3.7 Blocking TLR4-mTOR ameliorates inflammation via promoting autophagy in LPS-induced HT-29 cell.
According to the in vivo results, related mechanisms of autophagy regulation were subsequently explored in LPS-induced HT-29 cells. To explore whether the release of NF-κB-induced cytokines and oxidative stress were associated with autophagy, we chose 3-MA and rapamycin as the inhibitor and activator of autophagy, respectively. Firstly, pro-inflammatory cytokines were determined as shown in Fig. 8A. When cells were treated with 1 μg/mL LPS for 6 hours, IL-8 and IL-1β levels increased by nearly 3-fold; these levels ameliorated to a certain extent after rapamycin (an autophagy activator) treatment. When autophagy was inhibited by 3-MA, the release of LPS-induced inflammatory cytokines dramatically increased even higher than that of the LPS-induced group. Secondly, the expressions of p62, LC3B, and Beclin-1 were determined by Western blot analysis (Fig. 8B). In the second lane of Fig. 8, it can be seen that LPS blocked autophagy via the activation of mTOR and activated an inflammatory transcription factor, which is consistent with previous reports (Cosin-Roger et al., 2017; M. Zhou et al., 2018). While the expression of Beclin-1, degradation of p62, and conversion of LC3B were increased significantly in the rapamycin groups, this was also accompanied with the inhibition of NF-κB p65. As expected, suppression of autophagy by 3-MA significantly inhibited the level of autophagy and dramatically deteriorated the inflammation. The results above indicated that impaired autophagy was involved in LPS-induced inflammation in HT-29 cells.
Recent research has focused on Toll-like receptors (TLRs), which might be involved in orchestrating LPS-induced autophagy inhibition and inflammation aggravation via mTOR (M. Zhou et al., 2018). Thus, we next sought to investigate whether TLR4 is a potential target of governance of the activation of autophagy. As shown in Fig. 8, LPS significantly upregulated the expression of TLR4 and its downstream signaling molecules MyD88 and p38 MAPK in HT-29 cells, which paralleled with high phosphorylated level of mTOR and inhibition of autophagy. Autophagy-related proteins in TLR4 siRNA transfected cells further confirmed the regulation role of TLR4. TLR4 siRNA attenuated the autophagy impairment and inflammation activation induced by LPS, while 3-MA and rapamycin exhibited almost no effect on TLR4-MyD88. Corroborating the data above, we demonstrated that LPS-decreased autophagy was governed by the upstream TLR4-mTOR pathway. Additionally, autophagy was involved in inhibiting cytokine secretion and protecting cells from oxidative stress.