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益气活血解毒法抗溃疡性结肠炎复发的抗炎及修复作用机制研究
【作者】 于玫；
【导师】 王新月；
【作者基本信息】 北京中医药大学 ， 中医内科学， 2008， 博士
【摘要】 1研究目的本课题以溃疡性结肠炎(UC)慢性复发型活动期和慢性持续型患者作为研究对象,依据严格的随机、对照原则,以益气活血解毒为法的溃结复发方治疗,对其进行临床疗效及抗复发情况评价,同时结合动物实验研究从抑制黏膜炎症损伤、促进黏膜修复重建角度探讨其抗复发作用机制,寻找中医药作用的靶点,进一步明确其抗UC复发的作用机制,确立中药抗UC复发治疗的新思路和方法,为中药抗UC复发寻找理论根据。2研究方法2.1临床疗效及抗复发疗效评价研究本课题依据相关标准,进行严格的临床研究设计,结合具体临床实际,采用随机、阳性对照的方法,以东直门医院、301医院、306医院的门诊和住院患者为研究对象,选择溃疡性结肠炎慢性复发型活动期和慢性持续型病例85例,治疗组予益气活血解毒的溃结复发方(生黄芪30g、炒白术10g、赤白芍各10g、当归10g、炒五灵脂10g、生蒲黄10g、黄连10g、黄柏10g、焦槟榔10g、煨木香10g、连翘20g)加减治疗,对照组予柳氮磺胺吡啶(SASP)(4～6g/d)治疗。疗程均为3个月,对部分病例随访6个月,主要观察:临床综合疗效,复发率,中医证候疗效,临床活动指数、内镜指数的变化。2.2抗复发作用机制研究2.2.1抗复发作用机制临床研究本课题依据相关标准,进行严格的临床研究设计,结合具体临床实际,采用随机、阳性对照的方法,以东直门医院、301医院、306医院的门诊和住院患者为研究对象,选择溃疡性结肠炎慢性复发型活动期和慢性持续型病例85例,治疗组予益气活血解毒的溃结复发方加减治疗,对照组予柳氮磺胺吡啶(SASP)(4～6g/d)治疗。疗程均为3个月,对部分病例随访6个月,主要观察两组治疗前后及随访时的指标变化。主要指标:①血清肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)及血浆白细胞介素-13(IL-13)含量变化;②结肠黏膜组织表皮生长因子受体(EGFR)及核因子-κBp65蛋白(NF-κB p65)表达水平变化。2.2.2抗复发作用机制动物实验研究采用三硝基苯磺酸(TNBS)/乙醇法造模,将SD大鼠随机分为四组即正常组、模型组、中药治疗组、西药对照组。中药灌胃为溃结复发方原方,以奥沙拉嗪(畅美)为阳性对照药,正常组及模型组同步同量以蒸馏水灌胃作空白对照。疗程为30天,分别于给药10天、给药30天、停药10天部分杀检取材。观察以下指标:①一般情况及体重变化;②结肠黏膜病理形态学变化;③血清TNF-α、IL-8含量变化;④结肠黏膜组织EGF、IL-13、NF-κBp65及IL-1βmRNA表达水平变化。3结果3.1疗效及抗复发作用评价①治疗3个月后,治疗组临床综合疗效优于对照组(P<0.05),两组完全缓解率(67.44%vs42.86%)及总有效率(90.7%vs76.2%)亦经统计学检验,存在显著差异(P<0.05)。②疗程结束后对两组经治疗后完全缓解及有效的患者(治疗组39例,对照组32例)随访6个月,调查其复发情况。治疗组6个月复发率明显低于对照组(7.69%vs40.63%),经统计学检验,存在极显著差异(P<0.01)。③治疗组对中医证候积分的近期及远期改善均优于对照组(P<0.01)。④治疗组在临床活动指数改善方面,近期疗效及远期疗效均优于对照组(P<0.05vsP<0.01)。⑤治疗组在内镜指数改善方面,远期疗效优于对照组(P<0.01)。⑥治疗组在3个月的疗程中不良反应率为0,明显低于对照组(9.38%)。统计学检验有显著差异(P<0.05)。3.2抗复发作用机制研究3.2.1临床研究①炎性因子血清TNF-α、IL-8 UC患者血清TNF-α含量高于正常组(P<0.01),而复发组患者血清TNF-α含量高于未复发组(P<0.01),治疗后及随访时治疗组血清TNF-α含量低于对照组(P<0.05);UC患者血清IL-8含量高于正常组(P<0.01),复发组患者血清IL-8含量与未复发组无显著差异(P>0.05),虽然治疗后治疗组血清IL-8含量低于对照组(P<0.05),但两组在随访时含量无显著差异(P>0.05)。②转录调控因子结肠组织NF-κBp65 UC患者结肠NF-κBp65的表达水平高于正常组(P<0.01),复发组患者结肠NF-κBp65的表达水平与未复发组比较,无显著差异(P>0.05),治疗组在治疗后、随访时结肠NF-κBp65的表达水平与对照组比较,皆无显著差异(P>0.05)。③抗炎因子血浆IL-13 UC患者血浆IL-13含量低于正常组(P<0.01),而复发组血浆IL-13含量低于未复发组(P<0.01),治疗后及随访时,治疗组血浆IL-13含量皆高于对照组(P<0.05)。④黏膜修复因子结肠黏膜EGFR UC患者结肠EGFR表达水平高于正常组(P<0.01),而复发组黏膜EGFR表达水平低于未复发组(P<0.01),治疗后及随访时,治疗组黏膜组织EGFR表达水平皆高于对照组(P<0.05vsP<0.01)。3.2.2动物实验研究①一般情况、体重变化及黏膜形态学方面在改善大鼠一般情况及恢复体重方面,中药组从急性期即开始起作用,并在自主活动、皮毛光泽度改善方面早于西药组,可一直持续至损伤修复期。在抑制局部黏膜损伤及促进黏膜修复方面,中药抑制急性损伤效果较西药组略差,在炎症后期促进肉芽组织增生和肉芽肿形成方面优于西药组,恢复期两治疗组促进损伤修复程度差异不明显。②炎性因子血清TNF-α、IL-8在治疗10天时,两治疗组皆可明显降低TNF-α含量,组间无显著差异(P>0.05);治疗30天及停药10天时中药组降低TNF-α含量作用优于西药组(P<0.01vsP<0.05);三个阶段中药组含量与正常组皆无显著差异(P>0.05)。治疗10天、治疗30天及停药10天时,两治疗组皆可降低血清IL-8含量(P<0.01或P<0.05),但中药组在作用效果方面较西药组无显著差异(P>0.05)。③炎性因子结肠组织IL-1βmRNA治疗10天及治疗30天时,两治疗组皆可降低IL-1βmRNA在结肠局部的表达水平,组间无显著差异(P>0.05);但停药10天时,中药组降低IL-1βmRNA表达水平的效果优于西药组(P<0.01)。④抗炎性因子结肠组织IL-13治疗10天时中药组可明显提高IL-13在结肠黏膜局部的表达水平,与西药组存在极显著差异(P<0.01);但治疗30天及停药10天时作用与西药组比较,无显著差异(P>0.05)。⑤转录调控因子结肠组织NF-κBp65三阶段两治疗组皆可降低NF-κBp65在结肠黏膜的表达水平,与正常组比较,无显著差异(P>0.05);两组间作用效果亦无显著差异(P>0.05)。⑥黏膜修复因子结肠黏膜EGF治疗10天时中药组提高结肠黏膜局部EGF表达水平的作用优于西药组(P<0.01);治疗30天时作用逊于西药组(P<0.01);而停药10天时,中药组EGF表达水平升高,效果优于西药组(P<0.05)。4结论以益气活血解毒立法组成的溃结复发方治疗慢性复发型活动期和慢性持续型UC患者取得了满意的临床综合疗效。与柳氮磺胺吡啶对照组比较,本方有效地阻抑了愈后复发。其中本方在提高完全缓解率及总有效率、改善中医证候积分、临床活动指数、内镜指数方面作用皆优于后者,且未出现明显的毒副作用。其抗复发作用机制可能是:①抑制外周血炎性因子TNF-α及IL-1β的过度表达,减少炎性因子的大量合成,阻止炎性细胞趋化和募集,减轻黏膜局部炎性浸润;②提高外周血抗炎性因子IL-13的表达水平,以下调局部免疫反应状态,减轻黏膜炎症损伤;③增强黏膜局部EGF及EGFR的表达水平,早期即可加强黏膜上皮细胞增殖、减少凋亡,促进后期损伤黏膜的修复功能。
