【作者】 李燕；

【导师】 洪钦国；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2007， 博士

【摘要】 目的（1）总结中西医对狼疮性肾炎（LN）的发病机制和防治措施的研究进展；（2）观察清热解毒活血中药对瘀热痹阻证LN的临床疗效。（3）观察Ⅳ型LN患者血清对正常人肾小球系膜细胞（GMC）增殖的影响以及清热解毒活血中药对Ⅳ型LN血清干预下GMC增殖、细胞凋亡、GMC中Bcl-2蛋白合成和Bcl-2mRNA表达的影响，从细胞分子生物学水平探讨清热解毒活血中药治疗LN的机制，为临床应用提供实验依据。方法（1）文献研究：通过查阅大量古代和现代中、西医国内外文献，总结和评价有关LN的中医认识、病因病机、西医发病机制以及中西医防治措施方面的研究现状。（2）临床研究：用简单随机化、前瞻性的研究方法，将60例瘀热痹阻证LN患者按1：1比例分为对照组和治疗组。对照组采用常规西医治疗，治疗组在对照组的基础上加用清热解毒活血中药治疗，3月为一疗程。观察患者主要症状、证候（面部红斑、发热、口疮、双手红斑、关节疼痛、关节肿胀、肌肉疼痛、脱发、浮肿、胸痛、心悸、乏力、烦躁、手足心热、口干、失眠、耳鸣、痤疮、咽痛、月经血块）和实验室检查指标(白细胞、淋巴细胞、血红蛋白、血小板、尿白细胞、尿红细胞、尿蛋白、尿素氮、肌酐、血浆白蛋白、血沉、C₃、C₄、CH₅₀、抗核抗体、抗双链DNA抗体（抗ds-DNA抗体）、凝血功能等)的改善情况，计算治疗前后的狼疮活动评分（SLEDAI）和中医证候积分，评定疾病疗效和证候疗效。（3）实验研究：①将正常对照血清和不同浓度（5％、10％、15％、20％）的Ⅳ型LN患者的血清作用于GMC，观察其对GMC增殖的影响，并选择对GMC增殖影响最显著的一组血清（x％）用于下一步的实验。②结合血清药理学方法，分为5组进行观察（Ⅰ组为空白对照组、Ⅱ组为对照组血清+x％LN血清组、Ⅲ、Ⅳ、Ⅴ组分别为低、中、高浓度含药血清+x％LN血清组），分别采用MTT法检测GMC的增殖及抑制率，荧光法和流式细胞仪技术检测GMC细胞凋亡。③再将实验分为5组进行，1组为空白对照组、2组为对照组血清+x％LN血清组、3、4、5组分别为低、中、高浓度含药血清+x％LN血清组，采用western blot检测GMC中抑凋亡基因Bcl-2蛋白的合成、采用逆转录-多聚酶链反应（RT-PCR）测定Bcl-2mRNA的表达。结果（1）文献研究：①病名：根据狼疮性肾炎的临床表现可归属于中医“阴阳毒”、“红蝴蝶疮”、“阳毒发斑”、“痹证”、“水肿”、“虚劳（肾劳）”等范畴。②病因病机：肝肾阴虚、热毒血瘀为瘀热痹阻证LN的病机特点。③治疗：主要分为分型论治、分期论治、中成药治疗、自拟方治疗、单味药或中药提取物治疗、中药注射液治疗及其它疗法，取得较好的疗效，目前多数学者采用清热解毒活血法为主治疗LN。④现代医学研究：LN发病复杂，与遗传因素（遗传易感因素等）、GMC增殖及凋亡异常、免疫调节功能异常（细胞因子网络失衡、自身抗体如抗ds-DNA抗体、抗心磷脂抗体等的产生和免疫复合物清除能力下降等）以及神经、内分泌、代谢因素等有关，其中，肾小球固有细胞如GMC增殖、凋亡异常是LN发生、发展的重要机制，因此，防治措施力图抑制肾小球固有细胞如GMC的增殖、诱导细胞凋亡。治疗上有糖皮质激素、免疫抑制剂、其它疗法（血浆置换、免疫吸咐疗法、免疫球蛋白）等，但西医治疗存在着治疗方案不规范、部分患者对治疗不敏感或产生耐药、副作用大等缺点，因此，研究疗效确切、经济、副作用较小的中医药治疗方案成为当前临床研究的重点。（2）临床研究：治疗组疾病疗效评定总有效率和证候疗效评定总有效率分别为83.33％、93.33％，疾病疗效高于对照组，差异无统计学意义（P＞0.05），证候疗效明显高于对照组，差异有非常显著性意义（P＜0.01）。治疗组治疗后SLEDAI和中医证候积分较治疗前均明显减少，差异有非常显著性意义（P均＜0.01）；治疗组治疗后SLEDAI的降低优于对照组，但无统计学意义（P＞0.05），中医证候积分较对照组明显降低，差异有非常显著性意义（P＜0.01）。二组患者的LN主症较治疗前均可明显减轻，差异有非常显著性意义（P均＜0.01），且治疗组治疗后LN主症的改善作用优于对照组，但差异无统计学意义（P＞0.05），与对照组相比患者的烦躁、咽痛、痤疮、月经血块、口干、手足心热、耳鸣、心悸症状也明显减轻，差异有非常显著性意义（P均＜0.01）。在实验室检查方面，治疗组患者治疗后白细胞、淋巴细胞、血红蛋白、血小板、血浆白蛋白、C₃、C₄、CH₅₀明显升高，尿红细胞、尿蛋白、ANA滴度、血沉、血浆纤维蛋白原明显降低，抗ds-DNA抗体转阴的作用也较明显，与治疗前相比，差异均有非常显著性意义（P均＜0.01）；其中，在降低血沉、抗ds-DNA抗体转阴方面的作用优于对照组，差异有显著性意义（P＜0.05）。（3）实验研究：①Ⅳ型LN患者血清对GMC增殖的影响：发现不同浓度（5％、10％、15％、20％）的Ⅳ型LN血清均可明显促进GMC增殖，与对照血清相比有非常显著性差异（P＜0.01），其中以15％Ⅳ型LN患者血清最为显著。②观察清热解毒活血中药对GMC增殖和凋亡的影响：在15％Ⅳ型LN患者血清干预下，不同浓度（低、中、高浓度）的含药血清均可明显抑制GMC的增殖、诱导GMC的细胞凋亡，与对照组血清相比差异有非常显著性意义（P＜0.01），其作用呈较明显的量效关系，其中，以高浓度含药血清组的上述作用最为显著，与其余各组比较差异均有非常显著性意义（P均＜0.01）。③GMC中B细胞淋巴瘤／白血病-2（Bcl-2）蛋白合成、Bcl-2mRNA表达的检测：对照血清组的Bcl-2蛋白合成、Bcl-2mRNA的表达比空白对照组显著增强；而在15％Ⅳ型LN患者的血清干预下，不同浓度（低、中、高浓度）的清热解毒活血中药的含药血清与对照组血清相比均可明显抑制GMC中抑凋亡基因Bcl-2蛋白合成和Bcl-2mRNA的表达，其作用呈较明显的量效关系，其中，以高浓度含药血清组的上述作用最为显著。结论（1）LN的发病机制较为复杂，GMC增殖、细胞凋亡异常是LN发病的重要机制；西医治疗存在部分患者不敏感、副作用大等缺点，研究能抑制GMC增殖、诱导GMC细胞凋亡的中药具有广阔的研究的临床应用前景。（2）临床研究显示：清热解毒活血中药不仅可以显著改善瘀热痹阻证LN的主症，改善各项标示狼疮活动的实验室检查指标，而且可以明显减少SLEDAI和中医证候积分，对瘀热痹阻证LN有较好的治疗作用，副作用小。（3）实验研究发现：Ⅳ型LN患者血清可明显促进GMC的增殖，以15％Ⅳ型LN患者血清最为明显。而清热解毒活血中药的含药血清可明显抑制15％Ⅳ型LN患者血清干预下的GMC增殖、诱导GMC的细胞凋亡、抑制抑凋亡基因Bcl-2蛋白的合成、下调Bcl-2mRNA的表达，这可能是清热解毒活血中药治疗LN的分子机理，从而为临床广泛应用提供了实验和理论依据。更多还原

