[Protection of hepatocyte growth factor on neurons subjected to oxygen-glucose deprivation/reperfusion].

The present study was conducted to investigate the effect of hepatocyte growth factor (HGF) on cortical neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Primary cultured cerebral cortical neurons were prepared from Sprague-Dawley rats. The cells were used for experiments after culture for 12 d in vitro. To initiate OGD/R, the culture medium was replaced by glucose-free medium, and cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N(2) and 5% CO(2) at 37 °C for 2 h. Following this treatment, neurons were fed with glucose-supplemented (25 mmol/L) medium, and returned to the incubator under normoxic condition for 0-24 h. The cell viability was assessed by MTT assay, and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. The percentage of apoptotic cells was analyzed by flow cytometry and Hoechst 33258 staining. The expressions of c-Met mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. Oxygen-glucose deprivation for 2 h decreased the cell viability and increased LDH leakage rate in cultured cerebral cortical neurons. The cell viability declined and LDH leakage rate increased with the reperfusion time going on (0-24 h). To explore the influence of HGF on neurons under oxygen-glucose deprivation for 2 h/reperfusion for 24 h (OGD(2)/R(24)) condition, the cultures were pretreated with HGF at different concentrations (5-120 ng/mL) 2 h prior to OGD(2)/R(24). The results showed that OGD(2)/R(24) treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apopototic cells. Pretreatment with HGF at 5 ng/mL and 10 ng/mL did not affect the decrease in cell viability resulting from OGD(2)/R(24). In the presence of 20 ng/mL HGF, the increase in cell viability in cortical neurons exposed to OGD(2)/R(24) began to appear, and 80 ng/mL of HGF exhibited the maximal effect. HGF at 5, 10 and 20 ng/mL did not affect the increase in LDH leakage rate in cortical neurons exposed to OGD(2)/R(24). In the presence of 40 ng/mL HGF, the decrease in LDH leakage rate in cortical neurons subjected to OGD(2)/R(24) began to appear, and 80 ng/mL of HGF displayed the maximal effect. In addition, HGF at 80 ng/mL significantly attenuated cell apoptosis resulting from OGD(2)/R(24). As detected by semi-quantitative RT-PCR and Western blot analysis, c-Met mRNA and protein were expressed in cerebral cortical neurons cultured for 12 d in vitro. c-Met mRNA and protein expressions in cortical neurons exposed to OGD(2)/R(24) were significantly upregulated and were not affected by pretreatment of HGF at 80 ng/mL. Treatment with c-Met inhibitor SU11274 (5 µmol/L) completely eliminated HGF-mediated protection of cortical neurons subjected to OGD(2)/R(24). The results suggest that HGF directly protects cortical neurons against OGD/R-induced cell injury in a dose-dependent manner, and HGF has a potent anti-apoptotic action on neurons exposed to OGD/R.

HGF protects cultured cortical neurons against hypoxia/reoxygenation induced cell injury via ERK1/2 and PI-3K/Akt pathways.

Hepatocyte growth factor (HGF) has been revealed to exert multipotent activities on a variety of cells. In this study, we investigated whether HGF had a direct neuroprotection on cultured cerebral cortical neurons subjected to hypoxia/reoxygenation (H/R) and explored the intracellular signalings mediated the effects. The decrease in cell viability and increase in number of apoptotic cells resulting from H/R were significantly prevented by HGF pre-treatment. HGF stimulated both ERK1/2 and Akt activities in cortical neurons. Inhibition of ERK activation completely abolished the protective effects of HGF, and inhibition of Akt activation reduced, but did not completely eliminate the HGF mediated neuroprotection. It is suggested that the neuroprotection of HGF depend on ERK1/2 pathway, and, to a lesser extent, PI-3K/Akt pathway. In addition, we found that pre-treatment with HGF remarkably attenuated the decrease in expression of Bcl-2 and Bcl-xL induced by H/R, but failed to affect the amount of Bax. It is likely that Bcl-2 and Bcl-xL contribute to the protective effects of HGF.

