修回日期: 2003-07-01
接受日期: 2003-08-16
在线出版日期: 2004-01-15
目的: 评价根除H. pylori加疗胃煎剂对大鼠H. pylori相关性慢性胃炎病变的影响. 同时观察治疗前后环氧合酶-2(Cyclooxygenase-2, COX-2)表达的变化.
方法: 体质量80-100 g二级♂Wister大鼠80只, 采用H. pylori及MNNG综合方法制备大鼠H. pylori相关性慢性胃炎模型76只, 血清H. pylori抗体阳性54只随机分为4组: H. pylori根除与疗胃煎剂组(14只)、H. pylori根除与"麦滋林"组(阳性对照组)(14只)、自然恢复组(阴性对照组)(13只)、单纯H. pylori根除组(13只). 治疗结束后, 行胃窦黏膜涂片Gram染色和快速尿素酶试验, 检测H. pylori的定植情况, 并对胃窦黏膜组织学各项指标进行评定; 采用免疫组化染色方法, 检测胃窦黏膜的COX-2的表达.
结果: 自然恢复组大鼠均有H. pylori定植, 其他各组大鼠胃黏膜均未发现H. pylori定植; 自然恢复组的大鼠表现中度萎缩病变(2.0±0.20分), 伴有中度急性、慢性炎症(2.0±0.20分、1.90±0.39分); 而H. pylori根除联用疗胃煎剂组, 与自然恢复组比较, 萎缩病变(1.25±0.44分)轻度改善, 伴急性炎症(0.3±0.47分)消退, 慢性炎症(1.05±0.22 分)轻度改善, 差异显著(P<0.05); H. pylori根除与麦滋林组胃窦黏膜层萎缩病理积分为2.0±0.43分, 较自然恢复组无明显差异, 但急性炎症消退, 慢性炎症无改善. 单纯H. pylori根除组, 与自然恢复组比较, 萎缩病变和慢性炎症未见改善, 但活动性炎症消退；单纯根除H. pylori组、根除H. pylori与联用疗胃煎剂组、H. pylori根除与麦滋林组、自然恢复组COX-2表达率分别为14.0%±3.7%、10.0%±3.8%、13.0%±4.0%、19.0%±10.2%, 治疗组与自然恢复组比较, 胃窦黏膜COX-2表达率明显下降, 差异显著(P<0.05).
结论: 对H. pylori相关性慢性胃炎, 根除H. pylori可明显降低COX-2表达, 明显改善急性炎症, 但对慢性病变尤其萎缩病变无明显影响. 根除H. pylori的基础上应用疗胃煎剂治疗12 wk有较好的疗效.
引文著录: 姚希贤, 张琳, 王娜, 姚冬梅, 白文元, 冯丽英. H. pylori根除加中药疗胃煎剂对鼠慢性胃炎病变影响. 世界华人消化杂志 2004; 12(1): 97-100
Revised: July 1, 2003
Accepted: August 16, 2003
Published online: January 15, 2004
AIM: To evaluate the effects of H. pylori eradication with liaowei decoction on pathologic changes and COX-2 expression of H. pylori-associated chronic gastritis in rats.
METHODS: A total of 80 grade-II male Wister rats weighing 80-100 g were given H. pylori and MNNG to prepare H. pylori-associated chronic gastritis model with 4 mice dead. Among them, 54 rats with H. pylori serum antibody positive were randomly divided into 4 groups: H. pylori eradication with liaowei decoction (14 rats); H. pylori eradication with Maizilin (positive control, 14 rats); spontaneous recovery (negative control, 13 rats); and simple H. pylori eradication therapy (13 rats). After treatment, both Gram staining of gastric antrum mucosal smear and rapid urease testing were made to detect the H. pylori colonization of the gastric mucosa and to assess the histological indexes of gastric antrum mucosa. Immunohistochemical staining was also performed to detect COX-2 expression.
