Role Of Enteric Glial Cell In The Process Of Inflammation To Carcinoma In Ulcerative Colitis

Objective To explore the role of enteric glial cell in the process of inflammation to carcinoma in vivo and vitro by the model of AOM/DSS induced colitis associated colorectal cancer in mice and the co-culture system of EGC and IEC-6.Methods 1 180 SPF female Balb/c mice were random divided into 4 groups, experimental group(A), conditional control group(B), model group(C) and no-treatment control group(D). Group A, B and C were injected with AOM in abdominal and drank 2% DSS distilled water to build the model of colitis associated colorectal cancer, general condition was regularly daily recorded and disease activity index(DAI) was calculated. From third weekend, group A was injected with supernatant of EGC cultured 100 ul in abdominal cavity of mice everyday, group B was injected with DMEM. A sample of 5 mice was drawn from 4 groups and killed on 1W, 3W, 4W, 6W, 7W, 9W, 12 W, 15 W and 20 W randomly. Serum and colorectal tissues were collected. Level of inflammatory cytokine was detected by quantibody mouse inflammation array. Level of NGF and GDNF was detected by ELISA test. Colorectal tissues were subjected to haematoxylin and eosin(HE) staining and the colon macroscopic damage index(CMDI) and the histopathological score(HS) were evaluated. Expression of protein GFAP, S100β and PGP9.5 was detected by immunohistochemical in colorectal tissues. Expression of protein NF-κB, IKKβ, PI3 K, AKT, PTEN, BCL-2 and ZO-1 was detected by western blot analysis. 2 Intestinal epithelial cells(IEC-6) were co-cultured with enteric glial cell(EGC) and exposed to cytomix TNF-a and IL-6 for 36 hours. The effect of enteric glial cell for barrier function of intestinal epithelial cells was assessed by permeability to 150 k D FITC-dextran. The proliferation of IEC-6 was identified by immunostaining for Brd U, cycle and apoptosis of IEC-6 was analysed by flow cytometry. Changes in tight junction proteins ZO-1, E-Cadherin, Caspase-3 and Bcl-2 were assessed by western blot analysis.ResultsPart I Study in vivo 1) The DAI scores of model group were significantly higher than control group on 1W(0.67±0.62 vs 0), 4W(0.80±0.73 vs 0), 7W(1.00±0.85 vs 0), 9W(0.67±0.47 vs 0.07±0.15), 12W(0.93±0.43 vs 0.20±0.18), 15W(1.13±0.18 vs 0.20±0.45) and 20W(1.53±0.30 vs 0.07±0.15)(p<0.05). There was no significant difference between the experimental group and the sham group. 2) The CMDI scores of model group were significantly higher than control group on 1W(0.56±0.47 vs 0), 4W(2.37±1.57 vs 0), 7W(3.56±1.65 vs 0), 9W(2.86±0.99 vs 0), 12W(2.02±1.24 vs 0), 15W(3.26±1.13 vs 0) and 20W(3.88±0.90 vs 0)(p<0.05). The experimental group’s CMDI scores of 7W(1.32±1.38 vs 3.96±1.49,p<0.01)and 20W(2.63±0.64 vs 3.83±0.80,p<0.05)were significantly higher than the sham group. 3) The Histopathology Scores of model group were significantly higher than control group at different points(p < 0.05). The Histopathology Scores of experimental group were reduced at 4W(8.84±1.10 vs 10.66±1.25) and 7W(10.32±1.58 vs 13.96±2.49) compared with sham group(p<0.05). 4) Colon length shortening in model group compared with control group except for 3W(8.82±0.95 vs 9.56±0.66,p>0.05). There was no significant difference between the experimental group and the sham group. 5) There had appeared severe atypical hyperplasia after 6th week in experimental group, sham group and model group, in the 20 th week, the rate of tumor formation is 100% in three groups. 