【Abstract】 Objective （1） To sum up the progress of the research on the pathogenic mechanismand prevention measures of Lupus Nephritis （LN） in traditional Chinese andWestern medical sciences. （2） To observe the clinical therapeutic effects onLN of toxic heat and blood stagnation with the method of heat-clearing,detoxifying and promoting blood flow （HCDP）. （3） To observe the effects ofblood serum of type-ⅣLN patients on glomerular mesangial cell （GMC）proliferation of healthy people. To observe the effects of Chinese crude drugof HCDP on GMC proliferation and apoptosis, Bcl-2 protein synthesis andBcl-2mRNA expression. To explore the mechanism of the method of heat-clearing,detoxifying and promoting blood flow （HCDP） on the level of cell and molecularbiology and to provide experimental evidence for clinical application.Methods （1） Literature study: To consult a large number of ancient and modernresearch documents about Chinese and Western medical sciences, summarize andassess traditional Chinese medical science-oriented understanding,etiopathogenisis and Western medical science-oriented pathogenesis of LN, aswell as the status quo of researches into prevention and cure measures. （2）Clinical research: Sixty patients with LN of toxic heat and blood stagnationwere randomly and equally divided into a control group and a subject group.The control group adopted treatment of western medicine, while the subjectgroup was offered the method of heat-clearing, detoxifying and promoting bloodflow （HCDP） on top of western medicine. The period of treatment was three months.Cardinal symptoms included: erythema on face, febricity, aphtha, erythema onhands, arthralgia, arthrocele, myalgia, alopecia, puffiness, chest pain,palpitation, hypodynamia, dysphoria, feverish sensation in palms and soles, xerostomia, insomnia, tinnitus, ache, pharyngalgia and menstrual clots.Laboratory index contained: white blood cell （WBC）, lymphocyte （LYMN）,hemoglobin （HGB）, platelet （PLT）, urinary leukocyte （LEU）, urinaryerythrocyte （ERY）, urinary protein （PRO）, blood urea nitrogen （BUN）, serumcreatinine （Cr）, plasma albumin （ALB）, erythrocyte sedimentation rate （ESR）,C₃, C₄, CH₅₀, antinuclear antibody （ANA）, anti-ds-DNA antibody, and bloodcoagulation function （including fibrinogen, FIB）, etc. The Systemic LupusErythematosus Disease Activity Index （SLEDAI） and the integral syndrome ofTCM before and after the therapy were calculated. Therapeutic effect on thedisease and syndrome were respectively evaluated. （3） Empirical study:①Serum of healthy adults and serum of different concentration （5%, 10%, 15%and 20%） of type-ⅣLN patients were used to affect GMC, and their effectson GMC proliferation were observed. The serum （x%） whose effect on promotingGMC proliferation was the most significant was selected for the next experiment.②With the method of blood serum pharmacology, 5 groups were observed:group-Ⅰ: blank, group-Ⅱ: control serum+x% serum of type-ⅣLN patients,group-Ⅲ: low concentration serum which contained Chinese medicine of HCDP+x% serum of type-ⅣLN patients, group-Ⅳ: medium concentration serum whichcontained Chinese medicine of HCDP+x% serum of type-ⅣLN patients, group-Ⅴ: high concentration serum which contained Chinese medicine of HCDP+x%serum of type-ⅣLN patients. MTT method was adopted to detect theproliferation and inhibition ratios of GMC. Apoptosis of GMC were detectedwith the fluorometric method and flow cytometer respectively.③Afterwards,the methods of western blot and RT-PCR were respectively used to detectsynthesis of Bcl-2 protein and expression of Bcl-2mRNA , which was the genein GMC inhibiting apoptosis, by 5 groups （group-1: blank, group-2: controlserum+x% serum of type-ⅣLN patients, group-3、4、5: low、medium and highconcentration serum which contained Chinese medicine of HCDP+x% serum oftype-ⅣLN patients respectively）.Results （1）Study on publications:①Name: According to clinical manifestations,LN was discovered in diseases described in traditional Chinese medicalsciences, such as "toxin of Yin-Yang", "red butterfly-shaped sore","patch of Yang toxin", "arthromyodynia", "edema", "kidney deficiency"and so on.