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.