【作者】 李艳丽；

【导师】 张雷；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【摘要】 腹泻是许多疾病都可引起的一种症状,其病理基础为肠道在感染或非感染因素刺激下,黏膜发炎、水肿,肠分泌及运动功能亢进。短期剧烈腹泻可引起脱水和电解质紊乱,危及生命;持久腹泻则严重影响机体消化吸收功能。适当给予止泻药物,可尽快缓解症状、减少并发症的发生,研究治疗腹泻的药物对防治腹泻具有重要意义。复方双苓止泻散是一种纯中药制剂,具有抗炎、抑菌、免疫调节、治腹泻及腹痛等功效。万鑫母仔快康已在畜牧业中推广使用,临床主要应用于细菌、病毒等引起的肠炎及仔猪痢疾等病证。本研究参照周干南法建立SD大鼠腹泻模型,将60只SD大鼠随机分为:腹泻模型组(DM),低(0.31 g/kg,L)、中(0.62 g/kg,M)、高(1.24 g/kg,H)剂量复方双苓止泻散治疗组、万鑫母仔快康治疗组(阳性对照药物0.62 g/kg, PC)及正常对照组(NC),每组10只。用药48 h后,取材近段小肠组织,通过观察比较各组大鼠小肠黏膜组织形态及AQP4的表达水平,初步探讨复方双苓止泻散的作用机制、确定该有效部位的药用价值和适宜药量。旨在利用天然中草药替代抗生素治疗腹泻,减少排泄物对环境的污染,为畜牧业健康养殖技术的推广提供重要理论基础。本实验分为二部分:一复方双苓止泻散促进腹泻大鼠小肠黏膜组织形态恢复目的肠黏膜不仅是肠道发挥正常消化吸收功能的物质基础,还是保护机体免受细菌、毒素等有害物质侵袭的内在屏障。肠黏膜屏障功能首先依赖于肠黏膜的完整性,上皮细胞的形态结构和功能对肠黏膜机械屏障的完整性有重要作用。本部分研究观察比较各组大鼠小肠黏膜组织形态学变化,为揭示复方双苓止泻散的作用机制及适宜剂量提供实验依据。方法应用HE染色、扫描电镜和透射电镜技术分别观察腹泻模型及各治疗组大鼠小肠黏膜组织形态和上皮细胞的超微结构变化,并应用Motic 6.0数码医学图像分析系统测量并比较各组小肠绒毛的平均高度、宽度和面积。结果1 HE染色观察结果NC组大鼠近端小肠绒毛形态规整,绒毛中轴内少量间质,绒毛单层柱状上皮排列整齐、极少量嗜酸性变,上皮之间或绒毛顶端偶有淋巴细胞浸润。DM组中多数小肠绒毛明显水肿,部分绒毛融合;绒毛中轴间质明显水肿、充血、渗出、大量淋巴细胞等炎性细胞浸润;柱状上皮细胞之间界限不清、有较多嗜酸性变以及淋巴细胞浸润,部分绒毛上皮坏死、脱落;黏膜固有层肠腺之间的间质内也有较多炎性细胞浸润,但未见隐窝脓肿;肌层、浆膜完整。L组肠绒毛水肿、毛细血管充血和炎性细胞浸润现象仍较明显,肠腔内可见脱落的绒毛组织。PC组大鼠小肠绒毛受损结构明显恢复,部分绒毛顶端仍可见柱状细胞与间质分离,甚至缺失,少量毛细血管充血、淋巴细胞浸润。M组、H组大部分小肠绒毛内仅见极少量渗出及淋巴细胞浸润,柱状上皮细胞排列整齐。2小肠绒毛测量结果2.1绒毛宽度:DM组大鼠小肠绒毛宽度较其余5组,明显增大(P<0.01);各治疗组绒毛宽度均减小:M组、H组、PC组与NC组之间无显著性差异(P>0.05),但L组大鼠小肠绒毛宽度仍明显大于NC组(P<0.01)。2.2绒毛高度:DM组、L组、M组和PC组,四组大鼠小肠绒毛高度无显著性差异(P>0.05),均大于NC组小肠绒毛高度(P<0.01);而H组与NC组之间无显著性差异(P>0.05)。2.3绒毛面积:DM组大鼠小肠绒毛面积明显大于其余5组(P<0.01),差异显著;各治疗组小肠绒毛面积均减小,其中M组、H组与NC组无显著性差异(P>0.05),但L组、PC组大鼠小肠绒毛面积明显大于NC组(P<0.01);PC组大鼠小肠绒毛面积明显大于L组(P<0.01)。3扫描电镜观察小肠绒毛形貌特征NC组小肠绒毛大小较一致、排列整齐,表面光滑;柱状细胞分布均匀,微绒毛排列整齐。DM组大量小肠绒毛破损、结构紊乱;大量柱状细胞缺失,微绒毛结构破坏。经药物治疗后,各组小肠绒毛破损少见,L组肠绒毛明显水肿,表面附有较多粘液;PC组小肠绒毛水肿不明显,但排列欠规则;M组、H组小肠绒毛呈舌状,大小较一致、排列较规则,偶见绒毛破损;大部分柱状细胞微绒毛排列整齐。4透射电镜观察绒毛上皮细胞超微结构结果NC组小肠上皮细胞排列整齐,细胞器结构清晰,细胞微绒毛长而密集、排列整齐;偶见凋亡的上皮细胞。DM组小肠上皮细胞明显水肿,大部分线粒体明显肿胀,可见嵴断裂、减少、消失,甚至外膜溶解导致空泡化;部分内质网扩张;柱状细胞微绒毛断裂、脱落、数量减少。L组、PC组小肠黏膜上皮细胞的细胞器结构较DM组有较大改善:只有细胞游离端的胞质水肿、细胞器少;大部分线粒体双层单位膜清晰,嵴数量有所增加,但部分嵴模糊不清,部分线粒体仍明显肿胀;小肠上皮微绒毛较DM组数量增多,排列较整齐。M组、H组上皮细胞内大部分细胞器结构恢复,微绒毛结构恢复,排列整齐。二复方双苓止泻散对腹泻大鼠小肠黏膜AQP4表达的影响目的探讨AQP4在腹泻过程中的作用及复方双苓止泻散对AQP4表达的影响。方法本部分实验应用免疫组织化学、Western blot方法比较各组大鼠小肠黏膜内AQP4的表达。结果1免疫组织化学染色结果AQP4免疫阳性表达产物为棕黄色颗粒。全部实验大鼠近段小肠黏膜组织中均有AQP4表达的阳性细胞,AQP4蛋白极性表达在小肠黏膜深部肠腺细胞的细胞膜上,杯状细胞未见到阳性着色。DM组大鼠小肠黏膜中AQP4的表达明显减少;各治疗组大鼠小肠黏膜中AQP4表达增多。2 Western blot分析结果DM组小肠黏膜中AQP4表达最少(P<0.01),各药物治疗组大鼠小肠黏膜中AQP4表达增多。L组大鼠小肠黏膜中AQP4表达水平明显高于DM组(P<0.01),但低于M组和PC组(P<0.01)。M组和PC组大鼠小肠黏膜中AQP4的表达量低于NC组,具有显著性差异(P<0.01),H组大鼠小肠黏膜中的AQP4蛋白表达量增多最明显,与NC组无显著性差异(P>0.05)。结论复方双苓止泻散有效地使大鼠腹泻症状消失,能明显促进恢复小肠绒毛的损伤结构,其主要作用包括使炎性细胞浸润减少,绒毛中轴间质内充血、渗出减轻及绒毛水肿减轻;促进绒毛上皮细胞器的超微结构恢复正常,促进黏膜上皮细胞的增殖与恢复,从而加快腹泻大鼠小肠黏膜结构与功能的恢复。在促进小肠黏膜修复过程中,中、高剂量复方双苓止泻散及万鑫母仔快康的治疗效果优于低剂量复方双苓止泻散;中剂量与高剂量复方双苓止泻散的治疗效果无明显差异;但中、高剂量复方双苓止泻散的治疗效果优于万鑫母仔快康。AQP4蛋白极性表达在小肠黏膜深部肠腺细胞的细胞膜上,杯状细胞未见到阳性着色。AQP4参与调节SD大鼠小肠的液体转运。腹泻大鼠小肠黏膜中的AQP4表达明显减少。复方双苓止泻散有效停止腹泻,各治疗组大鼠小肠黏膜中的AQP4表达上调,推测AQP4还可能与参与腹泻大鼠肠黏膜的恢复过程。更多还原