RESULTS: The colonization of gastric mucosa by H. pylori was found in rats from spontaneous recovery group, as an indication of long-term continuous H. pylori infection, while no colonization was found in rats from other groups. Pathological changes of gastric antrum mucosa included moderate gastric mucosal atrophy in rats from spontaneous recovery group (pathologic integration 2.0±0.20 points), associatded with moderate acute or chronic gastric mucosal inflammation (pathologic integrations were 2.0±0.20 points and 1.90±0.39 points, respectively). In comparison with spontaneous recovery group, treatment effectiveness in the group of H. pylori eradication with liaowei decoction was remarkably different (P < 0.05). The gastric mucosal atrophy in the combination group (pathologic integration 1.25±0.44 points) was less severe, while acute gastric mucosal inflammation (pathologic integration 0.3±0.47 points) was extincted, and chronic inflammation (pathologic integration 1.05±0.22 points) was less severe; For the group of H. pylori eradication and "Maizilin", pathologic integration of gastric mucosal atrophy was 2.0±0.43 points. In comparison with spontaneous recovery group, there was no remarkable difference, however, acute inflammation was extincted and chronic inflammation was the same as before. Gastric mucosal atrophy and chronic inflammation were more severe in simple H. pylori eradication group than those in spontaneous recovery group, while active inflammatory changes extincted. COX-2 expression of gastric antrum mucosa in the group of simple H. pylori eradication, group of H. pylori eradication with liaowei decoction, group of H. pylori eradication and Maizilin as well as spontaneous recovery group were 14%±3.7%, 10%±3.8%, 13%±4.0% and 19%±10.2%, respectively. In comparison with spontaneous recovery group, COX-2 expression of the treatment groups were greatly decreased as an indication of remarkable difference (P < 0.05).
CONCLUSION: COX-2 expression and acute inflammation can be greatly decreased by applying H. pylori eradication therapy in the treatment of H. pylori-associated chronic gastritis, but the therapy cannot do much to chronic pathologic changes, especially to atrophic changes. Application of the combined therapy of H. pylori eradication with "liaowei decoction" for 12 weeks will have better treatment effectiveness on chronic pathologic changes, especially atrophic changes.
- Citation: Yao XX, Zhang L, Wang N, Yao DM, Bai WY, Feng LY. Effects of H. pylori eradication with liaowei decoction on pathology of H. pylori-associated chronic gastritis in rats. Shijie Huaren Xiaohua Zazhi 2004; 12(1): 97-100
- URL: https://www.wjgnet.com/1009-3079/full/v12/i1/97.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v12.i1.97
慢性萎缩性胃炎(CAG)为临床多发病, 其重症不典型增生被公认为癌前病变, 迄今对之尚乏有效治疗方法. H. pylori为慢性胃炎的重要病因, 但根除H. pylori对慢性胃炎病变有无治疗作用? 对慢性胃炎的肠化、不典型增生等萎缩性病变有无影响尚乏定论. 我们采用H. pylori及N甲基N硝基N亚硝基胍(MNNG, 胃癌诱导剂[1-2])综合方法制备大鼠H. pylori相关性慢性胃炎模型, 对根除H. pylori 联用疗胃煎剂对慢性病变的治疗作用及根除H. pylori后胃黏膜COX-2表达的情况进行观察.
体质量80-100 g二级♂Wister大鼠80只, 采用H. pylori及MNNG综合方法制备大鼠H. pylori相关性慢性胃炎模型, 制模实验结束后, 除去死亡的4只大鼠, 检测存活大鼠H. pylori血清抗体, 阳性54只, 随机分为4组: H. pylori根除与疗胃煎剂组(14只), 前2 wk采用传统三联疗法根除H. pylori, 继之灌胃疗胃煎剂12 wk. H. pylori根除与麦滋林组(阳性对照组)(14只), 前2 wk采用传统三联疗法根除H. pylori, 继之灌胃麦滋林12 wk, 自然恢复组(阴性对照组)(13只), 予蒸馏水灌胃, 正常饮食14 wk, 单纯H. pylori根除组(13只), 采用传统三联疗法根除H. pylori, 持续2 wk, 之后予正常饮食12 wk.
