芫花根水提物对肛瘘创面及肛缘肿胀的实验研究

发布时间：2018-07-02 08:57

本文选题：芫花根水提取物 + 炎性因子　；参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:通过芫花根水提物与空白对照及黄芩油膏对比,外用于大鼠肛瘘术后及肛缘水肿换药,观察创面水肿、炎症反应的变化情况及创面的愈合情况。以此验证芫花根治疗痔瘘疾病的有效性与其抗炎、消肿的药理作用相关,并且为芫花根的进一步探索研究提供动物实验研究基础。方法:实验一:在大鼠背上制备肛瘘术后创面模型,将造模成功的大鼠分成A组(生理盐水组)、B组(芫花根组)、C组(黄芩油膏组),分别使用生理盐水、芫花根水提物、黄芩油膏浸泡的纱布给大鼠创面换药。在用药3、7、14天时,取各组的创面组织进行HE染色和MASSON染色,观察肉芽组织变化、炎细胞浸润情况和纤维化变化,并应用PCR定量检测各组大鼠血清中IL-6、IL-12的表达及使用流式细胞术检测各组大鼠外周血中Treg/Th17比例。实验二:建立大鼠肛门巴豆油肿胀模型,造模成功后,将大鼠分成A组(正常组)、B组(生理盐水组)、C组(芫花根组)、D组(黄芩油膏组),A组不做任何处理,B、C、D组每隔4、24、48小时分别将生理盐水、芫花根水提物、黄芩油膏涂于肿胀的肛门处,48.5小时后,取各组大鼠肛周肿胀部位组织进行HE染色和MASSON染色,观察大鼠肛缘肿胀程度、血管生成情况、炎细胞浸润情况和纤维化变化,并行免疫组化检测SM22 α蛋白的表达,免疫荧光染色定位SM22 α蛋白,以及应用PCR和ELISA方法检测各组大鼠血清中IL-17和TNF-α的表达情况。数据均采用SPSS18.0分析。结果:实验一中,PCR定量检测结果显示:用药3天后,A、B、C组IL-12的表达量用X±S表示分别为1.02±0.28、2.52±1.01、2.78±1.83,经统计分析存在显著性差异,P0.05(P=0.011),而B、C组对比,统计学上无明显差异,P0.05(P=0.648)。三组中IL-6表达量用 X±S 表示分别为 1.04±0.46、0.84±0.29、0.69±0.33,P0.05(P=0.157),无统计学差异。用药7天后,三组IL-12的表达量分别为0.80±0.24、2.75± 1.13、3.02±2.21,经统计分析存在显著性差,P0.05(P=0.006),而B、C组对比,无明显差异,P0.05(P=0.700)。三组中IL-6表达量分别为1.86±0.56、0.62±0.21、0.53±0.23,经统计分析存在显著性差异,P0.05,而B、C组对比,则无明显差异。P0.05(P=0.589)。用药14天后,三组IL-12的表达量分别为0.49±0.21、3.48±1.45、4.19±1.48,经统计分析存在显著性差异,P0.05,而B、C组对比,则无明显差异,P0.05(P=0.237)。三组中IL-6表达量分别为2.84±0.99、0.49±0.23、0.31±0.14,经统计分析存在显著性差异,P0.05,而B、C组对比,则无明显差异P0.05(P=0.516)。HE染色显示:用药3天后A、B、C三组均有纤维脂肪组织中见大量急慢性炎细胞浸润,局部肌肉组织变性坏死,血管增生,管壁增厚,A组中还见间质水肿和血管闭塞,说明A组炎症表现更重,B、C较A组稍轻,两组炎症程度相当。用药7天后三组纤维脂肪组织中仍有大量急慢性炎细胞浸润,A、C组局部肌肉组织变性坏死、钙化,B、C组局部有肉芽组织形成,说明B、C组创面开始修复。用药14天后B、C组组织中炎细胞浸润较A组明显减少,且有玻璃样改变,说明创面开始有瘢痕组织形成。Masson染色显示:用药3天后创面组织中见大量粘液及有胶原纤维生成,并且B、C组中多于A组。用药7天后创面组织中胶原纤维增多,且B、C组比A组多。用药14天后A组创面组织中胶原纤维增减少B、C组中胶原纤维增多。流式细胞术检测外周血Treg/Th17比例的结果用X±S表示为:用药3天后,A、B、C组分别是0.663±0.042、0.770±0.009、0.926±0.002,经统计分析存在显著性差异,P0.05(P=0.004)。用药7天后,分别是0.565±0.001、1.077±0.033、1.372±0.065,经统计分析存在显著性差异,P0.05(P=0.001)。用药14天后,分别是0.295±0.011、1.722 ±0.074、2.362±0.187,经统计分析存在显著性差异,P0.05(P=0.001)。实验二中,PCR定量检测TNF-α及IL-17指标的结果显示:A、B、C、D四组中TNF-α 的表达量以 X±S 表示分别为 0.88±0.21、5.03±1.31、2.63±0.96、1.57±0.27,P0.05,存在显著差异,而C、D组对比,统计学上无明显差异,P0.05(P=0.073)。四组中IL-17表达量为0.92±0.26、3.37±0.85、2.15±0.45、1.48±0.39,经统计分析存在显著性差异,P0.05,而C、D组对比,统计学上无明显差异,P0.05(P=0.072)。ELISA定量检测TNF-α及IL-17指标的结果与PCR检测结果相符,P值均小于0.05,存在显著性差异。HE染色示:A组见正常的肛周粘膜、肌肉等组织。B、C、D组的肛周粘膜、肌肉组织中均见急慢性炎细胞浸润,C、D组较A组少,C组较D炎细胞浸润更少,且C组中未见肌肉组织坏死,并伴有小血管增生,说明C、D组用药后较A组炎症性水肿消退,且C组炎症、水肿程度最轻。MASSON染色观察肛缘纤维化程度的结果为:A组中见正常肛缘组织,B组见大量粘液及胶原纤维生成,C组和D组经药物治疗后见少量粘液及胶原纤维生成。免疫组化检测SM22α蛋白的表达用IOD值表示结果,A、B、C、D分别为8841.39±49841.88、4339.75±24700.33、7211.28±29230.33、7702.81±39702.19,四组经统计学分析,有显著性差异,P0.05,但是C、D两组比较,统计学上无明显差异,P0.05(P=0.166)。免疫荧光染色显示:SM22α蛋白及STIM1蛋白位于细胞质内,红色荧光位于细胞核中,所以Orail蛋白位于细胞质中。结论:芫花根水提物具有减轻痔瘘术后炎症反应、术后水肿的作用。在创面愈合的早中期减少炎细胞渗出,增加胶原纤维产生,提高SM22 α蛋白的表达,进而促进痔瘘术后创面的愈合。
[Abstract]:Objective: To compare the water extract of the root of the Daphne Daphne with the blank control and the Scutellaria baicalensis ointment, and to observe the edema, the changes of the inflammatory reaction and the healing of the wounds in the rats after the operation of anal fistula and the edema of the anal border, in order to verify the effectiveness of the root canal for the treatment of hemorrhoids and the pharmacological effects of the anti inflammation and swelling of the disease, and to be the root of the Daphne Daphne. Further exploration and research provide the basis of animal experiment. Experiment 1: to prepare the wound model after anal fistula