【Abstract】 ObjectiveUlcerative colitis (chronic nonspecific ulcerative colitis) is a disease of digestive tract characterised by chronic inflammation and ulceration of colonic mucous membrane. Clinical manifestation: mucus, pus and blood stool;diarrhea; abdominal pain; tenesmus and so on. It has anfractousive pathogenesy, which they affect each other and even emerge vicious cycle. It causes UC easily to relapse and last a very long period. At present, aminosalicylic acid, cortex steroid hormone and immunomodulator is the effective western medicine. In active stage of ulcerative colitis,these medicine can quickly control the symptoms.The clinical remission rate is high.But the disease is easily recur after drug withdrawal.And side reaction is multi with long-term medication, which causes very low compliance.Professor Wang Xinyue, my tutor who studies UC based on the theory of Traditional Chinese Medicine and Modern Medicine, expound it accurately. Kuijiefufafang is my tutor’s proved recipe based on treating with profiting Qi, promoting blood flow and detoxifying Du. The topic evaluate its therapeutic and resisting relapse effect. It meanwhile investigates the mechanism of resisting relapse effect from the view of restraining the reaction of inflammatory injury, promoting the repair of colonic mucous membrane combining with animal experiment. We deeply analyze the mechanism of repressing recur after henosis in order to offer the objective evidence of reasonably selecting the treating method and medicine.MethodsThe CR study was strictly designed under correlated standard. In combination with actual clinical situation, adopting random positive control method, on the basis of diagnosis criterion established on Inflammatory Bowel Disease Seminar at chengdu in 2000, 85 case of chronic recurrent type in UC active phase and chronic K/o type was selected from out-patient clinic (OPD) and ward of Dongzhimen Hospital, 301 Hospital, 306 Hospital, treated group received tutor’s proved recipe (parching excrementum pteropi 10g, raw pollen typhae 10g, angelicae 10g, red and white peony root 10g apiece, charred semen arecae 10g, roasted radix aucklandiae 10g, coptidis rhizoma 10g, amoorcorn tree bark 10g, raw astragali 30g, forsythia suspensa 20g, parching atractylodes macrocephala 10g), control group received sulfasalazine (SASP), (4-6g/d). Both treatment course was 3 months, and attended by 6 months partly, mainly observed index: 1. Therapeutic effect, including clinical general effect, change of TCM syndrome, the change of clinical activity index (CAI), endoscopic index (EI) and relapse rate. 2.serum Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8), serocym interleukin-13 (IL-13). 