[Effect of total triterpenes from Chaenomelis Fructus on mitigating indomethacin-induced gastric mucosal injury by inhibiting inflammation, oxidative stress, and NLRP3-mediated pyroptosis].

This study aims to investigate the protective effects and mechanisms of total triterpenes from Chaenomelis Fructus(TCS) on rats with GES-1 cells and gastric mucosal injury induced by indomethacin(IND). In vitro, MTT assay was used to test cell viability; scratch and Transwell assays were utilized to detect cell migration; immunofluorescence was used to measure mitochondrial membrane potential(MMP) and reactive oxygen species(ROS) levels; flow cytometry was used to test pyroptosis; ELISA methods were used to measure the levels of cyclooxygenase(COX)-1, COX-2, interleukin(IL)-4, IL-1β, IL-6, IL-10, IL-18, inducible nitric oxide synthase(iNOS), lactate dehydrogenase(LDH), prostaglandin E2(PGE2), and tumor necrosis factor-α(TNF-α) in the cell supernatant; the colorimetric method was utilized to determine the levels of catalase(CAT), glutathione(GSH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), and total antioxidant capacity(T-AOC) in the cells. In vivo, the gastric juice parameters(volume, pH value, and total acidity) and gastric mucosal volume in rats with gastric mucosal injury were measured; the ulcer area and ulcer inhibition rate were calculated; Hematoxylin-Eosin staining was used to observe the histomorphological changes in the gastric tissue, and the gastric mucosal injury score was calculated; colorimetric and ELISA methods were utilized to measure the levels of COX-1, COX-2, IL-1β, IL-4, IL-6, IL-10, IL-18, iNOS, LDH, PGE2, ROS, and TNF-α in the serum; the colorimetric method was used to determine the levels of CAT, GSH, MDA, MPO, SOD, and T-AOC in the gastric tissue; real-time polymerase chain reaction(PCR) was used to measure the mRNA expression levels of mitochondrial DNA(mtDNA), apoptosis-related spot like protein(ASC), caspase-1, claudin-5, COX-1, COX-2, glutamate cysteine ligase catalytic subunit(GCLC), heme oxygenase-1(HO-1), iNOS, Kelch-like ECH-associated protein 1(Keap-1), never in mitosis A-related kinase 7(NEK7), NOD-like receptor protein 3(NLRP3), phosphoamide adenine dinucleotide quinone oxidoreductase 1(NQO1), nuclear factor E2-related factor(Nrf2), occludin, prostaglandin E2(PGE2), tight junction protein-1(ZO-1), and thioredoxin-interacting protein(TXNIP) in the cells and gastric tissue; Western blot was utilized to determine the protein expression levels of ASC, caspase-1, claudin-5, GCLC, gasdermin D(GSDMD), N-terminal fragment of GSDMD(GSDMD-N), HO-1, Keap-1, NEK7, NLRP3, NQO1, nuclear Nrf2, total Nrf2, occludin, pro-caspase-1, pro-IL-18, pro-IL-1β, TXNIP, and ZO-1 in the cells and gastric tissue. The results showed that TCS prominently promoted proliferation and migration of IND-induced GES-1 cells, suppressed cell apoptosis, elevated mitochondrial MMP, and increased gastric mucosal volume, gastric juice pH, and ulcer inhibition rate in rats with gastric mucosal damage. It reduced the content of mtDNA in the cytoplasm, ulcer area, gastric juice volume, and total acidity, alleviated gastric mucosal edema, epithelial cell shedding, and inflammatory cell infiltration, and reduced scores and total scores for gastric mucosal bleeding, edema, epithelial cell loss, and inflammatory cell infiltration. It decreased the COX-2, IL-1β, IL-6, IL-18, iNOS, LDH, and TNF-α levels in the supernatant of IND-damaged GES-1 cells and rat serum, the ROS levels in IND-damaged GES-1 cells and rat serum, and the MDA and MPO levels in cells and gastric tissue, and elevated the COX-1, IL-4, IL-10, and PGE2 levels in the supernatant of IND-damaged GES-1 cells and serum, the GSH, SOD, and CAT activities, and T-AOC levels in GES-1 cells and gastric tissue. It also upregulated the expression of Nrf2, GCLC, HO-1, NQO1, COX-1, PGE2, ZO-1, occludin, and claudin-5 mRNAs and total Nrf2, nuclear Nrf2, GCLC, HO-1, NQO1, ZO-1, occludin, and claudin-5 proteins in IND-damaged GES-1 cells and gastric tissue, and downregulated the expression of Keap-1, TXNIP, NEK7, NLRP3, ASC, caspase-1, COX-2, and iNOS mRNAs and Keap-1, TXNIP, NEK7, NLRP3, ASC, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-18, and pro-IL-1β proteins. These aforementioned results demonstrated that TCS had a good preventive and therapeutic effect on IND-induced GES-1 cells and gastric mucosal injury in rats, and its mechanism was closely related to activating the Keap-1/Nrf2 pathway, reducing oxidative stress, and inhibiting the activation of the NLRP3 inflammasome pathway and pyroptosis, thereby restoring the structure and function of the gastric mucosal barrier.