基础研究 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-02-15; 12(2): 367-370
在线出版日期: 2004-02-15. doi: 10.11569/wcjd.v12.i2.367
当归多糖对大鼠乙酸性结肠炎的保护作用
刘少平, 董卫国, 余保平, 罗和生, 于皆平, 吴东方
刘少平, 董卫国, 余保平, 罗和生, 于皆平, 武汉大学人民医院消化内科 湖北省武汉市 430060
吴东方, 武汉大学人民医院药学部 湖北省武汉市 430060
刘少平, 男, 1973-11-08生, 湖北黄石市人, 汉族, 武汉大学人民医院消化内科硕士研究生, 主要从事炎症性肠病的防治研究.
基金项目: 湖北省科技攻关项目, No. 2001AA308B.
通讯作者: 董卫国, 430060, 湖北省武汉市解放路238号, 武汉大学人民医院消化内科. dongwg@public.wh.hb.cn
电话: 027-88074434
收稿日期: 2003-05-14
修回日期: 2003-05-20
接受日期: 2003-06-02
在线出版日期: 2004-02-15

目的: 研究当归多糖对大鼠乙酸性结肠炎损伤的保护作用及其初步机制.

方法: 建立大鼠乙酸性结肠炎模型. 实验设正常对照组, 模型对照组; 阳性药物对照组(5氨基水杨酸100 mg/kg); 当归多糖给药组(250, 500, 1 000 mg/kg), 每天灌肠给药一次, 用药7 d. 评价大鼠结肠黏膜损伤指数(CMDI)及粪便隐血实验(OBT), 检测结肠组织髓过氧化物酶(MPO), SOD活性, MDA、NO含量和TGF-β、EGF表达水平, 并作病理组织学观察.

结果: ASP灌肠明显降低模型组大鼠结肠显著升高的CMDI、OBT评分, MPO活性、MDA、NO含量(CMDI: 2.1±0.8, 1.8±0.6, 1.4±0.7 vs 2.9±0.6; OBT: 3.1±1.3, 2.7±1.1, 2.2±1.2 vs 3.8±0.8; MPO: 77.2±23.6, 72.5±16.8, 61.3±19.2 vs 98.1±26.9; MDA: 44.26±10.25, 38.72±14.84, 31.59±12.68 vs 31.59±12.68; NO: 0.252±0.041, 0.223±0.037, 0.217±0.032 vs 0.331±0.092, P<0.05或P<0.01), 明显升高模型组大鼠结肠显著降低的SOD活性与TGF-β表达水平 (SOD: 30.16±2.88, 31.27±2.73, 33.52±2.81 vs 28.33±1.17; TGF-β: 0.136±0.031, 0.153±0.036, 0.169±0.029 vs 0.105±0.021, P<0.05或P<0.01), 亦显著上调EGF: 表达(EGF: 0.178±0.021, 0.195±0.031, 0.191±0.022 vs 0.151±0.026, P<0.05或P<0.01). ASP用药呈一定量效关系. SF灌肠亦改善模型组大鼠结肠病理组织学表现.

结论: ASP灌肠明显缓解乙酸性结肠炎大鼠结肠损伤, 机制与促生长因子、抗氧化和一定的抗炎作用相关.

关键词: N/A
引文著录: 刘少平, 董卫国, 余保平, 罗和生, 于皆平, 吴东方. 当归多糖对大鼠乙酸性结肠炎的保护作用. 世界华人消化杂志 2004; 12(2): 367-370
Protective effects of Angelica sinensis polysaccharides on acetic acid-induced rat colitis
Shao-Ping Liu, Wei-Guo Dong, Bao-Ping Yu, He-Sheng Luo, Jie-Ping Yu, Dong-Fang Wu
Shao-Ping Liu, Wei-Guo Dong, Bao-Ping Yu, He-Sheng Luo, Jie-Ping Yu, Department of Gastroenterology, Wuhan University Renmin Hospital, Wuhan 430060, Hubei Province, China
Dong-Fang Wu, Department of Pharmacy, Wuhan University Renmin Hospital, 238 Jiefang Road, Wuhan 430060, Hubei Province, China
Supported by: Science and Technology Committee of Hubei Province, No. 2001AA308B.
Correspondence to: Wei-Guo Dong, Wuhan University Renmin Hospital, 238 Jiefang Road, Wuhan 430060, Hubei Province, China. dongwg@public.wh.hb.cn
Received: May 14, 2003
Revised: May 20, 2003
Accepted: June 2, 2003
Published online: February 15, 2004

AIM: To study the protective effects of Angelica sinensis polysaccharides (ASP) on colon injury in acetic acid-induced rat colitis and its mechanism.

