Data shown in (A, C, and D) are representative of 3 experiments. PIK3CA-AKT1-MTOR-RPS6KB1 pathway accounted for Andro-induced autophagy. Finally, Andro-driven inhibition of the NLRP3 inflammasome and amelioration of murine models for colitis and CAC were significantly blocked by knockdown, or by various autophagy inhibitors. Taken together, our findings demonstrate that mitophagy-mediated NLRP3 inflammasome inhibition by Andro is responsible for the prevention of CAC. Our data may help guide decisions regarding the use of Andro in patients with inflammatory bowel diseases, which ultimately reduces the risk of CAC. and BI-D1870 (Fig.?2D) in colonic tissue were remarkably downregulated by Andro treatment. Together, these results indicate that Andro administration reduces colitis-associated tumorigenesis in mice. Open in a separate window Physique?1. Andrographolide prevents colitis-associated tumorigenesis. Mice were injected i.p. with a single dose (7.5 mg/kg) of AOM followed by 3 cycles of 2.5% DSS given in the drinking water for 5 d. Andro (7.5 and 15 mg/kg) was given i.g. daily during the interval between DSS cycles NFKBIA as shown. Mice were sacrificed on d 95 after CAC induction. (A) Body weight was recorded. (B) The inside of the colon was photographed. (C) Colon tissues were fixed and stained with H&E. (D) Tumor numbers were counted. (E and F) Tumor diameter and distribution were measured. (G) The tumor load was determined by totaling the diameters of all tumors for a given animal. Values are mean SEM of 9 mice/group. * 0.05, ** 0.01 vs. AOM+DSS group. Andro, andrographolide. Open in a separate window Physique?2. Andrographolide inhibits inflammation in a colitis-associated colorectal cancer model. Mice were subjected to the AOM-DSS model. For other details, see the legend of Physique?1. (A) The expression of PCNA, p-STAT3, p-RELA/p-p65, and PTGS2/COX2 were analyzed by immunochemistry in paraffin-embedded colon sections. Data shown are representative of 3 experiments. (B) The expressions of PCNA, BI-D1870 p-STAT3, p-RELA, and PTGS2 in colonic tissues were examined by western blotting. (C) Statistical data of the expressions of protein from 3 mice were shown. (D and E) The mRNA expressions of in colon sections were determined by real-time PCR. Data are presented as means SEM (n = 6). * 0.05, ** 0.01 vs. AOM+DSS group. Andro, andrographolide. Andro attenuates inflammation in a colitis-associated colorectal cancer model In addition to the reduced colitis-associated tumorigenesis in AOM-DSS-treated mice, we found that the inflammation level was deeply decreased by Andro administration. BI-D1870 Phosphorylation of RELA/p65, the subunit of the key inflammatory transcription factor NFKB/NF-B, was markedly reduced by Andro as shown by immunochemistry and western blotting (Fig.?2ACC). Expression of proinflammatory cytokines such as (tumor necrosis factor), (interleukin 17A), and (interleukin 6) was also significantly suppressed (Fig.?2E). In addition, Andro remarkably inhibited the expression of PTGS2/COX2 (Fig.?2A and E), which is an important mediator of the inflammatory process.22 To determine whether Andro could inhibit tumors that had already formed in the AOM-DSS-induced tumorigenesis model, Andro was given to the mice from d 50 to d 120. Tumors had significantly formed at d 50, and Andro given to mice starting at this time point had only a minor effect on tumor growth (Fig. S1). Furthermore, we found that Andro at the dose of 15 mg/kg did not inhibit transplanted mouse colon carcinoma CT26 cell growth in mice (Fig. S2). Though a previous study reported that Andro inhibited tumor growth, the dose they used was as high as 100C200 mg/kg.21 Hence, the data obtained here strongly suggest that Andro prevents colitis-associated tumorigenesis by inhibiting inflammation rather than directly killing tumor cells. Andro ameliorates DSS-induced experimental colitis in mice Because Andro showed a strong effect in reducing inflammation in the AOM-DSS model, we hypothesized that Andro might prevent tumorigenesis in the AOM-DSS model by inhibiting inflammation. Next, we examined the effect of Andro on DSS-induced experimental colitis in mice. After being challenged with DSS in their drinking water, the mice showed an increasing severity of symptoms, including dramatic body weight loss, rectal bleeding, and diarrhea..