【作者】 王峥；

【导师】 谢桂权；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2010， 博士

【摘要】 目的本研究依据肾病综合征湿热证与感染关系密切以及前人对湿热证的研究基础,选择原发性肾病综合征湿热证为对象,从分子生物学水平探讨观察原发性肾病综合征湿热证与血清抗炎因子IL-4、IL-10、IL-13的相关性,对促进和揭示原发性肾病综合征湿热证的实质、湿热病因部分病理机制及清热利湿中药的作用机制研究具有一定理论意义与应用价值。方法本课题主要采用前瞻性、随机对照的临床研究方法,应用双抗体夹心酶联免疫吸附法(ELISA法)和荧光定量PCR的实验检测方法,通过对原发性肾病综合征湿热证、非湿热证患者体内主要抗炎因子IL-4、IL-10、IL-13的水平检测,利用SPSS软件进行统计处理,系统分析肾病综合征湿热证与抗炎细胞因子IL-4、IL-10、IL-13水平的关系。再通过对原发性肾病综合征湿热证患者经清热利湿药物黄葵胶囊治疗四周后,观察湿热证与抗炎细胞因子水平的变化情况,及清热利湿药物黄葵胶囊治疗PNS湿热证的临床疗效,利用SPSS软件进行统计处理,综合分析湿热证与抗炎症因子的相关性,并进一步分析具有清热利湿功效的黄葵胶囊治疗原发性肾病综合征湿热证的作用及机理。结果(一)PNS湿热证抗炎因子水平的实验研究按纳入标准选取肾综湿热证患者40例,非湿热证患者20例,共60例。经统计学检验,两组在性别、年龄、病理资料方面无显著性差异(P>0.05),具有可比性。采用双抗体夹心酶联免疫吸附法(ELISA法)检测抗炎因子IL-4、IL-10、IL-13的水平,荧光定量PCR法检测外周血单个核细胞中IL-13mRNA表达。结果如下：湿热证组患者体内的IL-4含量为45.67±8.27 pg/ml,高于非湿热证组患者体内的IL-4的含量32.73±9.01 pg/ml,经统计学检验有显著性差异(P<0.01)。湿热证组患者体内IL-10含量为29.97±4.22 pg/ml,高于非湿热证组患者体内IL-10的含量19.45±4.71 pg/ml,经统计学检验有显著性差异(P<0.01)。湿热证组患者体内IL-13含为25.92±6.94 pg/ml,高于非湿热证组患者体内IL-13的含量14.19±7.36 pg/ml,经统计学检验有显著性差异(P<0.01)。湿热证组患者的外周血单个核细胞中IL-13mRNA水平为0.65±0.11,明显高于非湿热证组IL-13mRNA水平0.35±0.09,经统计学检验有显著性差异(P<0.05)。与湿热证IL-13水平升高表现相同。湿热证组各患者的湿热证积分分别与IL-4、IL-10、IL-13含量相关性分析呈线性正相关(P<0.01)。(二)黄葵胶囊干预对PNS湿热证临床及抗炎因子水平影响的研究1、对症候的影响用药前治疗组和对照组中医症状积分比较无显著性差异(P>0.05),具有可比性。治疗组疗后对水肿、咽喉肿痛、胸腹痞闷、口干不思饮、口苦、口中粘腻、小溲黄赤、大便粘滞症状有明显改善(P<0.05或P<0.01)。对照组疗后对水肿、口干不思饮、口苦、小溲黄赤、大便粘滞症状显著改善(P<0.01)。治疗后两组中医症状积分组间比较,治疗组对水肿、皮肤疖肿、疮疡、咽喉肿痛、胸腹痞闷、口苦、口中粘腻、小溲黄赤、大便粘滞的改善作用有显著性差异(P<0.05或P<0.01)。表明在改善中医症状方面,治疗组优于对照组。2、对实验室指标的影响湿热证经治疗后,治疗组和对照组患者比较,治疗组的活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、总胆固醇、甘油三脂、低密度脂蛋白及24h尿蛋白定量指标的改善优于对照组(P<0.05或P<0.01)。3、对白细胞总数和超敏C反应蛋白的影响湿热证经治疗后,治疗组和对照组患者血中白细胞总数和超敏C反应蛋白均有所下降,经统计学分析,与疗前比较仅治疗组的差异有显著性意义(P<0.05或P<0.01)；治疗后两组组间比较,治疗组的白细胞总数、超敏C反应蛋白均值的降低更显著(P<0.05或P<0.01)。4、对抗炎因子水平的干预影响治疗组疗后IL-4水平明显下降(30.02±8.22pg/ml),与治疗前水平(45.63±8.30pg/ml)相比,有显著性差异(P<0.01)。对照组疗前后比较无显著性差异(P>0.05)。治疗后两组组间比较,治疗组IL-4含量的下降水平明显高于对照组(P<0.01)。治疗组疗后患者体内IL-10水平降低(19.21±4.78pg/ml),与治疗前水平(30.01±4.19pg/ml)相比,有显著性差异(P<0.01)。对照组疗前后比较无显著性差异(P>0.05)。治疗后两组组间比较,治疗组IL-10降低水平明显高于对照组(P<0.01)。治疗组疗后IL-13水平显著下降(14.11±7.20pg/ml),与治疗前的水平(25.94±6.91pg/ml)相比,有显著性差异(P<0.01)。对照组疗前后比较无显著性差异(P>0.05)。治疗后两组组间比较,治疗组IL-13下降水平明显高于对照组(P<0.01)。经药物治疗后,湿热证治疗组患者外周血单个核细胞中IL-13mRNA表达水平为0.34±0.06,明显低于对照组IL-13mRNA表达水平0.60±0.09,经统计学处理后,有统计学意义(P<0.05)。治疗组患者治疗前后中医症状总积分差值分别与IL-4、IL-10、IL-13水平含量差值相关性分析呈线性正相关(P<0.05)。结论1、由PNS湿热证抗炎因子水平及黄葵胶囊干预对PNS湿热证抗炎因子水平影响的研究,结果显示原发性肾病综合征湿热证患者抗炎因子IL-4、IL-10、IL-13水平异常增高；原发性肾病综合征湿热证与抗炎因子水平的增高具有一定的相关性,湿热证症候积分分别与IL-4、IL-10、IL-13水平呈线性正相关。2、由黄葵胶囊对PNS湿热证临床的干预影响结果显示：清热利湿黄葵胶囊具有下调原发性肾病综合征湿热证患者体内异常增高的抗炎因子水平的作用。3、清热利湿药物黄葵胶囊能较好的缓解患者的湿热症状,改善肾综湿热证患者蛋白尿、高脂血症及高凝状态等,可提高肾综湿热证的临床疗效,减轻肾综病情,其作用机理可能与该药的抗炎、清除抗原等作用有关。更多还原

【Abstract】 ObjectiveThis study based on nephritic syndrome with damp-heat and infection close relationship and the predecessors of the research basis about damp-heat syndrome. From the molecular biology level, observed the relevance of Primary nephrotic syndrome(PNS) Damp-heat syndrome and anti-inflammatory cytokines IL-4, IL-10, IL-13. It has a certain theoretical meaning and application value for Primary nephrotic syndrome Damp-heat syndrome to revealed evidence of Damp-heat syndrome substantial, part of the pathogenesis and mechanism of Chinese medicine about clearing away heat and dampness.MethodsThe main subject of a prospective, randomized, controlled clinical research methods, applied double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and fluorescence detection quantitative PCR experiments, through the primary nephritic