【Abstract】 Diarrhea is one symptom which many diseases may cause, and its pathological basis includes mucosal inflammation, edema, and transepithelial hypersecretion of fluid and hyperfunction of intestinal movement by the stimulation of infections or non-infective factors. Short-term severe diarrhea may cause dehydration and electrolyte disorders which are life-threatening; while lasting diarrhea can damage the function of digestion and absorption severely. To provide appropriate antidiarrhea medicine can allevate symptoms of diarrhea and reduce the incidence of complications as soon as possible, thus the research of drugs for the prevention and treatment of diarrhea is of great importance.Compound shuang-ling antidiarrhea powder is a pure Chinese traditional medical herb, with effects of anti-inflammatory, bacteriostasis, diuresis, immunoregulation and curing abdominal pain and emetocathartic. This study referred to Zhou Gannan’s research to establish the diarrhea model of Sprague-Dawley(SD) rats. The experimental design was as follows: 60 SD rats weighing between 180 and 220 g were randomly assigned into 6 groups(n = 10 in each group) including diarrhea model (DM)group, low dose-(0.31 g/kg, L), middle dose-(0.62 g/kg,M), and high dose-(1.24 g/kg,H) compound shuang-ling antidiarrhea powder treatment group, Wan-xin-mu-zi-kuai-kang(positive control drug 0.62 g/kg,PC)treatment group, and normal control (NC)group. After 48 hours’treatment,rats were sacrificed and the proximal small intestine was removed. The present study is to explore the possible mechanism of action and appropriate dose of compound shuang-ling antidiarrhea powder by comparing the small intestinal mucosal morpHology and the level of aquaporin-4 (AQP4) protein expression of different groups for the purpose of using pure natural plant ingredients to replace the use of antibiotics and reduce environmental pollution caused by the excreta. This study provides important theoretical basis for the promotion of animal husbandry healthy cultivation technology. The study is divided into two parts: 1 Compound shuang-ling antidiarrhea powder promotes the recovery of small intestinal villous morpHologyObjectiveSmall intestinal mucosa is not only the basis for digestion and absorption function of the body, but an internal mechanical barrier against bacteria, toxins and the invasion of other harmful substances. The mucosal barrier function depends on the integrity of intestinal mucosa. What’s more, the structure of intestinal epithelial cells is vital to the intestinal mucosal integrity. The objective of this part of research is to compare the small intestinal villus morphology in different groups in order to explore the possible mechanism of action of compound shuang-ling antidiarrhea powder on the recovery of the intestinal mucosae and provide an appropriate for its application.MethodsHematoxylin and eosin(HE)staining, the scanning electron microscope and transmission electron microscope are employed to observe small intestinal villus morpHology and the ultrastructure of small intestinal epithelial cells respectively. And Motic 6.0 digital medical image analysis system is used to measure and compare the average height, width and surface area.Results1.1 HE staining observationIn NC group, the small intestinal villi of the rats arrange regularly and a little of mesenchymal was seen in the central axis of the villi; the columnar absorptive cells align continuously with a small number of acidopHilic change; lympHocytes infiltration could be occasionally seen among the epithelial cells or at the top of villi. In DM group, however, the majority of small intestinal villi were edema, some villus fusion appeared and the mesenchymal in the central axis of the villi was edema, with capillary