operation on the back of the rat, the rats were divided into A group (physiological saline group), group B (Daphne root group), group C (Scutellaria baicalensis ointment group), using physiological saline, water extract of Daphne Daphne root, and gauze soaked in Scutellaria ointment to the wound of rats. After 3,7,14 days, the wound tissue of each group was stained with HE and MASSON, and the changes of granulation tissue, infiltration of inflammatory cells and fibrosis were observed. The expression of IL-6 and IL-12 in the serum of each group was detected by PCR and the proportion of Treg/Th17 in the peripheral blood of each group was detected by flow cytometry. Experiment two: establishment of experiment two: After the model was successful, the rats were divided into A group (normal group), group B (normal saline group), group C (Daphne root group), group D (Scutellaria baicalensis ointment group), A group did not do any treatment, B, C, D group every 4,24,48 hour respectively, the saline, Daphne root water extract, Scutellaria baicalin was coated on the swollen anus, after 48.5 hours, take each group big HE staining and MASSON staining were performed in the tissue of swollen perianal area. The swelling of the anal margin, angiogenesis, infiltration of inflammatory cells and fibrosis were observed in rats. The expression of SM22 alpha protein was detected by immunohistochemistry, SM22 alpha protein was detected by immunofluorescence staining, and IL-17 and TNF in serum of rats were detected by PCR and ELISA methods. The data were analyzed by SPSS18.0. Results: in the first experiment, PCR quantitative detection results showed that the expression of IL-12 in A, B and C group was 1.02 + 0.28,2.52 + 1.01,2.78 + 1.83 respectively after 3 days of drug use, and there were significant differences in statistical analysis, P0.05 (P= 0.011). 8) in the three group, the expression of IL-6 in the three group was 1.04 + 0.46,0.84 + 0.29,0.69 + 0.33, P0.05 (P=0.157), and there was no statistical difference. The expression of IL-12 in the three groups was 0.80 + 0.24,2.75 + 1.13,3.02 + 2.21 respectively after 7 days of drug use, and there was a significant difference in statistical analysis and P0.05 (P= 0.006). The expression of IL-6 was 1.86 + 0.56,0.62 + 0.21,0.53 + 0.23 respectively. There were significant differences in statistical analysis, P0.05, but there was no significant difference in.P0.05 (P=0.589) in B and C groups. The expression of IL-12 in the three groups was 0.49 + 0.21,3.48 + 1.48, respectively. The expression of IL-6 was P0.05 (P=0.237). The expression of IL-6 in the three groups was 2.84 + 0.99,0.49 + 0.23,0.31 + 0.14, respectively, and there was a significant difference by statistical analysis, P0.05, while B, C group contrast, there was no significant difference between P0.05 (P=0.516).HE staining: 3 days of A, three groups of fibrous adipose tissue were found to see a large number of acute and chronic inflammatory cells infiltration, local muscle tissue. Degeneration and necrosis, vascular hyperplasia and thickening of the wall of the tube, A group also found interstitial edema and vascular occlusion, which showed that A group was more severe in inflammation, B, C was slightly lighter than that in A group, and the two groups of inflammation were similar. There were still a large number of acute and chronic inflammatory cells in three groups of