Regulation of toll-like receptor-mediated innate immune response in chikungunya virus-infected and Fisetin-treated human hepatocellular carcinoma Huh7 cells / Rafidah Lani

Rafidah , Lani (2022) Regulation of toll-like receptor-mediated innate immune response in chikungunya virus-infected and Fisetin-treated human hepatocellular carcinoma Huh7 cells / Rafidah Lani. PhD thesis, Universiti Malaya.

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Abstract

Excessive inflammation caused by chikungunya virus (CHIKV) infection is manifested by incapacitating joint pain and prolonged arthritis in the chronic phase of CHIKV infection. Arthritis resulted from a large influx of infiltrating immune cells driven by pro-inflammatory cytokines and chemokines. Fisetin demonstrated anti-CHIKV activity and has long been known to possess anti-inflammatory and immunomodulatory properties. Modulating toll-like receptor (TLR)-mediated innate immune responses might be one of the mechanisms utilized by fisetin against CHIKV infection. This study was designed to assess fisetin capacity in modulating the TLR-mediated antiviral responses against CHIKV in Huh7 cells. The hallmarks of inflammation and cytopathological effects (CPE) were evaluated to test the hypothesis that fisetin promotes TLR-mediated antiviral responses and limit CPE in CHIKV-infected Huh7 cells. CHIKV-induced CPE was observed using bright-field microscopy. Viral infectivity was determined using a plaque-forming unit assay. CHIKV-E1 RNA copy number and antiviral genes mRNA level were quantified using qRT-PCR. The expression of TLR and CHIKV-E2 proteins was measured by performing an immunofluorescence assay. The level of the inflammatory cytokines was determined using a human cytokine array. The expression of the key transcription factors was determined using immunoblot assay. The results showed that fisetin induced the expression of the antiviral genes at an early time-point by promoting the phosphorylation of IRF3 and IRF7 in CHIKV-infected Huh7 cells. Fisetin hindered excessive inflammation in CHIKV-infected Huh7 cells by impeding over-phosphorylation of NF-κB and reducing pro-inflammatory cytokines levels. Fisetin also recovered Huh7 cells from CHIKV-induced CPE. The results also suggested that fisetin does not affect the level of the pro-and anti-inflammatory cytokines such as IL-1α, IL-1β, IL-8, IL-10, IL-13, GM-CSF, GRO, MCP-1, MIP-1β, and RANTES; in uninfected Huh7 cells. These suggested that fisetin is pharmacologically active only in CHIKV-infected Huh7 cells and not uninfected cells. Findings from the study supported the hypothesis that fisetin promotes antiviral responses and limit CPE in CHIKV-infected Huh7 cells. Fisetin's balancing act in CHIKV-infected Huh7 cells was subjected to fisetin modulating the key transcription factors manipulated by CHIKV infection. On this basis, further research into fisetin in targeting host inflammation as a therapeutic strategy is being considered.

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