广痛消泡沫气雾剂治疗肛门水肿模型的实验研究及炎性痔的临床观察

【作者】 张媛；

【导师】 赵宝明；

【作者基本信息】 北京中医药大学 ， 中医外科， 2014， 博士

【摘要】 1目的广痛消泡沫气雾剂是导师赵宝明教授开发研制,前期研究已表明该剂型在治疗混合痔、肛裂等肛肠疾病,既有中医药特色,又有外治优势,其剂型在国外也被广泛应用,该药止痛、解痉作用机理已得到证实。混合痔引起的肛门水肿在临床多见,患者痛苦症状明显,急待医者解决。本研究目的通过利用巴豆油致大鼠肛门水肿模型,研究并探讨广痛消泡沫气雾剂对此模型的抗炎、消肿、止痛作用及机制,并从临床观察门诊治疗和术后病理组织两方面,分析探讨广痛消泡沫剂对炎性痔临床症状的治疗作用,以及抑制炎性细胞因子和炎症反应的作用机制。2方法本研究分为三部分。第一部分为文献研究,首先通过归纳混合痔引起肛门水肿的中医病因病机,从宏观上探讨痔的发病学说以及水肿与血管内皮功能的微观认识,总结目前中西医非手术疗法治疗炎性痔的研究进展,从中找到广痛消泡沫剂消肿作用的研究切入点及相关因子的意义。其次以中药外治法治疗炎性痔为主题,进行Meta分析研究,探讨该方法的有效性及安全性。最后通过总结导师赵宝明教授临床经验,回顾该剂型的前期以及汇报本人跟师学习的体会。第二部分为动物实验部分,主要包括实验一巴豆油致SD大鼠肛门水肿模型的建立,实验二主要观察广痛消泡沫气雾剂对大鼠肛门水肿模型的止痛作用,实验三采用酶联免疫吸附法测定TNF-a和IL-6,实验四免疫组化法测iNOS,从以上四方面来证实广痛消泡沫气雾剂对大鼠肛门水肿有抗炎、止痛、消肿的作用。第三部分为临床观察部分,首先通过随机对照、单盲观察广痛消泡沫气雾剂治疗炎性痔门诊患者疗效,用药前后症状积分的改善情况,其次对炎性痔患者术前给予广痛消泡沫气雾剂治疗,术后用免疫组化法测定痔组织内iNOS、TNF-α、IL-6含量的变化等,客观反映该剂型的抗炎作用机制。3结果3.1动物实验结果3.1.1巴豆油致大鼠肛门水肿模型通过巴豆油致SD大鼠肛门肿胀的形态改变和病理组织切片,提示SD大鼠肛门肿胀模型符合急性炎症改变,与痔疮急性发作时炎性改变相似。广痛消泡沫气雾剂组的组织切片中肛门近端黏膜上皮及腺体从造模后缺失到给药后逐渐修复完整,粘膜下层水肿、血管充血、扩张程度也较模型组明显减轻。3.1.2广痛消泡沫气雾剂对大鼠肛门水肿模型的止痛作用各组造模前6h机械痛阈值和热痛阈值具有可比性。造模后6h各组间机械痛阈值比较P<0.05,差异具有统计学意义,提示各组间有差异。阳性对照组、广痛消泡沫气雾剂大剂量组、中剂量组给药前后机械性收缩反射阈值变化明显,且用药前后比较,P<0.05,差异显著并具有统计学意义。末次给药6h后MWT值均正态分布,且方差齐。末次给药后6h模型组、阴性对照组、广痛消泡沫气雾剂大剂量组和小剂量组与空白组MWT值比较,P<0.05,差异具有统计学意义；阳性性对照组与广痛消中剂量组比较,P>0.05,差异无统计学意义。造模前6h各组间TWL值比较P>0.05,无统计学意义,提示各组热痛阈值具有可比性。阳性对照组、广痛消泡沫气雾剂大剂量组和中剂量组给药前后热痛阈值变化明显,P<0.05,差异显著并具有统计学意义。末次给药后6h,空白组、广痛消大剂量组与阳性对照组比较,P>0.05,差异无统计学意义。3.1.3酶联免疫吸附法测定TNF-a和IL-6含量通过Elisa法测定动物肛周组织及直肠下段,正常组与模型组中’TNF-a和IL-6含量有明显差异,P<0.05,具有统计学意义,说明巴豆油致SD大鼠肛门水肿模型符合炎症改变。广痛消泡沫气雾剂各组与消炎痛栓组的TNF-a和IL-6含量无明显差异,P>0.05,差异无统计学意义；广痛消泡沫气雾剂各组之间两种因子的含量两两比较无明显差异,P>0.05,暂不能说明各个剂量无量效关系。3.1.4免疫组化法iNOS的表达通过免疫组化法观察大鼠肛周组织及直肠下段,广痛消泡沫气雾剂各剂量组iNOS的表达均少于模型组,较阳性对照组增多,以小剂量组增多明显。测定免疫组化染色光密度值,广痛消泡沫气雾剂各剂量组与模型组、空白组无统计学差异,P>0.05,暂不能说明广痛消泡沫气雾剂对iNOS的表达无抑制作用。3.2临床观察结果3.2.1门诊患者症状积分改变三组患者用药前性别、年龄、病程、混合痔分期,以及用药前疼痛、便血、坠痛、脱垂、痔黏膜糜烂情况、痔肿胀大小情况均具有可比性。用药治疗7天后,广痛消组和消炎痛组疼痛VAS积分明显降低；广痛消泡沫气雾剂组用药前便血症状积分最高,用药后便血积分最低；广痛消组坠痛积分下降幅度明显且第7天时积分最低；广痛消组对痔核黏膜的修复作用明显优于其它两组,普济痔疮栓对痔核表面的改善作用优于消炎痛栓组；广痛消泡沫气雾剂对痔核大小的消肿作用起效时间较消炎痛栓组慢,对痔核肿胀面积的改变与消炎痛栓组结果相一致,差异无统计学意义。三组总体有效率广痛消组(98.5%)明显高于消炎痛栓组(96.8%)和普济痔疮栓组(90.1%)。3.2.2免疫组化法测定IL-6、TNF-a、iNOS的表达利用免疫组化法观察水肿痔组织内的IL-6、TNF-a、iNOS的表达：三组图片中阳性表达最多的为空白组,表达较少的为广痛消泡沫气雾剂组,最少的是消炎痛栓组。广痛消泡沫气雾剂组与消炎痛栓组IL-6的免疫组化染色光密度值比较,P>0.05, TNF-a和iNOS的免疫组化染色光密度值比较P<0.05；与空白组IL-6、iNOS的免疫组化染色光密度值比较,P>0.05, TNF-a的OD值比较P<0.05。4结论广痛消泡沫气雾剂对于治疗巴豆油致SD大鼠肛门水肿有确切的抗炎、消肿、止痛的作用。临床观察广痛泡沫气雾剂对炎性痔引起的疼痛、便血等症状有明显的止痛、消肿、修复痔核黏膜、提升痔核位置的作用,总体效果明显优于消炎痛栓组和普济痔疮栓组。而且中药组在改善便血和修复痔核黏膜方面明显优于消炎痛栓组。广痛消泡沫气雾剂抗炎消肿、止痛的作用机制主要是该药能够降低TNF-a和IL-6的表达,抑制了iNOS的激活,减少NO的释放,从而达到抗炎、消肿、止痛的目的,其量效关系值得进一步探讨。更多还原

