从NF-κB、VEGF信号通路研究肠瑞灌肠剂治疗放射性直肠炎的分子机制

发布时间：2018-07-29 12:05

【摘要】：目的:基于“炎症诱导血管新生”假说从NF-κB及VEGF信号通路研究肠瑞灌肠剂治疗放射性直肠炎的分子机制,为放射性直肠炎的临床治疗提供新的药物作用靶点和新的研究思路方法。方法:以肠瑞灌肠剂7.2g/Kg、3.6g/Kg、1.8g/Kg高、中、低三个剂量灌肠6MV-X线直线加速器单次剂量27Gy照射致放射性直肠炎C57/BL小鼠,分别设地塞米松庆联合庆大霉素、八味锡类散为对照,灌肠给药,每日一次,连续2周。观察小鼠一般状况、粪便,剖取直肠,HE染色观察其病理改变,免疫组化分析放射性直肠炎小鼠模型直肠组织中NF-κB、VEGF的表达、PCR检测直肠组织NF-κB、VEGF表达以及ELISA检测小鼠血清中PKA的含量,探讨三者之间的关系。结果:1.肠瑞灌肠剂对放射性直肠炎C57/BL小鼠直肠组织病理学影响(1)光镜下直肠粘膜病变分级比较,照射后各组粘膜均有不同程度改变,与空白组正常粘膜相比(P㩳0.05)均有统计学差异,提示造模成功。(2)模型组与中药对照组、肠瑞低剂组比较(P0.05),差别无统计学意义;而与治疗高、中剂量组及西药对照组比较(P㩳0.05),差别有统计学意义,即可说明肠瑞高中剂量以及对照西药均能改善直肠粘膜组织炎症。(3)治疗高剂组与中、西药阳性对照组比较(P㩳0.05),差别均有统计学意义,说明高剂量肠瑞灌肠剂疗效优于对照西药地塞米松加庆大霉素以及对照中药八味锡类散。而与低剂组比较(P㩳0.05),差别有统计学意义,说明治疗放射性直肠炎高剂量肠瑞灌肠剂疗效优于低剂量药物。2.免疫组化分析肠瑞灌肠剂对放射性直肠炎C57/BL小鼠直肠组织中NF-κB、VEGF的表达的影响(1)空白对照组亦见少量NF-κB、VEGF表达,考虑与灌肠器械刺激有关,可以忽略。照射后各组均明显出现了NF-κB、VEGF表达,但与空白组比较比较(P㩳0.05)差别有统计学意义。说明NF-κB、VEGF表达是直肠粘膜受放射线照射刺激后产生的。(2)治疗组与模型组比较(P㩳0.05),差别有统计学意义,说明肠瑞组、地塞米松合庆大组以及八味锡类散组均能够抑制NF-κB以及VEGF的表达;肠瑞高剂组与西药阳性对照组比较(P㩳0.05),差别有统计学意义;肠瑞高、中剂量组与中药阳性对照组比较(P㩳0.05),差别有统计学意义。说明高剂量肠瑞灌肠剂较阳性对照组能够显著下调放射性直肠炎直肠组织NF-κB、VEGF表达水平,尚不能说明肠瑞中剂量组疗效优于西药阳性对照组。3.PCR检测肠瑞灌肠剂对放射性直肠炎C57/BL小鼠直肠组织中NF-κB、VEGF的表达的影响(1)统计结果显示,空白对照组可见极少量NF-κB、VEGF表达(考虑与灌肠器械刺激有关,可以忽略),其它各组均有不同程度的NF-κB、VEGF表达,组间比较,各组与空白组比较,差别有统计学意义(P㩳0.05),提示正常鼠直肠粘膜极少量或不表达NF-κB、VEGF,直肠粘膜受照射刺激后发生了NF-κB、VEGF基因表达的改变。(2)模型组NF-κB、VEGF表达明显增高,其它各组与之相比较,差别有统计学意义(P㩳0.05),说明经过治疗后出现了二者表达量的变化。(3)治疗高、中剂量组NF-κB、VEGF表达显著减少,与西药对照组、中药对照组比较,差别有统计学意义(P㩳0.05),说明肠瑞灌肠剂高、中剂量组能够显著下调放射性直肠炎直肠NF-κB、VEGF表达,因而能够控制炎症,抑制血管新生。4.ELISA法测定肠瑞灌肠剂对放射性直肠炎C57/BL小鼠血清中蛋白激酶A(PKA)的含量的影响(1)空白对照组及各治疗组与模型组相比,差别有统计学意义(p0.05),提示造模成功。(2)模型组、西药、中药阳性对照组以及肠瑞中、低剂量组与空白组比较,(p0.05)差别有统计学意义,肠瑞高剂量组与空白组比较(p0.05),差别无统计学意义。(3)肠瑞高剂量组分别与西药、中药阳性对照组比较(P㩳0.05),差别有统计学意义;而肠瑞中、低剂量组分别与阳性对照组比较,差别无统计学意义。说明肠瑞高剂量组能够明显抑制蛋白激酶A(PKA)的含量。结论:肠瑞灌肠剂能够迅速改善放射性直肠炎小鼠的一般状况,及时恢复进食与体重,及时保护和修复肠粘膜,促进溃疡愈合,能显著改善光镜下病理变化及结构异常。以炎症因子为靶点,从肠瑞灌肠剂通过降低蛋白激酶A(PKA)的含量进而下调IL-1β、NF-ΚB信号表达,从而明显抑制VEGF表达,达到控制炎症反应、止血,促进肠粘膜修复的目的,证实了“炎症诱导血管新生”的假说。高剂量肠瑞灌肠剂治疗小鼠放射性直肠炎方面疗效显著。
[Abstract]:Objective: Based on the "inflammatory induced angiogenesis" hypothesis from NF- kappa B and VEGF signaling pathway to study the molecular mechanism of enteral enema in the treatment of radionuclitis, to provide new drug targets and new ways of thinking for the clinical treatment of radionitis. Methods: three, 7.2g/Kg, 3.6g/Kg, 1.8g/Kg, middle and low. Dose enema 6MV- X-ray linear accelerator single dose 27Gy irradiation of radionuclitis C57/BL mice, respectively, dexamethasone combined with gentamicin, eight flavors of tin as the control, enema administration, once a day for 2 weeks, observation of the general state of the mice, feces, rectum, HE staining to observe the pathological changes, immunohistochemical analysis of radioactivity The expression of NF- kappa B, VEGF in rectal tissue of proctitis mice, PCR detection of NF- kappa B in rectal tissue, VEGF expression and the content of PKA in serum of mice detected by ELISA, and to explore the relationship between the three groups. Results: 1. the effect of enteral enema on the rectal histopathology of C57/BL mice of radionuclitis (1) comparison of the classification of rectal mucosal lesions under light microscope, illumination. The mucosa of each group changed in varying degrees, and compared with the normal mucosa of the blank group (P? 0.05), there were statistical differences, suggesting that the model was successful. (2) there was no statistical difference between the model group and the traditional Chinese medicine control group and the intestinal Rui lower agent group (P0.05), but the difference was statistically significant compared with the treatment group, medium dose group and Western medicine control group (P? 0.05). It showed that the dosage of Chang Rui high school and the control western medicine could improve the inflammation of rectal mucosa tissue. (3) the difference of the high dose group and the western medicine positive control group (P? 0.05) was statistically significant, indicating that the effect of high dose enteric enema was better than that of the control group of dexamethasone plus gentamicin and the eight flavour of the traditional Chinese medicine. Comparison (P? 0.05), the difference was statistically significant, indicating that the therapeutic effect of high dose enteric enema on radionitis is better than low dose.2. immunohistochemical analysis of the effect of intestinal rayon enema on the expression of NF- kappa B and VEGF in rectal tissue of C57/BL mice with radionuclitis (1) a small