Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial hyperplasia, persistent inflammation, and joint destruction. Targeted inhibition of intracellular signaling pathways, such as JAK-STAT, has improved RA treatment outcomes, though safety and selectivity remain as concerns. Brepocitinib, a dual TYK2/JAK1 inhibitor, has shown clinical efficacy in the management of autoimmune diseases, yet its mechanistic impact on synoviocytes remains underexplored. Objectives: To investigate the molecular and functional effects of brepocitinib on MH7A and RA-FLS synoviocytes, a key effector cell type in RA pathogenesis. Methods: MH7A and RA-FLS cells were treated with brepocitinib (0.5 µM, 1 µM, and 5 µM) for 24 hours. Cell viability was assessed. Western blotting was used to examine phosphorylation of TYK2, JAK1, STAT1/3, and apoptotic markers (BAX, BCL-2, caspase-3). Quantitative PCR and ELISA were performed to evaluate mRNA and protein levels, respectively, of IL-6, TNF-α, and IFN-γ. Wound healing assays measured synoviocyte migration. Results: Brepocitinib maintained ≥ 85% cell viability across all doses, compared with ~20% viability in doxorubicin-treated controls. At 5 µM, phosphorylation of JAK1 and STAT3 was suppressed by > 80%, while TYK2 and STAT1 inhibition reached ~70%. IL-6 and TNF-α transcripts were reduced by > 80% and IFN-γ by ~70%, with corresponding decreases in secreted cytokines (IL-6: 100 pg/mL to 20 pg/mL; TNF-α: 150 pg/mL to 15 pg/mL; IFN-γ: 41 pg/mL to 11 pg/mL). Brepocitinib shifted the BAX/BCL-2 ratio fourfold in favor of apoptosis and increased cleaved caspase-3 levels to ~80% of maximal response. Functionally, it reduced wound closure from ~75% in controls to ~20% at 5 µM, confirming potent inhibition of synoviocyte migration. Conclusions: Brepocitinib exerts multi-faceted effects on RA synoviocytes by simultaneously inhibiting inflammatory signaling, suppressing cytokine expression, restoring apoptotic sensitivity, and reducing migratory potential. These findings provide mechanistic support for brepocitinib as a targeted therapeutic agent in RA.

Background: Diabetic nephropathy is a major cause of end-stage renal disease, driven in part by molecular dysfunctions in podocytes. Sirtuin 2 (Sirt2), a cytoplasmic NAD⁺-dependent deacetylase, has emerged as a potential regulator of key metabolic pathways, but its specific role in podocyte biology remains poorly defined. Objectives: This study aimed to investigate the function of Sirt2 in human podocytes (hPodo), delineate its interaction with histone deacetylase 6 (HDAC6), and evaluate the therapeutic potential of Sirt2 inhibition in restoring metabolic balance and protecting against diabetic nephropathy-associated podocyte stress. Methods: Comparative expression analysis was performed between hPodo and HEK293T kidney cells. Pharmacological inhibition of Sirt2 was carried out using acylglycerol kinase 2 (AGK2), alongside siRNA-mediated Sirt2 knockdown. AMPK/AKT/mTOR signaling activity was assessed by Western blotting and functional assays to determine metabolic and growth responses. Results: Human podocytes exhibited significantly elevated Sirt2 expression and high levels of HDAC6, forming a unique Sirt2–HDAC6 regulatory complex. Inhibition or silencing of Sirt2 induced robust AMPK activation while suppressing AKT/mTOR signaling. This signaling reprogramming restored energy sensing and attenuated hyperactive growth pathways, alleviating podocyte stress. Acylglycerol kinase 2 treatment reestablished metabolic homeostasis by disrupting Sirt2-mediated repression of AMPK. Conclusions: Sirtuin 2 inhibition, particularly through AGK2, emerges as a novel pharmacological strategy to protect podocytes, restore metabolic regulation, and potentially slow the progression of diabetic nephropathy. Significance Statement By inhibiting one of the important intracellular signaling pathways in human kidney cells, we could reduce the cellular stress that is commonly observed in diabetic kidney injury. This could serve as a drug target to slow the progression of kidney disease associated with diabetes mellitus.

Background: Hepatitis B virus (HBV) establishes persistent infection through the formation of a covalently closed circular DNA (cccDNA) minichromosome, which enables immune evasion and sustained viral replication. While SIRT1, IFI16, and STING are known to modulate antiviral pathways, their cooperative role in regulating HBV transcription and host immunity remains poorly defined. Methods: We investigated the functional interplay between SIRT1, IFI16, and STING using confocal microscopy, chromatin immunoprecipitation (ChIP), gene silencing, and quantitative PCR in HBV-replicating cell models. Interactions with HBV cccDNA and effects on viral transcription and interferon-stimulated gene (ISG) expression were analyzed. Northern blotting and RT-qPCR were used to evaluate HBV RNA output, including total HBV RNA and 3.5 kb RNA, in order to provide both transcript-pattern and quantitative assessment of transcriptional changes. Results: SIRT1 physically interacted with IFI16, with increased colocalization during HBV replication. Both proteins were recruited to HBV cccDNA, promoting transcriptionally active chromatin. Silencing SIRT1 or IFI16 disrupted cccDNA binding and reduced viral RNA levels. Although STING was not cccDNA-associated, its antiviral activity via ISG induction was dependent on IFI16 knockdown produced the strongest reduction in both total HBV RNA and 3.5 kb RNA, supporting a cooperative role for these factors in sustaining HBV transcriptional output. Although STING was not cccDNA-associated, its antiviral activity via ISG induction was dependent on IFI16. Triple knockdown of SIRT1, IFI16, and STING showed the strongest suppression of ISG expression. Conclusion: SIRT1 and IFI16 enhance HBV gene expression through epigenetic regulation of cccDNA, while also supporting STING-mediated antiviral responses. These findings reveal a dual regulatory axis governing HBV persistence and identify potential targets for antiviral therapy.