Kinsenoside alleviates experimental acute pancreatitis by suppressing M1 macrophage polarization via the TLR4/STAT1 signaling pathway

Ethnopharmacological relevance: Acute pancreatitis (AP) is an inflammatory disease that can progress to systemic immune responses and multi-organ damage in its severe forms. Anoectochilus roxburghii (Wall.) Lindl. (AR), a traditional Chinese medicinal plant, has been reported to exhibit anti-inflammatory, hypoglycemic, hepatoprotective, and analgesic properties. Kinsenoside (KD), the primary bioactive glycoside in AR, is responsible for many of its therapeutic effects. Given its anti-inflammatory and immunomodulatory properties, KD may have the potential to mitigate pancreatic inflammation in AP. However, its protective role in AP has not yet been investigated. Aim of the study: This study aimed to investigate the protective effects of the natural active compound KD against acute pancreatitis (AP) and its associated molecular mechanisms. Materials and methods: Two AP mouse models were established: one by intraperitoneal injection of caerulein combined with lipopolysaccharide (LPS) and the other by retrograde injection of sodium taurocholate (NaT) into the biliopancreatic duct. KD (2.5, 5, 10 mg/kg) was administered as a pre-treatment 1 h before the induction of AP. The severity of AP was evaluated through histopathological analysis, while macrophage infiltration and phenotypic changes in pancreatic tissues were examined using immunofluorescence staining and flow cytometry. Bone marrow-derived macrophages (BMDMs) were polarized into the M1 phenotype through two distinct methods: stimulation with LPS and interferon-gamma (IFN gamma) and indirect co-culture with pancreatic acinar cells. Changes in macrophage phenotype after KD supplementation (100, 200, and 400 mu M) were analyzed using quantitative Reverse Transcription PCR (qRT-PCR) and flow cytometry. Network pharmacology and transcriptomic sequencing were utilized to identify potential targets and pathways affected by KD, with validation of key signaling pathways performed through qPCR and Western blot analysis. Results: In two models of AP mice, KD at a high dose (10 mg/kg) significantly alleviated pancreatic damage. It reduced pancreatic edema, necrosis, and inflammatory cell infiltration, with a notable decrease in macrophage infiltration. Furthermore, KD (10 mg/kg) administration significantly reduced serum lipase by 53.62% in the Caerulein + LPS model and 41.14% in the NaT model, as well as amylase by 28.13% and 27.99%, respectively. Additionally, KD (10 mg/kg) administration mitigated systemic inflammation and lung injury during AP. Both in vivo and in vitro experiments demonstrated that KD (400 mu M) significantly reduced the proportion of M1 macrophages. Furthermore, KD (400 mu M) downregulated the mRNA expression of M1-associated genes, including Nos2, Tnf, Ilub, and Il6, in macrophages stimulated by both LPS + IFN gamma and pancreatic acinar cell-conditioned media. Network pharmacology and transcriptomic analyses identified toll-like receptor 4 (TLR4) as a potential target of KD in the context of AP. KD (400 mu M) was shown to inhibit the activation of the TLR4/STAT1 signaling pathway in macrophages exposed to inflammatory stimuli. Conclusions: KD administration mitigated experimental AP induced by diverse etiologies through the inhibition of M1 macrophage polarization via the TLR4/STAT1 signaling pathway. These findings highlight KD as a promising therapeutic candidate with potential clinical applications in the management of AP.