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OBJECTIVE To investigate the effects of bilobalide (BB) on the myocardial tissue of rats with myocardial ischemia/reperfusion (Ml/R). METHODS An Ml/R model was prepared by ligating the anterior descending branch of the left coronary artery in rats for 30 min. BB 2, 4 and 8 mg-kg"1 was adminsterted for four weeks. The heart rate of rats was recorded and myocardial infarction area was detected. Detective kits were used to evaluate the concentrations of creatine kinase MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH). HE staining was performed to observe the myocardial injury, and TUNEL staining was performed to evaluate apoptosis. ELISA was performed to determine the concentrations of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), IL-1p, tumor necrosis factor-a (TNF-a) and IL-10. Immunohistochemistry was performed to evaluate the expressions of Ki67 and interleukin-6 (IL-6). Western blotting was performed to evaluate the expressions of Bcl-2, Bax, cleaved-caspase 3, nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2), heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQ01), NF-kB P65, phosphorylated inhibitor of NF-kB (P-IkBoc) and IkBcx kinase (p-IKK). RESULTS Compared with normal control group, the percentage of the myocardial infarction area and the concentration of CK-MB and LDH were increased in Ml/R model group, but heart rate was decreased (P<0.01) and myocardial tissue showed myocardial fiber rupture and inflammatory infiltration. Compared with Ml/R model group, the percentage of the myocardial infarction area and the concentration of CK-MB and LDH were decreased in Ml/R model+BB 2, 4 and 8 mg-kg"1 groups (P<0.05), but heart rate was increased (P<0.01) and myocardial tissue fibers tended to be regular while inflammatory cell infiltration was significantly reduced. Compared with normal control group, the percentage of apoptotic cells and Ki67 expression-positive cells and the expressions of Bax and cleaved-caspase 3 were increased in Ml/R model group (F<0.01), but the expression of Bcl-2 was decreased markedly (P<0.01). Compared with Ml/R model group, the percentages of apoptotic cells and Ki67 expression-positive cells and the expressions of Bax and cleaved-caspase 3 were decreased in Ml/R model+BB 2, 4 and 8 mg∗kg-1 groups (P<0.01), while the expression of Bcl-2 was increased (P<0.01). Ml/R could lower the activity of SOD and GSH-Px, but enhance MDA content. BB could weaken the effect of Ml/R on SOD, GSH-Px and MDA. Compared with normal control group, the concentrations of IL-1β, TNF-α and IL-10 in serum and the percentage of IL-6 expression-positive cells in myocardial tissue of Ml/R model group were increased (P<0.01). Compared with Ml/R model group, the concentrations of IL-1β and TNF-a and the percentage of IL-6 expression-positive cells were decreased in Ml/R model∗ BB 2, 4 and 8 mg-kg"1 group (P<0.01), but the concentration of IL-10 was increased(P<0.05). Compared with mormal control group, the protein levels of Nrf2, HO-1 and NQ01 significantly decreased (P<0.01), while the levels of NF-kB P65, p-IKK and p-kBa significantly increased (P<0.01). BB could effectively reverse protein changes (P<0.05, P<0.01). CONCLUSION BB can protect rats with Ml/R via anti-inflamma-tion and anti-oxidation.
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Objective To investigate the protective effect of ginkgolide A and ginkgolide B (GKAB) mixture on neurons of rats with permanent middle cerebral artery occlusion (pMCAO) and related molecular mechanisms. Methods Sixty male Sprague-Dawley rats were randomly divided into sham group, pMCAO permanent focal cerebral ischemia group and GKAB-treated low-, medium- and high-dose groups. In addition to the sham group (only isolated without interruption of the arteries), the rats in the remaining four groups were induced pMCAO by blocking the right middle cerebral artery. Rats in the GKAB-treated low-, medium- and high-dose groups were injected with GKAB 12.5, 25, and 50 mg/kg through sublingual vein at 10 min after pMCAO, while the sham and pMCAO groups were injected with saline of the same volume as the medium-dose group. After 12 h of treatment, the neuronal apoptosis was determined by TUNEL method, the level of phosphorylated c-Jun N-terminal kinase (p-JNK) was determined by immunohistochemistry, the expressions of p-JNK, Bcl-2, Bax, cytochrome C (Cyt C), caspase-9, caspase-3, cleaved caspase-9, and cleaved caspase-3 in brain tissues were detected by Western blotting. Results Compared with the sham group, the apoptosis rate and p-JNK expression of neurons in the pMCAO group were significantly increased (P<0.01), and the expressions of apoptosis-related proteins Bax, cleaved caspase-9 and cleaved caspase-3 in brain tissues were significantly increased (P<0.01), while the expressions of Bcl-2, caspase-9 and caspase-3 in brain tissues were significantly decreased (P<0.01). Compared with the pMCAO group, the apoptosis rate and p-JNK expression of neurons in GKAB-treated low-, medium- and high-dose groups were significantly decreased (P<0.01), the expressions of Bax, cleaved caspase-9 and cleaved caspase-3 protein were significantly decreased (P<0.01), and the expressions of Bcl-2, caspase-9 and caspase-3 were significantly increased (P<0.01) in a dose-dependent manner. Compared with the sham group, the expression of Cyt C in cytoplasm in the pMCAO group was significantly increased, and the expression of mitochondrial Cyt C was significantly decreased (P<0.01). Compared with the pMCAO group, the expressions of Cyt C in cytoplasm in the GKAB-treated low-, medium- and high-dose groups were significantly decreased in a dose-dependent manner, and the expressions of mitochondrial Cyt C were significantly increased (P<0.05, P<0.01). Conclusion GKAB can inhibit neuronal apoptosis after pMCAO in rats, and its mechanism may be related to the inhibition of JNK phosphorylation and JNK signaling pathway and the block of mitochondrial apoptosis pathway.
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Objective:To determine the neuroprotective effect of ginkgolides(Gin)on cultured rat embryos dorsal root ganglion(DRG)neurons injured by glutamate(Glu)in vitro.Methods:DRG neurons of Wistar rat embryos were cultured in vitro for 48 h and then exposed to Glu(200 μmol/L)with or without Gin(50 μmol/L).The living cells were observed with an inverted contrast microscope.The cultures were processed for detecting the apoptosis rate by using flow cytometry.The fluorescent intensity of intracellular Ca2+ was detected by confocal laser scanning microscope(CLSM).Results:The living status of DRG cells with Gin incubation was better than that of incubated cultures without Gin.The shape of neuronal cell bodies or neurite networks were almost the same in the glutamic acid group with Gin compared with that of the normal control group.The apoptosis rate of cells incubated with Glu for 24 h was 41.1% in DRG cultures.The apoptosis rate of cells incubated with Glu and 50 μmol/L of Gin for 24 h was 7.6% in DRG cultures.The fluorescent intensity was lower in Gin with Glu group than that in Glu group(P＜0.01).The fluorescent intensity was lower in the control group than that in Glu group(P＜0.01).There was no significant difference in the fluorescent intensity between Gin with Glu group and control group(P＞0.05).Conclusion:Ginkgolides may reduce intracellular Ca2+ concentration,and then protect DRG neurons from neurotoxicity induced by Glu.