| Inflammatory bowel disease(IBD) is a chronic gastrointestinal inflammatory disease including ulcerative colitis(UC) and Crohn’s disease(CD). One of the serious complications of IBD is colorectal cancer(CRC), the risk of which is 5-40/10 million.Tregs(Regulatory T cells, Tregs),as immunosuppressive cells, play a negative adjustment function in inhibiting effector T cell responses by secreting IL-10 and TGF-β and other anti-inflammatory cytokine aiming to maintaining immune homeostasis. Intestinal immune homeostasis disorder play an important role in the development of IBD, in which Th1, Th2, Th17 and other effector cell subsets are involved in mediating an inflammatory disorder. However, Treg can balance and negatively regulate the inflammatory reaction of the various effector T cells, which play an important role in the pathogenesis of IBD.Phosphatidylinositol 3-kinases(PI3Ks) are important molecules of growth factor signaling pathways, which play very important roles in the regulation of inflammation, proliferation, differentiation, apoptosis and glycometabolism. PI3 K can be divided into ClassI(IA and IB), II, III types. The Class IA PI3 K is a heterodimer composed of catalytic subunits(p110α/β/δ) and regulatory subunits(p85α/β, p55α/γ, p50α). p55 PIK is a regulatory subunit of PI3 Ks, the most difference between it and other regulatory subunit of PI3K(p85PIK, p50PIK) is the presence of 24 amino acids at the amino terminal. Our group synthesize TAT-N15 fusion peptide(N15 in N24 and recombinant HIV permeable membrane protein)using molecular biology techniques, which is a specific inhibitor of p55 PIK.The biological effects of non-classical regulatory subunit of PI3 K p55γ(ie p55PIK) has been rarely reported. Our group studying on p55 PIK signaling pathways and their relationship with tumor and inflammation researched more in depth, and had made the following progress: In tumor, we confirmed the difference of p55 PIK and p85 for the first time, and p55 PIK can recruit other protein molecules(such as Rb and PCNA) through N24, and causes phosphorylation of Rb and PCNA through the catalytic subunit p110 iSH2 domain, thereby promoting tumor cell proliferation and tumorigenesis; In inflammation, we firstly discovered that p55 PIK induced NF-κB signaling(p65 phosphorylation) through Akt-independent pathway in the LPS mediated inflammation, thus promoting secretion of pro-inflammatory cytokines by monocyte.It had been reported that the classic regulatory subunit of PI3K-p85 negatively regulated differentiation of Treg, but the effect p55 PIK on Treg regulation is unclear, and there are no domestic and foreign research reports.Combination of these preliminary results, we put forward the scientific question: whether can p55 PIK play a role by affecting Treg in the malignant transformation of chronic colitis or not?The research was based on the mouse model of AOM-DSS-induced colitis and associated colorectal cancer, and p55 PIK inhibitor(TAT-N15) was set as a therapeutic tool, saline(NS), control polypeptide(CP) were set as controls. The groups are as follows: NS group(model group), CP group(negative control group), TAT-N15 group(experimental group). In molding process, we detected weight and fecal occult blood every day from DSS inducing. We select the following time points in AOM-DSS model: acute inflammation period(Day8), chronic inflammation recovery period(Day37) and colorectal cancer period(Day70), mice were sacrificed to detect colon length and pathological changes. In the time point above-mentioned, the spleen, mesenteric lymph node and colon tissue are obtained from the mouse. And at the same time, Proinflammatory cytokines(TNF-a, IL-1β, IL-6) and anti-inflammatory cytokines(IL-10, TGF-β) in colon tissues were detected by ELISA. Treg(CD25+Foxp3+/CD4+cells) in the spleen and mesenteric lymph nodes were detected by FCM, Treg in the colon tissue was detected by immunohistochemistry. We aim to observe if p55 PIK inhibitor can relive colitis and colorectal cancer in AOM-DSS model, and to explore the possible mechanism of p55 PIK on colitis and colorectal cancer.In vivo experiments, the study based on Treg cell differentiation experiments in murine: under sterile conditions, spleen mesenteric/groin/armpit lymph nodes of C57BL/6 mice were obtained, then cell suspensions were prepared. CD44lowCD62 Lhigh Na?ve T cells were sorted by MACS and FCM. Firstly, Anti-CD3 and anti-CD28 co-stimulation were used to activate TCR; Secondly, we applied TAT-N15 or CP as stimulant, and at last, we added TGF-β and IL-2 for naive T cells differentiation into Treg in vitro. the cells were collected after 72 h, the proportion of Treg(CD4+ Foxp3+) were detected by FCM. We aim to observe the effect of p55 PIK on Treg differentiation. The results are as follows: I. p55 PIK promote the development of chronic colitis by modulating Treg 1. AOM-DSS-induced colitis in mice with chronic colitis carcinogenesis modelBody weight, fecal occult blood score shows regular fluctuations with or without 2.5% DSS water drinking, which is in line with change of chronic colitis symptoms. On this basis, we further successfully constructed mouse model of chronic colitis carcinogenesis induced by AOM-DSS. 2. p55 PIK inhibitors(TAT-N15) may relieve the basic traits of inflammation in AOM-DSS modelp55PIK specific inhibitor TAT-N15 can alleviate the basic traits of acute and chronic inflammation in AOM-DSS model: TAT-N15 can relieve weight loss in chronic colitis(p <0.01 or p <0.001), and had no effect on the fecal occult blood(p> 0.05). In acute inflammation(Day8) and chronic inflammation recovery(Day37), TAT-N15 can effectively alleviate reduced colon length induced by DSS(p <0.05) and pathological score of colitis(p<0.01, p<0.05). The results promoted that p55 PIK may be involved in promoting weight loss, colon’s shorten length and pathological score caused by chronic colitis in mice, thus contributing to the basic traits of acute and chronic inflammation AOM-DSS model. 