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.