肾小管间质损害的中医证候研究及通脉口服液的干预作用
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摘要
研究背景:
慢性肾脏病(CKD)是世界范围的医疗和公共卫生问题,美国肾脏数据系统(USRDS)的数据提示,CKD导致的终末期肾衰的发病率呈逐年上升趋势。CKD是一组临床综合征,可以由多种疾病引起,在我国主要由慢性肾小球肾炎引起,近年来,糖尿病肾病和高血压肾病所引起的CKD也呈上升趋势,是当今社会威胁人类健康和长寿的重要新流行疾病。因此,积极探讨和研究预防本病的有效措施及治疗方法具有非常重要的现实意义。
CKD属于中医学的“水肿”、“腰痛”等疾病范畴,后期可演变为“虚劳”“关格”等病证,导师杨霓芝教授根据对中西医理论的认识,结合多年的临床证候调查及治疗研究,认为CKD的病性属本虚标实,其中以气虚血瘀为关键病机之一,由此开展了以益气活血为主的方药治疗多种CKD的临床研究,取得了较好临床疗效。在此基础上,研制了具有益气活血功效的中药复方制剂“通脉口服液”用于治疗具有气虚血瘀证的慢性肾小球疾病等多种CKD,通过10余年的临床观察,证实有助于改善临床表现及相关实验室指标,延缓CKD的进展。进一步针对通脉口服液的配伍规律、有效部位、作用机制等科学问题申报了国家自然科学基金面上项目、国家中医药管理局新药开发项目、广东省中医药管理局项目、广东省科技攻关项目等一系列科研项目,研究明确了通脉口服液的有效部位、组分的最佳配伍比,而且体内、体外实验证明肾小球系膜细胞增生、防治肾小球硬化可能为其治疗CKD的作用机制之一。
长期以来,针对慢性肾小球肾炎、糖尿病肾病等CKD主要原发病的研究主要集中于其肾小球损害、肾小球硬化,肾小管间质损害(TIL)被认为是继发于肾小球损害的结果而未得到重视。随着对人类肾脏疾病病理改变和肾损害动物模型研究的深入,提示慢性肾小球疾病等CKD的发展和预后不仅与肾小球的损害有关,更取决于肾小管间质病变的有无及程度的轻重,TIL是决定十年肾脏存活率的最重要影响因素。肾小管间质损害发病机理十分复杂,早期表现可为肾间质局部炎症,随后出现细胞因子分泌,继而肾小管上皮细胞等表型转化、细胞凋亡或细胞过度增殖,细胞外基质(ECM)合成增多、降解减少,造成ECM过度沉积,最终导致肾间质纤维化(RIF),而后者几乎是所有CKD发展到后期的病理学特征。因此,通过早期防治TIL以阻止RIF的进展进而改善CKD的预后具有重大意义。
目的:
1.IgA肾病(存在不同程度TIL,尿蛋白定量＜3.5g/d,无高血压,肾功能正常)是我国CKD中的常见临床类型,其TIL对预后起着非常关键的作用。因此,本人在导师杨霓芝教授的指导下,选择上述病人为代表,开展中医证候调查,观察其中医证候特点及微观病理表现及宏观证候表现的关系,以探讨CKD肾小管间质病变的中医证候规律,指导临床辨证治疗和进一步的中医药治疗研究。
2.建立气虚血瘀证肾小管间质损害动物模型,从动物实验角度探讨通脉口服液对其宏观气虚血瘀证、病理形态学等微观表现的干预作用,进一步完善对通脉口服液防治CKD的治疗靶点及可能作用机制的研究,为将其开发为防治气虚血瘀证CKD的中药新药奠定基础。
方法:
第一部分临床研究
收集2002.9～2007.12在广东省中医院肾内科住院肾穿刺活检确诊IgA肾病(存在肾小管间质损害,尿蛋白定量＜3.5g/d,无高血压,肾功能正常)患者,总计87例;通过对中医证候学及病理学、血压、血肌酐、24小时尿蛋白定量等实验室检查资料调查分析,探索其中医证候的分布特点及与证候与肾小管间质病理改变的关系。
第二部分实验研究
实验一:采用洛汀新为的对照药物,选SD大鼠60只,分为空白对照组、模型对照组、洛汀新治疗组、通脉口服液高、中、低剂量治疗组组;造模组采用衰老+游泳疲劳+单侧输尿管结扎致气虚血瘀证肾小管间质损害病证结合模型:空白组予假手术处理;模型成功后予相应药物治疗4周。观测大鼠体重等一般状况;进行中医气虚证、血瘀证评分;检测血清中BUN、Scr、t-PA、PAI等指标水平;对梗阻侧肾组织病理切片进行HE、Masson染色,光镜下进行间质细胞浸润情况评分(CIS)及肾小管间质慢性病变(包括肾小管萎缩和间质纤维化评分(AFS),综合评价通脉口服液的疗效。
实验二:在实验一证明通脉口服液高剂量治疗组疗效显著的前提下,取空白对照组,模型对照组,通脉口服液大剂量治疗组,洛汀新治疗组梗阻肾组织,应用免疫组织化学染色法检测其HGF、TGF-β1、MMP-9、TIMP-1等指标表达;应用ELISA法检测梗阻肾匀浆PAI、t-PA等指标;以探讨其可能作用机制。
结果:
第一部分临床研究
1.研究发现此类病人中医本证:肺肾气虚证占39.1%,脾肺气虚证占35.6%,气阴两虚证占19.5%,肝肾阴虚证占5.7%,脾肾阳虚证占2.3%;主证中包含气虚证者占94.3%;标证:血瘀证占55.2%,湿热证占50.1%,湿浊证占17.2%;标证在各本证之间分布情况比较无显著性差异;
2.各证型间24h尿蛋白水平、血压、血肌酐无明显差异;
3.各证型肾小管间质损害平均积分排序:血瘀＞湿热＞湿浊＞肝肾阴虚＞肺肾气虚＞气阴两虚＞脾肾气虚＞脾肾阳虚。
第二部分实验研究
