| Objective: This experiment use in situ end labeling (TUNEL) technique and immunohistochemical method to detect the in situ cell apoptosis and different distribution of bcl-2, bax in mucosal layer and muscular layer of gastrointestinal tract.And also make it easy to understand the relationship be-tween apoptosis and lesions of constipation rat.Method:1 Fourty clean healthy adult Wister rats, half female and half male respectively, which were chosen and di-vided randomly into control group and experimental group. Marking weight and sex no significant difference after grouping. Leachate of rhubarb was intragastric administration in rhubarb group. At the beginning, the dosage is 800mg/kg.day (raw mate-rial), afterwards the dosage increased 200mg/kg.day (raw mate-rial)every day until half of rats'stool became watery, this dosage was maintained until 80 percent of rats'watery stool disap-peared, then the dosage continually increased until 50 percent of rats'stool become watery. One week after the 80 percent of watery stool disappear for the third time, the experiment was stopped. Distilled water was intragastric administration in con-trol group. 2 Take the specimens of rat gastric antrum, ileum, transverse colon from the each group. Using in situ end labeling (TUNEL) method to show the apoptosis, count the number of apoptotic index. Immunohistochemical staining.of bcl-2 bax Semi-quantitative analysis of results.The statistical analysis of experimental data with the application SPSS13.0 statistical software and Wilcoxon rank sum test ,test levelα=0.05. 3 Apoptosis determination , apoptosis index (AI) is calculated as follow : Random selected five high-power field (400 times) from each specimen,recognize apoptosis cell from at least 1,000 cells, and was represented by percentage. The positive result of bcl-2, bax is the brown particles appeared in the nuclei and cy-toplasm. Chose the good immunoreactive staining vision, where the positive cells were evenly distributed in region. Count the positive cells under high-power microscope vision in each one hundred cells. Represented by percentage. Immunoreactive cells more than 10% has been set for diagnostic criteria. colorless negative (—). Primrose was weakly positive (+). brown positive (++) brown color and strong positive (+++). Finally we use image analysis system (VIDAS) to measure mucosal and muscle in stomach antrum,ileum and transverse colon in optical density value of the average immunoreactive cells.Results:1 TUNEL-positive cells mainly present nucleus brown coloring, The positive cells is scattered in different gas-trointestinal level. The apoptosis cells in gastric mucosal in experimental group is more than that in control group (p<0.05); the apoptosis cells in stomach muscle in the experimental group is more than that in the control group (p<0.05); the apoptosis cells in ileum mucosal in the experimental group is more than that in the control group (P<0.05); the apoptosis cells in ileum muscle in experimental group showed no significant difference compared with the control group(p> 0.05); the apoptosis cells in transverse colon mucosa in the experimental group is more than that in the control group (P<0.05); the apoptosis cells in transverse colon muscle is more than the control group (p <0.05). 2 The result of bcl-2, bax : Coloring muscle cells could be seen, most of the color take place in the cytoplasm, but also in the nucleus, a membrane around prevalent. brown, and some of the performance was disseminated granular or small pieces. The expression of bcl-2 and bax in mucosa and muscle of gastric in experimental group is more than that in the control group(p <0.05). The ex-pression of bcl-2 and bax in mucosa of ileum in experimental group is more than that in the control group(p <0.05). The ex-pression of bcl-2 and bax in the muscular of ileum of the ex-perimental group shows no significant diffrence compare with the control group(p>0.05). The expression of bcl-2 and bax in mucosa of transverse colon in experimental group is more than that in the control group(p <0.05). The expression of bcl-2 in muscle of transverse colon of experimental group is greater than that in the control group(p <0.05), The expression of bax in the muscular of transverse colon of the experimental group shows no significant diffrence compare with the control group(p>0.05)3 Gastrointestinal anatomy and pathological changes in gastrointestinal tract show that there no significant difference between the two groups, No colonic mucosa pigmentation in both groups. There are no lesions in control group. Chronic inflammatory changes were found in experimental rats gastro-intestinal mucosa samples. a lot of eosinophil infiltration into gastrointestinal, mainly in lamina propria .muscular in submu-cosal layer get thickening, collagen fibrosis intrinsic membrane edema, muscle proliferation of collagen fibers, myenteric plexus nerve cell get degeneration,.Conclusion:1The lesions constipation rat include muscosal layer and muscular layer.2Apoptosis cells in gastric antrum, ileum, transverse colon in experimental group is much more than that in control group ,indicat that the impact of rhubarb is not confined to the colon, It involves the entire gastrointestinaltract.3 In situ detection of apoptosis shows that significantly increase in the number of apoptotic cells, and a large number of apop-totic bodies in experimental group. except ileum the rest of part of apoptotic cells is significantly more than that in control group. Rhubarb lead to the long-term cathartic colon, constipation and apoptosis in rat neurons in the degenerative changes are closely related. 4Long term consitipation lead to high risk of gastrointestinal turmor or cancer.
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