The Primary Research Of Synthesis And Biological Evaluation Of Novel Leonurine-cysteine Analogue Conjugates As Cardioprotective Agents

Objectives:Both of leonurine, a plant alkaloid present in Chinese motherwort, and cysteine analogues had been reported to have cardioprotective effects. In view of these, we designed, synthesized and biologically evaluated a serial of leonurine-cysteine analogues conjugates. We considered to compare all of these novel compound and to select the best one as a candidate of cardioprotective agent, then to investigate the mechanism of this novel compound assocated with anti-oxidative effect and anti-apoptosis, so as to research and develop one effective candidate for myocardaial ischemia disease in clinical.Methods:Part one:we made the caryophyllic acid as the mateial for the first step. we got the leonurine-cysteine analogue conjugates by carbonylation, esterification, hydrolization, then we identified these structure by MS, NMR and HRMS, the purity had been analized by HPLC. Part two:Methylthiazol tetrazoium (MTT) assay for cell viability, lactate dehydrogenase (LDH) release as an index of cytotoxicity, H2S level was mesured. Morphological changes of neonatal rat ventricular myocytes had been observed by Hoechst staining. Part three:Superoxide the activity of dismutase (SOD) and catalase (CAT) indicating clearing ability of active oxygen, malondialdehyde (MDA) and reactive oxygen species (ROS) content indicating lipid peroxidation, the activity of caspase-3 were measured. Gene expression of bcl-2, bax, caspase-3 and CSE had been observed by RT-PCR. Protein expression of Bcl-2, Bax, Caspase-3, CSE, Akt had been observed by western blot. Apoptotic changes of neonatal rat ventricular myocytes had been observed by Annexin V-FITC/PI staing used by flow cytometry. Mitochondrial transmembrane potential of neonatal rat ventricular myocytes had been observed by JC-1 staining used flow cytometry. Ultrastructure morphological changes of neonatal rat ventricular myocytes had been observed by transmission electron microscopeResults:Part one:We succesfully designed and synthesized a serial of leonurine-cysteine analogue conjugates and their structure and purity had been indified. Part two:Pharmacological evaluation had shown their potent cardioprotective effects. Both of la and 1b were able to modulate hydrogen sulfide production. la had better cardioprotective effects than those of 1b and 2, which indicated there was positive correlation between Leonurine-cysteine analogue conjugate and their precursors (SPRC, SAC and 3-(2-carboxy- ethyldisulfanyl)-propionic acid). la had potent cardioprotective effect at lower molar concentration (10-fold less than leonurine required and 100-fold less than SPRC required). 1a was able to increase cell viability, decrease leakage of LDH, modulate H2S level. Part three:1a was able to improve the activity of SOD and CAT, attenuate ROS and MDA level, protect some cell organs, attenuate the mitochondrial transmembrane potential of neonatal rat ventricular myocytes, up-regulate gene and protein expression of CSE, regulate apoptosis-associated genes and proteins expression mainly by Caspase-3 pathway, increase the phosphorylation of Akt during hypoxia.Conclusion:the novel compound 1a had the ability of cardioprotective effect in order to decrease the deth of neonatal rat ventricular myocytes. The mechanisms were related to increase the anti-oxidative enzymes activity so as to decrease oxidative stress, regulate the apoptosis-associated proteins by Caspase-3 pathway, up-regulate CSE pathway so as to increase H2S level and increase the phophorylation of Akt level. Furthermore these results provided us important clues for the future design and modification of this type of Leonurine-cysteine analogue conjugates and supported 1a to be a new class of multifunctional anti-myocardial ischemia agent.