The Protective Effects Of The Monomers In Fufangdanshen On Nervous Cells Induced By Oxidative Stress

Aim: The Chinese complex formula Fufangdanshen has been widely used to treat cardiovascular and nervous system diseases, and has been found to have beneficial effects on the nervous system including the effect of anti-Parkinson's disease, while the molecular mechanism still needs further study. Parkinson's disease (PD) is the most common movement disorder. While the cause of PD is not known, data obtained from familial disease and from animal models of PD support a pathogenic process that is closely linked to mitochondrial dysfunction and oxidative stress. In this research, the neuro-protective effects of the active monomers in Fufangdanshen were observed and the related mechanisms were also studied.Method: The PD in vitro model is built to quickly screen neuro-protective monomers from the Fufangdanshen formula in this experiment. Some researches focus on the molecular mechanisms of 6-OHDA induced damages and protective effects of salvianolic acid B and Isoborneol by means of cellular and molecular technologies.The human neuroblastoma cell line SH-SY5Y was culutred, and several parameters were observed after 6-OHDA (100μmol/L,24hr) induced damage by pharmaceutical methods, including cell viability and the change of intracellular ion level, etc. The protective mechanism of salvianolic acid B and Isoborneol were studied.Results: Experiments were carried to screen neuro-protective monoers from monomers in Fufangdanshen formula preliminary, and three of them show neuro-protective activies. Rg1, salvianolic acid B and Isoborneol showed protective effect dramatically. The neuro-protective mechanism of ginsenoside Rg1 we, so we focus on the protective mechanism of salvianolic acid B and Isoborneol.Pretreatment of SHSY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our Heochest 33258 staining and Annexin V-FITC/PI quantification data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. The related signal transduction pathway experiement showed that: Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C.Pretreatment of SH-SY5Y cells with isoborneol significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS) and 6-OHDA-induced increases in intracellular calcium. Furthermore, apoptosis induced by 6-OHDA was reversed by isoborneol treatment. Isoborneol protected against 6-OHDA-induced increases in caspase-3 activity and cytochrome C translocation into the cytosol from mitochondria. Isoborneol prevented 6-OHDA from decreasing the Bax/Bcl-2 ratio. We also observed that isoborneol decreased the activation of c-Jun Nterminal kinase and induced activation of protein kinase C (PKC) which had been suppressed by 6-OHDA.In summary, Salvianolic acid B and isoborneol-mediated cytoprotection is due, at least in part, to inhibition of the oxidative stress and the regulation of intracellular calcium balance. Protective effects of salvianolic acid B and isoborneol are involved in its free radical scavenging actions and inhibitory effect on cell apoptosis.Conclusion: Collectively, these results showed that the inhibition of oxidative stress and apoptosis may contribute to explain the the neuro-protective effect of monomers in Fufangdanshen formula. Furthermore, this research may explain the mechanism of traditional complex formula in the view of modern life science.