Studies on the protective effects of ergothioneine - rich mushroom extract against stress - induced premature senescence (SIPS) in neuronal cells / Yasaaswini Apparoo

Yasaaswini , Apparoo (2024) Studies on the protective effects of ergothioneine - rich mushroom extract against stress - induced premature senescence (SIPS) in neuronal cells / Yasaaswini Apparoo. PhD thesis, Universiti Malaya.

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Abstract

Healthy ageing is a crucial process that needs to be highlighted as it affects the quality of lifespan. An increase in mitochondrial oxidative stress along with ageing is the major factor related to the production of senescent cells which contributes to age-related diseases, especially neurodegenerative disorders. The beneficial effect of mushroom ergothioneine as an antioxidant is believed to counteract oxidative stress. Therefore, this study aimed to investigate the neuroprotective effect of ergothioneine (EGT)-rich mushroom extract against t-BHP-assaulted senescent HT22 cells. Among the 14 evaluated mushroom species, Lentinula edodes (LE), shiitake mushroom contains the highest EGT content and hence was used for the in-vitro studies. The cells were preincubated with ethanolic extract of EGT-rich mushroom (LE-ETH) in a dose-time dependent manner against t-BHP-induced HT22 cells. The equimolar concentration of EGT was also used to determine the efficacy of extract containing this compound directly on neuronal senescent cells. The extract was analysed for its free radical scavenging properties using DPPH and ABTS methods. The neuroprotective, anti-senescence, antioxidant and anti-inflammatory effects of LE-ETH and equimolar EGT concentration were also evaluated. Next, mitochondrial function was evaluated by measuring mitochondrial membrane potential (MMP), ATP levels and mitochondrial toxicity. The mechanisms of neuroprotection were also elucidated via the exploration of antioxidant and mitochondrial biogenesis signalling pathways. Our results revealed that a low dose of t-BHP successfully induces senescence and reduces cell viability in HT22 cells which was confirmed through β-galactosidase staining, expressions of P16INK4a, P21CIPL and mitochondrial functions which are the markers of cellular senescence. However, the pretreatment with ethanolic extract of LE and equimolar EGT concentration for 8 h significantly improved the cell viability, reversed the t-BHP-induced cellular senescence in the neuronal cells, which was evident by the significant reduction in positively blue-stained senescent cells and a healthy neuronal morphology. In addition, a significant reduction of the reactive oxygen species was visualized through DCFH-DA staining, confirming its role as an antioxidant. The pretreatment also significantly improves the MMP and ATP levels followed by the drastic reduction in mitochondrial toxicity and inflammation. Through gene expression studies, we demonstrated that pretreatment of LE-ETH significantly improves the antioxidant and mitochondrial biogenesis pathway via the Nrf2 signalling axis. However, the downstream genes of the mitochondrial biogenesis pathway were unaffected by equimolar EGT concentration. Correspondingly, GC-MS analysis has been carried out to identify the bioactive compounds that are present in LE-ETH extract which contributed to its better efficacy in improving the mitochondrial functions in t-BHP-induced senescent HT22 neuronal cells. A total of 23 compounds were identified in the ethanolic extract consisting of fatty acids, sterols and phenol. Some identified lead compounds include pentanoic acid, 4-methyl, 2- decenoic acid and ergosterol. These findings demonstrated that ergothioneine-rich mushrooms are a potential neuroprotective agent and a compelling therapeutic strategy for healthy ageing explorations through the elimination of senescent cells via the improvement of mitochondrial functions.

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