Fig. 4. GP inhibits inflammation via TLR4 pathway and activates
mTOR dependent autophagy in DSS-induced colitis in vivo . (A) (C)
Immunofluorescence for TLR4 and p62 was performed on colon sections. (B)
(D) The expressions of TLR4 and p62 were calculated relative to DAPI
staining from three independent experiments. (E) The expression of
autophagy-related and inflammation-related proteins measured by western
blot. (F) The expressions of proteins were quantified by the ratio of
phosphorylated protein/total protein and total amount protein/GAPDH. All
data shown are representative of 3 independent experiments. Bars in
graphs represent mean ± SD, # P<0.05,## P<0.01 VS Control group; *P<0.05,
**P<0.01 VS DSS group.
Activated TLR4 directly triggers the phosphorylation of mTOR. With the
increase of TLR4 in the DSS group, the phosphorylated mTOR was
upregulated followed by the dysfunction of autophagy. To investigate
whether autophagy was impaired in DSS-induced intestinal inflammation,
the expression of autophagy-related proteins including LC3B, p62, and
phosphorylated mTOR were evaluated by immunofluorescence and Western
blot. Compared with the blank group, the expression of p62, a cargo
protein degraded inside autolysosomes, was upregulated after DSS
stimulation, indicating the inhibition of autophagy. Defective autophagy
was further confirmed by the decrease in LC3BII/LC3BI when compared with
the blank group. Autophagy was recovered to a normal level after
treatment with GP, indicated by decreased p62 and increased LC3BII/LC3BI
levels. These results demonstrated that GP relieved intestinal
inflammation by promoting mTOR-dependent autophagy and blocking the
inflammatory cascade.