根除H. pylori药物: 德诺40 mg/kg, 阿莫西林166.7 mg/kg, 甲硝唑66.7 mg/kg, 每天灌胃1次, 疗程2 wk; 疗胃煎剂(以党参为君, 补脾养胃, 生津养血, 健运中气. 白术、云苓益气健脾, 丹参活血化瘀, 改善胃部血液循环与供养情况, 促进病变恢复, 木香、川朴、砂仁行气、除满): 50 mg/kg, 疗程12 wk; 麦滋林: 50 mg/kg, 每天灌胃1次, 疗程12 wk. 实验结束时, 采取颈椎脱位的方法处死动物后, 取出鼠胃, 沿大弯剪开胃腔, 用生理盐水洗净, 取一半胃黏膜组织做快速尿素酶实验, 黏膜涂片Gram染色; 取另一半胃黏膜组织, 用40 g/L多聚甲醛固定24 h后, 经石蜡切片、HE染色后, 分别进行病理组织学检测和免疫组化染色检测COX-2的表达. 观察动物的死亡情况、体质量、精神状态、活动情况、毛发光泽度及食欲. 处死动物后, 取其胃窦黏膜进行快速尿素酶试验, 并行胃窦黏膜涂片Gram染色检查, 两项检查均阳性判定H. pylori阳性.
1.2.1 胃黏膜病理组织学检查: 参照2000年全国慢性胃炎研讨会制定的标准对胃窦黏膜组织学各项指标进行评定. 组织切片由专人采取盲法阅片. 对慢性炎症、活动性、萎缩和肠化等形态学变量分成无、轻度、中度和重度4级(或0, +, ++, +++), 其对应分值为0, 1, 2, 3分. 分级的具体方法: (1)慢性炎症 根据慢性炎症细胞密集程度和浸润深度分级, 以前者为主. 正常: 单个核细胞每高倍视野不超过5个, 如数量略超正常而内镜无明显异常时, 病理可诊断为无明显异常. 轻度: 慢性炎症细胞较少并局限于黏膜浅层, 不超过黏膜层的1/3. 中度: 慢性炎症细胞较密集, 超过黏膜层的1/3, 达到2/3. 重度: 慢性炎症细胞密集, 占据黏膜全层. 计算密度程度要避开淋巴滤泡及其周围的淋巴细胞区. (2)活动性慢性炎症背景上有中性粒细胞浸润. 轻度: 黏膜固有层少数中性粒细胞浸润. 中度: 中性粒细胞较多存在于黏膜层, 并在表面上皮细胞间、小凹上皮细胞间或腺管上皮间可见. 重度: 中性粒细胞较密集, 或除中度所见外还见小凹脓肿. (3)萎缩指胃的固有腺体减少, 幽门腺萎缩是幽门腺减少或由肠化腺体替代, 胃底(体)腺萎缩是指胃底(体)腺假幽门腺化生、肠上皮化生或腺体本身减少. 萎缩程度以胃固有腺减少各1/3来计算. 轻度: 固有腺体数减少不超过原有腺体的1/3, 大部分腺体仍保留. 中度: 固有腺体数减少超过1/3, 但未超过2/3. 残存腺体不规则分布. 重度: 固有腺体数减少超过2/3, 仅残留少数腺体, 甚至完全消失. 标本过浅未达到黏膜肌层的不可能诊断萎缩, 要剔除. 胃窦部少数淋巴滤泡不算萎缩, 但胃体黏膜层出现淋巴滤泡要考虑为萎缩. (4)肠腺化生 肠化部分占腺体和表面上皮总面积1/3以下的为轻度、1/3-2/3的为中度, 2/3以上为重度.
1.2.2 免疫组化染色检测COX-2的表达: 仅胃窦部的标本进行免疫组化检测, 采用链霉卵白素蛋白-过氧化物酶连结法(streptavidin-peroxidase conjugated method). COX-2多克隆抗体购自美国Stanta Cruz公司, SP 免疫染色试剂盒购自北京中山生物技术公司. 石蜡切片(3-5 m)常规脱蜡、梯度水化后, 切片置30 mL/L过氧化氢孵育5-10 min清除内源性过氧化物酶活性. 蒸馏水冲洗, PBS浸泡5 min. 置电炉煮沸进行抗原修复15-20 min. 冷却室温后滴加封闭用血清15 min, 倾去血清, 滴加1: 100稀释COX-2多克隆抗体, 4 ℃过夜；PBS冲洗3次, 每次3 min, 滴加生物素化二抗, 37 ℃孵育15 min, PBS冲洗3次, 每次3 min, 滴加辣根酶标记链霉卵白素, 37 ℃孵育15 min, PBS冲洗3次, 每次3 min, DAB显色5 min, 自来水充分冲洗, 苏木精复染, 中性树脂封片. 以PBS替代一抗作为阴性对照, 用已知强阳性的结肠癌切片作为阳性对照, 对每个标本中胞质阳性的黏膜表面上皮和固有腺上皮进行记数, 以阳性细胞率即阳性细胞数占总细胞数的比例进行比较, 记数10个高倍视野细胞, 取其平均值.