[Effects of hypoxic preconditioning on hypoxia tolerance of astrocytes in vitro].

AIM: To explore the mechanisms of hypoxic preconditioning on protecting cultured astrocytes from hypoxia injury. METHODS: Cultured astrocytes were divided randomly into several groups: control(C), hypoxia(H) and hypoxic preconditioning (HP). Cells MTT metabolic activity, qualitation of apoptosis and modality to explore the protection effects of hypoxic preconditioning. Immunocytochemistry of Bcl-2 and Bax to explore the mechanisms of hypoxic preconditioning on protecting astrocytes from hypoxia. RESULTS: Compared with H group there was marked increase of MTT metabolic activity in HP48 and HP72 groups. Immunocytochemistry of Bcl-2 and Bax showed that compared with H group, expression of Bcl-2 was increased in HP group, while expression of Bax was decreased in HP group. CONCLUSION: Hypoxic preconditioning can protect astrocytes from hypoxia. One possible mechanism maybe concerned with inhibition of Bax and maintain of Bcl-2 to depress apoptosis procedure.

[Effect of thrombin on cultured rat cerebral astrocyte injured by hypoxia/reoxygenation and its relationship with iNOS].

OBJECTIVE: To observe the effect of thrombin on the cytotoxicity of astrocytes injured by hypoxia/reoxygenation(H/R) and to explore its relationship with inducible nitric oxide synthase (iNOS). METHODS: Primary astrocytes were cultured in DMEM with 10% approximately 15% calf serum and divided into 6 groups: a control group, a Tm control group, an H/R group, a Tm+H/R group, a hirudin (HR) control group, and a Tm+HR+ H/R group. The cell damage and viability were detected by the 3-(4, 5-di-methyl-thazol-2-yl)-2, 5 diphenyl-tetrazol-iumbromide (MTT) conversion method. The NO level in the cultured cell supernatant was assayed by Griess reagent. The flow cytometry was performed to evaluate the apoptosis rate of astrocytes. The iNOS mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemistry was used to observe the expression of iNOS protein. RESULTS: The cell viability injured by H/R was lower than that of the control group, the NO production and apoptosis rate in the cell of H/R group were higher than those of the control group. Incubation of H/R cell with 10kU/L Tm enhanced the cytotoxicity of H/R stimulation compared with the cells injured by H/R. Hirudin can reverse the effect of thrombin. RT-PCR and immunocytochemistry analysis demonstrated that the levels of iNOS mRNA and iNOS protein increased in the cells treated by H/R. Tm enhanced the expression of iNOS mRNA and iNOS protein in the cells treated by H/R. Hirudin blocked the effect of Tm. CONCLUSION: Increasing the level of iNOS and enhancing the production of NO may be the mechanism of thrombin cytotoxicity in astrocytes injured by H/R.

[Protective effect of thrombin precondition on astrocytes insulted by oxygen-glucose deprivation].

OBJECTIVE: To explore the effect of thrombin precondition (TPC) on the rat cerebral astrocytes(As) cultured in oxygen-glucose deprivation (OGD). METHODS: Astrocytes were pretreated with thrombin (TB) at various concentrations (0.005 approximately 5.000 kU/L), and then insulted by OGD. The cell damage and viability were evaluated by the lactate dehydrogenase (LDH) effusion rate and the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion method. Detection of apoptotic cells was determined by the flow cytometry technique. The glutamate uptake of astrocytes was studied with [3H]-glutamate incorporation. RESULTS: OGD increased the LDH, decreased the cell viability, increased the number of apoptotic astrocytes, and decreased the glutamate uptake (P<0.01). While preconditioned with thrombin at the same condition, the LDH decreased, the cell viability increased, the percentage of apoptotic cells decreased, and the glutamate uptake increased (P<0.05). The maximum protective effect of thrombin was observed at 0.1 kU/L. CONCLUSION: Low concentration of thrombin precondition (TPC) can protect the astrocytes from oxygen-glucose deprived injury, and attenuate its apoptosis in a dose-dependent manner.

