仙茅苷对溃疡性结肠炎大鼠JAK/STAT/NF-κB信号通路的影响

Effects of Curculin on JAK/STAT/NF-κB Signaling Pathway in Rats with Ulcerative Colitis

目的:探究仙茅苷对溃疡性结肠炎(UC)大鼠Janus激酶(JAK)/信号转导和转录激活因子(STAT)/核因子-κB(NF-κB)信号通路的影响。方法:SD大鼠随机选取15只大鼠为正常对照组,其余大鼠采用5%葡聚糖硫酸钠(DSS)诱导建立慢性UC模型。取造模成功大鼠60只随机分为模型组、托法替尼组(4 mg·kg^(-1))、仙茅苷低(25 mg·kg^(-1))、中(50 mg·kg^(-1))、高(100 mg·kg^(-1))剂量组,每组12只。正常对照组和模型组不进行药物干预,仅灌胃蒸馏水;托法替尼组和仙茅苷各剂量组灌胃相应剂量的药物,1次/d,连续给药2周。给药期间,观察大鼠的一般状态和疾病活动指数(DAI);异硫氰酸荧光素-葡聚糖(FITC-Dextran)示踪法检测各组大鼠肠黏膜通透性;检测各组大鼠血浆二胺氧化酶(DAO)活性和D-乳酸(D-LA)、内毒素(ET)含量;检测结肠长度及结肠黏膜损伤指数(CMDI);HE染色观察结肠组织病理学变化;ELISA法检测结肠组织匀浆中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-6含量;免疫组织化学法检测结肠组织磷酸化细胞核因子κB p65(p-NF-κB p65)阳性表达;Western blot检测结肠组织JAK/STAT/NF-κB通路相关蛋白的表达。结果:正常对照组大鼠状态良好,饮食正常,无腹泻、便血发生;模型组大鼠出现活动减少、厌食症状,且体质量下降,有腹泻、便血情况。与正常对照组相比,模型组大鼠DAI评分、血清FITC-Dextran浓度、血浆DAO活性和D-LA、ET水平、CMDI评分、结肠组织病理学评分、TNF-α、IL-1β、IL-6含量、p-NF-κB p65的阳性表达、JAK1、JAK3表达和p-STAT3/STAT3比值显著升高(P<0.05),结肠显著缩短,NF-κB p65(胞质)和IκBα表达显著降低(P<0.05);与模型组相比,托法替尼组和仙茅苷中、高剂量组大鼠一般状态明显好转,较活跃,DAI评分、血清FITC-Dextran浓度、血浆DAO活性和D-LA、ET水平、CMDI评分、结肠组织病理学评分、TNF-α、IL-1β、IL-6含量、p-NF-κB p65的阳性表达、JAK1、JAK3表达和p-STAT3/STAT3比值显著降低(P<0.05),结肠长度增加,NF-κB p65(胞质)和IκBα表达显著升高(P<0.05),且仙茅苷各组呈一定的剂量依赖性。结论:仙茅苷对UC大鼠结肠损伤的保护作用可能与抑制JAK/STAT/NF-κB信号通路,降低促炎细胞因子释放有关。

Objective: To explore the effects of curculigoside on the Janus kinase(JAK)/signal transducer and activator of transcription(STAT)/nuclear factor-κB(NF-κB) signaling pathway in ulcerative colitis(UC) rats. Methods: Fifteen SD rats were randomly selected as the normal control group, and the other rats were induced to establish chronic UC model with 5% dextran sodium sulfate(DSS). Sixty rats successfully modeled were randomly divided into model group, tofacitinib group(4 mg·kg^(-1)), curculigoside low(25 mg·kg^(-1)), medium(50 mg·kg^(-1)) and high(100 mg·kg;) dose groups with 12 rats in each group. The normal control group and the model group were without drug intervention, only treated with distilled water by gavage;tofacitinib group and curculigoside groups were given corresponding dose of drug, once daily for 2 weeks. In the administration period, the general state and disease activity index(DAI) of rats were observed;the intestinal mucosal permeability of rats in each group was detected by FITC-Dextran tracing method;the plasma diamine oxidase(DAO) activity, D-lactic acid(D-LA) and endotoxin(ET) contents of rats in each group were detected;the colon length and colonic mucosal injury index(CMDI) were detected;the histopathological changes in colon were observed after HE staining;the contents of TNF-α, IL-1β and IL-6 in colon tissue homogenate were detected by ELISA method;the positive expression of p-NF-κB p65 in colon tissue was detected by immunohistochemical method;the expression of JAK/STAT/NF-κB pathway related proteins in colon tissue was detected by Western blot. Results: The rats in the normal control group were in good conditions with normal diet and without diarrhea or blood in stool;the rats in the model group showed such symptoms as reduced activity, anorexia, weight loss, diarrhea and blood in stool. Compared with those in the normal control group, the DAI score, serum FITC-Dextran concentration, plasma DAO activity and D-LA, ET levels, CMDI score, colonic histopathology score, TNF-α, IL-1β and IL-6 contents, positive expression of p-NF-κB p65, expression of JAK1, JAK3 and the ratio of p-STAT3/STAT3 were significantly increased in the model group(P<0.05), colon was significantly shortened, and the expression of NF-κB p65(cytoplasm) and IκBα was significantly reduced(P<0.05). Compared with those in the model group, the general state of rats in tofacitinib group, curculigoside medium and high dose groups were improved, and the rats were more active, the DAI score, serum FITC-dextran concentration, plasma DAO activity and D-LA, ET level, CMDI score, colonic histopathology score, TNF-α, IL-1β, IL-6 contents, positive expression of p-NF-κB p65, expression of JAK1, JAK3 and ratio of p-STAT3/STAT3 were significantly reduced(P<0.05), the length of colon increased, and the expression of NF-κB p65(cytoplasm) and IκBα increased significantly(P<0.05), and the effects of curculigoside were dose-dependent.Conclusion: The protective effects of curculigoside on colon injury in UC rats may be related to inhibiting JAK/STAT/NF-κB signaling pathway and reducing the release of pro-inflammatory cytokines.