| Background Ulcerative colitis(UC) and Crohn’s disease(CD), two clinical types of inflammatory bowel disease(IBD), are chronic intermittent or progressive intestinal inflammation. CD exhibits the inflammation of the whole gastroenteric tract, while UC only the colonic mucosa. Increasing evidences support that the pathogenesis of Although the etiology of IBD is unknown, the dominant hypothesis suggests the inflammation results from sustained immune response towards altered or pathogenic microbiota within a genetically susceptible host during which innate immune plays a central role. NOD2/CARD15 has been the firstly found loci associated with human Crohn’s disease in Caucasians, but it is not the same in Asians. Since then, increasing experiments both in animals and cells demonstrate that inflammasome plays an important role in inflammatory and autoimmune diseases including IBD. NLRP3 has been the mostly characterized NLR because of its various agonists. IL-1β and IL-18,the effectors of NLRP3 inflammasome, play a crucial role in the etiology and development of IBD. However, it is still not sure about the regulation of NLRP3 inflammasome. Oxidation stress is also associated with IBD. Recently, some reports show that reactive oxygen species(ROS) can activate the assembly of NLRP3 inflammatory. So, it is worth further reseach about the interactivation of Oxidation stress and NLRP3 inflammatory and their involvements in IBD.Objective After taking N-Acetyl-L-cysteine(NAC) and diethyldithiocarbamate(DDC) to change the level of ROS in mice with colitis, the expressions of NLRP3, Caspase-1,IL-1β, IL-18 and their m RNA were measured. In addition, the disease activity index(DAI) and histochemistry index(HI) were evaluated to discuss these signals’ involvement in colitis.Methods1.To build the model of IBD, we obtained eight-weeks-aged, male C57BL/6mice and orally supplied them with 3% DSS solution for free drinking. After 10-days drinking, DSS solution was exchanged with autoclaved water. After another 2-days drinking, all the mice suffered from colitis.2.All the mice were randomly divided to four groups. And each group concludes twelve mice. Control group was supplied with autoclaved water for drinking. The group of colitis model was supplied with 3% DSS for drinking. NAC group was supplied with DSS and received oral gavage with NAC(250mg/kg/d) every other day. DDC group was supplied with DSS and received oral gavage with DDC(250mg/kg/d)every other day.3.The feces and body weight were examined everyday. All the mice were executed on the twelfth day through eye blood sampling to collect serum and colon specimen.4. DAI and HI were used to help assessment the severity of colitis. enzyme linked immunosorbent assay(ELISA) was used to measure the expression of IL-1βand IL-18 in serum. Immunohistochemistry(IHC) and western blot were used to detect the level of NLRP3 and Caspase-1 in colon. Q-PCR was used to examine the relative expressions of NLRP3 m RNA and IL-1βm RNA.5. All data were expressed as the means ± standard deviation(SD) and analyzed with Prism 5.0v software(Graph Pad). The statistical differences between the groups were compared using two-tailed Student’s t test or Fisher’s exact test; those among groups were compared using one-way analysis of variance(ANOVA) or Chi-square test. For all statistical tests, p< 0.05 was considered significant.Results1. disease activity index(DAI)Compared with the control group, mice with DSS-induced colitis showed diarrhea, bloody stools and weight loss. Symptoms in NAC group were lighter than the model group while Symptoms in DDC group were severer compared with the model group. There were differences in DAI among four groups(F = 51.8, P <0.05).DAI in model group was higher than in control group(7.86 ± 0.98 vs 1.49 ± 0.30).NAC group was lower compared with the model group(5.82 ± 1.15 vs 7.86 ± 0.98).DDC group was higher than the model group(9.92 ± 1.63 vs 7.86 ± 0.98). The differences were statistically significant(P <0.05).2. histochemistry index(HI)Compared with the control group, the colonic lamina propria of model group showed a large number of lymphocytes infiltration, angiogenesis and crypt destruction. NAC group was lighter compared with the model group and DDC group was severer compared with the model group under mic ROScopy. There were differences about HI among four groups(F = 69.58, P <0.05), namely the model group higher than control group(6.52 ± 0.67 vs 0.50 ± 0.31), NAC group lower than the model group(4.58 ± 1.12 vs 6.52 ± 0.67), DDC group higher than the model group(8.14 ± 1.15 vs 6.52 ± 0.67). All the differences were statistically significant(P<0.05).3. the expressions of IL-1βand IL-18 in serum(ELISA)There were differences about the amount of IL-1βin serum among four groups(F = 23.96, P <0.05), namely the model group higher than control group(86.5±7.88 vs70.20±7.04), NAC group lower than the model group(74.66±3.43 vs 86.5±7.88),DDC group higher than the model group(99.15±4.17 vs 86.5±7.88). All the differences were statistically significant(P <0.05).There were differences about the amount of IL-18 in serum among four groups(F = 71.98, P <0.05), namely the model group higher than control group(118.60±2.70 vs 102.20±6.50), NAC group lower than the model group(89.40±3.58 vs118.60±2.70), DDC group higher than the model group(128.40±4.56 vs118.60±2.70). All the differences were statistically significant(P <0.05).4. the expession of NLRP3 in colon(IHC)There were differences about the expession of NLRP3 in colon among four groups(χ2=16.67,P<0.05), namely the model group higher than control group(75.0%vs 16.7%), NAC group lower than the model group(25.0% vs 75.0%), DDC group higher than the model group(83.3% vs 75.0%). All the differences were statistically significant(P <0.05).5. the expession of Caspase-1 in colon(western blot)There were differences about the expession of Caspase-1 in colon among four groups(F=22.08, P<0.05), namely the model group higher than control group(0.65±0.13 vs 0.33±0.13), NAC group lower than the model group(0.36±0.12 vs 0.65±0.13), DDC group higher than the model group(0.89±0.13 vs 0.36±0.12).All the differences were statistically significant(P <0.05).6. the level of NLRP3 and IL-1βm RNA in colon(Q-PCR)There were differences about the expession of NLRP3 m RNA in colon among four groups(F=145, P<0.05), namely the model group higher than control group(6.17±0.56 vs 1.35±0.44), NAC group lower than the model group(3.11±0.34vs6.17±0.56), DDC group higher than the model group(7.63±0.71 vs 6.17±0.56). All the differences were statistically significant(P <0.05).There were differences about the expession of IL-1β m RNA in colon among four groups(F=22.53, P<0.05), namely the model group higher than control group(1.34±0.27 vs 0.41±0.15), NAC group lower than the model group(0.97±0.15 vs 1.34±0.27). All the differences were statistically significant(P <0.05).Conclusion1. The supplement of 3% DSS could induce colitis in eight-weeks-aged, male C57BL/6 mice.2. NLRP3, Caspase-1, IL-1βand IL-18 play a crucial role in the pathology of colitis. NAC could inhibit this signaling and attenuate colitis, while DDC showed the opposite effects. |
PDF Full Text Request