雷公藤多苷对溃疡性结肠炎大鼠模型炎症及TLR4／MyD88信号通路的作用

Effect of Tripterygium wilfordii polycoride upon inflammation and TLR4/MyD88 signaling pathway in ulcerative colitis rats model

目的观察雷公藤多苷（TWP）对溃疡性结肠炎（UC）的作用，以及对Toll样受体4（TLR4）／髓样分化因子88（MyD88）信号通路的干预作用，以探讨其可能的作用机制。方法采用三硝基苯磺酸（TNBS）／乙醇灌肠法建立UC大鼠模型。采用随机数字表法将90只雄性Wistar大鼠随机分为正常对照组、模型对照组及TWP低、中、高剂量（3、6、12mg／kg）组、硫唑嘌呤（AZA）组（6mg／kg），每组15只。造模后第4天，各组分别给予相应药物连续灌胃14d。观察各组大鼠疾病活动指数（DAI）、结肠大体形态损伤以及组织学评分，采用反转录（RT）-PCR法和Western印迹法检测各组大鼠肠道组织中TLR4／MyD88信号通路相关蛋白——TLR4、MyD88、肿瘤坏死因子受体相关因子6（TRAF-6）、核因子κB（NF—κB）、肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）的表达。结果模型对照组大鼠DAI、结肠大体形态损伤及组织学评分均显著高于正常对照组（均P〈0．01），大鼠造模成功；TwP高剂量组的DAI评分、结肠大体形态损伤评分及组织学评分均低于模型对照组[（0．87±0．25）比（1．60±0．76）分，（3．93±1．94）比（5．40±2．21）分，（5．45±2．73）比（13．27±3．50）分，均P〈0．05]。模型对照组大鼠TLR4、MyD88、TRAF-6、NF—κB、TNF-α、IL-1βmRNA及蛋白表达明显高于正常对照组（均P〈0．01）；TWP高剂量组上述指标mRNA及蛋白表达均明显低于模型对照组（mRNA：2．166±0．475比5．647±0．275、1．295±0．087比3．774±0．418、1．125±0．188比2．535±0．320、1．201±0．152比2．082±0．077、1．525±0．218比3．094±0．022、1．797±0．257比17．152±0．145；蛋白：0．252±0．010比0．277±0．008、0．172±0．002比0．213±0．005、0．233±0．006比0．248±0．003、0．099±0．003比0．122±0．007、0．238±0．002比0．252±0．005、0．235±0．003比0．245±0．006，均P〈0．05），AZA组上述指标mRNA及蛋白表达明显低于模型对照组（均P〈0．01），TWP高剂量组与AZA组之间差异无统计学意义（均P〉0．05）。结论TWP能减轻肠道炎症反应，促进黏膜愈合，对UC具有治疗作用；抑制TLR4的表达，影响MyD88引发信号通路下游的TRAF-6表达，从而抑制NF—KB的活化，减少炎症因子TNF-α、IL-1β的释放是其作用机制之-。

Objective To observe the effect of Tripterygium wilfordii polycoride （TWP） on ulcerative colitis （UC）, and its intervention effect on toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88） signaling pathway, thus to investigate its possible mechanism. Methods Trinitrobeuzene sulfouie acid （TNBS）/ethanol enema method was used to set up the UC rat model. With random number table, 90 male Wistar rats were divided into normal control group, model group, TWP low, medium and high dose group （3, 6, 12 mg/kg, respectively） and azathioprine （AZA） group （6 mg/kg）, with 15 rates in each group. Four days after enema, rates in each group were given corresponding drug lavage for 14 consecutive days. Disease activity index （DAI）, colon gross morphological damage and histological grading of each group were observed. Using Western blot and reverse transcription （ RT）-PCR method, the TLR4/MyD88 signaling pathway-related proteins in UC rat intestinal tissue were detected, namely TLR4, MyD88, tumor necrosis factor receptor related factor 6 （ TRAF-6）, nuclear factor kappa B （ NF-KB）, tumor necrosis factor alpha （ TNF-α）, and interleukin-1 beta （IL-1β）. Results The DAI, colon gross morphological damage, and histological grading of the model group were significantly higher than that of the normal control group （ all P 〈0. 01 ）, indicating successful establishment of UC model. The DAI, colon gross morphological damage and histological grading of the TWP high dose group were lower than those of the model group （0. 87 ± 0. 25 vs 1.60 ± 0.76, 3.93 ± 1.94 vs 5.40 ± 2. 21, 5.45 ± 2. 73 vs 13.27 ± 3.50, P 〈 0. 05）. Compared with the normal control group, the mRNA and protein expressions of TLR4, MyD88, TRAF-6, NF-κB, TNF-α, and IL-1β in the model group rats were significantly increased （all P 〈 0. 01 ）; which were significantly decreased in the TWP high dose group compared with model group rats （ mRNA ：2. 166 ± 0. 475 vs 5. 647 ± 0. 275, 1. 295 ± 0. 087 vs 3. 774 ± 0. 418, 1. 125 ± 0. 188 vs 2. 535 ± 0. 320, 1. 201 ± 0. 152 vs 2. 082 ± 0. 077, 1. 525 ± 0. 218 vs 3. 094 ± 0. 022, 1. 797 ± 0. 257 vs 17. 152 ± 0. 145;protein：0. 252 ±0.010 vs 0.277 ±0.008, 0. 172 ±0.002 vs 0.213 ±0.005, 0.233 ±0.006 vs 0.248 ±0.003, 0.099 ±0.003 vs 0. 122 ±0.007, 0.238 ±0.002 vs 0.252 ±0.005, 0.235 ±0.003 vs 0. 245 ± 0. 006, all P 〈 0.05 ）, also decreased in the AZA group （ all P 〈 0. 01 ） ; and there were no significant differences between the TWP high dose group and the AZA group （ all P 〉 0. 05 ）. Conclusions TWP can alleviate intestinal inflammation, promote healing of mueosa, showing a therapeutic effect for UC. One of its mechanisms may be through inhibiting the expression of TLR4, affecting the expression of TRAF- 6, which is downstream to MyD66 signaling pathway, thus to suppress the activation of NF-αB and reduce the release of inflammatory factor such as TNF-α and IL-1β.