amount of NF- kappa B, VEGF expression, and the consideration of enema in the blank control group NF- kappa B, VEGF expression appeared in all groups after irradiation, but compared with the blank group (P? 0.05), the difference was statistically significant. It indicated that NF- kappa B, VEGF expression was produced by radiation exposure of the rectal mucosa. (2) the treatment group was compared with the model group (P? 0.05), the difference was statistically significant, indicating the intestinal Rui group, The expression of NF- kappa B and VEGF in the group of dexamethasone Heqing and the group of eight stannic groups could inhibit the expression of kappa kappa B and VEGF. The difference was statistically significant between the intestinal reagents group and the western medicine positive control group (P? 0.05), and the middle dose group was compared with the positive control group of traditional Chinese medicine (P? 0.05), and the difference was statistically significant. The group can significantly reduce the NF- kappa B and VEGF expression in rectal tissue of radionuclitis, and the effect of the intestinal Rui middle dose group is better than that of the western medicine positive control group. The effect of.3.PCR detection on the expression of NF- kappa B and VEGF in rectal tissue of C57/BL mice of radioactive proctitis (1) shows that a small amount of NF in the blank control group shows a very small amount of NF. - kappa B, VEGF expression (considering the irritation of the enema device, neglecting), the other groups had different degrees of NF- kappa B, VEGF expression, compared with the blank group, the difference was statistically significant (P? 0.05), suggesting that the normal rat rectum mucosa was very small or non expression of NF- kappa B, VEGF, and the rectal mucosa was irradiated with NF- kappa B, VEGF base occurred. (2) the expression of NF- kappa B and VEGF in the model group was significantly higher than that in other groups (P? 0.05), indicating the changes in the expression of two. (3) the treatment was high, the medium dose group NF- kappa B, the VEGF expression decreased significantly, and the difference was statistically significant compared with the western medicine control group and the traditional Chinese medicine control group (P). 0.05), indicating that the enteric enema is high, the medium dose group can significantly down regulate the expression of NF- kappa B and VEGF in rectum of radionuclitis, and thus can control the inflammation and inhibit the effect of the neovascularization.4.ELISA method on the content of protein kinase A (PKA) in the serum of C57/BL mice of radionuclitis (1) blank control group and each treatment group and model The difference was statistically significant (P0.05). (2) the model group, the western medicine, the Chinese medicine positive control group and the Chang Ruizhong, the low dose group and the blank group were compared with the blank group, and the difference was statistically significant. The difference between the high dose group and the blank group (P0.05) was not statistically significant. (3) the intestinal RRH dose group was positive for the western medicine and the Chinese medicine positive. Compared with the control group (P? 0.05), the difference was statistically significant, but the low dose group had no statistically significant difference compared with the positive control group. It was indicated that the content of protein kinase A (PKA) could be significantly inhibited by the intestinal RI dose group. Conclusion: the intestinal reagents can quickly improve the general condition of the radionuclitis mice and restore the food in time. And weight, timely protection and repair of intestinal mucosa, promote ulcer healing, can significantly improve the pathological changes and structural abnormalities under the light microscope. Targeting the inflammatory factors, the expression of IL-1 beta and NF- B signal is down regulated by the intestinal reagents by reducing the protein kinase A (PKA) content, thus obviously inhibiting the expression of VEGF, controlling the inflammatory reaction, stopping bleeding and promoting the effect. The purpose of intestinal mucosal repair is to confirm the hypothesis of "inflammation inducing angiogenesis". High dose enema has a significant effect on the treatment of radiation proctitis in mice.
【学位授予单位】：山西省中医药研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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