Euphorbium triterpenes ameliorate LPS induced RAW264.7 macrophage inflammatory response, with potential links to the TLR4/MyD88/NF-κB pathway and NLRP3-related signaling.
ETHNOPHARMACOLOGICAL RELEVANCE Euphorbium, the dried latex from Euphorbia resinifera, is classic traditional medicinal material used to treat various diseases in Uyghur medicine and has demonstrated significant efficacy in the intervention of inflammatory disorders. Total triterpenes extracted from Euphorbium (TTE) and its major monomer euphol possess obvious anti-inflammatory activities, but their systematic mechanism regulating macrophage inflammation remains insufficiently clarified. AIM OF THE STUDY The present work was designed to verify the in vivo anti-inflammatory efficacy of TTE using a carrageenan-induced acute inflammatory model, and further clarify the anti-inflammatory mechanisms of TTE and its major active monomer euphol in LPS-triggered RAW264.7 macrophages and acute inflammatory animals models, focusing on the TLR4/MyD88/NF-κB signaling cascade and NLRP3-related signaling. MATERIAL AND METHODS In vivo, a carrageenan-induced acute rat paw edema model was established to preliminarily evaluate the anti-inflammatory effect of TTE and observe paw tissue histopathological damage. On the basis of confirmed efficacy of TTE, in vitro, RAW264.7 macrophages experiments were further conducted to exclude the molecular mechanism. Both TTE and its major active component euphol were applied in cell experiments. Cells were pretreated with TTE and euphol respectively followed by LPS stimulation. The secretion of multiple inflammatory mediators and cytokines was quantified via biochemical detection and ELISA assays. The protein and transcriptional profiles of core molecules associated with TLR4/MyD88/NF-κB pathway and NLRP3-related signaling were determined by Western blot and qRT-PCR, respectively. Furthermore, immunofluorescence staining was conducted to qualitatively observe the regulatory effects of TTE and euphol on LPS-induced NF-κB p65 nuclear translocation. RESULTS In vivo results showed that TTE obviously alleviated carrageenan-induced acute paw edema and reduced tissue inflammatory infiltration. Further in vitro cellular experiments demonstrated that both TTE and euphol markedly suppressed the overproduction of NO, PGE2, MCP-1 and core pro-inflammatory cytokines, downregulated the expression of iNOS and COX2 at both protein and mRNA levels. Mechanistically, both interventions coincided with decreased protein levels of TLR4, MyD88, NLRP3, ASC and caspase-1, alongside reduced phosphorylation of NF-κB p65 and IκBα. Correspondingly, the transcriptional levels of key genes involved in NF-κB and NLRP3-related signaling were also dramatically decreased. Consistent with the altered p65 phosphorylation status, immunofluorescence observations further verified that TTE and euphol correlated with weakened LPS-triggered NF-κB p65 nuclear accumulation, providing direct phenotypic evidence consistent with attenuated NF-κB signaling activation. CONCLUSION TTE exerts prominent in vivo anti-inflammatory effects in acute inflammatory injury. Both TTE and its active component euphol exert potent anti-inflammatory effects in vitro by dual regulation of the TLR4/MyD88/NF-κB inflammatory cascade and NLRP3-related signaling. These findings provide a basis for the verification of the traditional efficacies of Euphorbium.