Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII α in epileptic hippocampal neurons.

PURPOSE: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated. METHOD: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg(2+) free medium for 3 hours; 3) Model group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg(2+) free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca(2+) turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca(2+) turnover was observed under a laser scanning confocal microscope. RESULTS: The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca(2+) fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes. CONCLUSION: GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.

Intervention effects of ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of neurotrophin-4 and N-cadherin.

Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg(2+) free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg(2+) free medium for 3 hours), iii) GLS group I (incubated with Mg(2+) free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg(2+) free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.

Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro.

Radix Lithosperm eyrthrorhizon is a common prescription compound in traditional Chinese medicine. Shikonin is a major component of Radix Lithospermi and has various biological activities. We have investigated the inhibitory effect of shikonin on the growth of adenovirus type 3 (AdV3) in vitro. The antiviral function of shikonin against AdV3 and its virus inhibition ratio were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT). The expression of hexon protein in AdV3 was determined by immunofluorescence assay using laser scanning confocal microscopy (LSCM) and Western blot analysis. In addition, the rate of apoptosis in cells infected by AdV3 was determined by flow cytometry. Shikonin (0.0156-1 µM) inhibited growth of AdV3 in a concentration-dependent manner with a virus inhibition rate of 23.8-69.1%. Expression of hexon protein in AdV3 was higher in the virus control group than in the shikonin-treated groups as determined by immunofluorescence assay and Western blotting (p<0.05). The rate of shikonin-treated HeLa cell apoptosis had a statistically significant decrease with increasing concentration of drug (p<0.05). Our data demonstrate that shikonin possesses anti-AdV3 capabilities and that the potential antiviral mechanism might involve inhibiting the degree of apoptosis and hexon protein expression of AdV.

[Intervention effect of ganoderma lucidum spores on the changes of XOD, MPO and SDH in the testis tissue of NIDDM rats].

OBJECTIVE: To investigate the changes of xanthine oxidase (XOD), myeloperoxidase (MPO) and mitochondrial succinate dehydrogenase (SDH) in the testis and the protective effect of ganoderma lucidum spores on the testicular tissue of rats with non-insu- lin-dependent diabetes mellitus (NIDDM). METHODS: Fifty male Wistar rats were divided randomly into a model, a ganoderma and a normal control group, the first two groups injected with 2% STZ (25 mg/kg) through the peritoneum, and the last one with half-and-half sodium citrate/citrate buffer solution. Two weeks after normal diet, glucose tolerance tests were performed and the rats with abnormal glucose tolerance in the model and ganoderma groups received high-fat and high-carbohydrate food, the latter given ganoderma lycium spores (250 mg/kg x d) in addition, both for 10 weeks and all rats fed alone. Glucose tolerance tests were repeated 1 day before the end of the experiment and the testes of the rats were harvested for the determination of XOD, MPO and SDH. RESULTS: SDH was significantly lower (P < 0.05) while XOD and MPO significantly higher in the model group than in the ganoderma and control groups (P < 0.05). The model rats exhibited abnormal convoluted seminiferous tubules, indistinct parietal layers, decreased or abolished gonepoiesis, luminal peripheral fibrous tissue (interstitial substance) accrementition, basal lamina thickening, and vessel wall fibrous tissue accrementition and sclerosis. CONCLUSION: Ganoderma lucidum spores can protect the testis of diabetic rats by reducing free radical-induced damage to the testicular tissue and enhancing the activity of SDH.