实验一:
1治疗后,与空白组相比,各组大鼠体重明显下降(P＜0.001);与模型组比较,通脉口服液高、中剂量组均可显著改善UUO大鼠体重(P＜0.05)。治疗前造模各组与空白组相比气虚证、血瘀证积分值明显升高(P＜0.001);治疗后中药高、中、低剂量组气虚证、血瘀证积分值较模型组、西药组显著降低(P＜0.01),西药组和模型组比较差异无显著差异,中药高、中、低剂量组之间无显著性差异。
3治疗后与空白组相比,各造模组大鼠血清t-PA含量均有不同程度的降低趋势,血清PAI有不同程度的升高趋势;与模型组比较,各用药组血清t-PA有所升高,血清PAI有所降低,但均无显著性差异。治疗后模型组、通脉低剂量组BUN、Scr与空白组有升高趋势,但无统计学意义。
4光镜下,模型组大鼠HE、Masson染色可见:大部分肾小管上皮细胞颗粒、空泡变性,部分肾小管上皮细胞坏死脱落,呈裸基底膜,管腔扩张,部分管腔内可见脱落坏死组织及细胞形成颗粒管型及细胞管型,可见灶性肾小管萎缩;间质区明显增宽,炎性细胞浸润,纤维组织增生,呈条索状、片状纤维化;肾小球除部分有肾小囊轻-中度扩张外,无其他明显病变。中药各剂量组和西药组也有以上类似的改变,但病变与模型组相比有不同程度的减轻。
5治疗后与空白组相比,各造模组炎症细胞浸润积分均明显增高(P＜0.001);与模型组比较,各用药组炎症细胞浸润积分均有不同程度的降低,其中中药高剂量组与西药组下降显著(P＜0.05);与西药组比较,中药高剂量组均无显著性差异(P＞0.05)。与空白组相比,各造模组肾小管萎缩和间质纤维化积分均明显增高(P＜0.001);与模型组比较,各用药组肾小管萎缩和间质纤维化均有显著降低(P＜0.01),其中中药高剂量组降低最明显,且与西药组比较具有显著性差异(P＜0.05),与中药中、低剂量组相比也具有显著性差异(P＜0.05)。
实验二:
1空白组肾组织无明显TGF-β1表达,治疗结束后与空白组相比,各造模组肾小管及间质TGF-β1的表达明显增高(P＜0.001);与模型组比较,各用药组TGF-β1表达均有不同程度的降低,洛汀新组下降显著(P＜0.01),通脉大剂量组下降明显(p＜0.05),但不如西药组(p＜0.05);空白组肾组织无明显HGF表达,治疗后与空白组相比,各组HGF的表达都明显增高(P＜0.001);与模型组比较,通脉高剂量组表达明显增高(P＜0.05):与西药组比较,通脉高剂量组表达明显增高(P＜0.05)。
2治疗后与空白组相比,各造模组MMP-9、TIMP-1的表达均明显增高(P＜0.001),MMP-9/TIMP-1比例明显下降(P＜0.001);与模型组比较,各用药组MMP-9表达均有不同程度的升高,TIMP-1表达均有不同程度的降低,MMP-9/TIMP-1均有不同程度的升高,且具有显著性差异(P＜0.001),与西药组比较,中药高剂量组在升高MMP-9、降低TIMP-1及MMP-9/TIMP-1方面优势明显(P＜0.001)。
3治疗后与空白组相比,各造模组梗阻侧肾组织内t-PA含量均有不同程度的降低,,PAI含量均有不同程度的升高,但无统计学差异;与模型组比较,各用药组梗阻侧肾组织内t-PA有所升高,但无显著性差异(P＞0.05),PAI含量均有不同程度的下降,但仅有中药高剂量组同模型组比较具有显著性差异(P＜0.05)。
结论:
1 IgA肾病(存在TIL,尿蛋白定量＜3.5g/d,无高血压,肾功能正常)患者本证以肺肾气虚证,脾肺气虚证,气阴两虚证多见,肝肾阴虚证,脾肾阳虚所占比例较少;标证以血瘀证、湿热证为主。本证中包含气虚证者占94.3%,标证中血瘀证占55.2%;因此可以推论:气虚证、血瘀证是此类疾病的常见临床证候,血瘀、湿热、湿浊等标实证特别是血瘀证可能与肾小管间质损害存在较直接的相关性,提示益气活血方药在此类疾病有较广泛适用性和针对性。同时血瘀、湿热、湿浊等标证及本证中肝肾阴虚证的存在提示肾小管间质损害可能较其他证为重的结果,可为根据临床表现推测病理损害所借鉴。
2衰老+游泳疲劳+单侧输尿管结扎致气虚血瘀证肾小管间质损害病证结合模型大鼠与空白对照组比较,体重等一般状况明显下降;气虚证、血瘀证积分值明显升高;大鼠血清t-PA含量均有不同程度的降低趋势,PAI含量均有不同程度的升高趋势;具有典型的肾小管间质损害病理改变;益气活血方药对上述表现都一定程度的改善作用;可见衰老+游泳疲劳+单侧输尿管结扎致气虚血瘀证肾小管间质纤维化病证结合模型是相对成功的,可为类似研究参考应用。
3通脉口服液可改善改善模型大鼠体重等一般情况;改善气虚证、血瘀证积分等宏观整体表现;明显减轻其肾小管间质损害的微观局部病理改变,其中降低肾小管间质炎症细胞浸润积分与洛汀新相当,降低肾小管萎缩和间质纤维化积分优于洛汀新,而且具有量效依赖性,其中以通脉高剂量最佳。
4通脉口服液治疗肾小管间质损害可能与:上调梗阻侧肾组织HGF表达;下调梗阻侧肾组织TGF-β1表达;上调梗阻侧MMP-9表达,下调梗阻侧肾组织TIMP-1表达,上调MMP-9/TIMP-1比值;降低肾组织PAI含量等机制有关,特别在上调作为主要的抑制纤维化因子HGF及调节细胞外基质酶降解系统MMP-9/TIMP-1,t-PA/PAI比例失衡方面较洛汀新有优势,值得进一步深入研究。
