A mechanistic study of the neurite stimulative and cyto-protective properties of sclerotial extracts of Lignosus rhinocerotis Ryvarden / Tan Yong Hui

Tan , Yong Hui (2022) A mechanistic study of the neurite stimulative and cyto-protective properties of sclerotial extracts of Lignosus rhinocerotis Ryvarden / Tan Yong Hui. PhD thesis, Universiti Malaya.

	PDF (The Candidate's Agreement) Restricted to Repository staff only Download (232Kb)
	PDF (Thesis PhD) Download (2822Kb)

Abstract

Neuritin, a member of the neurotrophic factor family, is important in neuritogenesis, neurite arborization, and neurite extension. Although the neurite stimulatory effect of Lignosus rhinocerotis sclerotia extracts are well-documented in literature, the role of neuritin and its correlation to neurite outgrowth and cyto-protection in L. rhinocerotis treated pheochromocytoma (PC12) cells remained unknown. Therefore, this study aims to investigate the involvement of neuritin protein in L. rhinocerotis-induced neurite outgrowth and cyto-protection against hydrogen peroxide-induced oxidative stress. Freeze-dried L. rhinocerotis sclerotia powder was subjected to aqueous and ethanolic extraction. Pheochromocytoma cells were cultured in low serum and treated with nerve growth factor (5 ng/ml) and extracts (20-1280 μg/ml) for 48 hr. Cytotoxicity of the extracts was assessed and the average length of neurites from 20-30 cells was calculated based on 5 microscopic fields at 200x total magnification by measuring their axon-like extensions with each extension defined as double the length of cell body diameter. Neuritin concentration was quantified using an enzyme-linked immunosorbent assay. Ribonucleic acid (RNA) extraction was carried out to investigate differential expression of genes involved in neurogenesis via PCR array profiling and real-time PCR. The viability of PC12 cells upon hydrogen peroxide-induced oxidative stress in pre- and co-treatment with extracts was determined before selecting the best neuroprotection model to proceed with oxidative stress, mitochondrial membrane potential, and mitogen-activated protein kinase phosphorylation quantification using flow cytometry. GCMS analysis was carried out to identify the compounds that are present in the ethanolic extract. The IC50 values obtained from the tetrazolium (MTT) assay showed no toxic effects following 24 hours of exposure of PC12 cells to the extracts. There was a significant difference (p ≤ 0.05) in neuritin protein abundance in 640 μg/ml aqueous-treated (5.00 ± 0.83 ng/ml) and 50ng/ml NGF-treated PC12 cells (5.00 ± 0.96 ng/ml) as compared to untreated cells (1.86 ± 0.65 ng/ml) with average neurite length of 98 ± 12 μm, 106 ± 13 μm, and 73 ± 9 μm, respectively. Neuritin protein abundance is directly proportional to the average neurite length in PC12 cells treated with L. rhinocerotis aqueous extracts. Seventeen genes out of 84 genes in the PCR array were commonly expressed in positive and extract-treated cells as compared to untreated cells. These genes were found to involve cell differentiation, synaptic transmission, as growth factors and cytokines during neurogenesis. In terms of cyto-protection, pre-treatment and co-treatment for 24 hours with aqueous extract recorded viability of more than 80%. No significant difference in neuritin concentration in the cyto-protection experiment. It was also interesting to note that cyto-protection properties of extract were not via reactive oxygen species reduction but through prevention of mitochondrial membrane depolarization. Based on ApoTox-Glo triplex assay, treatment of PC12 cells with extracts mitigates cells by reducing apoptosis events. Fatty acids, phenol, phytosterol, and dicarboxylic acid were found in L. rhinocerotis ethanolic extract. In conclusion, the mechanistic action of neurite outgrowth in PC12 cells upon L. rhinocerotis treatment could be facilitated by an upregulation in neuritin protein. L. rhinocerotis extracts protect PC12 cells from hydrogen peroxide-induced oxidative stress via the reduction of mitochondria depolarized cells and reduction in caspase 3/7 activity but no via regulation of neuritin expression.

Actions (For repository staff only : Login required)