syndrome damp, non-damp patients is the main anti-inflammatory factor IL-4, IL-10, IL-13 levels detected using the SPSS software to analyze the data, systems analysis nephritic syndrome damp-heat and anti-inflammatory cytokines IL-4, IL-10, IL-13 Levels. Then through the primary nephritic syndrome patients by clearing away heat and damp of drug capsules for four weeks treated by Huang Kui capsule, observed the change of damp-heat and anti-inflammatory cytokines and evaluated clinical efficacy clearing away damp-heat drug-Huang Kui Capsule in treatment of PNS with damp-heat syndrome Clinical. Use of SPSS software to analyze the data, comprehensive analysis of damp- heat syndrome and related anti-inflammatory factors, and further analysis of the efficacy of Huang Kui Capsule-clearing away damp- heat drug in treatment of primary nephritic syndrome with damp-heat syndrome about the effect and mechanism.Result1. PNS with damp-heat experimental study of anti-inflammatory cytokinesSelect the kidney by the inclusion criteria 40 patients comprehensive damp-heat, non-damp-heat 20 patients, total 60 patients. The difference in the two groups in gender, age, pathological information was no significant difference(P>0.05), comparable. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection of anti-inflammatory factor IL-4, IL-10, IL-13 levels, quantitative PCR, peripheral blood monocyte cells of IL-13mRNA expression. The results are as follows:(1)PNS with damp-heat patients the level of IL-4 was 45.67±8.27 pg/ml, higher than patients of non-damp-heat IL-4-32.73±9.01 pg/ml, the difference was statistically significant(P<0.01).(2) PNS with damp patients the level of IL-10 was 29.97±4.22 pg/ml, higher than the patients of non-damp-heat IL-10-19.45±4.71 pg/ml, the difference was statistically significant(P<0.01).(3)PNS with damp patients the level of IL-13 was 25.92±6.94 pg/ml, higher than the patients of non-damp-heat IL-13-14.19±7.36 pg/ml, the difference was statistically significant(P<0.01).(4)PNS damp-heat patients peripheral blood monocyte cells of IL-13mRNA level of 0.65±0.11, significantly higher than non-damp-heat syndrome group IL-13mRNA level of 0.35±0.09, were statistically significant difference test (P<0.05).In comparison with PNS with damp-heat syndrome elevated levels of IL-13 was the same performance.(5)The group PNS with damp- heat syndrome scores and the level of IL-4, IL-10, IL-13 were correlative (P<0.01).2. Study the effect of Huang Kui Capsule on clinical and inflammatory cytokines in PNS with damp-heat symptoms(1)The impact of the symptomsBefore treatment the comparison between the study group and the control group symptom score was not statistically significant (P>0.05). The study group after treatment edema, sore throat, distension and nausea in chest and belly, dry mouth and not think to drink, bitter mouth, sticky mouth, yellow red urine, sticky stool symptoms improved significantly (P<0.05 or P<0.01). After treatment, there was statistically significant difference between the study group and the control group in clinical symptoms (P<0.05 or P<0.01). These clinical symptoms were oedema, swelling of skin boils, sore, sore throat, distension and nausea in chest and belly, bitter mouth, sticky mouth, yellow red urine, sticky stool. That improve the clinical symptoms, the study group was better than the control group.