Background:
Chronic kidney disease(CKD)is a worldwide medical and public health issues, The data of U.S.Renal Data System(USRDS)prompted that the incidence of end-stage renal failure caused by the CKD rised year by year.Chronic kidney disease is a clinical syndrome which can be caused by various diseases,but in China mainly caused by chronic glomerulonephritis.In recent years,chronic kidney disease caused by diabetes,hypertension and other kidney disease also showed an upward trend,which is a new an important new epidemic threat to human health and longevity in today' s society which lead to cardiovascular disease that is a threat to human health and an important reason 1 to death. Therefore,actively exploring and studying the prevention and effective treatment of the disease has very important and practical significance.
Chronic kidney disease belongs to the TCM medicine edema,back pain and other disease areas in the latter part of which can be evolve into Xulao,guange and other related diseases.According to the theory of knowledge of Chinese and Western Medicine with many years of clinical investigation and syndrome study Mentor Professor Yang Nizhi considers that the chronic kidney disease is a disease of deficiency in origin and excess in superficiality,in which qi deficiency and blood stasis is one of the critical machines.Professor Yang carried out a variety of clinical research related to chronic kidney disease with the treatment of supplementing Qi and activating blood circulation method and achieved good effect.On this basis,"Tongmai oral solution" had been developed which has the efficacy of supplementing Qi and activating blood circulation.Through more than 10 years of clinical research,"Tongmai oral solution" could interfere with the progress of chronic kidney disease and improve their prognosis through various mechanisms.To further adopt various science funding,and in the in vivo/in vitro studies,we found that Tongmai oral solution through a variety of mechanisms could inhibit mesangial cell proliferation which indicated that Tongmai oral solution can improve the prognosis of chronic kidney disease through control glomerulosclerosis.
