Nrf2-NF-κB-NOS2 axis in RSA59 induced oxidative-inflammatory stress & therapeutic intervention of Methanolic Neem Bark Extract

Bhattacharyya, Debina (2025) Nrf2-NF-κB-NOS2 axis in RSA59 induced oxidative-inflammatory stress & therapeutic intervention of Methanolic Neem Bark Extract. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Debina Bhattacharyya (20RS008)) 20RS008.pdf - Submitted Version Restricted to Repository staff only Download (16MB)

Abstract

Murine β-coronavirus Mouse Hepatitis Virus, MHV-A59 is the best-explored hepato-neurotropic experimental animal virus to investigate virus-induced neuroinflammation and demyelination mimicking the pathobiology of human neurological disease Multiple Sclerosis (MS). Very recently, MHV-A59 induced neuroinflammation served as an prototype experimental model to understand COVID-19-associated neuropathogenesis. Previous studies have explored that MHV-A59 and its isogenic recombinant strain RSA59 elicit a host response through the XBP-DJ-1-Nrf-2 axis as well as MMP-inflammatory axis in four-week-old mice inflamed brains. Parallel studies demonstrated that the deficiency of a pro-inflammatory molecule, inducible nitric oxide synthase, iNOS or NOS2, was found to be a key player in aggravating demyelination in RSA59-infected male mice. Observing the potential of induction of arrays of stress pathways in β-CoV infection & pathogenesis, my current study for my PhD thesis is focused on the interplay of oxidative and inflammatory stress pathways in RSA59 infection through Nrf-2 -NOS2-NF-κB axis. In the holistic approach, the RSA59 infected wild-type C57BL/6 male mice CNS were analyzed for the induction of oxidative inflammatory stress pathways. In the reductionist approach, we used neuronal cell line Neuro2A, astrocytoma cell line DBT, and a secondary macrophage cell line RAW 264.7 cells to understand cell-specific response upon RSA59 infection. With RSA59 infection, cellular Reactive Oxygen Species were induced in the brains with a robust up-regulation of antioxidant transcriptional regulator Nrf-2 at day 7 p.i. Nrf-2 up-regulation further activated the anti-oxidant defense through HMOX-1 and Catalase. By day 10 p.i. when the infectious RSA59 particles were cleared from the system there were reduction of expression of Nrf-2, HMOX-1, and Catalase. Inflammatory marker NF-κB fold changes were persistent from day 7 to 10 post RSA59infection. Neuro2A cells responded with active antioxidant defense with delayed inflammatory response, whereas DBT and RAW cells showed impaired antioxidant activation and robust inflammatory response. NOS2 were differentially active in these cell lines, indicating its role in the interplay of oxidative and inflammatory stress pathways. Furthermore, to investigate the central role of iNOS or NOS2 in this nexus, we compared the stress induction in wild-type and NOS-2 deficient mice. In the absence of NOS2, viral mRNA persisted in mice brains with an impaired induction of oxidative stress pathways. The inflammatory pathways were triggered aggravatedly as observed with TNF-α activation in NOS2-/- mice brains. Interestingly, NF-κB & Arginase-1 were persistently downregulated despite enhanced inflammation, implying a complex and context-dependent role of NOS2 in immune regulation. These observations may explain the early and severe demyelination seen in NOS2-deficient mice, underscoring NOS2’s dual role in antiviral defense and immune homeostasis. To further investigate a therapeutic convention against MHV-RSA59-induced oxidative inflammatory stress, we used methanolic neem bark extract or MNBE and its bioactive compound in this system. MNBE significantly ameliorated the replication and spread of RSA59, reduced oxidative& inflammatory stress induction, and significantly induced antioxidant expression. With the clearance of the virus from mice brains, MNBE also successfully restricted leukocyte infiltration. Overall, these findings highlight the role of Nrf-2-NOS2-NfκB in the nexus of RSA59-induced oxidative-inflammatory stress and the transitional role of MNBE as a promising therapeutic intervention against RSA59-induced oxidative stress and inflammation.

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