Abstract 1288 The Anti-Inflammatory Effect of Small Molecular Compound CYD0630 Occurs Through the Canonical NF-κB-Regulated STAT3 Pathway in Hepatic Stellate Cells

Introduction.Hepatic fibrosis arises from dysregulated wound healing, primarily driven by the activation of hepatic stellate cells (HSCs).This activation is triggered by various inflammatory stimuli, including signaling through the TNFa, NF-kB, MAPK, and STAT3 pathways.Among these, the NF-kB and STAT3 pathways are pivotal pro-inflammatory signaling routes that are significantly upregulated in fibrogenesis within the liver.However, the precise interaction between NF-kB and STAT3 activities in HSCs remains incompletely understood, particularly in the context of hepatic fibrosis.In prior research, we demonstrated that oridonin, a naturally occurring diterpenoid compound, and its synthetic analogs exhibit potent anti-inflammatory properties, with a remarkable capacity to inhibit both the NF-kB and STAT3 pathways.Building on these findings, our current study explores the therapeutic potential of CYD0630, a novel oridonin analog designed to enhance potency and specificity in targeting inflammatory signaling in HSCs.CYD0630 has shown potent antiproliferative effects and improved specificity against multiple cancer types in vitro, compared with oridonin, however, its efficacy in limiting inflammatory signaling is unknown.In this study, we will examine effect of CYD0630 on inflammation and hypothesize that CYD0630 will minimize inflammation in the liver through the NF-kB and STAT3 pathways.Methods.The activated human HSC line LX-2 was treated with either CYD0630 or other chemical inhibitors, then exposed to TNFa.Nuclear and cytosolic proteins were isolated for Western blots or immunofluorescence assay.The secretion of cytokines were measured by ELISA.Results.CYD0630 treatment suppressed TNFa-induced pro-inflammatory factors ICAM-1, IL-6 and IL-1b in dose-dependent manner in LX-2 cells.These TNFainduced pro-inflammatory factors were similarly suppressed by both STAT3 and NF-B chemical inhibitors.Our data showed that CYD0630 inhibited TNFa-induced NF-Bp65 nuclear translocation and DNA binding.TNFa-induced phosphorylation of NF-B inhibitory protein IBa and its upstream factors IKKa/b were suppressed by CYD0630.NF-B has been reported to interact with other major signaling pathways.Our results revealed that, in LX-2 cells, TNFa-induced Stat3 phosphorylation of T705 and its nuclear translocation, DNA binding were suppressed by CYD0630.Notably, NF-B inhibitors Bay11-7082, PDTC and p65 siRNA also suppressed TNFa-induced Stat3 activation, while Stat3 inhibitors did not affect NF-B activation by TNFa, indicating that STAT3 activation occurs downstream of NF-B activation by TNFa.Conclusion.The anti-inflammatory effect of CYD0630 is similar to NF-B inhibitors and may occur through inhibition of canonical NF-B-regulated STAT3 pathway.