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Murine β-coronavirus Mouse Hepatitis Virus, MHV-A59, is the best-explored hepato-neurotropic experimental animal model to investigate virus-induced neuroinflammation and demyelination mimicking certain pathobiology of human neurological disease, Multiple Sclerosis (MS). Very recently, MHV-A59 induced neuroinflammation, also serving as a prototype model to understand the COVID-19-associated neuropathogenesis among the SARS-CoV-2 survivors. From the mechanistic understanding of the neuropathogenesis, our previous studies demonstrated that MHV-A59 and its isogenic recombinant strain, RSA59, elicit an acute host response by activating the expression of UPR pathway molecule XBP, Oxidative stress pathway activator -DJ-1, and Nrf-2 axis. Further studies showed that downstream activation of MMPs is also involved in the process of neuroinflammation and demyelination in four-week-old mice. Parallel studies also 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 mice during the bridging of the acute innate and chronic adaptive immunity. Though individual studies showed the involvement of such molecules in MHV-A59/ RSA59 infection, their holistic interaction in the process of neuroinflammation is yet to be studied. Given that my study for my PhD thesis is focused on the interplay of oxidative stress molecules and inflammatory stress pathways molecules in RSA59 infection through the Nrf-2 -NOS2-NF-κB axis. RSA59-infected 4-week-old wild-type C57BL/6 male mice were infected with RSA59 and analyzed for the induction of oxidative inflammatory stress pathways across the innate immune responses (Until day 10 p.i). Results demonstrated that upon 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 or resolved from the system, there was a significant reduction in the expression of Nrf-2, HMOX-1, and Catalase. There is a significant persistence of p65/NF-κB signaling until day 10 p.i even when the viral particles were resolved, demonstrating the negative Nrf2-NF-κB feedback loop in oxidative inflammatory stress. While it is difficult to understand the complex feedback loop in the CNS, in an inflamed brain, I also took a reductionist approach to study the cell-specific response upon RSA59 infection effects and their complex arrays in the Neuronal cell line Neuro2A, the Astrocytoma cell line DBT, and the secondary macrophage cell line RAW 264.7. Neuro2A cells responded with active antioxidant defense pathways by activating Nrf-2 expression with a delayed inflammatory response, whereas DBT and RAW cells showed impaired antioxidant activation and a robust inflammatory response. iNOS or NOS2 were differentially active in these cell lines, indicating their 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 the brains of mice with an impaired induction of oxidative stress pathways. The inflammatory pathways were triggered by 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 (MNBE) and its bioactive compound Nimbin 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-Nf-κB in the nexus of RSA59-induced oxidative-inflammatory stress. |