Effect of Baitouweng Decoction on mTORC1-STAT3-COX-2 Signaling Pathway on Colon Tissue of DSS-Induced Ulcerative Colitis Mice
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摘要:
目的 探讨白头翁汤治疗溃疡性结肠炎(UC)的可能作用机制。 方法 40只C57BL/6雌鼠随机分为空白组、模型组、白头翁汤给药组及雷帕霉素给药组。采用3.5%葡聚糖硫酸钠(DSS)溶液构建小鼠UC模型。造模同时进行给药处理。白头翁汤组每日给予6.83 g·kg-1白头翁汤灌胃, 雷帕霉素组每日用2 mg·kg-1雷帕霉素药液进行腹腔注射, 空白组和模型组每日给予等体积0.5%羧甲基纤维素钠溶液灌胃。记录小鼠每日体质量变化及粪便性状, 7 d后处死小鼠。测量结肠长度并观察结肠组织病理学变化。采用流式细胞术检测小鼠血清炎症因子含量, 采用免疫印迹法检测结肠组织中哺乳动物雷帕霉素靶蛋白(mTOR)、转录激活因子3(STAT3)、核糖体S6蛋白激酶(P70S6K)及其磷酸化蛋白表达, 免疫组化法检测结肠组织中磷酸化P70S6K的表达, qPCR检测结肠组织环氧化酶(COX-2) mRNA表达水平。 结果 与空白组相比, 模型组小鼠体质量减轻, 结肠长度缩短、疾病活动指数(DAI)和组织病理学评分升高(P < 0.001), 血清中白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)含量升高(P < 0.001), 结肠组织中COX-2 mRNA表达水平、p-mTOR/mTOR、p-P70S6K/P70S6K、p-STAT3/STAT3均升高(P < 0.01，P < 0.001)。与模型组相比, 白头翁汤组和雷帕霉素组小鼠上述表型症状均得到改善, 血清中IL-6和TNF-α含量下降(P < 0.01，P < 0.001), 结肠组织中p-mTOR/mTOR、p-P70S6K/P70S6K、p-STAT3/STAT3和COX-2 mRNA表达减少(P < 0.05，P < 0.01，P < 0.001)。 结论 白头翁汤能有效预防DSS诱导的小鼠结肠炎症, 其作用机制可能与抑制mTORC1-STAT3-COX-2信号通路有关。 -
关键词:
- 溃疡性结肠炎 /
- 白头翁汤 /
- mTORC1-STAT3-COX-2信号通路 /
- 雷帕霉素
Abstract:OBJECTIVE To investigate the possible mechanism of Baitouweng decoction (BTW) in the treatment of ulcerative colitis. METHODS The UC mice were established by 3.5% dextran sodium sulfate (DSS) and randomly divided into four groups: control group, model group, BTW group and rapamycin group. The DSS administration and drug treatments were carried out simultaneously. The BTW group was given Baitouweng decoction 6.83 g·kg-1by gavage, the rapamycin group was treated with rapamycin 2 mg·kg-1 by intraperitoneal injection, and the other two groups received the same amount of 0.5% sodium carboxymethyl cellulos. Daily body weight loss and stool characteristics were recorded and the mice were sacrificed 7 days later. Colon length was measured and histopathological changes were observed. The serum inflammatory cytokines were detected by flow cytometry. The expression of mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3), ribosomal S6 protein kinase (P70S6K) and corresponding phosphorylated protein were detected by Western blot. The immunohistochemical detection of phosphorylated P70S6K protein expression was explored in colon tissue, and the level of cyclooxygenase-2 (COX-2) was evaluated by RT-PCR. RESULT Compared with the control group, the body weight, the colon length, the disease activity index (DAI) and the histopathological score were decreased in colitis mice (P < 0.001). The contents of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum, the expression of COX-2 mRNA in colon tissue, p-mTOR/mTOR, p-STAT3/STAT3 and p-P70S6K/P70S6K values were increased (P < 