The Effect And Mechanism Of A Novel MyD88 Inhibitor And Its Function On Preventing And Treating Colitis Associated Colorectal Cancer

[Objective] To explore the effect and mechanism of a novel MyD88 inhibitor TJ-M2010-5 on MyD88 homodimerization and heterodimerization and provide evidence for computer associated pharmaceutical design.[Methods] The plasmids of Flag-MyD88 pcDNA3.1-, HA-MyD88 pcDNA3.1-and Flag-TIRAP pcDNA3.1-were constructed in vitro. Flag-MyD88 pcDNA3.1-and HA-MyD88 pcDNA3.1-were co-transfected to HEK293T cells. Flag-TIRAP pcDNA3.1-and HA-MyD88 pcDNA3.1-were also co-transfected to HEK293T cells. After transfection, HEK293T cells were treated with different doses of TJ-M2010-5 (0?M, 10?M,40?M). The combination of MyD88 and MyD88 as well as TIRAP and MyD88 was measured 48 h after transfection. Immature BMDCs of BALB/c were induced in vitro. Seven days after induction, BMDCs were treated with different doses of TJ-M2010-5 (0?M,10?M,40?M) for 2 h followed by stimulation with LPS for 48 h. After that, the translocation of NF-?B in BMDCs was measured by EMSA and the levels of TNF-? and IL-1? in supernatants were measured by ELISA.[Results] TJ-M2010-5 does dependently inhibited the homodimerization of MyD88-MyD88 and heterodimerization of TIRAP-MyD88. BMDCs of BALB/c were successfully induced in vitro. After the administration of TJ-M2010-5 and LPS, translocation of NF-?B and the levels of TNF-? and IL-1? were inhibited by TJ-M2010-5 in a dose dependent manner.[Conclusion] TJ-M2010-5 inhibits MyD88 dimerization by interfering with MyD88-MyD88 homodimerization and TIRAP-MyD88 heterodimerization. Furthermore, TJ-M2010-5 inhibits the TLR/MyD88 signaling pathway in BMDCs. Therefore, TJ-M2010-5 is a powerful MyD88 inhibitor to interfere with TLR/MyD88 signaling, which lays the foundation of clinical practice.[Objective] To explore the effect and mechanism of a novel MyD88 inhibitor TJ-M2010-5 on preventing and treating AOM/DSS induced CAC model of mice and provide the experimental data for clinical practice.[Methods] The CAC model of mice, which was treated with TJ-M2010-5, was induced by using AOM and DSS. BALB/c were injected intraperitoneally with high dose of TJ-M2010-5 to evaluate the acute toxicity of TJ-M2010-5 by observing physical activities or behavior and with normal dose of TJ-M2010-5 to evaluate the sub-acute toxicity of TJ-M2010-5 by measuring the hematological parameters and pathological changes of kidney and liver. There were three experimental groups:Normal group, AOM/DSS group and TJ-M2010-5 group. The changes of weight and survival of mice were monitored for 10 weeks. RAW264.7 cells were cultured in vitro. The phosphorylated levels of IRAK4, p38 and Erk in RAW264.7 cells were measured after the administration of TJ-M2010-5 followed by stimulating with LPS. Meanwhile, translocation of NF-?B in colon tissues was measured by EMSA. Therefore, the effect of TJ-M2010-5 on TLR/MyD88 signaling of CAC mice could be further assessed. The tumor formation of colon tissues was detected by gross observation. The tumor progress of colon tissues was detected by HE staining and COX2 analysis using IHC and Western bolt. The inflammatory degree of colon tissues was evaluated by HE staining and the infiltration of neutrophils was detected by MPO staining. The proliferation of epithelial cells was measured by incorporation of BrdU and Ki-67 staining. The apoptosis of epithelial cells was measured by active caspase-3 and TUNEL staining. The protein levels of cytokines of serum were analyzed by Bio-Plex and ELISA. The mRNA levels of cytokines of colon tissues were analyzed by qPCR. The CD68+ macrophages of colon tissues were detected by IHC staining for CD68. The percentages of macrophages, DCs and CD4+T cells in LPMC were analyzed by flow cytometer. The mRNA levels of IL-6 in macrophages were analyzed by qPCR.[Results] The CAC model of mice was successfully induced by combinational usage of AOM and DSS. Neither acute toxicity nor sub-acute toxicity was observed in mice treated with TJ-M2010-5. TJ-M2010-5 inhibited the TLR/MyD88 signaling in CAC mice. Compared with AOM/DSS group, the administration of TJ-M2010-5 decreased the mortality from 53% to 0% and the incidence of colon cancer form 100% to 0%. Furthermore, the treatment of TJ-M2010-5 inhibited the weight loss, decreased the proliferation and increased the apoptosis of epithelial cells, and ameliorated the progress of CAC. Meanwhile, TJ-M2010-5 regulated the inflammatory microenvironment of colon by interfering with production of cytokines and infiltration of neutrophils and immune cells.[Conclusion] Aberrant activation of TLR/MyD88 signaling induces the formation of pro-tumorigenesis inflammatory microenvironment. The administration of TJ-M2010-5 inhibits the formation of tumor microenvironment through regulating inflammatory microenvironment and inhibiting infiltration of immune cells, thus, TJ-M2010-5 prevents CAC initiation and progress. TJ-M2010-5 remarkably inhibits the initiation and progress of CAC, which exhibits high value for clinical practice and provides more therapeutic protocols for patients suffering from colitis or CAC.