Diterpene ginkgolides meglumine injection inhibits oxidative stress induced by oxygen-glucose deprivation by activating Akt/Nrf2 pathway in SH-SY5Y cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the protective effects and mechanism of diterpene ginkgolides meglumine injection (DGMI) against oxidative stress induced by oxygen-glucose deprivation (OGD) in SH-SY5Y cells. METHODS SH-SY5Y cells were divided into five groups: normal control, model control (OGD group) and drug(25 mg · L- 1) administration groups including DGMI group, extract of ginkgo biloba leaves injection group (EGBLI) and lactones ginkgo biloba injection group (LGBI). The cells suffered from oxygen-glucose deprivation (OGD) for 4 h, followed by reoxygenation with drugs for 6 h. Then, cell viabilities were detect using CCK-8 assays, reactive oxygen species (ROS) levels using fluorescence probe DCFH-DA and superoxide dismutase (SOD) activities using WST-1 test. Western blotting was used to detected protein levels of hemeoxygenase-1(HO-1), NAD(P)H, quinone oxidore?ductase l (Nqo1), protein kinase B (Akt), phosphorylated Akt (p-Akt), nuclear factor-E2-related factor2 (Nrf2) and phosphorylated Nrf2 (p-Nrf2). The cells were induced by OGD for 4 h, followed by reoxygen?ation and DGMI for 1 h, combined with different concentrations of PI3K inhibitor (LY294002) (at the final concentration of 12.5, 25 and 50 μmol · L-1) before the protein levels of AKT, p-AKT, Nrf2 and p-Nrf2 were detected by Western blotting. RESULTS SH-SY5Y cells induced by OGD for 4 h resulted in an increase in ROS(P<0.01), but a decrease in cell viabilities(P<0.01), SOD activities(P<0.01), and antioxidant protein levels ( Akt, p-Akt, Nrf2, p-Nrf2, HO-1 and Nqo1) (P<0.01). Compared with OGD group, treatment with reoxygenation and drugs (DGMI,EGBLI and LGBI respectively) for 6 h resulted in a decrease in ROS (P<0.01), but an increase in cell viabilities, SOD activities and antioxidant protein levels of p-Nrf2, HO-1, Nqo1 and p-Akt(P<0.05,P<0.01). DGMI group showed the best efficiently. Moreover, after OGD for 4 h, compared with DGMI group, combining reoxygenation and DGMI with LY294002 for 1 h resulted in a concentration-dependent inhibition of the protein levels of p-AKT and p-Nrf2(P<0.01). CONCLUSION DGMI 25 mg · L-1 can inhibit oxidative stress in SH-SY5Y cells induced by OGD by increasing the activity and expression of Nrf2 through PI3K/Akt pathway, which may be one of the mechanisms by which DGMI protects neurons from stroke.

Diterpene ginkgolides meglumine injection attenuates oxygen-glucose deprivation-induced apoptosis of nerve cells via inhibition of calpain signaling pathway / 中国药科大学学报

@#To investigate the anti-apoptotic effect of diterpene ginkgolides meglumine injection(DGMI)on SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation(OGD/R), and to explore its mechanisms. After 4 h of OGD, the SH-SY5Y cells were treated with 25 mg/L DGMI for 1 h. The release of lactic dehydrogenase(LDH)was measured by cytotoxicity detection kitplus. Cell apoptosis was detected by caspase-3/7 assays. Cell death was detected by ELISA. The concentration of ［Ca2+］i in cytoplasm was measured by Fluo-3 AM and the levels of calpain and cleaved capaease-12 were evaluated by western blot. As we expected, DGMI significantly decreased the release of LDH, the concentration of ［Ca2+］i, the protein levels of calpain and cleaved caspase-12. Furthermore, DGMI injection also attenuated the activities of caspase-3/7 and the contents of cytoplasmic histone-associated- DNA-fragments. These data demonstrated that the DGMI injection showed good anti-apoptotic effect in SH-SY5Y cells induced by OGD/R. The mechanisms may be associated with the inhibition of Ca2+/calpain/caspase-12/caspase-3 signaling pathway.

Study on NO Inhibitory Activity of Total Lactones fromAndrographis paniculata / 世界科学技术-中医药现代化

This study was aimed to explore the suppression of nitric oxide (NO) production in RAW264.7 cells by total lactones fraction from Andrographis paniculata. The inflammatory model in vitro was established by stimulating the RAW264.7 cells with lipopolysaccharide (LPS). The NO production and inhibitory rate were determined by Griess assay. Cytotoxicity was evaluated by MTT method. The results showed that total lactones fraction ofA. paniculata suppressed NO production in a concentration-dependent manner in the concentration range from 5 to 50μmol·L-1 and their IC50 values were 8.58μmol·L-1, 11.52μmol·L-1 and 8.94μmol·L-1, respectively. In this condition, major constituents were andrographolide (5-60μmol·L-1), dehydroandrographolide (5-100μmol·L-1) and neoandrographolide (5-100μmol·L-1) inhibited NO production in a dose-dependent manner with an IC50 values of 17.54μmol·L-1, 49.54μmol·L-1 and 41.80μmol·L-1, respectively. It was concluded that the NO inhibitory activity of total lactones fromA. paniculata was better than three active ingredients, which may be able to provide a theoretical foundation and scientific basis for the preparation and clinical application of total lactones fraction fromA. paniculata.