统计学处理 采用成组设计, 计量资料用mean±SD表示, 用t检验, 多个样本均数间两两均数比较, 采用方差分析和q检验, 当P < 0.05时有显著差异.
自然恢复组一般情况差, 被毛松散蓬乱, 毛色偏黄无光泽, 两眼无神, 活动迟钝, 食量仍小, 形体瘦小, 多数大鼠有便溏和拉尾现象. H. pylori根除与疗胃煎剂组较自然恢复组改善, 食量较前增加, 体重渐回升, 粪便成型. 单纯H. pylori根除组及H. pylori根除与麦滋林组仅略改善. 自然恢复组大鼠均有H. pylori定植, 提示H. pylori长期持续感染. 其他各组大鼠胃黏膜均未发现H. pylori定植, 提示H. pylori已被根除.
自然恢复组动物胃窦黏膜呈慢性胃炎改变. 胃窦黏膜色偏白, 未见血管显露, 少数大鼠有糜烂及溃疡. 其他各组未见糜烂及溃疡, 其他表现与自然恢复组相似. 自然恢复组: 胃窦黏膜固有层有较多淋巴细胞和中性粒细胞浸润, 腺体中度减少, 无肠化及不典型增生. 活动性炎症、慢性炎症、萎缩的病理积分分别为2.0±0.20分, 1.90±0.39分, 2.0±0.20分(表1). H. pylori根除与疗胃煎剂组胃窦黏膜层几乎无中性粒细胞浸润, 故急性炎症消退, 较自然恢复组明显改善；慢性炎症和萎缩病理积分分别为1.05±0.22分, 1.25±0.44分, 较自然恢复组轻度改善(P<0.05). H. pylori根除与麦滋林组胃窦黏膜层几乎无中性粒细胞浸润, 故急性炎症消退, 较自然恢复组明显改善; 慢性炎症病理积分为1.70± 0.40分, 较自然恢复组略有改善 , 但P>0.05. 萎缩性病变, 在少数动物表现轻度改善, 但病理积分为2.0±0.43分, 较自然恢复组无明显差异. 单纯H. pylori根除组胃窦黏膜基本无中性粒细胞浸润, 固有层较多量淋巴细胞浸润, 腺体中度减少. 提示除活动性炎症明显改善轻度改善外, 与自然恢复组比较萎缩和慢性炎症未见改善.
治疗结束时, 各组的COX-2阳性细胞率如下: 单纯H. pylori根除组为14.0%±3.7%; H. pylori根除和应用疗胃煎剂组为10.0%±3.8%; H. pylori根除与麦滋林组13.0%±4.0%; 自然恢复组为19.0%±10.2%. 单纯H. pylori根除组、H. pylori根除与疗胃煎剂组与自然恢复组比较, 胃黏膜表面上皮和固有腺上皮细胞胞质COX-2表达明显下降, 差异显著 (P<0.05).
