Xuanfudaizhe Decoction Alleviate Reflux Esophagitis through Inhibition of NLRP3/Caspase-1 Pathway

Abstract

Background: Reflux esophagitis (RE) is a clinically common digestive disease, and the main pathological manifestation of RE is esophageal mucosa inflammatory damage. Xuanfu Daizhe (XFDZ) decoction is a traditional Chinese herbal compound that is famous for RE treatment, but the pharmacological and molecular mechanism of XFDZ remains largely unknown. Methods: The active ingredients of XFDZ were detected using the ultra-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). The rat model of RE was established with pylorus clamp and 2/3 fundus of stomach ligated. XFDZ (8.55 g/kg) and Omeprazole + Mosapride (1.35 g/kg) were orally administered for 14 days. Pathology of esophageal mucosal inflammation was evaluated under microscopy by hematoxylin-eosin (HE) staining. In vitro, by inducing cellular inflammatory response with glycocholic and taurocholic acid mixture (PH 4.7 acid medium added 500 μmol/L concentration of mixed bile acids) in human esophageal epithelial cells(HEEC). The mitochondrial membrane potential was detected using JC-1 fluorescence mitochondrial imaging. The mtDNA copy number was determined via quantitative real-time polymerase chain reaction (qRT-PCR). Fluorescent probe DCFH-DA was used to detect ROS. The relative fluorescence expression of NLRP3 inflammasome was determined by high-intension cell imaging and quantitative fluorescence techniques. ELISA was used to detect and quantify inflammatory cytokines related to the NLRP3 inflammasome. Western blot analysis was performed to investigate proteins that are associated with the NLRP3 inflammasome. Results: Twenty chemical components such as alkaloids and flavonoids were identified in the analysis of XFDZ, which may be the material basis for XFDZ to exert its effect. XFDZ can significantly reduce the contents of Caspase-1, IL-1β and IL-18 in serum of rats, and down-regulate the protein expression levels of NLRP3, Caspase-1, IL-1β and IL-18 in esophageal tissue. The simulated reflux could decrease the membrane potential, increase the ROS production, decrease the relative expression of mtDNA and activate the NLRP3/Caspase-1 signaling pathway in vitro. XFDZ had no obvious protective effect on the membrane potential and mtDNA, but could inhibit ROS production and the activation of NLRP3/Caspase-1 signaling pathway. Conclusion: We concluded that XFDZ could reduce the inflammatory damage in RE by inhibiting NLRP3/Caspase-1 signaling pathway both in vitroand in vivo, indicating the capability of XFDZ as a promising drug for the treatment of RE.