(2)The impact of laboratory testDamp-heat syndrome after treatment, the difference between the study group and the control group about activated partial thromboplastin time (APTT), fibrinogen (FIB), total cholesterol, triglyceride, low density lipoprotein and 24h urinary protein indicators was better than the control group (P<0.05 or P<0.01).(3)The impact of WBC total and hs-CRPDamp-heat syndrome after treatment, the study group and the control group of patients with white blood cell count and high-sensitivity C-reactive protein were decreased, by statistical analysis, and in comparison with before treatment was significant only in the study group (P<0.05 or P<0.01);after treatment between the two groups, study group, the total number of leukocytes, high sensitive C-reactive protein more significantly reduced the mean (P<0.05 or P<0.01).(4)The impact of inflammatory cytokines levelStudy group, IL-4 levels after treatment were significantly decreased (30.02±8.22pg/ml), in comparison with before (45.63±8.30pg/ml), was significant (P<0.01). The control group before and after treatment showed no significant difference (P>0.05). Between the two groups after treatment, the study group IL-4 level decreased more significantly than control group (P<0.01).Study group, IL-10 levels after treatment were significantly decreased (19.21+4.78pg/ml), in comparison with before (30.01+4.19 pg/ml), was significant (P<0.01). The control group before and after treatment showed no significant difference (P>0.05). Between the two groups after treatment, the study group IL-10 level decreased more significantly than control group (P<0.01).Study group, IL-13 levels after treatment were significantly decreased (14.11±7.20pg/ml), in comparison with before (25.94±6.91pg/ml), was significant (P<0.01). The control group before and after treatment showed no significant difference (P>0.05). Between the two groups after treatment, the study group IL-13 level decreased more significantly than control group (P<0.01).After drug treatment, the damp-heat syndrome group and in peripheral blood monocyte cells of IL-13mRNA expression level of 0.34±0.06, significantly lower than the control group, the expression of IL-13mRNA 0.60±0.09, after treatment was statistically significant(P<0.05).The IL-4、IL-10、IL-13 levels difference between before and after treatment for patients respectively correlated linearly with the syndrome integral difference of Damp-heat syndrome patient in the study group (P<0.05).Conclusion1.The study PNS with damp-heat syndrome inflammatory cytokines treated by Huang Kui capsule, the results showed that patients with primary nephritic syndrome damp-heat inflammatory cytokines level IL-4, IL-10, IL-13 increased abnormally; primary nephritic syndrome with damp-heat and increased levels of inflammatory cytokines have close relationship, symptoms of damp-heat syndrome scores and the level of IL-4, IL-10, IL-13 correlative.2. Used Huang Kui Capsule to treat of PNS with damp-heat syndrome the results showed that clearing away damp- heat drug Huang Kui Capsule adjusted down markedly inflammatory cytokines in PNS damp-heat syndrome patients with abnormal increase of inflammatory cytokines.3. Clearing away damp- heat drugs Huang Kui capsule can better alleviate the symptoms of patients with damp-heat and improve proteinuria in patients with renal comprehensive damp-heat, hyperlipidemia, and hypercoagulable state, etc., could improve the clinical efficacy of comprehensive damp-heat in PNS patient, reduced kidney integrated disease, the mechanism may be related to the anti-inflammatory drug as the role of the antigen clearance.更多还原