| Objective:Damp-Heat syndrome of spleen-stomach (DHSS for short), a common type of excess syndromes of spleen and stomach, is a significant task of the study on syndrome of Traditional Chinese Medicine(TCM).The balance of Th1/Th2cells, together with the proliferation and apoptosis of the gastric surface epithelium reflects the mutual opposition and mutual effect of yin and yang in human body to some degree, which has a close relation with the characteristics of DHSS, including the compound evils of yin and yang, the struggle of healthy atmosphere and pathogenic factor and the dynamic balance of yin and yang. According to the clinical features of DHSS, we established the animal models based on the composite element of high calorie diet, artificial environment simulation and microbiological infection to observe the effects of Sanren decoction on the body mass (BM), rectal temperature (RT), water and food intake, histopathological changes of the gastrointestinal mucosa, serum expression of inerleukin (IL)-4and interferon (IFN)-γ by by enzymelinked assay (ELISA) and protein expression of Fas、Bcl-2by SABC immunohistochemical method. These contribute to deepen the understanding of the biological foundation and pathogenesis of DHSS, which aims to elevate the research level of DHSS.Methods:Fifty male SD rats were randomly allocated into the normal control group, DHSS model group, and3Sanren Decocotion groups (high, medium and low doses). Rat models with DHSS were established by composite element of high calorie diet, artificial environment simulation (dry bulb temperature (Td)33±0.5℃, relative humidity (rh)65±5%) and salmonella typhimurium infection. Then three Sanren decoction groups (high, medium and low doses) administered intragastrically with2ml/200g Sanren decoction for7days, the control group and model group received equal volume of saline once a day. Water drinking and food intake were recorded everyday, the body weight (BM) and rectal temperature (RT) were measured in the first, tenth, eighteenth and twentysixth days. After the treatment, all rats were executed, the whole blood(5ml) from abdominal aortic was remained and serum (500μl) was obtained after centrifuge, then the level of serum IFN-γ and IL-4were measured by enzymelinked assay (ELISA). Pathological changes of gastric, duodenal, colon tissues were oberved using paraffin-eft section(H.E.) by the light microscope. The expression of Fas, Bcl-2were detected by SABC immunohistochemical method in50gastric mucosa tissues. The data was dealt with statistical analysis of repeated measure ANOVA and One-way AN OVA by SPSS13.0software and made0.05the boundary of statistical significance.Results:1.Effects of Sanren decoction on the general state, foodintake and water quantity changes of rats with DHSSThe rats in the control group were in the state of good mental health, had a shiny coat, sensitive reaction, water and food intake equilibrium, while Spleen-Stomach Damp-Heat rats gradually appeared to be sluggishness, moveless, fur lackluster, unresponsive, loose stools, periproctitis, and took less food (P<0.001) and water (P>0.05). There was a reduced trend of food and water intake in the models-making process of three Sanren Decoction groups (high, medium and low doses) rats (P>0.05). After the treatment, compared with model rats, the general state of rats was gradually improving, the food intake was significantly increased in the High-dose (P<0.001), medium-dose (P=0.024, P<0.05) and low-dose groups (P=0.022, P<0.05). The amount of water had little increased (P>0.05).2.Effects of Sanren Decoction on body weight (BW) and rectal temperature (RT) of rats with DHSS.Body weight increased slowly (P>0.05) and rectal temperature rised rapidly (P<0.001) in the molding process while rats in the control group showed progressive growth. All three doses of Sanren decoction showed a tendency to increase weight (P>0.05) and significantly reduced rectal temperature in rats with DHSS (P<0.05).3.Effects of Sanren Decoction groups on the expression of IFN-γ in rats with DHSS.Compared with the control group, the serum levels of IFN-γ in model group and Sanren Decoction groups(high, medium and low doses) increased significantly (P<0.001). Low-dose and medium-dose Sanren Decoction obviously decreased the production of IFN-γ cytokine (P<0.001). The levels of IFN-γ in High-dose Sanren Decoction group showed a decreasing tendency (P>0.05)4.Effects of Sanren Decoction groups on the expression of IL-4in rats with DHSS.The serum levels of IL-4in the model group significantly increased compared with those in the control group(P<0.001). Compared with model group, all three doses of Sanren Decoction has obviously reduced the production of IL-4(P<0.001), low dosage proves the most obvious effect.5.Effects of Sanren Decoction on the IFN-γ/IL-4ratio in rats with DHSS.The serum levels of IFN-γ and IL-4in the model group significantly increased compared with those in the control group, with a slightly increased IFN-γ/IL-4ratio(P>0.05). High-dose Sanren decoction caused more markedly decreased IL-4level to result in a significantly increase IFN-γ/IL-4ratio(P=0.028, P<0.05). Low-dose Sanren Decoction showed a decreasing tendency in IFN-γ/IL-4ratio, but there were no significant difference(P>0.05).6.Effects of Sanren Decoction on the pathology of gastric, duodenal, colonic mucosal tissue.Under light microscope, the gastric, duodenal, colonic epithelial mucosa in the control group were smooth and no erosion. In the model group, the main histological mucosal changes of stomach, duodenum, colon were congestion, edema and a great of inflammation cell invasion in lamina propria and submucous layer. After the treatment with three doses of Sanren decoction the inflammation in mucosa was alleviated while the gastric gland, duodenal villous and colonic epithelium were recovered and organized in good order.7.Effects of Sanren Decoction on the expression of Fas protein in rats with DHSS.A quantity of brown positive granules which appeared within the gastric surface epithelium showd the moderate expression of Fas protein in the control group, while weakly positive expression in the model group(P=0.005, P<0.01). The mean OD value were significantly increased in all three dose of Sanren decoction groups (P<0.001).8.Effects of Sanren Decoction on the expression of Bcl-2protein in rats with DHSS.Weak expression of Bcl-2protein was noted in a few scattered gastric surface epithelium in control group, while deep brown strong expression of the protein in a great number of cells was found in the model group (P=0.016, P<0.05). All three dose of Sanren Decoction has remarkably reduced the expression of Bcl-2protein, the mean OD value were significantly lower than that of the model group (P<0.05), low dosage proves the most obvious effect.Conclusion:1. Application of high calorie diet, artificial climate chamber, biological pathogenic factor can be successfully induced the animal model with damp-heat syndrome of spleen-stomach which is similar to clinical feature with the molding time.2. Over-expression of both IFN-γ and IL-4indicates that rats with Spleen-Stomach Damp-Heat syndrome are in a state of violent struggle between pathogenic factor and healthy qi. It is suggested that the secretory disturbance of cell factors with Thl response tendency to drift and balancing disorder of immune network may be one of the important mechanisms in DHSS.3. A dense inflammatory infiltration in the gastric, duodenal, colonic mucosa of rats with DHSS indicates that damp-heat syndrome of Spleen-Stomach is on the basis of morphology.4. The over-expression of Bcl-2protein and low-expression of Fas protein that inhibits cell apoptosis and excessive cell proliferation promote the formation of damp-heat syndrome in the spleen and stomach.5. Sanren decoction has functions of inhibiting inflammation and regulating balance between Thl and Th2that contributes to improve symptoms in rats with DHSS, as up-regulated expression of Fas protein and down-regulated expression of Bcl-2protein to induce gastric epithelial cells apoptosis contributes to repair and protect of gastric mucosa. |
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