METHODS: The colitis model of rats was produced by intracolon enema with acetic acid. The experimental animals were divided into 6 groups: normal, model, 5-ASA (100 mg/kg), and ASP (250, 500, 1 000 mg/kg), and treated intracolonically with saline, 5-ASA, and ASP respectively once a day for 7 days. The colon mucosa damage index(CMDI) and occult blood test (OBT) were evaluated. The activities of MPO and SOD, the contents of MDA and NO, the expression levels of TGF-β and EGF in the colon tissue were detected. H-E stained section was also observed.

RESULTS: Intracolon enema with ASP decreased the significanctly elevated extents of CMDI, OBT and levels of MPO, MDA, and NO in the model group (CMDI: 2.1±0.8, 1.8±0.6, 1.4±0.7 vs 2.9±0.6; OBT: 3.1±1.3, 2.7±1.1, 2.2±1.2 vs 3.8±0.8; MPO: 77.2±23.6 , 72.5±16.8, 61.3±19.2 vs 98.1±26.9; MDA: 44.26±10.25, 38.72±14.84, 31.59±12.68 vs 31.59±12.68; NO: 0.252±0.041, 0.223±0.037, 0.217±0.032 vs 0.331±0.092, P < 0.05 or P < 0.01), increased the significantly reduced level of expression of TGF-β and the activity of SOD in the model group (SOD: 30.16±2.88, 31.27±2.73, 33.52±2.81 vs 28.33±1.17; TGF-β: 0.136±0.031, 0.153±0.036, 0.169±0.029 vs 0.105±0.021, P < 0.05 or P < 0.01), and also increased significantly the expression of EGF (EGF: 0.178±0.021, 0.195±0.031, 0.191±0.022 vs 0.151±0.026, P < 0.05 or P < 0.01). The histological changes were also alleviated with ASP treatment.

CONCLUSION: Enteroclysis with ASP markedly relieve the colon injury in acetic acid-induced rats colitis, which is related with promoting growth factors, decreasing oxygen free radicals and some anti-inflammation effects.

Key Words: N/A
0 引言

当归多糖(angelica sinensis polysaccharides, ASP)具有调节免疫、抗肿瘤、抗感染、促进造血等广泛药理作用[1]. 近年研究发现, ASP体外通过促生长因子合成, 上调c-myc蛋白表达、升高鸟氨酸脱羧酶活性等作用, 促进大鼠胃上皮细胞增生与迁移, 加速其损伤模型的修复; ASP灌胃明显缓解大鼠实验性胃溃疡损伤, 促进溃疡愈合; ASP可拮抗大鼠胃肠道大量中性粒细胞浸润引起的炎症损伤; ASP亦能抑制 MDA, NO生成, 减少谷光甘肽的清除, 表现出抗氧化特性, 拮抗CCI4诱导的大鼠氧化性肝损伤[2-7]. 为探讨ASP对IBD肠组织损伤是否亦具有缓解保护作用机制, 我们建立大鼠乙酸性结肠炎模型, 首次观察ASP灌肠给药对其结肠损伤程度及氧自由基、生长因子水平的影响.

1 材料和方法
1.1 材料

健康Sprague-Dawley大鼠, 雌雄兼用, 体质量250±30 g, 由湖北预防医学科学院实验动物中心提供; ASP由武汉大学人民医院药学部参照文献[5]方法提取、纯化, 所用当归产于甘肃岷县, 使用前溶于生理盐水, 高压消毒; 5-ASA原料药, 国怡药业有限公司提供, 批号0209477; 分析纯乙酸, 上海生化试剂厂产品; MPO, SOD, MDA, NO检测试剂盒购于南京建成生物工程研究所; OB试纸, Tonyar Biotech公司产品; TGF-β, EGF多抗分别购自美国Santa Cruz和Zymed公司; SP试剂盒由北京中山生物技术有限公司提供; 其余试剂均为国产分析纯.

