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).