neo japonicum. The current function reports the study of neuritogenic ef fects of aqueous extracts of medicinal mushrooms basidio carps, namely H. erinaceus, G. lucidum, G. neo japonicum and G. frondosa on Pc twelve cells. Additionally, the results of cellular signaling pathways, MEK ERK1 2 and PI3K Akt in the potentiation of neuritogenic activity in Pc 12 cells through the use of precise pharmacological inhibitors have been investigated. Strategies Products and chemical compounds The H. erinaceus and G. lucidum basidiocarps have been obtained from Ganofarm in Tanjung Sepat, Selangor. Ganoderma neo japonicum basidiocarps were collected from a forest in Ulu Grik, Perak and G. frondosa basidiocarps were obtained from a hypermarket in Selangor, Malaysia. The mushrooms had been recognized and authenticated by authorities within the Mushroom Study Centre, University of Malaya.
Voucher specimens are de posited during the University of Malaya herbarium. Rat pheochromocytoma cell line was pur chased from American Sort selleck chemicals Culture Collection. Kaighns Modification of Hams F 12 Medium, NGF 7S from murine submaxillary gland, 3 2,5 brom ide, phosphate buffered saline, dimethyl sulfoxide, MEK inhibitor, PI3K inhibitor, anti neurofilament 200 antibody produced in rabbit and Anti Rabbit IgG Fluorescein isothiocyanate antibody generated in sheep had been obtained from Sigma Co. ProLong Gold Antifade Reagent with DAPI was purchased from Existence Technologies Corporation. Fetal bovine serum and horse serum have been pur chased from PAA Laboratories. Preparation of aqueous extracts The aqueous extracts have been prepared in accordance to Eik et al.
Briefly, the fresh basidiocarps of H. erinaceus and G. frondosa CH5424802 have been sliced, weighed and freeze dried although G. lucidum and G. neo japonicum were air dried. The dried basidiocarps were then ground into powder by a Waring business blender. The powder was then soaked in distilled water at a ratio of 1,20 and 150 rpm at area temperature. After 24 h, the mixture was double boiled within a water bath for thirty min and soon after cooling was filtered as a result of Whatman no. four filter paper. The resulting aqueous extracts were freeze dried and stored at twenty C just before use. In vitro cell culture The rat pheochromocytoma cells were sustained in ATCC formulated F twelve K medium and supplemented with 15% of heat inactivated HS and 2. 5% of heat inactivated FBS with ultimate pH 6. eight seven. two. The cells had been subcultured each 2 to 3 days and in cubated at 37 2 C inside a 5% CO2 humidified incubator. Cell viability and cytotoxicity assay Cell viability was assessed by the mitochondrial dependent reduction of MTT to purple formazan. Pc 12 cells have been plated in 96 well plates at a density of five ? 103 cells nicely and incubated overnight at 37 C inside a 5% CO2 humidified incubator. Then, the aqueous extracts had been added to the cells.