1.2 方法

参照文献[Gut 1996; 39: 407-415]制备动物模型, 乙醚麻醉固定大鼠, 橡胶输液管由肛门轻缓插入结肠内约8 cm, 推入80 mL/L乙酸2 mL, 作用20 s后, 立即注入5 mL生理盐水, 冲洗2次. 实验设正常对照组、模型对照组、阳性药物对照组、ASP给药组（250, 500, 1 000 mg/kg）, 每组8只, 均灌肠给药, 1次/d, 给药时间从制备模型后24 h开始, 用药7 d. 正常对照组及模型对照组均给予等量生理盐水灌肠. 实验完成后用OB试纸检测粪便隐血实验(OBT), 显色评分标准按OB试纸说明进行. 处死动物, 参照文献[8]评价CMDI, 评分标准为: 0 = 无损伤; 1 = 轻度充血、水肿, 表面光滑, 无糜烂或溃疡; 2 = 充血水肿, 黏膜粗糙呈颗粒状, 有糜烂或肠粘连; 3 = 高度充血水肿, 黏膜表面有坏死及溃疡形成, 溃疡最大纵径小于1 cm, 肠壁增厚或表面有坏死及炎症; 4 = 在3分基础上溃疡最大纵径大于1 cm, 或全肠壁坏死. 在距肛门7-9 cm处取适量结肠新鲜标本, 快速置于液氮中速冻, 待测SOD, MDA, NO, MPO, 检测操作按相应试剂盒说明进行; 用40 mL/L多聚甲醛溶液固定结肠组织标本, 常规石蜡包埋、切片, HE染色并光镜观察. TGF-β、EGF免疫组化检测步骤严格按试剂盒说明进行, 均以非免疫性山羊血清封闭非特异性抗原, 一抗分别为1: 100, 1: 150的TGF-β与EGF兔IgG多抗, 二抗均为生物素化羊抗兔IgG工作液, 以PBS代替一抗做阴性对照, 以细胞核蓝色为阴性, 胞质内或核膜上呈棕黄色为阳性. 每张切片选取10个400倍视野, 采用全自动图像分析仪与HPIAS-2000图像分析软件, 分别扫描记录阳性细胞的吸光度A值, 取其平均值, 作为此切片TGF-β, EGF的相对含量.

统计学处理 数据以mean±SD表示, 采用SPSS医学统计软件处理, 两组间均数差异的比较采用t检验.

2 结果
2.1 结肠损伤程度和炎症反应

模型组大鼠CMDI, OBT与MPO活性较正常对照显著升高(P<0.01), 不同剂量ASP灌肠显著降低CMDI, 中、大剂量ASP亦使OBT评分, MPO活性明显降低(P<0.05或 P<001). 大剂量ASP改善CMDI, OBT作用与100 mg/kg 5-ASA相当(表1). 结肠标本HE染色亦显示, 模型组黏膜高度充血水肿, 上皮溃疡形成, 局部见坏死, 大量炎性细胞浸润, 腺体中杯状细胞显著减少而大剂量ASP用药组黏膜充血水肿, 上皮溃疡坏死明显减轻, 炎性细胞浸润亦有所缓解.

表1 ASP对结肠炎大鼠CMDI, OBT的影响 (mean±SD, n = 8).
分组剂量(mg/kg)CMDI(分)OBT(分)MPO(nkat/g)
正常对照00.0±0.00.0±0.0413±192
模型对照02.9±0.6b3.8±0.8b1 635±448b
5-ASA1001.6±0.9d2.6±1.1c493±180d
ASP2502.1±0.8c3.1±1.31 287±393
ASP5001.8±0.6d2.7±1.1c1 209±280c
ASP1 0001.4±0.7d2.2±1.2d1 022±320d
bP<0.01 vs 正常对照组;
cP<0.05,
dP<0.01 vs 模型对照组.
2.2 结肠氧由基和生长因子

模型组大鼠MDA, NO含量显著升高, TGF-β表达、SOD活性显著降低(P<0.01), EGF表达无明显变化, ASP灌肠显著降低MDA, NO含量, 升高TGF-β, EGF表达水平, SOD活性(P<0.05或P<0.01), 而100 mg/kg 5-ASA对模型组TGF-β, EGF表达水平无明显影响. ASP用药呈一定量效关系(表2, 图1-4).

