Qingyi decoction and its active ingredients ameliorate acute pancreatitis by regulating acinar cells and macrophages via NF-κB/NLRP3/Caspase-1 pathways

Abstract

Background and Purpose

Macrophage infiltration and activation is a critical step during acute pancreatitis (AP). NLRP3 inflammasomes in macrophages plays a critical role in mediating pancreatic inflammatory responses. Qing-Yi Decoction(QYD)has been used for many years in clinical practice of Nankai Hospital combined with traditional Chinese and western medicine treatment of acute pancreatitis. Although QYD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects. Here, we elucidate the therapeutic effects of QYD against acute pancreatitis and reveal its mechanism of action.

Methods

The main components of QYD were identified using UHPLC-Q-Orbitrap MS. Network pharmacology was employed to predict potential therapeutic targets and their mechanisms of action. C57BL/6 mice were randomly divided into control group, model group, low, medium and high dose (6, 12, 24 g/kg) QYD groups, with 10 mice in each group. The therapeutic effect of QYD on cerulein-induced acute pancreatitis. (CER-AP) was evaluated by histopathological score, immunohistochemistry, serum amylase and cytokines detection by ELISA. The protein expressions of MyD88/NF-κB/NLRP3 signaling pathway were detected by Western blotting. Along with molecular docking of key bioactive compounds and targets, RAW264.7 cells stimulated with 1μg/ml LPS is used to screen components with more potent effects on target proteins. AR42 J cells were stimulated with 100 nM dexamethasone (dexa) combined with 10 nM cerulein (CN) as s a cell-culture model of acute pancreatitis. Inhibitory effects of the main chemical composition Wogonoside on NLRP3 inflammasomes were analyzed by qRT-PCR and Western blots.

Results

Using UHPLC-Q-Orbitrap MS, 217 compounds were identified from QYD, including Wogonoside, Catechins, Rhein, etc. A visualization network of QYD-compounds-key targets-pathways-AP show that QYD may modulate PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, Ras signaling pathway and Apoptosis signaling pathway by targeting TNF, IL1β, AKT1, TP53 and STAT3 exerting a therapeutic effect on AP. QYD administration effectively mitigated CER-induced cytokine storm, pancreas edema and serum amylase. QYD (12 mg/kg) showed better effect. The protein expression levels of MyD88, NF-κB, NLRP3, Caspase-1 and GSDMD in pancreatic tissue were significantly decreased. Through molecular docking and LPS-RAW264.7 inflammation model, the selected Wogonoside significantly decreased IL-1β mRNA. The expression levels of NLRP3/Caspase-1/GSDMD pathway-related proteins were also decreased on AR42J-AP.

Conclusion

The results of network pharmacology indicate that QYD can inhibit AP through multiple pathways and targets. This finding was validated through in vivo tests, which demonstrated that QYD can reduce AP by inhibiting NLRP3 inflammasomes, additionally, it should be noted that 12mg/kg was a relatively superior dose. One of the main chemical compositions Wogonoside regulated NLRP3 inflammasome activation to protect against AP. This study is the first to verify the intrinsic molecular mechanism of QYD in treating AP by combining network pharmacology and animal experiments. The findings can provide evidence for subsequent clinical research and drug development.