15 W tumor number(≥2mm) experimental group vs sham group(3.17 ± 1.72 vs 5.83 ± 2.04, p < 0.05). 20 W tumor number( ≥ 2mm) experimental group vs sham group(2.67±1.37 vs 5.67±2.58, p<0.05). 6) Mouse inflammation array demonstrate that the serum levels of TIMP-1(A/B reduced from 4W 5.99 to 20 W 0.00), M-CSF(A/B reduced from 4W 3.48 to 20 W 0.42), G-CSF(A/B reduced from 4W 3.48 to 20 W 0.39), IL-6(A/B reduced from 4W 2.60 to 20 W 0.44) were reduced gradually, however the level of MIP-1a(A/B increased from 4W 1.00 to 20 W 67.32), IL-2(A/B increased from 4W 0.78 to 20 W 3.67)and BLC(A/B increased from 4W to 20 W 2.95)were increased gradually(p < 0.05). 7) ELISA detection showed that NGF of experimental group were significantly higher than sham group at 4W,9W,15 W and 20W(p<0.05).GDNF of experimental group were increased compared with sham group in 6W-20 W. 8) Immunohistochemistry showed that the expression of GFAP and S100β in the experimental group were higher than sham group at 20W(p<0.05), model group lower than control group in 6-7W(p<0.05), and the expression of PGP9.5 were reduced at 7W(p<0.05). 9) Western blot showed that the expression of ZO-1 were increased in 4W-6W(p<0.05), and the expression of PTEN were increased in 4W-7W(p<0.05), but the expression of PI3 K, AKT were decreased(p<0.05) for experimental group vs sham group. 15 W experimental group vs sham group the expression of PTEN were increased(p < 0.01), and the expression of PI3 K were decreased(p < 0.05). Experimental group vs sham group the expression of IKKβ were decreased(p<0.05) in 6W, but the expression of NF-κB has no significant difference. The expression of both IKKβand NF-κB were decreased(p<0.05) in 7W. Part II Study in vitro 1) Fluorescein leakage test show that IEC-6+IL-6+EGC vs IEC-6+IL-6(724.33±22.08 vs 1451.50±36.18,p<0.01),IEC-6+TNF-a+EGC vs IEC-6+TNF-a(1261.17±53.08 vs 2623.50±62.77,p<0.01)。 2) Brd U incorporation and measurement:IEC-6+IL-6+EGC vs IEC-6+IL-6(0.168±0.075 vs 0.023±0.015, p<0.05), there was no significant difference between the other groups. 3) IEC-6 cell’s apoptosis was detected by flow cytometry after co-culture of EGC and IEC-6 cells. IEC-6+TNF-a+EGC vs IEC-6+TNF-a and IEC-6+IL-6+EGC vs IEC-6+IL-6 IEC-6 cell’s apoptosis was reduced in the early stage(p<0.01). 4) IEC-6+IL-6+EGC vs IEC-6+IL-6 the number of cells in G0/G1 phase are increasing(p<0.05), and S phase are decrease(p<0.01). IEC-6+TNF-a+EGC vs IEC-6+TNF-a the number of cells in S phase are decrease(p<0.05), but the number of cells in G0/G1 phase have no significant difference between two groups. 5) Western blot show that IL-6 can induce ZO-1 and E-cadherin upregulation in IEC-6 cells(p<0.01), and TNF-a induces the over expression of Bcl-2(p<0.05).Conclusions 1 The model of colorectal cancer by AOM/DSS induced were constructed successfully in mice. 2 In the process of inflammation to carcinoma in ulcerative colitis, the serum levels of inflammatory cytokines, NF-κB, IKK-β,PI3 K and AKT proteins were reduced,NGF and GDNF levels were elevated in the serum, ZO-1 and PTEN proteins’ expression were upregulated with EGC culture supernatant was injected in abdominal cavity of mice. All of these changes protect the function of intestinal mucosal barrier and delay the occurrence of colorectal cancer. 3 In the inflammatory environment, EGC can promote the proliferation of IEC-6 cells, and upregulate the expression of ZO-1 and E-cadherin, what’s more, the normal barrier function of IEC-6 was maintained.