| Inflammatory bowel disease(IBD) includes ulcerative colitis(UC) and crohn’s disease(CD) and its pathogenesis is still not very clear. Now it is thought that IBD results from intestine mucosal dysimmunity, primarily. Th9 cells are newly found subgroup belonging to CD4+T cells, mainly secretes IL-9. In recent years, many studies suggested that Th9 cells and IL-9 play a vital role in the development of IBD with more and more attention.IL-9 has extensive biological activities. It plays an important role in allergic disease, rheumatoid arthritis and other autoimmune diseases.Although playing an important role in many autoimmune diseases, the research of Th9 cells in the field of IBD is rare so far. The study showed that Th9 cells and IL-9 levels in patients with UC and animal colitis model were significantly higher. They were positively correlated with the disease severity.The bowel inflammation degree of experimental colitis was lighter in IL-9knockout mice, compared to the wild type(WT) group. While the intestinal inflammation of acute colitis mice with the treatment of IL-9 antibodies got better. These studies remind that Th9 cells and IL-9 may play an important role in the pathogenesis of IBD.Tumor necrosis factor ligand-related molecule1A(TLlA) is a novel member of TNF-superfamily, which causes intestinal inflammation by combining with death receptor 3(DR3). A report showed that TL1A could enhance the generation of Th9 cell in allergic lung disease models. However,the relationship between TL1A and Th9 cells in IBD is still unknown.Astragaloside Ⅳ(AS- Ⅳ) is one of the main materials of Astragalus membranaceus. Astragalus has been widely applied in the treatment of IBD.The decoction of Atractylodes and Astragalus and Shenqi decoction oral treatment of UC has proved effective. It has been found that AS- Ⅳ canimprove myocardial damage through down-regulating TL1A expression.Unfortunately, there is currently no report focusing on the role of AS-Ⅳ in IBD.Objective: To explore the effects of AS- Ⅳ on expression of IL-9 in Chronic Colitis of Sustained TL1A Expression Mice.Methods:1 LCK-CD2-TL1A-GFP transgenic(Tg) mice were identified and selected by RT real-time PCR.2 Chronic colitis was induced by administration of dextran sodium sulfate(DSS) drinking water. WT and lymphoid TL1A-Tg C57BL/6 mice(20-22 g, 8-10 weeks old) were grouped as follows: Control group(n=10),DSS group(n=10), AS- Ⅳ low dose group(n=10) and AS- Ⅳ high dose group(n=10). The Mice in control group received regular water, the mice in DSS group received 2% DSS drinking water on day 1-5, 8-12, 15-19, 22-26,27 and 28, distilled water during intermission, day-29 was the sacrifice day.Mice in low dose group and high dose group were administrated by AS-Ⅳ 20mg/kg·d and 30mg/kg·d on day-14 until sacrificed.3 Severity of colitis was observed by body weight(BW) changes, disease activity index(DAI), colon length and damage store.4 Colon histology changes and pathology score, Myeloperoxidase(MPO)were measured in each group.5 The expression of TL1A 、 DR3 and IL-9 in colon mucosa was detected by western blot and real-time Q-PCR, respectively.6 Cells were extracted from colon, MLN and spleen then counted one by one. And the percentage of Th9 cells in CD4+T cells was measured by flow cytometry. The concentrations of IL-9 from LPMC, MLN monocytes, spleen monocytes supernatant and serum were tested by enzyme-linked immune sorbent assay(ELISA).Results:(1)The TL1A transgenic mice were identified by PCR.The TL1A DNA located at 192bp in Tg mice,while it did not express at 192 bp in WT mice;(2)The results of severity of chronic colitis showed that comparedto that in the control