3.5 TBⅡ mitigated LPS+ATP-induced inflammation via suppressing NLRP3
Considering that co-treatment TBⅡ with NLRP3 inhibitor MCC950 had a marginal effect on TBⅡ-mediated the amelioration of anti-inflammatory effect, we aim to explore whether the protective mechanism of TBⅡ is largely dependent on NLRP3 inhibition or not. In line with the results from Figure 5D and E, we found that, as expected, both TBⅡ and MCC950 treatment substantially reduced LPS+ATP-induced the generation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) (Figure 6A). Correspondingly, the expression of NLRP3 and the pro-inflammatory genes including Tnf-α, Il-1β, Il-6 and Il-10 was marked inhibited by TBⅡ and MCC950 treatment (Figure 6B and C). However, co-treatment TBⅡ and MCC950, likewise as Figure 5D and E, had not further decrease the expression of NLRP3 and the release of pro-inflammatory cytokines. To further corroborate the mechanism of TBⅡ-mediated NLRP3 inhibition, an adenovirus overexpressing NLRP3 (Ad-NLRP3) were generated. BMDMs were infected with Ad-NLRP3 or a control Ad-GFP adenovirus (Figure S2A and B) and then stimulated with LPS+ATP, and followed by TBⅡ treatment. Consistently, in Ad-GFP-infected control BMDMs, TBⅡ treatment could markedly abate LPS+ATP-induced elevated the expression of pro-inflammatory genes (Nlrp3, Tnf-α, Il-1β, Il-6 and Il-10 ) and the generation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) via repressing NLRP3 protein expression, an effect that was not observed in LPS+ATP-induced BMDMs infected with Ad-NLRP3 (Figure 6D-F), suggesting that overexpression of NLRP3 may significantly impair the anti-inflammatory effect of TBⅡ. These results demonstrated that the anti-inflammatory mechanism of TBⅡ was largely dependent on the inhibition of NLRP3.