(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.

(1) Background globe. The etiology of CHDs is complex and involves both genetic and non-genetic factors. Although, significant progress has been made in deciphering the genetic components involved in CHDs, recent reports have revealed that mutations in Nk2 homeobox5 (NKX2-5) and actin alpha cardiac muscle1 (ACTC1) genes play a key role in CHDs such as atrial and ventricular septum defects. Therefore, the present study evaluates the role of key hotspot mutations in NKX2-5 and ACTC1 genes of congenital cardiac septal defect (CCSD) in ethnic Kashmiri population. (2) Methods: A total of 112 confirmed CHD patients were included in the current study, of which 30 patients were evaluated for mutational analysis for hotspot mutations of NKX2-5 and ACTC1 genes. The total genomic DNA was extracted from the samples (cardiac tissue/blood) and were subjected to amplification for NKX2-5 (exon 1 and 2), and ACTC1 (exon 2) genes by using PCR specific primers to analyze the hotspot mutations in respective exons. The amplified products obtained were sent to Macrogen Korea for sequencing by Sanger’s method. (3) Results: Our results confirmed that not a single mutation was found in either hotspot exon 1 and 2 of NKX2-5 and exon 2 of ACTC1 in the patients included in the current study. Interestingly, a novel synonymous nucleotide variation leading to G > C transversion (GCG > GCC) was found in exon 2 of NKX2-5 gene of CCSD patient. (4) Conclusions: The current findings demonstrated the role of NKX2-5 and ACTC1 in cardiac development. The study will provide an insight in understanding the genetic etiology and highlights the role of newly identified mutations in patients with CDS’s in ethnic Kashmiri population. In silico findings revealed amino acid changes, splice site variation and the creation of new site. Furthermore, the study warrants complete screening of genes involved in CCSDs.