根除H. pylori 可消除或明显改善H. pylori相关性慢性胃炎急性活动性炎症, 此已为学者所共识. 但根除H. pylori 对慢性病变有无影响, 是否需要对之进行治疗, 以及如何治疗, 目前尚乏明确结论, 相关临床结果并不一致[3-10]. 多数学者认为H. pylori 相关性慢性胃炎的发生发展与H. pylori密切相关, 但单纯根除H. pylori, 对治疗萎缩性病变无效[11-18]. 动物实验方面, 胡品津 et al[19]报道单纯使用SS1感染小鼠12 mo, 造成轻度CAG之后根除H. pylori, 可明显消除黏膜的炎症反应, 降低细胞的高增生状态, 但萎缩病变并未消失. 本实验结果显示: 单纯根除H. pylori后, 大鼠急性活动性炎症消失或明显改善, 慢性炎症无明显改善; H. pylori根除和应用疗胃煎剂急性炎症消退, 慢性炎症轻度改善; H. pylori根除与麦滋林组急性炎症消退, 慢性炎症略有改善, 后者与自然恢复组比较, 无显著性差异(P>0.05). 同时本实验采用免疫组化的方法检测COX-2的表达变化, 发现COX-2表达率的下降与慢性活动性炎症改善一致. 其中自然恢复组COX-2表达率为19.0%±10.2%, 单纯根除H. pylori组为14.0%±3.7%, H. pylori根除与麦滋林组13.0%±4.0%, 根除H. pylori与疗胃煎剂组为10.0%±3.8%, 治疗组胃窦黏膜COX-2表达率较自然恢复组有明显下降. 实验结果与相关的临床研究结果相似, 提示COX-2的表达可能参与H. pylori相关性慢性胃炎病变产生和演变过程[20-22]. 故根除H. pylori, 可改善慢性活动性炎症, 降低COX-2表达率. 但单纯根除H. pylori, 萎缩病变(2.1±0.32)分较自然恢复组(2.0±0.2)分并无明显变化.
Correa et al[23]追踪调查了780例具有正常胃黏膜或慢性浅表性胃炎的患者, 平均5.1年重复1次胃镜检查, 有284例患者发展成萎缩性胃炎, 每年7.5%的转变率. 根除H. pylori后进行长达6年随访研究证实, 患者的萎缩性病变得到了改善. 如能延长实验时间, 进行更深入的研究, 对回答单纯根除H. pylori能否改善或逆转萎缩性病变这一问题将更有意义.
近年来中医中药在治疗慢性胃炎方面显示了可喜的势头[24-30]. 姚希贤et al 报道采用健脾益气活血方剂治疗H. pylori相关性慢性胃炎, 除能明显改善症状, 对慢性胃炎的萎缩性病变有一定治疗作用. 我们在根除H. pylori后, 继续采用疗胃煎剂治疗慢性病变, 调节胃功能, 经治12 wk, 萎缩病变轻度改善, 病理积分较自然恢复组的(2.2±0.2)分下降, 为(1.25±0.44)分, 差异有显著性, 而H. pylori根除与麦滋林组萎缩性病变较自然恢复组并无明显改善. 提示疗胃煎剂治疗H. pylori相关性慢性胃炎具有良好的前景.
编辑: N/A
| 3. | Sepulveda AR. Improvement of gastric atrophy after cure of Helicobacter pylori. What we know, what we don't know, and why care? J Clin Gastroenterol. 2003;36:382-384. [PubMed] [DOI] |
| 4. | Schuster MJ. Helicobacter pylori. Update 2002. Schweiz Rundsch Med Prax. 2002;91:2093-2099. [PubMed] [DOI] |
| 5. | Vucelic B, Mesihovic R, Bratovic I, Vanis N, Gribajcevic M, Selak I. Helicobacter pylori infection and chronic gastritis as extra-esophageal factors in gastroesophageal reflux disease. Med Arh. 2003;57:39-44. [PubMed] |
| 6. | Han SU, Kim YB, Joo HJ, Hahm KB, Lee WH, Cho YK, Kim DY, Kim MW. Helicobacter pylori infection promotes gastric carcinogenesis in a mice model. J Gastroenterol Hepatol. 2002;17:253-261. [PubMed] [DOI] |
| 7. | Hahm KB, Song YJ, Oh TY, Lee JS, Surh YJ, Kim YB, Yoo BM, Kim JH, Han SU, Nahm KT. Chemoprevention of Helicobacter pylori-associated gastric carcinogenesis in a mouse model: is it possible? J Biochem Mol Biol. 2003;36:82-94. [DOI] |
| 8. | Tian SF, Xiong YY, Yu SP, Lan J. Relationship between Helicobacter pylori infection and expressions of tumor suppressor genes in gastric carcinoma and related lesions. Ai Zheng. 2002;21:970-973. [PubMed] |
| 9. | Zhuang X, Lin S, Zheng J, Wang L, Sun G, Li Y. The prognostic research of expression and relationship between Helicobacter pylori of c-met oncogene correlation with gastric mucosal proliferation. Zhonghua Neike Zazhi. 2001;40:381-384. [PubMed] |