【Abstract】 GuangTongXiao Foam Aerosol(GTX) is invented by professor Zhao Baoming. Previous studies have shown the dosage form in the treatment of mixed hemorrhoids, anal fissure and other anorectal diseases has both features of traditional Chinese medicine and external treatment advantage. The dosage form has been widely used in foreign countries. GTX’s mechanism of relieving pain and spasmolysis has been confirmed. Anal edema caused by mixed hemorrhoid in clinical and pain in patients with symptoms,is an pressures for doctors to solve.The purpose of this study is repeating anal edema model in rats by croton oil, researching and discussing its anti-inflammation, detumescence and analgesic effects and mechanism on the anus edema model. And from the two aspects of conservative treatment and surgical treatment of clinical observation, to analyze that GTX can improve the situation of anal edema caused by inflammatory mixed hemorrhoid symptoms, to discuss inhibition of inflammatory cytokines and the mechanism of action of inflammatory mediators.2MethodsThis study is divided into three parts.The first part is the literature research. First concluding TCM etiology and pathogenesis of anal edema caused by mixed hemorrhoid, Western medicine theories of hemorrhoids, microscopic understanding of edema and vascular endothelial function, summarizing the current research progress of non-surgical therapy of traditional Chinese and western medicine treatment of inflammatory mixed hemorrhoid, these will help us find the point of animal experiment and clinical observation research on GTX foaming agent and the related factors of the meaning. Then including a meta analysis of external treatment of traditional Chinese medicine to inflammatory hemorrhoids. The last part of the literature research by summarizing the tutor professor Zhao Baoming’s clinical experiences, we can remember the review of GTX in the early period and my learning experiences. The second part is animal experiment parts. It mainly includes the experiment of building SD rats anal edema model by croton oil, the second is observing how GTX can treat the pain of the model, the third one is using enzyme-linked immune sorbent assay to measure TNF-a and IL-6,the last is using immunohisto-chemistry to measure iNOS. From these four aspects to prove GTX foam aerosol has the effect of analgesia, anti-inflammatory and detumescence to anal edema of rat model of inflammatory pain.The third part is the part of clinical observation. The first is an randomized controlled single-blind trial, to observe GTX foam aerosol using as a conservative treatment to outpatients who has the symptoms of anal edema by mixed hemorrhoids. and to record the symptoms integral before and after medication. The second clinical observation is using GTX foam aerosol to anal edema patients caused by mixed hemorrhoid before surgery, then measuring the content of iNOS,IL-6and TNF-a in postoperative hemorrhoid tissues and comparing them.3Results3.1the results of animal experiments3.1.1anal swelling model of SD rats caused by croton oilAfter the morphological changes and pathological section from SD rats which is anal swelling caused by croton oil, it can infer that the change of the model is in accordance with acute inflammatory change and also similar to acute onset hemorrhoids inflammatory change. The biopsy of GTX group is significantly changed, especially for mucosal epithelium and gland of the proximal anus. The lack of epithelium and glands just after molding can gradually complete after using GTX. The edema of submucosa, congestion of blood vessel. the expansion degree is also significantly reduce which is compared with model group.3.1.2analgesic effect of GTX foam aerosol to anal edema of rat modelMechanical withdrawal threshold and thermal withdrawal latency are comparable for each group before six hours.Mechanical withdrawal threshold comparison of each group six hours after building the model,P<0.05, the difference is statistically significant. It suggests there are differences between groups. Positive control group, the large and the middle dose of GTX group are changed after using medication, P<0.05, their significant difference is statistically significant. Six hours after giving medicine. MWT of each group are normal distribution, and variance. Model group, negative control group, GTX foam aerosol high-dose group and small dose group’s MWT value compared with the blank group, P<0.05, the difference is statistically significant. Positive control