Anti-ulcerogenic activity of Citrus medica var. sarcodactylis fruit extract nano-emulsion in gastric ulcer rat model through modulating HMBG1/TLR4/NF-κB and Nrf2/HSP70/PGE2 signaling pathways

Abstract

One of the most prevalent gastrointestinal multi-etiological diseases is gastric ulcer, with several consequences and serious side influences. Nowadays, nanotechnology is one of the advanced tools to enhance drug delivery and bioavailability. Hence, the prime goal of this study was to create safe gastro-protective therapy using methanol fruit extract of Citrus medica var. sarcodactylis (CMF) and its prepared nano formula (Nano-CMF). CMF or its nano form was given to rats at dose 600 mg/kg/day for a week along with a single oral administration of ethanol (5 ml/kg) at the 7th day. The particle size distribution of formulated CMF reflected the successful preparation of nano form, with average size 65 nm. Pretreatment with CMF or its nano formula to ethanol challenged rats significantly controlled the inflammatory response through down-regulated gastric HMBG1/TLR4/NF-κB signaling pathway and its downstream mediators at p<0.001. Moreover, CMF or nano-CMF lessened oxidative stress incidence via modulating Nrf2 signaling pathway, sustaining prostaglandin content, and reducing gastric juice acidity with a significant p value less than 0.001, as compared to the EtOH-intoxicated rats. In addition, nano form effectively preserved the gastric mucosal integrity and alleviated apoptosis in gastric cells as validated by ulcer index (p<0.001), and the histological and immunohistochemical findings, when matched to the EtOH-treated group. The metabolomics analysis revealed the characterization of 36 compounds. GC/MS of CH2Cl2 fraction led to the identification of 34 compounds, the major category was fatty acid esters amounting 43.92%. The TLC technique for methanol extract led to isolation of 8 compounds: (4,5-O-Dicaffeoylquinic acid, ferulic acid, hesperidin, limettin, 5-demethylsinensetin, brassicasterol, campesterol and β-sitosterol). Brassicasterol is newly reported from species while 5-demethylsinensetin was newly reported from the Citrus genus. Furthermore, the docking experiments revealed strong binding affinity interactions of these compounds with TLR-4, Nrf2-Keap1 complex, and pNF-κBp65. In conclusion, mechanism of action of CMF or its nano formula exerting anti-ulcer effect was postulated based on the synergistic action of its reported compounds which possesses anti-inflammatory, antioxidant, and anti-apoptotic activities. To be noted, nano CMF showed a superior potent anti-ulcer activity than CMF and conventional therapy, omeprazole.