摘要
本试验主要探讨四君子汤煎剂防治小鼠大肠杆菌性腹泻的作用机制。试验方法:通过腹腔注射大肠杆菌建立动物腹泻模型,将试验小鼠分为预防组、治疗组、自愈组和空白对照组,攻菌小鼠每只腹腔注射2.6亿大肠杆菌O101诱发腹泻。预防组提前5d开始用四君子汤煎剂灌胃直到试验结束,每次灌服0.5mL,早晚各一次;治疗组:造模后开始用四君子汤煎剂灌胃,早晚各一次;自愈组,仅用大肠杆菌进行造模;空白对照组,四君子汤煎剂和大肠杆菌肉汤悬液均用生理盐水代替。每天观察小鼠的临床症状,各组剖检3只小鼠,观察记录剖检变化,应用常规切片技术、电镜技术对各组小鼠的病理组织学变化以及超微结构变化进行观察,并应用免疫组织化学和PCR方法对小鼠的肠粘膜修复基因的表达情况进行研究。
试验结果如下:
临床症状:自愈组、治疗组和预防组小鼠攻菌后8h左右出现腹泻症状,小鼠精神沉郁,倦怠,饮食量减少,被毛逆立,腹式呼吸,排黄色稀便,眼睑红肿,有分泌物流出,96h后,症状有所好转并逐渐恢复;与自愈组相比治疗组小鼠腹泻率较低,临床症状较轻;预防组临床症状明显减轻,小鼠腹泻率显著降低,恢复时间明显缩短;空白对照组小鼠未见异常。
剖检变化:攻菌后,小鼠肝脏、肾脏、脾脏肿大,十二指肠有散在出血点,肠壁变薄,肠管内充满黄色稀薄的内容物,胆囊充满胆汁,胃鼓气严重。120h后,剖检变化较轻;治疗组在96h后剖检变化不明显;预防组小鼠病变明显减轻,肝脏、肾脏、脾脏病变消失,十二指肠出血点减少;空白对照组剖检未见异常。
病理组织学变化:小鼠攻菌后,小肠绒毛断裂,绒毛上皮细胞肿胀,部分坏死脱落,粘膜下层充血水肿,有少量炎性细胞浸润,杯状细胞数量增多,肠腺变性明显。120h后,病变有所减轻并逐渐恢复。与自愈组相比治疗组杯状细胞数量变化不显著,120h后病理变化逐渐减轻;预防组杯状细胞数量和粘膜下层充血水肿变化不显著,96h后逐渐恢复;空白对照组肠道组织结构正常,绒毛排列整齐,肠腺轮廓清晰。
超微结构变化:扫描电镜显示攻菌后,绒毛肿胀,杯状细胞形态异常,数量增多,顶端微绒毛肿胀脱落,排列杂乱稀疏。96h后,绒毛结构逐渐恢复;治疗组较自愈组病变较轻;预防组超微结构病变较轻,微绒毛排列紊乱,无脱落现象,72h后逐渐恢复;空白对照组小肠粘膜绒毛高度一致,排列整齐,微绒毛分布均匀致密整齐,杯状细胞数量、形态及分布均正常。透射电镜观察显示,攻菌后,粗面内质网扩张形成许多小空泡,存在胞吐现象和吞饮小泡。上皮微绒毛排列紊乱。线粒体空泡化,嵴和膜消失。杯状细胞内含有大量的粘原颗粒;96h后,超微结构病变好转并逐渐恢复;治疗组与自愈组基本相同,预防组超微结构病变较轻;空白对照组小肠吸收上皮细胞微绒毛排列整齐,线粒体结构正常,细胞器完整。
免疫组织化学检测结果显示:腹泻初期,预防组、治疗组和自愈组较空白对照组PCNA、TGFβ1表达量增加,其中预防组与空白对照组相比差异显著(P<0.05)。
粘膜细胞修复相关基因表达检测结果显示,腹泻初期,预防组、治疗组和自愈组TGFβ1、EGFR表达量较空白对照组差异极显著(P<0.01),其中预防组,治疗组与自愈组相比差异显著(P<0.05)。
结论:预防组小鼠在临床症状、剖检变化、病理组织学变化以及电镜观察等方面结果均显示四君子汤预防效果显著。预防组粘膜修复基因PCNA、EGFR、TGFβ1的表达量明显增加。结果表明四君子汤对小鼠大肠杆菌性腹泻的防治效果可能与调控粘膜修复基因PCNA、EGFR、TGFβ1的表达密切相关。
The aim of this paper was to clarify the prevention and cure mechanism of sijunzitang on E.coli-induced diarrhea. Methods:Mice were devided into Prevention group, treatment group, self-healing group and Control group. Infected mice were Intraperitoneal injection 2.6 billion E.coli O101 to build Diarrhea model. Prevention group mice were lavaged with sijunzitang 5 days in advance,after build model keep lavaging with 0.5ml sijunzitang in the morning and evening. Treatment group mice were lavaged with 0.5ml sijunzitang in the morning and evening. Self-healing group mice were to build model with e.coli only. Control group mice were lavaged with Saline. Everyday the clinical symptoms were observed and selected 3 mice for autopsy in each group. The changes of Histopathological and Ultrastructure were observed by the Conventional slicing and Electron microscopy methods, the expression of mucosal repair gene were analysised by the Immunohistochemistry and PCR methods.