group,body weight had distinctly greater loss,DAI score were higher,colon length became shorter,histological score(WT mice:13.5±1.06 vs 3.25±0.46,P<0.05;Tg mice:16.25±1.03 vs 3.38±0.51,P<0.05)and MPO activity were much higher in DSS group(P<0.05).Compared to WT mice,the colitis bowel inflammation degree of Tg mice was higher(P<0.05).Compared with DSS group,AS-Ⅳhigh dose group and AS-Ⅳlow dose group injury was in a marked improvement(WT mice histological score:8.75±0.88 vs 13.5±1.06,P<0.05;6±1.06 vs 13.5±1.06,P<0.05;Tg mice histological score:11.38±1.06 vs 16.25±1.03,P<0.05;9.12±0.83 vs16.25±1.03,P<0.05),especially in the ASⅣhigh dose group(P<0.05);(3)The proteins and mRNA levels of TL1A and IL-9 in DSS group were higher compared with control group(WT mice:TL1A mRNA:0.58±0.038 vs0.01±0.02,P<0.05;TL1A protein 0.47±0.03 vs 0.15±0.05,P<0.05;IL-9mRNA:0.13±0.009 vs 0.01±0.002,P<0.05;IL-9 protein 0.44±0.02 vs0.10±0.02,P<0.05.Tg mice:TL1A mRNA:1.5±0.05 vs 0.26±0.045,P<0.05;TL1A蛋白0.74±0.02 vs 0.33±0.04,P<0.05;IL-9 mRNA:0.16±0.011 vs0.02±0.005,P<0.05;IL-9 protein 0.73±0.02 vs 0.30±0.02,P<0.05).The proteins and mRNA levels of TL1A and IL-9 in the AS-Ⅳlow dose group and AS-Ⅳhigh dose group were significantly decreased(P<0.05).The proteins and mRNA levels of DR3 changed slightly after AS-Ⅳtreatment(P>0.05).Compared to the AS-Ⅳlow dose group,the proteins and mRNA levels of IL-9 in AS-Ⅳhigh dose group significantly lower(P>0.05),but the levels of TL1A had little difference(P>0.05).Compared to Tg mice,the proteins and mRNA levels of TL1A,DR3 and IL-9 were significantly lower after AS-Ⅳtreatment(P<0.05);(4)Compared to the control group,monocytes cells from colon,MLN and spleen raised in DSS group,while in the AS-Ⅳlow dose group and the AS-Ⅳhigh dose group,they descended significantly(P<0.05),especially in the AS-Ⅳhigh dose group(P<0.05).The monocytes cells from colon,MLN and spleen were significantly increased in the Tg group compared with the WT group and down-regulated in the both AS-Ⅳgroup(P<0.05);(5)Compared to the control group,the percentage ofTh9 cells in total CD4+T cells raised in DSS group(WT mice:the percentage of Th9 cells in MLN 3.64%±0.22%vs 1.75%±0.021%,P<0.05;the percentage of Th9 cells in spleen 5.84%±0.1%vs 3.38%±0.29%,P<0.05;Tg mice:the percentage of Th9 cells in MLN 9.63%±0.28%vs 1.64%±0.13%,P<0.05;the percentage of Th9 cells in spleen 9.53%±0.27%vs 2.36%±0.21%,P<0.05),while in the AS-Ⅳlow dose group and the AS-Ⅳhigh dose group,they descended significantly(P<0.05),especially in the AS-Ⅳhigh dose group(P<0.05).The percentage of Th9 cells were significantly increased in the Tg group compared with the WT group and down-regulated in the both AS-Ⅳgroup(P<0.05);(6)Inflammatory markers of IL-9 in LPMC,monocytes cells from MLN and spleen culture supernatants and serum were tested by ELISA.Compared to the control group,the IL-9 level raised in DSS group(WT mice:the concentrations of IL-9 from LPMC 246.16 pg/mL±6.14 pg/mL vs 26.16pg/mL±2.48 pg/mL,P<0.05;Tg mice:the concentrations of IL-9 from LPMC285 pg/mL±6.78 pg/mL vs 30.16 pg/mL±3.65 pg/mL,P<0.05),while in the AS-Ⅳlow dose group and the AS-Ⅳhigh dose group,it descended significantly(P<0.05),especially in the AS-Ⅳhigh dose group(P<0.05).The IL-9 level was significantly increased in the Tg group compared with the WT group and down-regulated in the both AS-Ⅳgroup(P<0.05).Conclusion: TL1A could promote the development of Th9 cells that secrete IL-9 to drive intestinal inflammation. AS-Ⅳ could improve intestinal inflammation in a dose-dependent way, probably through the down-regulation of TL1A and suppression of IL-9. |
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