group and GTX middle-dose groups compared, P>0.05, there was no statistically significant difference.Thermal withdrawal latency comparison of each group six hours before building the model,P>0.05, no statistical significance, it prompts that each thermal pain threshold comparable. Thermal withdrawal latency of positive control group, GTX foam aerosol large dose group and middle dose group are changed significantly after using medicine,P<0.05, significant difference is statistically significant.Six hours after the treatment,the blank group and the GTX foam aerosol high-dose group compared with positive control group,P>0.05, there was no statistically significant difference.3.1.3the content of TNF-a and IL-6by using Elisa methodWith Elisa method, TNF-a and IL-6content of the normal group and model group serum have obvious differences, P<0.05. It is statistically significant. Anal edema of SD rats model by croton oil conforms to the inflammatory change.GTX has no obvious difference with indomethacin suppository group, P>0.05.It shows that indomethacin suppository effect is more obvious than GTX. The three groups of GTX compare with each other have no significant differences, P>0.05, temporarily it can’t explain each dose limit effect relationship. 3.1.4the content of iNOS by using immunohisto chemical methodUsing immunohistochemical method to observe the rats’crissum tissues, the expression of iNOS of each does GTX foam aerosol group are less than model group, more than control group. Especially the small dosage group increases obviously. Each dose GTX group comparing with model group and the blank group, there is no statistical difference by the study optical density value of immunohistochemical. GTX foam aerosol cannot infer it has no inhibition of iNOS.3.2the results of clinical observations3.2.1the clinic symptoms integral changesTo observe the clinic symptoms integral changes:the sex, age, course, mixed hemorrhoid stages of three groups are comparable, so are the pain, bloody, bearing-down pain, the prolapsed, the erosion of hemorrhoids mucosa and swelling of the situation before using treatments. GTX and indomethacin groups’scores of VAS decreased obviously after seven days’ therapy. The bloody symptom is the most obvious before using GTX foam aerosol, then it becomes the lowest one of the three groups. After using GTX for seven days,bearing-down pain decreased obviously than others. And GTX group for repairing hemorrhoids mucosal is superior to other two groups, the PUJI hemorrhoids suppository is better than indomethacin one.GTX is slower than indomethacin bolt on the size of the haemorrhoids detumescence effect, and is consistent with the changes of haemorrhoids swelling area.There is no statistically significant difference between these two groups.The overall efficient of GTX group (98.5%) is obviously higher than indomethacin suppository group (96.8%), and PUJI group (90.1%).3.2.2the expression of IL-6, TNF-a and iNOSby using immunohistochemical methodUsing immunohistochemical method to observe the expression of IL-6, TNF-a and iNOS from edema hemorrhoid tissues. The blank group are more positive expressed in three sets of pictures, GTX foam aerosol express less, the least is indomethacin suppository group.The immunohistochemical study optical density value comparison:GTX foam aerosol group compare with indomethacin suppository group for IL-6, P>0.05, for TNF-a and iNOS, P<0.05.Comparing with the blank group forIL-6and iNOS, P>0.05,for TNF-a,P<0.05.4ConclusionGuangTongXiao foam aerosol has the exact anti-inflammation, detumescence and analgesic effect in treating anal swelling SD rats model caused by croton oil.It also can stop the pain and reduce the swelling, repair the haemorrhoids mucosa, lift the location of haemorrhoids position of anal edema caused by mixed hemorrhoid,such as anal edema, pain, bloody and other symptoms. And the effect is better than indomethacin suppository group and PuJi group. We can conclude that Chinese traditional medicine in improving the bloody symptom and repairing the haemorrhoids mucosa is obviously better than indomethacin suppository. The mechanism of anti-inflammatory swelling and stoping the pain may be related to reducing the expression of TNF-a and IL-6for GuangTongXiao foam aerosol,Inhibiting the activation of iNOS,reducing the release of NO,so as to achieving the purpose of anti-inflammation, detumescence and analgesia. And the concentration-response relationships of each does of GTX deserve to be further discussed.更多还原