姜黄素对大鼠肠纤维化的作用及其机制研究

Effects of curcumin in intestinal fibrosis of rats and its mechanism

目的 探讨姜黄素在三硝基苯磺酸（TNBS）诱导的大鼠结肠纤维化中的抗纤维化作用和机制.方法 SD大鼠40只随机分组,模型组（10只）、治疗组（10只）和对照组（10只）分别于第1、8、15、22和29天予TNBS 10 mg、15 mg、20 mg、25 mg和30 mg灌肠,另取10只大鼠给予50%乙醇灌肠,作为阴性对照（正常组）.从实验周期第1天起,治疗组大鼠每日予姜黄素30 mg/kg腹腔注射,对照组每日予0.9%NaCl腹腔注射,模型组和正常组不予处理.采用HE染色及Masson胶原三色染色观察大鼠结肠组织损伤和纤维化变化,采用ELISA法检测结肠黏膜中Th1/Th2型细胞因子IL-2、TNF-α、IL-4、IL-17的含量,采用荧光定量PCR法检测结肠黏膜中肠纤维化相关细胞因子如转化生长因子（TGF）-β1、结缔组织生长因子（CTGF）、Smad3、胶原Ⅰ、ⅢmRNA的表达.结果 模型组与对照组大鼠结肠组织大体损伤评分[（6.14±1.07）分,（6.17±1.47）分]及组织损伤评分[（8.42±1.40）分,（8.17±1.47）分]、胶原面积比（36.59%±4.07%,37.18%±4.05%）较正常组[分别为（2.13±0.64）分,（2.25±1.28）分和25.43%±5.39%]明显升高（均P＜0.05）.模型组与对照组大鼠黏膜组织中IL-2[（378.25±29.90）ng/L,（410.06±64.74）ng/L]、TNF-α[（87.11±23.85）ng/L,（100.41±12.59）ng/L）]、IL-17[（47.80±5.62）ng/L,（41.45±2.12）ng/L]含量及TGF-β1（4.71%±2.71%,4.12%±3.01%）、CTGF（10.33%±6.99%,11.46%±4.72%）、Smad3（9.35%±7.32%,10.11%±3.80%）、胶原Ⅰ（1.52%±1.11%,1.57%±1.35%）、胶原Ⅲ（3.04%±1.33%,3.03%±3.53%）mRNA表达量较正常组[分别为（179.74±20.73）ng/L,（35.47±7.13）ng/L,（14.48±7.52）ng/L和0.90%±1.13%,0.53%±0.47%,0.62%±0.44%,0.16%±0.09%,0.18%±0.10%]均明显升高（均P＜0.05）.而治疗组大鼠结肠组织大体损伤评分[（4.00±1.07）分]及组织损伤评分[（5.13±1.46）分]、胶原面积比（30.01%±7.56%）,IL-2[（223.91±28.04）ng/L]、TNF-α[（44.19±4.77）ng/L]、IL-17[（14.89±4.31）ng/L]含量和TGF-β1（0.85%±0.76%）、CTGF（1.56%±1.13%）、Smad3（3.62%±3.03%）、胶原Ⅰ（0.40%±0.31%）、胶原Ⅲ（0.60%±1.02%）mRNA表达量较模型组及对照组明显降低（P＜0.05）,与正常组无明显差异（P＞0.05）.结论 姜黄素可通过降低细胞因子的过度表达,减轻大鼠结肠炎症,从而抑制过度＂损伤-修复＂所致的组织纤维化.

Objective To investigate the anti-fibrotic effects of curcumin in trinitrobenzene sulphonic acid （TNBS） induced intestinal fibrosis in rats and its mechanism. Methods Forty SD rats were randomly divided into model group, treatment group, control group and normal group with 10each. Except the normal group, the other three groups were given 10, 15, 20, 25 and 30 mg of TNBS enema on the 1st, 8 th, 15th, 22nd and 29th days,respectively. The rats in treatment group were intraperitonealy injected with 30 mg/kg of curcumin daily. Control group was injected with 0. 9%NaCl solution and normal group received an equal volume of 50% ethanol enema without any treatment. The damage and fibrosis of colon were detected with HE staining and Masson collagen staining, respectively. The contents of interleukin （IL） -2, tumor necrosis factor （TNF） -α, IL-4 and IL-17 in colon were measured by enzyme-link immunosorbent analysis （ELISA）. The expressions of intestinal fibrosis related cytokines such as transforming growth factor （TGF） -β1, connective tissue growth factor （CTGF）, Smad3, collagen Ⅰ and collagen Ⅲ mRNA were determined by FQ-PCR.Results The macroscopic and micrpscopic colonic damage scores and collagen area were significantly higher in model group （6.14 ± 1.07, 8. 42 ± 1.40 and 36. 59% ± 4.07%, respectively） and control group （6.17 ± 1.47, 8. 17 ±1.47 and 37.18 %±4.05 %, respectively） than those in normal group （2.13±0.64, 2.25±1.28 and 25.43%±5.39% ,respectively）（P〈0.05）. Contents of IL2, TNF-α, IL-17, as well as expressions of intestinal fibrosis related cytokines including TGF-β1, CTGF,Smad3, collagen Ⅰ and Ⅲ mRNA were also higher in model group [（378. 25±29. 90） ng/L,（87.11±23.85） ng/L, （47.80±5.62） ng/L, 4.71%±2.71%,10.33%±6.99%,9.35%±7.32%,1.52% ± 1.11% and 3.04% ±1.33%, respectively] and control group [（410. 06 ± 64.74） ng/L,（100.41±12.59） ng/L, （41.45±2. 12） ng/L, 4. 12%±3.01%,11.46%±4.72%,10. 11%±3.80%,1. 57% ± 1. 35% and 3. 03% ± 3. 53%, respectively] in comparision with normal group [（179.74±20. 73） ng/L, （35. 47±7. 13） ng/L, （14. 48±7. 52） ng/L and 0. 90%± 1. 13%,0.53%±0.47%, 0. 62%±0. 44%, 0. 16%±0. 09% and 0. 18%±0. 10%, respectively] （P〈0.05）. While in treatment group, the macroscopic （4.00 ± 1.07 ） and micrpscopic （5. 13 ± 1.46）colonic damage scores, collagen area （30.01%±7.56%）, contents of IL-2 [（223.91±28.04） ng/L],TNF-α [（44.19±4. 77） ng/L] and IL-17 [（14.89±4. 31） ng/L], expressions of TGF-β1 （0.85%±0.76%）, CTGF （1.56%±1.13%）, Smad3 （3.62%±3.03%）, collagen Ⅰ （0.40%±0.31%） and Ⅲ （0.60 % ± 1.02 % ） mRNA were much lower than those in model group and control group （P〈0.05 ）, but similar to those in normal group （P〉 0.05 ）. Conclusions Curcumin can inhibit intestinal fibrosis caused by excessive ＂wound-healing＂ reaction via reducing the overexpression of cytokines in colonic mucosa and attenuating the inflammation of colon.