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.