| Purpose:Ischemic stroke has become a very harmful desease and the leading cause of death in China. Sanqi Tongshu Capsule, the major material of which is Panaxatriol Saponins (PTS) is an exactly effective Chinese medicine product for the treatment of ischemic stroke in clinic. At present there are so many experimental and clinical studies on the efficacy of the PTS, but its mechanism is still lack of systematic elaboration. Oxidative stress and platelet aggregation play important roles in cerebral ischemia development. We intend to use PC12cells and platelet aggregation in vitro as the models of cerebral ischemia and following platelet aggregation, respectively. The active substances and mechanisms of PTS remedying of cerebral ischemia was revealed through the regulation of Nrf2signaling pathway, protection against OGD-Rep and anti-platelet aggregation. This study based on compatibility of components could supply an effective scientific explanation of Sanqi Tongshu Capsule’s clinical effect, and could be a valuable reference for exploitation, while laying a foundation for the further development of new drugs.Method:1. Protection effect of Sanqi Tongshu Capsule’s main ingrients against OGD-Rep. CCK-8kit was used for detection of the cytotoxic effect of PTS and its main ingredients ginsenoside Rg1, ginsenoside Re and Notoginsenoside R1on PC12cells. PC12cells cultured in Tri-Gas incubator was used to mimicking the OGD-Rep model and the protection effect of PTS was determined by CCK-8kit. The regulation of PTS on Nrf2signaling pathway was determined by Western blotting detection. The significance of Nrf2activation by PTS to remedy of OGD-Rep was proved using an inhibitor of PI3K/Akt and decoy oligonucleotides technology.2. The study on activity of combination of three main ingredients. Tri-Gas incubator combined CCK-8kit was used to detect the induction of phase Ⅱ enzymes and protection against OGD-Rep of combination with different proportion. The effect of propotion of three main ingredients on pharmacological activity was assessed. 3. The machenism of Sanqi Tongshu Capsule inhibiting platelet aggregation. The inhibition of platelet aggregation induced by a variety of inducers of PTS and the main components was observed using a microplate reader. The inhibiton of platelet calcium influx of PTS was determined using a fluorescence spectrophotometer. Western blotting was used for detection of the regulation of P-Erk and P-p38MAPK during platelet aggregation. The side effect of PTS that prolonged bleeding time was observed through mouse tail bleeding experiment.Results:1. Protection effect of Sanqi Tongshu Capsule’s main ingrients against OGD-Rep. PTS at concentration of40μg·l-1, Rgl, Re and R1at concertration of50μmol·L-1, or even lower concertration for all of them did not show significant cytotoxic effects on PC12cells. PTS significantly protected PC12cells against OGD-Rep and induced HO-1expression in a concentration and time dependent manner, while16μg·ml-1for24h incubation was the best treatment. PTS activated Nrf2via Akt phosphorylation induction primarily. Blockage of PI3K pathway and inhibition of Nrf2transcription could significantly inhibited the induction of HO-1expression and anti-OGD-Rep activity of PTS.2. The study on activity of different combination of three main ingredients. Three ingredients of PTS could not protected PC12cells against OGD-Rep when used alone at concentration of2.5μM. The combination of the three main components showed a significant protection effect against OGD-Rep. All of three ingredients showed significant effect on HO-1induction at2.5μM. The combination treatment had obvious inducing effect on HO-1and NQO1at2.5μmol·L-1. But the combination of them according to natural proportion showed weak HO-1induction and cell protection against OGD-Rep.3. The machenism of Sanqi Tongshu Capsule inhibiting platelet aggregation. PTS and its major components could inhibit platelet aggregation induced by a variety of inducers in a concentration-dependent manner. There is no synergistic or additive effect among three components. PTS could significantly inhibit thrombin-induced platelet calcium influx. PTS had a dual-directional regulation effect on Erk phosphorylation but no effect on p38MAPK phosphorylation. PTS did not show the side effect of prolonging bleeding time.Conclusion:PTS and its main components could protect PC12cells against OGD-Rep through synergistic induction of HO-1expression. PTS mainly activated Nrf2nuclear translocation followed by the induction of Akt phosphorylation, and then regulated the expression of the downstream phase II enzymes to exert an antioxidant and anti-OGD-Rep activity. All of three major components of PTS are effective constituents and the proportional relation could affect the potency significantly. The natural proportion is not the best and the most effective proportion is still not clear. PTS and three ingredients inhibited platelet aggregation induced by a variety of inducers via regulating the expression of P-Erk and inhibiting calcium influx. This systematic research of PTS’s function substance basis and the machanism based on Nrf2signaling pathway and anti-platelet aggregation effect provided a scientific explanation for the treatment of cerebral ischemic diseases with Sanqi Tongshu Capsule. Meanwhile, these studies could also lay the foundation for the further development of Chinese herb extracts. |
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