hyperemia, exudation and a large number of inflammatory cells infiltrations. And the boundaries between columnar epithelial cells became unclear; acidopHilic change of epithelial cells increased; lympHocytes infiltration could be frequently seen among the epithelial cells; and a lot of epithelial cells got necrosis and exfoliated. What’s more, lots of inflammatory cells infiltrated in the mesenchymal of intestinal glands in lamina propria. But muscularis mucosae and serosa were not damaged. In the rats of L group, it was still obvious that intestinal villi edema, capillary hyperemia and inflammatory cells infiltration; and a little of exfoliated villi tissue could be seen in the lumens. In PC group, the damaged structure of intestinal villi recovered a lot; some columnar cells separated from the musenchymal at the top of the villi, even exfoliated; capillary hyperemia and inflammatory cells infiltration decreased greatly. Columnar epithelial cells aligned continuously in M and H groups, with a small number of inflammatory cells infiltration and exudation in the intestinal villi.1.2 Intestinal villous measurement results1.2.1 Comparison of villus width: The intestinal villous width of DM group increased significantly compared with that of the other 5 groups(P<0.01). The intestinal villus width of each treatment group decreased greatly and there was no significant difference among M、H、PC and NC groups (P>0.05). But the villus width of L group was still wider than that of NC group(P<0.01). 1.2.2 Comparison of villus height: There was no significant difference among the villus height of PC、L、M and DM groups(P>0.05), either of which was significantly higher than that of NC group(P<0.01). And the difference of the villus height between H group and NC group was not remarkable(P>0.05).1.2.3 Comparison of villus surface area: The intestinal villous surface area of DM group increased strikingly compared with that of the other 5 groups(P<0.01). The intestinal villus surface area of each treatment group decreased obviously and there was no significant difference among M、H and NC groups(P>0.05), though either the villus surface area of L group or PC group was significantly larger than that of NC group(P<0.01).1.3 Intestinal villi morpHology shown by the scanning electron microscopeIn NC group, the sizes of small intestinal villi were consistent; the surface of the villi was neat and the absorptive cells distributed evenly; villi and microvilli arranged regularly. In DM group, however, most of the villi and microvilli were damaged with a lot of mucus adhesion and massive epithelial cells exfoliated. The damaged structure was improved a lot after drug treatment. Though the villi were still edema and they arranged less regularly in L group, the villus edema was not obvious in PC group. The villi of M group and H group were tongue-shaped with good arrangement, damaged villi could be seen seldom and most microvilli aligned regularly.1.4 The ultrastructure of epithelial cells shown by transmission electron microscopeIn NC group, the epithelial cells aligned continuously and their ultrastructure was normal; the microvilli were long and arranged well; apoptosis of epithelial cells could be seen occasionally. But in DM group, the majority of epithelial cells were obviously edema and most mitochondrial