②Etiopathogenisis and pathogenesis: The etiopathogenisis andpathogenesis of LN of toxic heat and blood stagnation was summarized as Yin asthenia of liver and kidney, toxic heat and blood stagnation.③Therapy: Themain TCM therapeutic methods of LN include: treatment by types, by stages,by Chinese patent medicine, by private prescription, by a single medicinalherb, by extract from or injection of traditional Chinese medicine, etc. Thetherapeutic effects on LN are satisfactory. Nowadays, most academicians adoptthe method of HCDP to cure LN.④The study of modern medical science: It isconsidered that the incidence of LN is very complicated. It is in connectionwith genetic factors （genetic predisposing factors, etc）, abnormal GMCproliferation and apoptosis, dysfunction of immunoregulation （imbalance ofcytokine network, declining ability to generate autoantibody like anti-ds-DNAantibody and anticardiolipin antibody and clean immune complex）, as well asneural, endocrine and metabolic factors, etc. The abnormal generation andapoptosis of intrinsic glomerular cells such as GMC is an important mechanismof the incidence and evolvement of LN. Therefore, prevention and cure methodsare to inhibit proliferation and induce apoptosis of intrinsic glomerularcells like GMC. Therapeutic methods contain glucocorticoid, immunosuppressiveagent, other treatments such as plasmapheresis, immunoadsorption （IA）,immunoglobulin, etc. But Western medical science-oriented therapy has manydisadvantages, such as non-standard therapeutic programs, insensitivity ordrug tolerance of some patients, considerable side effects, and so on.Therefore, researches focus on studying TCM therapeutic methods of tangibleeffects, economy and few side effects. （2）Clinical research: In the subjectgroup, the total effective rates of the disease and syndrome of TCM were 83.33%and 93.33% respectively. The effective rate of the disease was higher thanthe control group, but the difference had no statistical significance （P＞0.05）. The effective rate of the syndrome was also higher compared with thecontrol group and the discrepancy was extremely significant （P＜0.01）. In thesubject group, SLEDAI and the integral syndrome of TCM after therapy wereremarkably lower than before, and the discrepancies were extremely significant（P＜0.01）. After therapy, the reduction of SLEDAI in the subject group wasmore satisfactory compared with the control group but the difference was notsignificant （P＞0.05）; while the integral syndrome of TCM was remarkably lowerthan the control group, and the difference was extremely significant （P＜0.01）. After therapy, main symptoms of the patients in the two groups wererelieved notably compared with symptoms before therapy and the differences were extremely significant （P＜0.01）. In the subject group, the effect ofmoderating main symptoms of LN was better than the control group, but thediscrepancy was not significant （P＞0.05）. Moreover, in the subject group,symptoms such as dysphoria, pharyngalgia, acne, menstrual clots, xerostomia,feverish