葛根提取物对溃疡性结肠炎大鼠肠黏膜损伤影响及机制

Effect and mechanism of Pueraria lobata extract on intestinal mucosal injury in rats with ulcerative colitis

目的探讨葛根提取物对溃疡性结肠炎(UC)模型大鼠肠黏膜损伤的影响及可能机制。方法 SPF级SD大鼠90只,除对照组外均建立UC大鼠模型,成模大鼠随机分为模型组、葛根提取物低、中、高剂量组(1.75、3.50、5.25 g/kg)、柳氮磺胺吡啶片(SASP)组(0.3 g/kg),对照组、模型组每天灌胃生理盐水10 mL/kg,连续14 d;实验期间观察大鼠一般状况;对大鼠进行疾病活动指数(DAI)评分,结肠黏膜损伤指数(CMDI)评分,组织学损伤(TDI)评分;采用ELISA方法检测大鼠血清白细胞介素1β(IL-1β)、IL-4、IL-6水平;qRT-PCR法检测p38丝裂原活化蛋白激酶(p38 MAPK)、过氧化物酶增殖物激活受体–γ(PPARγ)、核因子–κB(NF-κB)mRNA表达;Western blot检测结肠组织蛋白表达情况。结果与对照组比较,模型组大鼠出现厌食、嗜睡、排脓血便等病理状态、体重增加量显著降低,DAI评分明显升高(P <0.05);葛根提取物组和SASP组大鼠在干预第2周病理症状开始缓解,体重逐渐增加,DAI评分逐渐降低(P <0.05);对照组大鼠结肠组织无水肿、溃疡及出血,模型组大鼠结肠黏膜水肿、出血、散在溃疡,镜下可见上皮细胞坏死、脱落,炎症细胞浸润;葛根提取物组、SASP组大鼠肉眼及镜下结肠损伤明显减轻;与对照组比较,各组大鼠CMDI、DAI评分明显升高(P <0.05);与模型组比较,葛根提取物组、SASP组大鼠CMDI、DAI评分明显降低(P <0.05)。与对照组比较,模型组大鼠血清IL-6、IL-1β水平明显升高,IL-4水平明显减低(P <0.05);与模型组比较,葛根提取物组、SASP组大鼠血清IL-6、IL-1β水平明显降低,IL-4水平明显升高(P <0.05);与对照组比较,模型组大鼠结肠组织p38 MAPK、NF-κB mRNA与蛋白表达水平均升高,PPARγmRNA与蛋白表达明显降低(P <0.05);与模型组比较,葛根提取物组、SASP组大鼠结肠组织p38 MAPK、NF-κB mRNA与蛋白表达水平均明显降低,PPARγmRNA与蛋白表达均明显升高(P <0.05)。结论葛根提取物具有改善UC模型大鼠一般状况及黏膜保护作用,其机制可能与下调p38 MAPK、NF-κBp65和上调PPARγ蛋白表达有关。

Objective To investigate the effect of Pueraria lobata extract on intestinal mucosal injury via p38 mitogenactivated protein kinase(p38 MAPK)-peroxidase proliferator-activated receptor-γ(PPARγ)-nuclear factor-κB(NF-κB)regulation pathway in rats with ulcerative colitis(UC). Methods UC model was established in 75 specific pathogen free Sprague-Dawley(SD) rats and a control group in another 15 rats. The UC model rats were assigned into a model group(gavaged with saline) and low, moderate, high Pueraria lobata extract(gavaged at dosages of 1.75, 3.5, and 5.25 g/kg)groups, and a senescence-associated secretory phenotype(SASP)(0.3 g/kg) group;the gavages were performed once a day continuously for 14 days. General condition of all the rats were observed during the experiment. At the end of the treatments,all the rats were weighed;the disease activity index(DAI), colon mucosal damage index(CMDI), and histological damage index(HDI) were determined;serum interleukin-1β(IL-1β), interleukin-4(IL-4), and interleukin-6(IL-6) were detected by enzyme-linked immunosorbent assay(ELISA);the expressions of p38 mitogen-activated protein kinase(p38 MAPK),peroxidase proliferator-activated receptor-γ(PPARγ), and nuclear factor-κB(NF-κB) were detected with quantitative reversetranscription PCR(qRT-PCR);Western blot was used to detect the expression of protein in colon tissues. Results In the rats of model group, anorexia, lethargy, and purulent stool were observed and weight gain decreased but DAI score increased in comparison with those of the control rats(P < 0.05). Alleviated pathological symptoms were observed and weight gain increased but DAI score declined significantly in the rats one week after Pueraria lobata extract and SASP treatment(P <0.05). No pathological changes were observed in the rats of control group;while there were edema, hemorrhage, and scattered ulcer in colon tissues of model rats and microscopic observations of(hematoxylin-eosin) HE staining specimens revealed necrosis and exfoliation of epithelial cells and infiltration of inflammatory cells in colon tissues of the model rats;both gross anatomic and microscopic observations indicated obviously alleviated pathological changes in colon tissues of the rats treated with Pueraria lobata extract and SASP compared to the model rats. The model rats showed significantly higher CMDI and DAI than those of the control rats(both P < 0.05) but the CMDI and DAI of the rats treated with Pueraria lobata extract and SASP were significantly lower than those of model rats(both P < 0.05). Significantly increased IL-6 and IL-1βbut decreased IL-4 were detected in the rats of model group compared to those of the control rats(P < 0.05 for all);compared to the model rats, the rats with Pueraria lobata extract and SASP had significantly decreased IL-6 and IL-1β but increased IL-4(both P < 0.05). Compared to the control rats, the model rats had significantly increased protein and mRNA expressions of p38 MAPK and NF-κB and decreased protein and mRNA expression of PPARγ in colon tissues(all P < 0.05);while the protein and mRNA expressions of p38 MAPK and NF-κB decreased but the protein and mRNA expression of PPARγincreased significantly in colon tissues of the rats with Pueraria lobata extract and SASP treatment in comparison with those in the model rats(P < 0.05 for all). Conclusion Pueraria lobata extract can improve the survival of UC model rats and play a protective role in intestinal mucosa by down-regulating p38 MAPK, NF-κBp65 and up-regulating PPARγ protein expression.