Ganoderic Acid\nA Alleviates Severe Acute Pancreatitis\nby Modulating Gut Homeostasis and Inhibiting TLR4-NLRP3 Signaling

Background Severe\nacute pancreatitis (SAP) manifests as a critical\nstate marked by acute abdominal symptoms, often associated with intestinal\nbarrier dysfunction, exacerbating SAP retroactively. Ganoderic acid\nA (GAA) demonstrates anti-inflammatory properties in various inflammatory\ndisorders. Nonetheless, its potential therapeutic impact on SAP and\nthe underlying mechanisms remain unexplored. Methods In both wild-type\nand TLR4<sup>–/–</sup> mice, experimental SAP was induced\nusing caerulein plus lipopolysaccharide. Caerulein injections were\nadministered intraperitoneally following 7 days of intragastric GAA\nadministration. Additionally, the potential mechanisms by which GAA\nameliorates SAP were further investigated using fecal microbiota transplantation\nand TLR4-overexpressing IEC-6 cells. Results We observed that GAA\ntreatment significantly ameliorated serum levels of amylase, lipase,\nand pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α)\nin SAP mice. Pretreatment with GAA mitigated pathological injuries\nand reduced M1 macrophage and neutrophil infiltration in pancreatic\nor ileal tissues. Additionally, GAA treatment down-regulated TLR4-MAPK/NF-κB\nsignaling and NLRP3 inflammasome activation in the pancreatic and\nileal tissues of SAP mice. The results further revealed that the gavage\nof GAA decreased bacterial translocation (Escherichia\ncoli and EUB338), repaired intestinal barrier dysfunction\n(ZO-1, occludin, DAO, and FITC), increased lysozyme and MUC2 expression,\nand raised the levels of short-chain fatty acids. Analysis of the\ngut microbiome showed that the beneficial effects of GAA treatment\nwere associated with improvements in pancreatitis-associated gut microbiota\ndysbiosis, characterized by notable increases in α-diversity\nand the abundance of probiotics such as <i>Akkermansia</i>, <i>GCA-900066575</i>, and <i>Parvibacter</i>. Fecal transplantation experiments further confirmed that GAA exerts\nprotective effects by modulating intestinal flora. The protective\nrole of GAA in intestinal and pancreatic injuries is mediated by the\ninhibition of TLR4 signaling, as further evidenced in TLR4-deficient\nmice and TLR4-overexpressed IEC-6 cells. The results of docking indicated\nthat GAA interacts with TLR4 via a hydrophobic interaction. Conclusions\nThe study demonstrates that GAA significantly alleviates SAP through\nits anti-inflammatory and antioxidant capacities, as well as by restoring\nintestinal homeostasis, thereby providing insights into novel treatments\nfor SAP.