3.colon mucous membrane Nuclear factor-κBp65 (NF-κBp65) and Epidermal growth factor receptor (EGFR).In animal experiment , the rats were randomly divided into 4 groups: normal group, model group, treated group and Olsalazine sodium group( the controlled group). The last 3 groups are UC rats induced by 2,4,6-trinitrobenzenesulfonic Acid (TNBS) /ethanol mixture. We took serum and colon tissues from 1/3 of the animals every time after giving medicine for 10 days, 30 days and 10 days after stopping treatment. Mainly observed index are: 1.the effects on living state of the rats, the general behaviors and the weight involved. 2. the local pathological change, both gross and microscopic observations. 3. serum TNF-αand IL-8. 4. mucous membrane NF-κBp65, IL-13, Interleukin-1βmessenger Ribonucleic acid (IL-1βmRNA) and Epidermal growth factor (EGF).ResultsEvaluation of therapeutic effect and resisting relapse effect①Clinical general therapeutic effect of treated group is better than control group;Including the total remission percentage of two group, total effective percentage of two group, the statistic significant is difference, treated group is obviously better than control group.②After treatment, the relapse rate in six months of treated group is obviously lower than that of control group.③Syndrome scores of treated group was dropped apparently, the forward improvement of treated group is better than control group.④CAI and EI of treated group significantly dropped after treatment, the forward improvement of control group was better than control group (P<0.01).⑤There was no obvious ill-effect on treated group, which is obviously lower than control group.Researching on mechanism of resisting relapseClinical index:①Serum TNF-αconcentration of patients in UC active phase were remarkablely higher than normal value.Serum TNF-αconcentration in relapse phase of UC patients were significantly higher than that of not relapse patients.After treatment and after six months of attending, serum TNF-αconcentration of treated group were significantly lower than that of control group.②Serum IL-8 concentration of patients in UC active phase were remarkablely higher than normal value.Serum IL-8 concentration in relapse phase of UC patients had no difference from that of not relapse patients.After treatment, serum IL-8 concentration of treated group were significantly lower than that of control group. But after six months of attending, the value of two sets were similar.③Serocym IL-13 concentration of patients in UC active phase were remarkablely lower than normal value.Serocym IL-13 concentration in relapse phase of UC patients were significantly lower than that of not relapse patients.After treatment and six months of attending, Serocym IL-13 concentration of treated group were significantly higher than that of control group. ④Colon mucous NF-κBp65 concentration of patients in UC active phase were remarkablely higher than normal value.Colon mucous NF-κBp65 concentration in relapse phase of UC patients had no difference from that of not relapse patients.After treatment and six months of attending, the value of two sets were similar.