Research On The Material Basis Of The Protective Effects Of Guanxin Danshen Formula On Oxidative Stress-induced Cardiomyoblast Injuries And Underlying Mechanisms

Ischemic heart disease (or coronary artery disease, CAD) is one of the most dangerous diseases threatening human's health due to its high morbidity and high mortality, which has attracted the attention of medical workers around the world.Increasing evidences show that cardiomyocyte apoptosis plays an important role in the development of coronary artery diseases. There are two major signaling pathways that could trigger cellular apoptosis, the intrinsic pathway (or the mitochondrial pathway) and the extrinsic pathway (or the death receptor pathway). The intrinsic pathway is triggered by the changes of mitochondrial membrane permeability caused by various stimuli, which leads to the release of proapoptotic factors such as cytochrome C from the interemembrane space of mitochondria and activates caspase-9. While in the extrinsic pathway, the combination of ligands to the corresponding death receptors on the membrane activates caspase-8. Both of the pathways cause the activation of caspase-3 and eventually result in the characteristic changes of apoptotic cell death. Oxidative stress is one of the leading factors causing cardiomyocyte apoptosis in the pathological progress of these cardiovascular diseases. Oxidative stress is defined as the imbalanced state that the overproduction of reactive oxygen species (ROS) exceeds the detoxification and/or the utility ability of cells. The over accumulation of intracellular ROS causes oxidative modification of important biological macromolecules, which results in the functional and structural changes of cells through various signaling pathways, eventually leads to cell death. Therefore, it is of great importance to search for effective antioxidant to prevent the apoptosis of cardiomyocytes and the treatment of CAD.Guanxin Danshen Formula is consisted of Salvia miltiorrhiza Bunge, Panax notoginseng and Lignum Dalbergiae Odoriferae, which shows the effects of promoting blood circulation by removing blood stasis and promoting qi circulation and relieving pain, and is clinically used to treat cardiovascular diseases. Previous researches have shown that the antioxidative effects contribute most to its cardiovascular protective effects. Therefore, we researched on the material basis of the protective effects of Guanxin Danshen Formula on H2O2-induced H9c2 cardiomyoblasts injuries and further investigated the underlying mechanisms to provide fundamental experimental basis for the explanation of the effectiveness and mechanisms of Traditional Chinese formula by modern molecular biological methods.PartⅠResearch on the material basis of the protective effects of Guanxin Danshen Formula on H2O2-induced H9c2 cardiomyoblast injuryObjectiveTo investigate the effects of major components of Guanxin Danshen Formula on H2O2-induced H9c2 cells injuries.MethodsH9c2 cells were pretreated with different concentrations of major components including sodium tanshinone IIA sulfonate and salvianolic acid B from Salvia miltiorrhiza Bunge, Ginsenoside Rbl and notoginsenoside R1 from Panax notoginseng, luteolin from Lignum Dalbergiae Odoriferae for indicated period of time followed by 150μM H2O2 incubation for 6 hours. The cell viability was measured by MTT assay.ResultsSodium tanshinone IIA sulfonate, salvianolic acid B, ginsenoside Rbl and luteolin showed protective effects on H2O2 induced cell injury after 4 hours of pretreatment, while notoginsenoside R1 showed no protective effects. Luteolin showed significant protective effects when pretreated for 1 hour. Notoginsenoside R1 also exhibited protective effects on H2O2-induced H9c2 cell injuries while pretreated for 24 hours.ConclusionLuteolin, one of flavonoid from Lignum Dalbergiae Odoriferae showed significant protective effects on the H2O2-induced H9c2 cardiomyoblast injuries and might contribute most to the antioxidative effects of Guanxin Danshen Formula.PartⅡProtective effects of flavonoids on H2O2-induced H9c2 cell injuries and structure-effects relationshipObjectiveTo investigate the effects of flavonoids on H2O2-induced H9c2 cells injuries and analyze the structrure-effects relationship.MethodsH9c2 cells were pretreated with representative aglycon compound from five flavonoid categories including luteolin, quercetin, naringenin, taxifolin, catchin for 1 hour and then exposed to 150μM H2O2 for 6 hours. The cells were incubated with glycosides including cynaroside, baicalin, rutin, myricitrin, liquiritin, naringin for 4 hours before treated with 150μM H2O2 for 6 hours. Cell viability was measured by MTT assay to determine the effects of flavonoids.ResultsThe protective effects of aglycon compounds on H2O2 induced H9c2 cells injury were shown in descending order as follows:quercetin> luteolin> catchin> taxifolin> naringenin; while the protective effects of glycosides in descending orders were as follows:cynaroside> rutin> baicalin> myricitrin> liquiritin and naringin.ConclusionOf the five aglycon compounds investigated, quercetin from flavonols and luteolin from flavones showed significant protective effects on oxidative stress-induced H9c2 cells injuries; while cynaroside from flavones showed the best protective effects on H2O2-induced H9c2 cells injuries of the six glycosides tested. Structure-activity relationship analysis indicated that certain structural properties affected the protective effects, including the numbers of hydroxyl, the presence of 3-OH in C ring,2,3-double bond in C ring,4-carbonyl in C ring and the glycosyl.PartⅢProtective Effects of Cynaroside against H2O2-Induced Apoptosis in H9c2 CardiomyoblastsObjectiveTo investigate the protective effects of cynaroside against H2O2-induced apoptosis in H9c2 cardiomyoblasts and the underlying mechanisms.MethodsH9c2 cells were pretreated with cynaroside for 4 h before exposure to 150μM H2O2 for 6 hours. Cell morphology was analyzed by Hoechst33342 and TUNEL assay. Flow cytometry analysis of Annexin V/PI staining and caspase activities was used to assess the effects of cynaroside on H2O2-induced H9c2 cell apoptosis. The possible mechanisms were investigated by measurement of activities of endogenous antioxidative enzymes including SOD, GSH-Px, CAT; the intracellular ROS level; the mitochondrial membrane potential; the expressions of apoptosis-related proteins were analyzed by Western blot.ResultsH2O2 treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous antioxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H2O2-induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. The blockage of mitochondrial ATP-sensitive K+ channel and Ca2+ -activated K+ could diminish the protective effects of cynaroside in various degrees. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H2O2-induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression.ConclusionCynaroside from Lignum Dalbergiae Odoriferae prevents H2O2-induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.