Rapamycin Alleviates 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis through Autophagy Induction and NF-κB Pathway Inhibition in Mice

Abstract

Background. Recent genetic studies indicated that variants of autophagy genes were associated with the predisposition of Crohn’s disease (CD). The autophagy deficiency may affect the innate and adaptive immunity, which is related to persistent and excessive inflammation of the bowel. However, it remains unclear how autophagy modulates the expression of immune response regulator NF-κB and proinflammatory cytokine TNF-α in CD. Aim. We aimed to investigate the role of rapamycin on the expression of NF-κB p65 and TNF-α in 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis and lipopolysaccharide (LPS)-induced HT-29 cells. Methods. TNBS-induced colitis mice were treated with saline or rapamycin, and the disease activity index (DAI) and histological scores of colonic mucosa were evaluated. The expressions of p65, ATG16L1 and LC3 were detected by western blot and immunohistochemistry staining. The monodansylcadaverine (MDC) staining and transmission electron microscopy were developed to study the autophagy in LPS-induced HT-29 cells. Expression of TNF-α from colon tissue and HT-29 cells were detected by ELISA. The expressions of p65, ATG16L1 and LC3 in active CD patients were also investigated. Results. Significantly more autophagosomes were observed in rapamycin-treated cells than in controls. Rapamycin remarkably upregulated the expression of ATG16L1 and LC3II, inhibited p65 nucleus translocation and secretion of TNF-α both in vivo and in vitro. The expression of both ATG16L1 and LC3II increased in mild to moderate CD specimens, while no significant difference was noted between severe CD and normal controls. The expression of p65 increased notably in severe CD compared to those in mild to moderate patients. Conclusions. In LPS-treated HT-29 cells and TNBS-induced colitis, p65 is overexpressed, which results in exaggerated secretion of TNF-α and induce or worsen the inflammation in the bowel. Rapamycin protects against colitis through induction of autophagy, thus inhibiting the activation of NF-κB pathway and secretion of TNF-α.

Rapamycin extenuates experimental colitis by modulating the gut microbiota

“…Rapamycin is used to regulate autophagy in vivo and in vitro, especially to induce and sustain responses in IBD patients resistant to conventional anti‐tumor necrosis factor alpha (anti‐TNF‐α) therapy 11,31 . In our experiments, after administration of rapamycin to mice with colitis, the symptoms reduced significantly, which is in line with the reports in the literature suggesting that rapamycin inhibits inflammation and strengthens the intestinal barrier 21,32 …”

“…11,31 In our experiments, after administration of rapamycin to mice with colitis, the symptoms reduced significantly, which is in line with the reports in the literature suggesting that rapamycin inhibits inflammation and strengthens the intestinal barrier. 21,32 Besides, we found that activation of autophagy improved inflammation due to colitis by regulating the gut microbiota. Previous studies have mostly focused on the effects of changes on specific genes involved in autophagy (Atg5 and Atg7) 33,34 on the gut microbiota, while we observed changes in gut microbiota by activating or inhibiting autophagy levels, which has never been reported previously.…”

“…Following rapamycin intervention, the levels of IL-6 (P = 0.0187) and TNF-α (P = 0.0154) decreased significantly, consistent with previously reported findings. 21,22 However, the levels of IL-6 (P = 0.08) and TNF-α (P = 0.5177) did not differ significantly between the hydroxychloroquine and DSS groups.…”

Rapamycin inhibits B16 melanoma cell viability in vitro and in vivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway

“…Induction of apoptosis is key in the suppression of tumors. Rapamycin is a classic autophagy inducer, which is able to induce autophagy in most vital cells (38)(39)(40)(41). However, some studies have also found that it can induce the apoptosis of certain types of cells, including Kaposiform hemangioendothelioma primary cells, human MG-63 osteosarcoma cells and SHSY5Y neuroblastoma cells (19,20,42,43).…”