Applied Biochemistry and Biotechnology, Vol.191, No.2, 555-566, 2020
Anthraquinone from Edible Fungi Pleurotus ostreatus Protects Human SH-SY5Y Neuroblastoma Cells Against 6-Hydroxydopamine-Induced Cell Death-Preclinical Validation of Gene Knockout Possibilities of PARK7, PINK1, and SNCA1 Using CRISPR SpCas9
Parkinson's disease (PD) results from the degeneration of the nervous tissue brought about by ecological and hereditary components which affects nerve cells in the brain. It is the world's second most normal neurodegenerative issue, which can essentially weaken the personal satisfaction, make reliance, and trigger untimely mortality of affected people. The commonness pace of PD is 0.5-1% among individuals in the age group of 65-69 years and 1-3% among those 80 or more. Clinical appearances incorporate bradykinesia, tremors, unbending nature, and postural unsteadiness; spectrums of non-motor symptoms include psychological hindrance and passionate and behavioral brokenness. In this study, 6-OHDA-induced neurotoxicity was analyzed for various cytotoxicity analyses. The genes identified were PINK1 (PTEN-induced kinase 1), PARK7 (Parkinsonism-associated deglycase) and SNCA 1 (alpha synuclein1) validated using CRISPR spcas9 genome editing tool. In this study, Anthraquinone isolated from Pleurotus ostreatus was treated against a dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), which induced neurotoxicity in SH-SY5Y cells. Experimental groups in SH-SY5Y neuroblastoma cells were treated with anthraquinone (50 nM) and 6-OHDA (100 nM). MTT and ROS assays were performed to assess the cell viability and oxidative stress within the cells, followed by mixed-member proportional (Mitochondrial membrane potential), dual staining, and immunoblotting. 6-OHDA-induced cell death in SH-SY5Y cells was dose-dependently attenuated by treatment with anthraquinone. The genes responsible for mutation were studied and the mutated RNAs knockout possibilities was studied using CRISPR spcas9 genome editing tool. Treatment with anthraquinone attenuated the level of oxidative stress and reduced the mitochondrial dysfunction associated with 6-OHDA treatment. Immunoblot analysis carried out with apoptotic markers showed that cytochrome C and caspase-3 expression increased significantly in anthraquinone-treated cells, whereas 6-OHDA-treated group showed a significant decrease when compared with an experimental control group. The mutated genes PARK7, PINK1, and SNCA1 were analyzed and found to exhibit four gene knock possibilities to treat PD. Reports demonstrate that other than following up on the biosynthesis of dopamine and its metabolites, these mixes counteract D2 receptors' extreme touchiness. It is proposed that further examinations need be directed to better understand the activity of the bioactive mixes circulated in these edible fungi Pleurotus ostreatus. The gene knockout possibilities identified by CRISPR SpCas9 will pave a way for better research for PD treatment.