⑤Colon mucous EGFR concentration of patients in UC active phase were remarkablely higher than normal value.Colon mucous EGFR concentration in relapse phase of UC patients were significantly lower than that of not relapse patients. After treatment and after six months of attending, colon mucous EGFR concentration of treated group were significantly higher than that of control group.Animal experiment index:①On the aspect of improving the living state, treated group could have obvious effect since the acute period. And improving independence activity, glossiness of coat emerged earlier than control group. Its effect could last to the chronic period and injury recovery period. As the observation of the local colon mucosa, both treated and control group had the effects of inhibiting the injury and and improving the plerosis of mucosa tissues. In acute period, effect of the former inferiored to the latter. But treated group’s effect of improving gt hyperblastosis and forming granuloma surpassed that of control group.②Serum TNF-αconcentration in both treated and control group cut down at the time of treating 10 days. Their effects were similar. Effect of treated group at the time of 30 days and 10 days after stopping treatment was significantly better than control group. Among three periods, value of Serum TNF-αconcentration in treated group was similar to normal group.③Serum IL-8 concentration in both treated group and control group cut down in three periods. Their effects were similar.④Colon mucous IL-1βmRNA concentration in both treated and control group cut down at the time of treating 10 days and 30 days. Their effects were similar. But colon mucous IL-1βmRNA concentration in treated group was significantly lower than that in control group.⑤Colon mucous IL-13 concentration in treated group was significantly higher than that in control group. But effects of cutting down colon mucous IL-13 concentration in both groups at the time of 30 days and 10 days after stopping treatment were similar.⑥Colon mucous NF-κBp65 concentration cut down in both groups at three periods. And effects in both groups were similar.⑦Colon mucous EGF concentration in treated group is significantly higher than that in control group at the time of 10 days. Effect of raising colon mucous EGF concentration in treated group inferiored to that in group at the time of 30 days. Meanwhile, at the time of 10 days after stopping treatment, colon mucous EGF concentration in treated group is significantly higher than that in control group. ConclusionWe have got satisfied therapeutic effect in treating UC by using the methods of profiting Qi, promoting blood flow and detoxifying Du guided by determination of treatment based on differentiation of syndromes. And the therapeutic effect was obviously better than SASP. Especially in the TCM syndrome, CAI, EI, and resisting relapse, showed its superiority. Compared with western medicine, it had no obvious ill-effect. The following is the contra-relapse mechanism of Chinese medicine: 1. Restraining the multi expression of TNF-αand IL-1β, preventing the aggregation of inflamed cell; 2. Raising the expression of IL-13, down regulating the condition of local immunity; 3. Raising the expression of Colon mucous EGF and EGFR since nonage so as to enhance mucosa epithelial cell proliferation, lessen apoptosis, promoting the repair of injuried mucous membrane in later stage.