swelled and cristae and outer membrane were damaged; many endoplasmic reticulums expanded; the microvilli varied in length and got sparse. It was obvious that the intestinal epithelium edema was attenuated in L and PC groups and the microvilli of them became better than that of DM group, though some mitochondria were still edema. Most ultrastructure of the epithelial cells recovered and microvilli arranged regularly in M and H groups.2 Compound shuang-ling antidiarrhea powder promotes the expression of AQP4 in small intestinal mucosa of diarrhea rats ObjectiveThe objective of this part of the study is to explore the possible role of AQP4 in the process of diarrhea and the effect of compound shuang-ling antidiarrhea powder on the expression of AQP4.MethodsThe expression of AQP4 in small intestinal tissue of rats in different groups is determined by immunohistochemistry and Western blot. Results2.1 Immunohistochemical staning resultThe immunoreactive protein was detected as yellow-brown deposits. AQP4 was immunolocalized to the basolateral membrane of deep glands of the small intestine of all the rats, but positive staining could not be observed in the goblet cells. The expression of AQP4 in intestinal mucosa of rats of DM group was obviously less than that of each treatment group.2.2 Western blot analysis resultThe expression of AQP4 in intestinal mucosa of rats in DM group strikingly decreased compared with that of each treatment group(P<0.01). The expression of AQP4 in intestinal mucosa of rats in L group was significantly lower than that in M or PC group respectively(P<0.01), and the expression of AQP4 in M or PC group was significantly lower than that in NC group( P<0.01 ) . There was no significant difference in the expression of AQP4 between H group and NC group(P>0.05). ConlusionCompound shuang-ling antidiarrhea powder can stop the symptoms of diarrhea effectively and promote the recovery of damaged structure of intestinal villi. Its main mechanism includes reducing the inflammatory cells infiltration and capillary hypermia and exudation in the mesenchymal of the villi and villus edema, promoting the restoration of ultrastructure of epithelial cells and the proliferation of epithelium in order to restore the structure and function of intestinal mucosa as soon as possible. And the effect of middle dose- or high dose-compound shuang-ling antidiarrhea powder or Wan-xin-mu-zi-kuai-kang (positive control drug) on the restoration of intestinal mucosa is better than that of low dose-compound shuang-ling antidiarrhea powder; the effect of middle dose-compound shuang-ling antidiarrhea powder on the restoration of intestinal mucosa is better than that of Wan-xin-mu-zi-kuai-kang; but there is no remarkable difference of the effect between middle dose- and high dose-compound shuang-ling antidiarrhea powder.AQP4 has been immunolocalized to the basolateral membrane of deep glands of the small intestine, but positive staining could not be observed in the goblet cells. AQP4 may play a role in the water transport of small intestine. The expression of AQP4 in the rats of DM group decreased strikingly, while compound shuang-ling antidiarrhea powder upregulates the expression of AQP4 protein. These results indicate that AQP4 may also participate in the restoration process of intestinal mucosa.更多还原