3.4 TBⅡ alleviates LPS+ATP-induced intestinal epithelial cell
damage and inflammatory response via blocking the communication between
epithelial cells and macrophages
Given the permeability-alleviating and anti-inflammatory effects of TBⅡ
in the DSS-induced colitis mice, we next investigated whether TBⅡ can
protect intestinal epithelial cell damage and inflammatory response
mediated by LPS+ATP-induced NLRP3 activation in a cell model. Consistent
with mice results, the expression levels of E-CADHERIN and OCCLUDIN were
reduced in HCoEpiC cells that treat with LPS+ATP than that of cells
treat with control (Figure 4A and B), and thereby causing to higher
production of pro-inflammatory cytokine (TNF-α, IL-1β, IL-6 and IL-10)
in cell supernatant (Figure 4C). Accordingly, the expression of genes
involved the generation of pro-inflammatory cytokine (TNF-α,
IL-1β, IL-6 and IL-10 ) was prominently elevated in
LPS+ATP-induced cells compared with control-treated cells (Figure 4D).
In contrast, these effects were significantly reversed by TBⅡ treatment
(Figure 4A-D). As expected, the expression levels of proteins (NLRP3,
ASC, CASP1 and IL-1β) were markedly enhanced by LPS+ATP stimulation, an
effect that was dramatically attenuated by TBⅡ treatment (Figure 4E).
Similarly, a prominent anti-inflammatory effect of TBⅡ was also observed
in LPS+ATP-treated BMDM cells via inhibiting NLRP3 signaling (Figure
5A-C).
Mounting data have evidenced that the crosstalk between epithelial cells
and macrophages is crucial for the homeostasis of the intestinal barrier
(Spalinger et al., 2020). To investigate whether the alleviative effect
of TBⅡ in intestinal barrier homeostasis is associated with the mutual
communication between colon cells and macrophages, conditional media
(CM)-associated culture were performed. First, THP-1 cells were
stimulated with LPS+ATP and supernatant CM were collected, and then
cultured for HCoEpiC cells. The expression levels of pro-inflammatory
genes (TNF-α, IL-1β, IL-6 and IL-10 ) and intestinal
barrier genes (E-CADHERIN and OCCLUDIN ) were significantly
elevated or decreased in THP-1 cells cultured to the CM of HCoEpiC cells
stimulated with LPS+ATP compared with those with DMSO control,
respectively (Figure 5D), an effects that was markedly reversed in THP-1
cells cultured to the CM of HCoEpiC cells treated with TBⅡ compared with
those with vehicle control, respectively (Figure 5D). Second, similarly,
we also observed the anti-inflammatory effects on THPs cells that
stimulated with supernatant CM of HCoEpiC cells that previously
pretreated with or without TBⅡ for 24h, and then followed by LPS+ATP for
another 24 h (Figure 5E). However, Co-treatment TBⅡ with MCC950, a NLRP3
inhibitor, did not further reduce the anti-inflammatory effect and
improve the intestinal barrier compared with MCC950 treatment alone
(Figure 5E), suggesting a potential role of NLRP3 in TBⅡ-mediated the
alleviation of intestinal permeability. These results suggest that the
disruption of colonic epithelial integrity triggered by LPS+ATP-induced
NLRP3 activation was inhibited by TBⅡ treatment via hindering the
interaction between epithelial cells and macrophages, probably in an
NLRP3 inhibition mechanism.