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Objective: To study Pulsatilla alcohol extract trinitrobenzene sulfonic acid (trinitro-benzen-sulfonic acid, TNBS) induced colitis intestinal epithelial cells tight junctions protein regulation further explore Pulsatilla alcohol extract on rats with experimental colitis intestinal mucosal barrier protection. Methods: SD rats (both male and female) 38, divided into four groups: normal control group (control group, n = 8), by TNBS in model group (model group, n = 10), Pulsatilla alcohol extract treatment group (Pulsatilla group , n = 10), Jinshuangqi treated group (Jinshuangqi group, n = 10). Establish TNBS-induced colitis model: of SD rats preoperative fasting 24h model groups, Pulsatilla the group and Jinshuangqi of of the group with a diameter of about 2mm, about 12cm silicone tube from the anus gently insert depth of about 8cm to contain 100mg / kgTNBS equal volume of 50% ethanol solution was slowly into the colon to allow the animals to keep the anal high awake by volume of saline control group slowly into the colon. Animal model, the control group and the model group for 1-7 days each rat daily with 1ml of distilled water enema, and free access to water; Pulsatilla group the first 1-7 days Pulsatilla alcohol extract each rat enema, the Pulsatilla alcohol extract dosage 2g/kg prepared with distilled into 1ml, once a day, and free access to water; Jinshuangqi group each rat 1-7 day Jinshuangqi 2Co / only ground into powder with distilled water preparation into 1ml enema, and free access to water. Observed daily rat mental state, such as eating, activity, stool and weight. Eight days after modeling orbital venous determination of endotoxin in the blood, and All rats were sacrificed, free, and remove the entire colon cut longitudinally along the mesenteric observation the colon specimens congestive edema after cleaning the intestines dirt the remaining specimens liquid nitrogen, ulcer size, distribution, and the presence or absence of active bleeding; take 1cm diseased tissue, HE staining and immunohistochemistry method detection Occludin expression and disease activity index (DAI) and histological injury score; cryopreservation, for ELISA assay colon tissue of TNF-alpha, IL-10. Results: ① general: 24 hours after modeling, the model rats suffer from mental activity, diet further reduce diarrhea with gross blood in the stool or fecal occult blood test was positive, the first 2-5 days of diarrhea 5-6 days The blood will gradually reduce food intake recovery. Pulsatilla group, the symptoms of Jinshuangqi rats compared with the model group significantly improved, 24 hours after the modeling began to appear loose stool or shapeless, bloody stools and more occult blood positive, diarrhea slightly 2-3 days, 3-4 days after diarrhea blood will gradually reduce, the activity gradually increased, the appetite is slowly coming back. Colonic mucosal erosions and ulcers of the model group in histology to form a larger area, many involving the myometrium, glandular distorted part of the glandular epithelium atypical hyperplasia; Pulsatilla group Jinshuangqi groups colon, mucosal erosions and ulcers smaller, and more involved to the submucosa, duct distorted, partial gland dysplasia is not obvious; complete intestinal mucosa of rats of the control group, a clear structure, glands arranged rules the intrinsic membrane goblet cells, no abnormal myometrial occasional trace inflammation cell infiltration. ② disease activity index (DAI) and histological injury score: model group Pulsatilla group Jinshuangqi group and the normal group DAI, respectively, 9.20 ± 1.75,6.50 ± 1.27,5.90 ± 1.67 and 0.75 ± 0.71; histologic injury score 7.10 ± 0.99,5.00 ± 1.05,4.80 ± 1.23 and 0.25 ± 0.46, compared with the normal group, the differences were statistically significant (P lt; 0.05) the Pulsatilla the group and Jinshuangqi of group difference had no statistical significance (P gt; 0.05 ). ③ colon tissue TNF-α, IL-10 and endotoxin content: TNBS - induced colitis in rats, colon tissue TNF-α levels elevated IL-10 levels decreased and serum endotoxin levels, while Pulsatilla alcohol mentioning matter and Bifidobacterium Lactobacillus acidophilus Enterococcus triple viable cells after treatment, the colon tissue TNF-α levels lower (657.54 ± 149.60,521.24 ± 109.38,514.73 ± 163.47 and 205.40 ± 72.49, P lt; 0.05), blood endotoxin The lower level (0.213 ± 0.023,0.148 ± 0.093,0.153 ± 0.106 and 0.071 ± 0.034, P lt; 0.05) and IL-10 levels (42.92 ± 23.74,92.19 ± 30.09,89.77 ± 28.48 and 123.00 ± 36.16, P lt ; 0.05), the Pulsatilla the group and Jinshuangqi of group comparison showed no significant difference (P gt; 0.05). ④ colon intestinal epithelial tight junction protein Occludin expression: model group Pulsatilla group Jinshuangqi group and normal group were 25.80 ± 10.28,50.70 ± 17.02,52.40 ± 16.15 and 81.62 ± 11.39, and the normal group comparison, differences between groups statistically significant (P lt; 0.05), Pulsatilla Group and Jinshuangqi group difference was not statistically significant (P gt; 0.05). Conclusion: Pulsatilla ethanol extract of TNBS-induced colitis in rats intestinal mucosal barrier has a significant protective effect and possible mechanism by upregulating intestinal epithelial cells tight junction protein Occludin expression, mitigate the damage of the intestinal mucosa and protect the intestinal mucosa, inhibition of endotoxin into the systemic circulation through the colon tissue TJ, thereby reducing colon tissue content of TNF-α, IL-10 levels, to achieve the anti-inflammatory effects.
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