Discovery And Research Of Natural Products Against Cardiomyocytes Injury

Objects: To obtain natural compounds or highly effective derivatives/analogues that protected cardiomyocytes from oxidative stress or lipotoxicity, and investigate their anti-apoptotic mechanisms.Methods: 1) H9c2 cells were treated with H2O2 at different concentrations, cell viability was tested by MTT to determine the most appropriate experiment concentration. 2) H9c2 cells were divided into 4 groups according to different treatments(control group, CGA-6 group, H2O2 group, H2O2+CGA-6 group), cell viability was assayed with MTT. The lactate dehydrogenase(LDH) release and catalase(CAT) activity measurement were performed according to kit protocols. The intracellular reactive oxygen species, Ca2+, mitochondrial membrane potential(MMP), were measured with CM-H2 DCFDA, Fluo-4 and JC-1 fluorescent probes, respectively; The DNA was stained with Hoechst 33342 to observe cell apoptosis; Expression level of proteins related to mitochondrial apoptotic pathways and MAPKs were analyzed by western blot. 3) Time and dose-dependent palmitate-induced H9c2 cells viability were tested by MTT and the most appropriate experiment condition was determined; 4) The efficacy of curcumin analogues on protecting H9c2 cells from pamitate induced apoptosis were evaluated by MTT, Hoechst 33342 staining and western blot analysis of major apoptotic proteins, cleaved caspase-3, p-JNK and p-NFκB expression.Results: 1) H9c2 cells were treated with 0.3 m M H2O2 for 3 h to induce oxidative stress. We used this in vitro cell model to screen natural compounds and obtained 14 promising natural compounds including CGA-6. 2) H9c2 cells were pretreated with CGA-6 for 1 h and then co-incubated with 0.3 m M H2O2 for 3 h. After H9c2 cells were treated with H2O2, the cell viability and CAT activity decreased, while LDH release, intracellular ROS production and Ca2+ increased. Meanwhile, MMP loss appeared and apoptotic rate rised. Treatment with different concentrations of CGA-6 protected H9c2 cells from all above detrimental effects. Furthermore, CGA-6 inhibited H2O2-induced H9c2 cell apoptosis by suppressing mitochondrial apoptotic pathways and suppressed the activation of JNK and ERK MAPKs signal pathway. 3) H9c2 cells were treated with 0.4 m M palmitate for 20 h to build an in vitro cell model of FFA-induced cardiomyocytes apoptosis for compound screening. 4) We obtained 17 curcumin analogues, which were more effective than curcumin on antagonizing palmitate-induced H9c2 cell injury, among which C16, C20, C37, C40 protected cardiac cells from PA-induced apoptosis. Furthermore, C20 and C40 may protect H9c2 cells from PA-induced apoptosis through inflammation signal pathway.Conclusions: 1) We investigated the possible mechanism by which CGA-6 protected H9c2 cardiac cells from H2O2-induced apoptosis, which involved mitochondrial apoptotic pathway and suppression of the activation of ERK and JNK through ROS scavenging. 2) 4 promising curcumin analogues against palmitate-induced cardiac apoptosis were found.