| 10. | Hiyama T, Haruma K, Kitadai Y, Miyamoto M, Tanaka S, Yoshihara M, Sumii K, Shimamoto F, Kajiyama G. B-cell monoclonality in Helicobacter pylori-associated chronic atrophic gastritis. Virchows Arch. 2001;438:232-237. [DOI] |
| 11. | Santacroce L, Bufo P, Latorre V, Losacco T. Role of mast cells in the physiopathology of gastric lesions caused by Helicobacter pylori. Chir Ital. 2000;52:527-531. [PubMed] |
| 12. | Testino G, Cornaggia M, Testino R, Valentini M. The physiopathological aspects of chronic gastritis, Helicobacter pylori and precancerous gastric changes. Recenti Prog Med. 2000;91:186-190. [PubMed] |
| 13. | Li S, Lu AP, Zhang L, Li YD. Anti-Helicobacter pylori immunoglobulin G (IgG) and IgA antibody responses and the value of clinical presentations in diagnosis of H. pylori infection in patients with precancerous lesions. World J Gastroenterol. 2003;9:755-758. [PubMed] [DOI] |
| 14. | Yamada T, Miwa H, Fujino T, Hirai S, Yokoyama T, Sato N. Improvement of gastric atrophy after Helicobacter pylori eradication therapy. J Clin Gastroenterol. 2003;36:405-410. [PubMed] [DOI] |
| 15. | Annibale B, Di Giulio E, Caruana P, Lahner E, Capurso G, Bordi C, Delle Fave G. The long-term effects of cure of Helicobacter pylori infection on patients with atrophic body gastritis. Aliment Pharmacol Ther. 2002;16:1723-1731. [PubMed] [DOI] |
| 16. | Ito M, Haruma K, Kamada T, Mihara M, Kim S, Kitadai Y, Sumii M, Tanaka S, Yoshihara M, Chayama K. Helicobacter pylori eradication therapy improves atrophic gastritis and intestinal metaplasia: a 5-year prospective study of patients with atrophic gastritis. Aliment Pharmacol Ther. 2002;16:1449-1456. [PubMed] [DOI] |
| 17. | Kokkola A, Sipponen P, Rautelin H, Harkonen M, Kosunen TU, Haapiainen R, Puolakkainen P. The effect of Helicobacter pylori eradication on the natural course of atrophic gastritis with dysplasia. Aliment Pharmacol Ther. 2002;16:515-520. [PubMed] [DOI] |
| 20. | Kimura A, Tsuji S, Tsujii M, Sawaoka H, Iijima H, Kawai N, Yasumaru M, Kakiuchi Y, Okuda Y, Ali Z. Expression of cyclooxygenase-2 and nitrotyrosine in human gastric mucosa before and after Helicobacter pylori eradication. Prostag Leukot Essent Fatty Acids. 2000;63:315-322. [PubMed] |
| 22. | 郜 恒骏, 白 剑峰, 彭 延申, 孙 谷, 赵 翰林, 缪 锟, 吕 秀珍, 赵 志泉, 萧 树东. 幽门螺杆菌感染胃黏膜病变基因表达和细胞生物学行为. 中华消化杂志. 2001;21:18-21. |
| 23. | Correa P, Fontham ET, Bravo JC, Bravo LE, Ruiz B, Zarama G, Realpe JL, Malcom GT, Li D, Johnson WD. Chemoprevention of gastric dysplasia: randomized controlled trial of antioxidant supplements and anti-Helicobacter pylori therapy. J Natl Cancer Inst. 2000;92:1881-1888. [PubMed] [DOI] |
| 25. | 姚 金锋, 姚 希贤, 郝 桂敏, 宫 心鹏, 张 新暖. "疗胃煎剂"胃黏膜保护作用的实验研究. 中国中西医结合脾胃杂志. 2000;8:330-332. |
| 27. | 王 丙信, 李 进根, 杨 同占, 姚 希贤. 中西医结合治疗幽门螺杆菌相关胃炎81例. 中国中西医结合脾胃杂志. 1998;6:215-216. |
| 28. | 陈 飞松, 施 波, 车 建途, 屈 志炜, 任 蜀兵, 刘 晋生, 徐 春军, 张 晓辉, 赵 德骥, 危 北海. 芪龙方防治大鼠胃癌癌前疾病的作用. 中国中西医结合脾胃杂志. 1999;7:68-71. |