Clinical experience in treatment of Amanita mushroom poisoning with Glossy Ganoderma Decoction and routine Western medicines.

OBJECTIVE: To assess the effects of treatment of Amanita mushroom poisoning with Glossy anoderma Decoction (, GGD). METHODS: Twelve patients with acute Amanita mushroom poisoning received conventional treatment (penicillin and reduced glutathione) combined with oral administration of GGD (treated group), which was prepared out of 200 g Glossy ganoderma decocted in water to 600 mL, and 200 ml was given once, three times a day for 7 successive days; while conventional treatment alone was given to the other 11 patients assigned to the control group. The therapeutic efficacy and changes in serum levels of total bilirubin (TBil), bile acids (BA), alanine transaminase (ALT), and aspartate transaminase (AST) activities in the two groups were compared. RESULTS: The cured-markedly effective rate in the treated group was more significant than that in the control group (P<0.01). Elevation in TBil, BA, ALT, and AST activities were observed in both groups 3 days after poisoning, which progressively increased thereafter in the control group. In the treated group, they reached their peak on the 3rd day and then declined gradually. The differences between pre-treatment and post-treatment in both groups were obviously significant (P<0.01), so were the differences between the two groups at corresponding time points (P<0.01). CONCLUSION: GGD shows excellent clinical efficacy in the treatment of acute Amanita mushroom poisoning and can reduce mortality significantly.

[Effect of hepatocyte growth factor on oxygen-glucose deprived injury of astrocytes].

OBJECTIVE: To explore the effect of hepatocyte growth factor (HGF) on oxygen-glucose deprived injury and apoptosis of astrocytes. METHODS: The injury of primary cultured rat cerebral cortical astrocytes was induced by oxygen-glucose deprivation. Astrocytes were treated with HGF at various final concentrations of 20 - 100 ng/mL. The cell damage and viability were evaluated by the lactate dehydrogenase (LDH) released rate and the 3- (4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion method. Detection of apoptotic cells was determined by the flow cytometry, and the ultrastructure was observed by the transmission electron microscope. RESULTS: Oxygen-glucose deprivation increased the LDH release rate, decreased the cell viability and increased the number of apoptotic astrocytes. While exposed to HGF at the same condition, the LDH release rate decreased, the cell viability increased, and the percentage of apoptotic cells decreased (P <0.05). The maximum protective effect of HGF was observed at 60 ng/mL. CONCLUSION: HGF can protect cultured astrocytes from oxygen-glucose deprived injury, and attenuate the apoptosis of astrocytes in a dose-dependent manner.

[Clinical observation on treatment of Russula subnigricans poisoning patients by Ganoderma lucidum decoction].

OBJECTIVE: To observe the effect of Ganoderma lucidum decoction in treating Russula subnigricans poisoning (RSP) patients. METHODS: The 14 patients of RSP in the treated group were treated with GLD (GLD, one dose was prepared by 100 g of Ganoderma lucidum decocted with water to 600 ml), on the base of conventional treatment, and 11 patients received conventional therapy in the previous year were taken as control. The clinical efficacy and parameters in them were compared, including the urine N-acetyl-D-glucosaminidase (NAG, which reflects the injury of kidney), the red blood cell and protein in urine, the alanine transaminase (ALT, which reflects the injury of liver), and the aspartate aminotransferase (AST, which reflects the injury of heart). RESULTS: A better clinical cure-markedly improving rate was showed in the treated group as compared with the control group, P < 0.01. In the treated group, red blood cell in urine disappeared after 24 hrs treatment in the majority of patients, urinary protein reduced obviously and the other three parameters reached the peak at the 3rd day then lowered gradually. In the control group, all the parameters increased continuously. Comparison between the parameters at corresponding time in the two groups showed significant difference (P < 0.01), those in the treated group were markedly lower than those in the control group respectively. CONCLUSION: GLD has good effect in treating RSP, could obviously lower the fatat rate of RSP.