fibrous adipose tissue after 7 days, A, C group muscle tissue degeneration and necrosis, calcification, B, and C group local granulation tissue The formation of B, C group wound began to repair. After 14 days, B, the infiltration of inflammatory cells in group C was significantly lower than that in the A group, and there was a glass like change. It showed that the wound began to have scar tissue to form.Masson staining showed that a large number of mucus and collagen fibrinolysis were found in the wound tissue after 3 days of drug use, and B and C group were more than the A group. 7 days after the drug use, the wound group was used. The collagen fibers in the fabric increased, and the B, C group were more than the A group. The collagen fibers in the wound tissue of group A decreased B, and the collagen fibers increased in group C 14 days later. The result of flow cytometry for detecting the proportion of Treg/Th17 in peripheral blood was expressed as X + S: 3 days after medication, A, B, and C groups were 0.663 + + 0.002, respectively. The statistical analysis was significant. Difference, P0.05 (P=0.004). 7 days after medication, 0.565 + 0.001,1.077 + 0.033,1.372 + 0.065 respectively, statistically significant differences, P0.05 (P=0.001). 14 days after drug use, 0.295 + 0.011,1.722 + 0.074,2.362 + 0.187 respectively, statistically significant differences, P0.05 (P=0.001). The results showed that the expression of TNF- alpha in A, B, C, D four groups was 0.88 + 0.21,5.03 + 1.31,2.63 + 0.96,1.57 + 0.27 and P0.05, and there were significant differences. There was no significant difference between P0.05, C and D group. The results of P0.05 (P=0.072).ELISA quantitative detection of TNF- A and IL-17 were consistent with the results of PCR detection, P values were less than 0.05, and there was a significant difference in.HE staining. Chronic inflammatory cell infiltration, C, D group less than the A group, C group less infiltration than D inflammatory cells, and C group no muscle tissue necrosis, accompanied by small vascular hyperplasia, indicating C, D group after the use of A group inflammatory edema subsided, C group inflammation, edema degree of the least.MASSON staining observation of the degree of anal fibrosis in the case of the normal anal margin in the A group A large number of mucus and collagen fibers were produced. A small amount of mucus and collagen fibers were found in group C and group D after treatment. The expression of SM22 alpha protein was expressed with IOD values by immunohistochemistry. A, B, C, D were 8841.39 + 49841.884339.75 + 24700.337211.28 + 39702.19, respectively. The four groups were statistically analyzed with significant differences. 05, but C, D two groups compared, statistically no significant difference, P0.05 (P=0.166). Immunofluorescence staining showed that the SM22 alpha protein and STIM1 protein in the cytoplasm, red fluorescence in the nucleus, so the Orail protein in the cytoplasm. Conclusion: Daphne root water extract has the effect of alleviated inflammatory reaction after hemorrhoids operation, postoperative edema. In the wound. In the early and middle stages of healing, the inflammatory cells exudate, the production of collagen fibers is increased, the expression of SM22 alpha protein is increased, and the wound healing after hemorrhoid fistula operation is promoted.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】