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 Mg2+ 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 Mg2+ free medium for 3 hours), iii) GLS group I (incubated with Mg2+ 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 Mg2+ 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

Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used