0.01, P < 0.001); Compared with the model group, all above clinical symptoms of mice in the BTW group and rapamycin group were improved. The contents of IL-6 and TNF-α in serum (P < 0.01, P < 0.001), the level of COX-2 mRNA in colon tissue, p-mTOR/mTOR, p-STAT3/STAT3 and p-P70S6K/P70S6K values were significantly decreased (P < 0.05, P < 0.01, P < 0.001). CONCLUSION Baitouweng decoction can effectively prevent DSS-induced colitis in mice by inhibiting mTORC1-STAT3-COX-2 signaling pathway. -
图 1 白头翁汤改善了DSS诱导的结肠炎症状
注: Control.空白组; DSS.模型组; BTW.白头翁汤组; RAPA.雷帕霉素组; 与空白组比较, ###P < 0.001;与模型组比较, ***P < 0.001, **P < 0.01。
Figure 1. Baitouweng decoction ameliorated the symptoms of DSS-induced colitis
图 2 各组小鼠结肠组织的代表性HE染色图(×200)
Figure 2. Representative hematoxylin-eosin (HE) staining of colon tissues of mice in each group (×200)
图 4 各组小鼠结肠组织p-P70S6K免疫组化图片(×200)
Figure 4. The immunohistochemical staining of p-P70S6K in mouse colon tissues (×200)
表 1 各组小鼠结肠组织病理学评分(x±s)
Table 1. Histological scores of colon tissues of mice in each group(x±s)
组别 n 病理学评分 空白组 8 0.25±0.46 模型组 8 6.75±1.49### 白头翁汤组 8 4.13±0.99*** 雷帕霉素组 8 4.63±1.30** 注: 与空白组相比, ###P < 0.001;与模型组相比, ***P < 0.001, **P < 0.01。 
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表 2 各组小鼠血清IL-6和TNF-α的含量(x±s，pg·mL-1)
Table 2. Contents of IL-6 and TNF-α in mouse serum (x±s, pg·mL-1)
组别 n IL-6 TNF-α 空白组 5 25.72±5.90 3.61±1.26 模型组 5 111.30±26.52### 9.12±1.94### 白头翁汤组 5 25.56±6.44*** 3.71±0.86** 雷帕霉素组 5 13.79±3.70*** 3.68±1.27*** 注: 与空白组相比, ###P < 0.001;与模型组相比, **P < 0.01, ***P < 0.001。
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表 3 各组小鼠结肠组织p-P70S6K/P70S6K、p-STAT/STAT3和p-mTOR/mTOR值比较(x±s)
Table 3. Comparison of p-P70S6K/P70S6K、p-STAT/STAT3 and p-mTOR/mTOR in colon tissue of mice in each group(x±s)
组别 n p-P70S6K/ P70S6K p-STAT3/ STAT3 p-mTOR/ mTOR 空白组 3 0.22±0.31 0.15±0.05 0.47±0.28 模型组 3 1.22±0.05### 1.18±0.18### 1.14±0.03## 白头翁汤组 3 0.51±0.11** 0.58±0.21** 0.68±0.15* 雷帕霉素组 3 0.76±0.05** 0.31±0.08*** 0.48±0.18** 注: 与空白组相比, ##P < 0.01，###P < 0.001;与模型组相比, *P < 0.05, **P < 0.01，***P < 0.001。
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表 4 各组小鼠结肠组织p-P70S6K免疫组化评分(x±s)
Table 4. The immunohistochemical score of p-P70S6K in mouse colon tissues(x±s)
组别 n 组化评分 空白组 5 1.6±0.74 模型组 5 4.5±0.71### 白头翁汤组 5 1.8±0.67*** 雷帕霉素组 5 1.7±0.45*** 注: 与空白组相比, ###P < 0.001;与模型组相比, ***P < 0.001。
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表 5 各组小鼠结肠组织COX-2 mRNA表达情况(x±s)
Table 5. The mRNA level of COX-2 in mouse colon tissues (x±s)
组别 n COX-2 空白组 4 1.00±0.13 模型组 4 3.96±0.79### 白头翁汤组 4 0.47±0.18*** 雷帕霉素组 4 0.93±0.26*** 注: 与空白组相比, ###P < 0.001;与模型组相比, ***P < 0.001。
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