3. p55 PIK inhibitor may inhibit the expression of pro-inflammatory cytokines in mice with chronic colitisIn acute inflammation period(Day8): TNF-a and IL-6 of colon homogenates in TATN15 group were lower than the CP group and NS group(p<0.05), while the expression of IL-1b had no significant difference among the three groups; In chronic inflammation recovery period(Day37): IL-1b and IL-6 levels in TAT-N15 group were significantly lower than the CP and NS group(p<0.05), the expression of TNF-a had no significant difference among the three groups; These results suggest that: TAT-N15 inhibit proinflammatory cytokine TNF-a and IL-6 expression in acute inflammation period(Day8), inhibit IL-1b and IL-6 expression in chronic inflammation recovery period(Day37). The results promoted that p55 PIK can promote expression of TNF-a and IL-6 in acute inflammation and IL-1b and IL-6 in chronic inflammation in the AOM-DSS model. 4. p55 PIK inhibitor(TAT-N15) may promote the expression of anti-inflammatory cytokines in mice with chronic colitisIn acute inflammation(Day8) and chronic inflammation recovery(Day37), compared to the CP and NS group, the expression of IL-10 of colon homogenates in TATN15 group of were not statistically significant; However, the expression of TGF-β of colon homogenates in TAT-N15 group were significantly higher than the CP group and NS group(p<0.001). These results show that: in acute inflammation(Day8) and chronic inflammation recovery(Day37), TAT-N15 can promote colon tissue anti-inflammatory cytokine TGF-b expression. The results suggest that: p55 PIK can inhibit the expression of anti-inflammatory cytokine TGF-β in acute and chronic inflammation period. 5. p55 PIK inhibitors(TAT-N15) increase Treg ratio in mice with chronic colitisIn chronic inflammation recovery period of AOM-DSS model(Day37), compared to the CP and NS group, TAT-N15 can increase the spleen and colon tissue Treg ratio(p<0.05, p<0.01), while There was no significant difference in the mesenteric lymph node. There were no significant difference in Treg ratio(spleen, mesenteric lymph node and colon tissue) in acute inflammation(p>0.05). This result indirectly showed: In AOMDSS chronic inflammation period, p55 PIK may play a negative role regulating Treg(spleen and colon tissue). p55 PIK had no effect on Treg ratio(spleen, mesenteric lymph node and colon tissue) in acute inflammation. II. p55 PIK promote the incidence of colitis associated cancer by regulating Treg 1. p55 PIK inhibitors(TAT-N15) can reduce the number of colitis associated cancerIn tumor stage(Day70), the number of tumors were detected in each group of mice, the results show: TAT-N15 can inhibit the occurrence of colitis associated cancer and reduce the number of tumors(p<0.001). 2. p55 PIK inhibitor increase the proportion of Treg in mice with colitis associated cancerTAT-N15 can increase Treg ratio of spleen(p<0.001), mesenteric lymph nodes(p <0.01) and colon tissue(p<0.05) in colitis associated cancer stage. The results prompted that, p55 PIK promote colitis associated tumor development possibly through negative regulating Treg in AOM-DSS model. Ⅲ. P55 PIK negatively regulate differentiation of Treg in vitroOn the basis of an experimental Treg differentiation in murine, we aim to observe the effect of TAT-N15 on Treg differentiation. The results showed that: TAT-N15 can promote na?ve T cells into Treg(p<0.05), which reminded that p55 PIK may play a negative role in Treg differentiation. Conclusion: 1. p55 PIK specific inhibitor TAT-N15 can relieve inflammatory basic traits in acute and chronic inflammation of AOM-DSS model. The mechanism may be that TAT-N15 promoted colitis by inhibiting/promoting the expression of pro/antiinflammatory cytokines, increasing Treg ratio of the spleen and colon tissue in the period of chronic inflammation.2. In the AOM-DSS model of tumor, TAT-N15 inhibit colitisassociated tumor development. The mechaniscm may be that TAT-N15 can increase the proportion of Treg in the spleen, mesenteric lymph node and colon tissue. 3. TAT-N15 can promote na?ve T cells differentiation into Treg cells in vitro. These results also suggest that: 1. p55 PIK may promote the expression of pro-inflammatory cytokines, and inhibit anti-inflammatory cytokines in acute and chronic inflammation, and promote chronic inflammation by negatively regulating Treg proportion in AOM-DSS model of inflammation. 2. p55 PIK may promote tumors occurrence and colitis-colorectal carcinoma malignant transformation by negatively regulating Treg. 3. p55 PIK negatively regulated differentiation of Treg cells in vitro. 4. TAT-N15, as a target of regulatory subunit PI3 K disturbed inflammatory bowel disease and related tumors in vivo, which may be expected to provide new strategies and ideas for the treatment of inflammatory bowel disease and related tumors.This issue proved that TAT-N15 may inhibit malignant transformation of chronic colitis by increasing the proportion of Treg in vivo, and TAT-N15 can promote differentiation of Treg in vitro for the first time. This research will not only clarify the role of PI3 K non-classical pathway in Treg differentiation, but also provide new targets and strategy for the prevention and treatment of IBD and other autoimmune diseases. |
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