PHI improved LPS and ATP-induced BMDM inflammation by inhibiting the activation of NLRP3 inflammasome by TLR4/MyD88/NF-κB pathway
Macrophages are known to play a critical role in both the progression of inflammation and the eventual remission of disease during intestinal mucosal repair. To investigate the efficacy of PHI in vitro , bone marrow-derived macrophages (BMDM) were utilized in an in vitroexperiment due to their significant impact on macrophages compared to other myeloid cells. In the initial phase of the experiment, the impact of PHI on cell viability was examined, and three safe concentrations were selected (100, 50, and 25 μg/mL) for subsequent research (Fig. 6A). BMDM was then stimulated with lipopolysaccharide (LPS), a typical TLR4 agonist, to induce inflammatory cytokine production. PHI displayed potent anti-inflammatory properties on BMDM, which was reflected in a reduction in genetic and protein expression levels of IL-1β and IL-6 under LPS induction (Fig. 6B-C).
Based on these findings, we tested the effect of PHI on NLRP3 inflammasome in vitro . The NLRP3 inflammasome was activated by LPS and NLRP3 agonist adenosine triphosphate (ATP). As with the results in UC mice colon, PHI inhibited the expression of NLRP3 and Caspase1, especially NLRP3, which was also demonstrated by using cellular immunofluorescence (Fig. 6D-F). The influence mechanism was also proven, as it inhibited the upstream pathway as a depressant. By combining the results in both in vivo and in vitro experiments, the conclusion was that PHI had a positive effect on ulcerative colitis, and it inhibited inflammation by preventing NLRP3 inflammasome activation through the TLR4/MyD88/NF-κB pathway (Fig. 7).