the indicated compounds of SSNX (SAA, RA, THP and DHC) had obvious protective effect on cardiomyocytes after H/R injury, while Rg1and Re had obvious protective effect on cardiomyocytes after hypoxia injury. Rg1and Re can improve cellular energy status effectively after hypoxia injury, while SAA, RA and THP markedly inhibited cardiomyocytes apoptosis induced by H/R injury, and the above protection of Rg1, Re, SAA and RA were association with the activation of Akt signaling pathway. Furthermore, SAA may inhibited the opening of mPTP induced by H/R injury by increasing p-GSK-3β(Ser9) expression and subsequent inhibiting GSK-3β activity, and SAA exerted the protective effect via Akt-dependent signaling pathway.