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.