| 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. |
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