图1
图1 TGF-β在模型组大鼠结肠组中的表达SP×400.
表2 ASP对结肠炎大鼠结肠氧自由基与生长因子的影响(mean±SD, n = 8).
分别剂量(mg/kg)SOD(ukat/g)MDA(umol/g)NO(mmol/g)TGF-β(A×10-3)EGF(A×10-3)
正常对照0602±329.2±3.8176±45146±25162±23
模型对照0472±20b57.5±12.4b331±92b105±21b151±26
5-ASA100548±49d34.7±9.4d194±35d97±27159±28
ASP250503±4844.3±10.2c252±41c136±31c178±21c
ASP500521±46c38.7±14.8c223±37d153±36d195±31d
ASP1000559±47d31.6±12.7d217±32d169±29d191±22d
bP<0.01 vs 正常对照组;
cP<0.05,
dP<0.01 vs 模型对照组.
图2
图2 TGF-β在ASP大剂量用药组大鼠结肠组中的表达SP×400.
图3
图3 EGF在模型组大鼠结肠组中的表达SP×400.
图4
图4 EGF在ASP大剂量用药组大鼠结肠组中的表达SP×400.
3 讨论

乙酸诱导的大鼠实验性结肠炎是成熟经典的IBD模型, 因其发病机制、病理表现与人类IBD相似, 常用于IBD治疗药物的筛选与评价[8-9]. 本研究显示, ASP灌肠显著减轻模型组大鼠结肠黏膜损伤程度, 明显缓解便血症状, 表现出明显的结肠损伤保护作用. MPO活性是间接反映IBD炎症程度的重要指标[10], 本研究亦显示中、大剂量ASP明显降低模型组MPO活性, 显示出一定的抗炎效果, 但其作用仍明显弱于100 mg/kg 5-ASA (P<0.01). ASP抗炎机制之一可能为其含有药理特性类似于肝素的成分, 可通过与一系列炎症前化学激动反应相互作用, 产生拮抗炎症效应[4]. IBD肠黏膜中OFR水平明显升高, 抗氧化系统存在缺陷, 大量OFR对自身组织产生攻击作用, 充当或激活炎性递质, 使结肠炎症损伤进一步加重[11-14]. 过量的弱氧自由基NO不仅趋化中性粒细胞和单核细胞, 还使血管扩张, 通透性增加, 有助于炎症的始动与发展, 且可与超氧阴离子反应生成具有高度细胞毒性的过氧亚硝酸盐, 损伤结肠上皮细胞的功能和结构, 破坏肠道黏膜屏障的完整性[15-18]. 抗氧化剂与抑制NO生成, 可有效缓解IBD结肠损伤[8,18-19]. 本研究显示模型组大鼠OFR与NO大量生成, ASP灌肠明显降低MDA、NO含量, 升高SOD活性, 表现出较强抗OFR作用, 从而缓解结肠氧化损伤与炎症反应. IBD肠黏膜中iNOS活性显著升高是NO大量病理性生成的主要原因[20]. 研究证实ASP可剂量依赖性抑制iNOS活性[5-6], 这可能是其减少炎症结肠组织NO含量的机制. ASP通过抗氧化、清除氧自由基作用, 从而抑制氧自由基对IBD重要炎症调控因子NF-κB的激活, 亦可能是其抗炎效应的机制[5,21-22]. TGF-β, EGF是对多种组织损伤修复和再生具有重要意义的细胞生长因子, 具有相互协同促进作用[23-28], 且均可阻止氧化剂诱导的肠黏膜屏障损伤与功能低下, 对肠道黏膜屏障的完整性及功能维持具有重要意义[29-32]. 研究证实TGF-β不足可加重IBD肠道损伤, 外源性给予TGF-β, EGF均可明显缓解IBD肠道损伤[33-37]. 本研究显示模型组大鼠结肠TGF-β水平显著降低, EGF无明显改变; ASP灌肠显著升高二者表达水平, 表明其明显促进TGF-β、EGF的合成分泌, 其促生长因子作用与新近研究报道相似[2-3], 而5-ASA对二者表达无明显影响. 结肠组织中显著增加的TGF-β、EGF可通过促多种组织细胞增生、分化、迁移作用, 直接加速结肠上皮细胞、黏膜屏障及血管损伤的修复与再生, 并发挥细胞保护作用, 这可能是ASP缓解模型组大鼠结肠损伤及肠道出血的主要机制.

编辑: N/A

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