Evaluation of the Cytotoxic, Anti-Inflammatory, and Immunomodulatory Effects of Withaferin A (WA) against Lipopolysaccharide (LPS)-Induced Inflammation in Immune Cells Derived from BALB/c Mice

Abstract

(1) Background: Inflammation is one of the primary responses of the immune system and plays a key role in the pathophysiology of various diseases. Recent reports suggest that various phytochemicals exhibit promising anti-inflammatory and immunomodulation activities with relatively few undesirable effects, thus offering a viable option to deal with inflammation and associated diseases. The current study evaluates the anti-inflammatory and immunomodulatory effects of withaferin A (WA) in immune cells extracted from BALB/c mice. (2) Methods: MTT assays were performed to assess the cell viability of splenocytes and anti-inflammatory doses of WA. Under aseptic conditions, the isolation of macrophages and splenocytes from BALB/c mice was performed to investigate the anti-inflammatory effects of WA. Analysis of the expression of proinflammatory cytokines and associated signaling mediators was performed using proinflammatory assay kits, real-time polymerase chain reaction (RT-PCR), and immunoblotting, while the quantification of B and T cells was performed by flow cytometry. (3) Results: Our results demonstrated that WA exhibits anti-inflammatory and immunomodulatory effects in LPS-stimulated macrophages and splenocytes derived from BALB/c mice, respectively. Mechanistically, we found that WA promotes an anti-inflammatory effect on LPS-stimulated macrophages by attenuating the secretion and expression of proinflammatory cytokines TNF-α, IL-1β, IL-6, and the inflammation modulator NO, both at the transcriptional and translational level, respectively. Further, WA inhibits LPS-stimulated inflammatory signaling by dephosphorylation of p-Akt-Ser473 and p-ERK1/2. This dephosphorylation does not allow IĸB-kinase activation to disrupt IĸB–NF-ĸB interaction. The consistent interaction of IĸB with NF-ĸB in WA-treated cells attenuates the activation of downstream inflammatory signaling mediators Cox-2 and iNOS expression, which play crucial roles in inflammatory signaling. Additionally, we observed significant immunomodulation of LPS-stimulated spleen-derived lymphocytes by suppression of B (CD19) and T (CD4+/CD8+) cell populations after treatment with WA. (4) Conclusion: WA exhibits anti-inflammatory and immunomodulatory activity by modulating Akt/ERK/NF-kB-mediated inflammatory signaling in macrophages and immunosuppression of B (CD19) and T cell (CD4+/CD8+) populations in splenocytes after LPS stimulation. These results suggest that WA could act as a potential anti-inflammatory/immunomodulatory molecule and support its use in the field of immunopharmacology to modulate immune system cells.

Combined Anti-Angiogenic and Anti-Inflammatory Nanoformulation for Effective Treatment of Ocular Vascular Diseases

“…Lipopolysaccharide (LPS) was used to induce the inflammatory response. 32 HRVECs were seeded onto a 24-well plate and incubated for 24 h to allow cell attachment. Then, they were stimulated with LPS (100 ng/mL).…”

Effects of Mitragyna Speciosa (Korth.) on macrophage immune responses

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Evaluating anticancer properties of Withaferin A—a potent phytochemical

“…In another study, 0.25–2.0 μg/ml Withaferin A treatment of mouse and human islets resulted in prevention of cytokine induced cell death and inhibited NF-κB activation ( SoRelle et al, 2013 ). Immunomodulatory and anti-inflammatory effects of Withaferin A has been noted in macrophages and splenocytes ( Alnuqaydan et al, 2022 ). Lipopolysaccharide (LPS) stimulated macrophages treated with Withaferin A showed reduced expression and secretion of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in a dose dependent manner ( Alnuqaydan et al, 2022 ).…”

“…Immunomodulatory and anti-inflammatory effects of Withaferin A has been noted in macrophages and splenocytes ( Alnuqaydan et al, 2022 ). Lipopolysaccharide (LPS) stimulated macrophages treated with Withaferin A showed reduced expression and secretion of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in a dose dependent manner ( Alnuqaydan et al, 2022 ). Inhibitory immunomodulatory effects of Withaferin A were also noted in BALB/c mice spleen derived CD4/CD8 T cells and CD19 B cells in a dose dependent treatment ( Alnuqaydan et al, 2022 ).…”