sensation in palms and soles, tinnitus and cardiopalmus werealleviated more remarkably than the control group, and the difference wasextremely significant （P＜0.01）. Regarding laboratory detection, in thesubject group, WBC, LYMN, HGB, PLT, ALB, C₃, C₄, CH₅₀ rose notably after therapy,and the discrepancy was extremely significant （P＜0.01）. Furthermore, ERY,PRO, ANA, ESR and FIB fell remarkably, and negative conversion of anti-ds-DNAantibody was visible. The discrepancies were extremely significant （P＜0.01）after treatment. The function of reducing ESR and converting anti-ds-DNAantibody to negative were better than the control group, and the differencewas significant （P＜0.05）. （3）Empirical study:①The effects of serum oftype-ⅣLN patients on GMC: Different concentration （5%,10%,15% and 20%）serumof type-ⅣLN patients could promote GMC proliferation more markedly comparedwith control serum, and the difference was extremely significant （P＜0.01）.The 15% serum of type-ⅣLN patients made the most significant impact.②Theeffects of traditional Chinese medicine of HCDP on GMC proliferation andapoptosis: Being intervened by the 15% serum of type-ⅣLN patients, differentconcentration （low, medium and high） serum containing traditional Chinesemedicine of HCDP could inhibit the proliferation of GMC and induce theapoptosis of GMC more notably than the control group, showing extremelysignificant difference （P＜0.01）.The function indicated an obviousdose-effect relationship. The effect of high concentration serum was the mostnotable compared with other groups, the discrepancies were extremelysignificant （P＜0.01）.③The synthesis of Bcl-2 protein and the expressionof Bcl-2mRNA in GMC: Being intervened by the 15% serum of type-ⅣLN patients,different concentration （low, medium and high） serum containing traditionalChinese medicine of HCDP could inhibit the synthesis of Bcl-2 protein and theexpression of Bcl-2mRNA, a gene inhibiting apoptosis, more remarkably thanthe control group. The function also showed an apparent dose-effectrelationship. The effect of high concentration serum was the most notable.Conclusions （1）The pathogenesis of LN is very complicated. Abnormalproliferation and apoptosis of GMC is an important mechanism. Western medical science-oriented therapy has disadvantages such as insensitivity of somepatients, many side effects, etc. Studying traditional Chinese medicine thatcan inhibit GMC proliferation and induce GMC apoptosis is tremendouslypromising in clinical application. （2）Clinical research shows: TraditionalChinese medicine of HCDP can relieve main symptoms of LN of toxic heat andblood stagnation, improve laboratory detection indices, and reduce SLEDAI andthe integral syndrome of TCM. The therapeutic effects are satisfactory andthe side effects are fairly few. （3）Empirical study discovers: The serum oftype-ⅣLN patients can remarkably promote the proliferation of GMC and the15% serum has the most notable impact. Being intervened by the 15% serum oftype-ⅣLN patients, the serum containing traditional Chinese medicine of HCDPcan inhibit the proliferation of GMC, induce the apoptosis of GMC, and inhibitthe synthesis of Bcl-2 protein and the expression of Bcl-2mRNA, a geneinhibiting apoptosis remarkably. This may be the molecular mechanism of TCMof HCDP curing LN and provides experimental and theoretical evidence forclinical application.更多还原