The results as follow:
Clinical symptoms: self-healing group, the treatment group and the prevention group appeard the Diarrhea after Infection 8 hours, mice gloomy spirit, fatigue, reduced food intake, hair reverse lap, abdominal breathing, yellow excretion, the eyelids swelling and secretion increase, 96h later, the symptoms gradually improved and recovery; treatment groups mice had the same symptoms, but the incidence was slightly decreased; prevention group mice symptoms decreased and the number of diarrhea mice was reduced, the restoration time was significantly reduced; control group mice had any clinical symptoms.
Autopsy changes: after Infect, the mice liver, kidney, spleen swelled, duodenal have scattered bleeding points, intestinal wall became thiner, intestine tract was full of yellow thin contents, gall bladder was full of bile, Stomach flatulence severely; The treatment group had the similar changes, but the repair time was reduced; The lesion of prevention group mice were decreased, and bleeding points in Duodenal were reduced, and repair time was reduced; control group mice had any visible pathological change.
Histopathological changes: After Infection, the small intestine villi appeared partial ruptured, villous epithelial cell were swelled and necrosis partly, submucosa were congestion, edema and some inflammatory cell infiltration. The numbers of goblet cells were increased. The intestinal gland became degeneration obviously. The number of goblet cells did not increased significantly in treatment group, and other lesions consistent with the self-healing group. in prevention group, the number of goblet cells and submucosal edema had no significant changes, after 96h the mice recovery gradually. Control group mice had the normal intestinal organizational structure.
Ultrastructural Changes: under SEM, the villous were edema severely, the goblet cell had abnormal form and the number was increased. The top microvilli were swollen, necrotic, sparse and clutter. In treatment group, the ultrastructural changes were relatively better than that of self-healing group. In prevention group, ultrastructural have lighter lesions and the microvilli were in disorder. Control group, small intestinal villous and microvilli were arranged normally.
By TEM observe, rough endoplasmic reticulums were expansion, many small vacuoles and pinocytotic vesicles were existed. The epithelial microvilli were disordered. Mitochondria appeared vacuolization, mitochondrial cristae and membrane disappeared. Goblet cells contain a large number of secretory granules. In the treatment group and prevention group, the changes of ultrastructural were relatively better than that of self-healing group. In control group, the small intestine epithelium microvilli arranged neatly, the mitochondrial structure were normal, and the epithelium cell were integrity.
Immunohistochemistry results showed that, in the early diarrheal period, the expression of PCNA, TGFβ1 were most significantly increased in prevention group than that of the normal group, and it were significantly increased in prevention group than that of the normal group (P<0.05).
The result by RT-PCR methods showed that the expression of Repair gene TGFβ1、EGFRwere most significantly increased in prevention group than that of the normal group, and it were significantly increased in treatment group and the self-healing group than that of the normal group (P<0.01) , it were significantly increased in prevention group and treatment group than that of the self-healing group (P<0.05).
Conclusion: The Sijunzitang had the significantly effects in improved clinical symptoms, pathologic changes, histopathological changes and the ultrastructural pathologic changes of diarrheal mice. The sijunzitang significantly increased the expression of TGFβ1、EGFR、PCNA. The results suggest that the preventive effect of Sijunzitang on diarrhea maybe have the relationship with the upregular the expression of mucosal repair gene PCNA、EGFR、TGFβ1 and promote the damaged mucosa repaired.
引文
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