黄连-厚朴配伍抑制PI3K/Akt信号通路改善TNBS诱导的大鼠溃疡性结肠炎研究

Effect of Coptidis Rhizoma-Magnoliae Officinalis Cortex on TNBS-induced ulcerative colitis in rats by inhibiting PI3K/Akt signaling pathway

目的研究黄连-厚朴配伍对溃疡性结肠炎(ulcerative colitis,UC)大鼠及磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路的调控作用。方法利用高通量基因表达数据库获取UC基因表达谱数据,并综合生物信息学分析,预测黄连-厚朴治疗UC的潜在机制。采用2,4,6-三硝基苯磺酸(TNBS)/乙醇法建立UC大鼠模型,SD大鼠随机分为对照组、模型组、黄连(2g/kg)组、厚朴(2g/kg)组、黄连-厚朴配伍(4g/kg)组和柳氮磺胺吡啶(0.4 g/kg)组,每组8只。造模24 h后,各给药组ig相应药物,1次/d,连续6 d,采用试剂盒检测各组大鼠血清中白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平和髓过氧化物酶(myeloperoxidase,MPO)活性;计算并评价各组大鼠结肠质量/长度、脾脏指数和肠黏膜损伤指数(colon macroscopic damage index,CMDI)评分;采用苏木素-伊红(HE)染色法观察各组大鼠结肠组织病理变化并进行病理损伤评分;采用免疫组化法检测各组大鼠结肠组织PI3K、Akt和剪切型半胱氨酸蛋白酶-3(cleaved Caspase-3)表达情况;采用Western blotting法检测各组大鼠结肠组织磷酸化Akt(p-Akt)、B淋巴细胞瘤2(B-cell lymphoma 2,Bcl-2)和cleaved Caspase-3蛋白表达情况。结果生物信息学预测显示,黄连-厚朴抗UC的潜在靶点有49个,可能与调控生物过程及过氧化物酶体增殖物活化受体(peroxisome proliferator-activated receptor,PPAR)信号通路、核因子κB(nuclear factor-κB,NF-κB)信号通路和PI3K/Akt信号通路等有关。实验验证结果显示,与模型组比较,各给药组大鼠血清中IL-1β、IL-6、TNF-α水平和MPO活性显著降低(P<0.01);结肠质量/长度、脾脏指数、CMDI评分、结肠组织病理损伤评分均显著降低(P<0.01);结肠组织PI3K、Akt和cleaved Caspase-3阳性表达显著减少(P<0.01);结肠组织中p-Akt/Akt、cleaved Caspase-3蛋白表达水平显著降低(P<0.01),Bcl-2蛋白表达水平显著升高(P<0.01),其中黄连-厚朴配伍组疗效最佳。结论黄连、厚朴及其配伍能够有效缓解TNBS诱导的UC,其作用机制可能与抑制PI3K/Akt通路相关,且黄连-厚朴配伍疗效最佳,体现了中药"相使"增效的配伍关系。

Objective To study the effect of Huanglian(Coptidis Rhizoma)-Houpo(Magnoliae Officinalis Cortex)on regulation of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway in rats with ulcerative colitis(UC).Methods The potential mechanism of Coptidis Rhizoma-Magnoliae Officinalis Cortex on treating UC was predicted based on UC gene expression profile data obtained from high-throughput gene expression database combined with comprehensive bioinformatics analysis.UC rats model was established by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ethanol method.SD rats were randomly divided into control group,model group,Coptidis Rhizoma(2 g/kg)group,Magnoliae Officinalis Cortex(2 g/kg)group,Coptidis Rhizoma-Magnoliae Officinalis Cortex(4 g/kg)group and sulfasalazine(0.4 g/kg)group,with eight rats in each group.The rats in each administration group were ig corresponding drugs 24 h after modeling,once a day for 6 d.Levels of interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α)and myeloperoxidase(MPO)activity in serum of rats in each group were detected by kits;Colon mass/length,spleen index and colon macroscopic damage index(CMDI)were calculated and evaluated;Hematoxylin-eosin(HE)staining method was used to investigate the pathological changes and pathological damage scores of colon tissue in rats;Immunohistochemical method was used to detect PI3K,Akt and cleaved Caspase-3 expression in colon of rats;Western blotting was used to detect expressions of phosphorylated Akt(p-Akt),B-cell lymphoma 2(Bcl-2)and cleaved Caspase-3 in colon of rats.Results Bioinformatics predictions showed that there were 49 potential targets of Coptidis Rhizoma-Magnoliae Officinalis Cortex on UC,which may be related to the regulation of biological processes and pathways such as peroxisome proliferator-activated receptor(PPAR)signaling pathway,nuclear factor-κB(NF-κB)signaling pathway and PI3K/Akt signaling pathway.Experimental validation results showed that compared with model group,IL-1β,IL-6,TNF-αlevels and MPO activity in serum of rats in each administration group were significantly reduced(P<0.01);Colon mass/length,spleen index,CMDI score,and colon tissue pathological damage score of rats were significantly reduced(P<0.01);Positive expressions of PI3K,Akt and cleaved Caspase-3 in colon tissue was significantly reduced(P<0.01);Expressions of p-Akt/Akt and cleaved Caspase-3 in colon were significantly reduced(P<0.01),Bcl-2 expression was significantly increased(P<0.01),and Coptidis Rhizoma-Magnoliae Officinalis Cortex had the best curative effect.Conclusion Coptidis Rhizoma,Magnoliae Officinalis Cortex and their compatibility can effectively alleviate TNBS-induced UC,of which mechanism may be related to the inhibition of PI3K/Akt pathway,and Coptidis Rhizoma-Magnoliae Officinalis Cortex has the best effect,which reflects the compatibility of traditional Chinese medicines with"combination".