All along,for the primary diseases of CKD such as chronic glomerulonephritis, diabetes,kidney disease,and other diseases,the related research main focus on glomerular damage,glomerulosclerosis.The previous view considered that tubulointerstitial lesions(TIL)was not so important because it was only in the results of glomerular lesions.But in recent years,with the In-depth study of kidney pathological changes of human and animal model of kidney damage,the new view prompted that the development of chronic glomerular diseases and the prognosis was not only with the glomerular damage on its own, but also with the existence and extent Of the severity of the tubulointerstitial lesions.The latest point of view considered that tubulointerstitial damage is a most important factor which determined the the kidney survival rate in 10 years.The mechanism of TIL is very complicated,in the early stage the kidney mesenchymal became local inflammatory,subsequently cytokines secreted,Then the tubular epithelial cells and other forms transformed, the cell apoptosised or excessive proliferated,extracellular matrix (ECM)synthesis increased,and degradation reduced,resulted in excessive sedimentation of ECM,eventually led to renal fibrosis(RIF),the latter was the pathological features which performanced by almost all chronic kidney disease in the end stage.Therefore,early prevention TIL to stop the progress of RIF could improve the prognosis of CKD that was of great significance.
Objective:
Therefore,under the guidance of Professor Yang Nizhi,to further conduct the investigation of TCM Clinical Syndrome of IgA nephropathy-the most common chronic kidney disease in china,and investigate the TCM Syndrome characteristic of the tubulointerstitial lesions in order to illustrate the chinese medical pathogenesis of the tubulointerstitial lesions to guide clinical research and treatment,and to further conduct the the study of Tongmai oral solution intervention on experimental Qi blood stasis tubulointerstitial damage of animal,to discuss the possible treatment target and the mechanism of the Tongmai oral solution to chronic kidney disease for further development of chinese medicine to the prevention and treatment of chronic kidney disease.
Method:
The first part clinical research
The patients confirmed IgA nephropathy by renal biopsy in Chinese medicine hospitals of Guangdong Province from 2002.9 to 2007.12 who had tubulointerstitial damage,urine protein <3.5 g/d,without hypertension,renal function was normal,ther toatle numbe of the patients were 87.Through investigations of Chinese medicine and pathology Syndrome,blood pressure,serum creatinine,a 24-hour urine protein quantitative information etc we explored the relationship of distribution of TCM syndrome with tubuloin- terstitial pathological changes.
The second part experimental study
Experiment 1:
Lotensin for the use of as control medicine,the election of SD 60,into the control group,model,Lotensin group,Tongmai Solution high,medium and low-dose group.The models were blood stasis Qi Syndrome combining tubulointerstitial fibrosis model caused by swimming fatigue +aging + unilateral ureteral ligation.The rats in the control group were treated with sham treatment.After the model successed drug was treated for four weeks. To Observe the general body weight;Chinese Qi Deficiency Syndrome,blood stasis score;serum BUN,Scr,t-PA,PAI,and other indicators;Obstruction side kidney HE,Masson stain,light microscope to stromal cells infiltration score(CIS)and chronic tubulointerstitial change(including tubular atrophy and fibrosis)score(AFS),and other indicators on the base of which Evaluate the effect of Tongmai oral solution.
Experiment 2:
On the basis of experiment one proving that high-dose oral solution Tongmai was effective,Applicating immunohistochemistry to detected HGF,TGF-β1, MMP-9,TIMP-1 and other targets by ELISA serum PAI,t-PA,and other indicators, and to explore its possible mechanism.
Result:
The first part clinical research
1.Study found that Qi deficiency of Fei and Shen accounted for 39.1%,Qi deficiency of Fei and pi accounted for 35.6%,Qi and Yin deficiency accounted for 19.5%,Yin deficiency of Gan and Shen accounted for 5.7%,Yang deficiency of Pi and Shen accounted for about capital syndroms in selected patients. This shows that Qi deficiency accounted for 94.3%in capital syndromes. Among biao syndrom,blood stasis syndrome accounted for 55.2%,damp-heat syndrome accounted for 50.1%,damp-zhuo syndrome accounted for 17.2%.There is no significant difference about distribution of biao syndrom of no significant difference among every capital syndrom.
2.There was no significant difference about urine protein,blood pressure, serum creatinine among TCM syndrome types.
3.The sorting of tubulointerstitial damage average points of every TCM syndrome type:blood stasis> damp-heat> damp-zhuo> Yin deficiency of Gan and Shen> Qi deficiency of Fei and pi> Qi and Yin deficiency> Qi deficiency of Fei and Shen> Yang deficiency of Pi and Shen.
The second part empirical study
Experiment 1:
1.Post-treatment,compared with the control group,body weight decreased significantly in other groups(P＜0.001).Compared with the model group,high, medium-dose Tongmai Oral Solution treatment group rats can be signif- icantly improved in weight(P＜0.05).Before treatment,each made-modle group Qi deficiency and blood stasis syndrome' s score increased significantly compared with the control group(P＜0.001).Post-treatment,Qi deficiency and blood stasis syndrome' s score of high,medium,medium and low-dose Tongmai oral solution treatment group significantly decrease compared with the model group and lotensin treatment group(P＜0.01),there are no significant differentce between the model group and lotensin treatment group,no significant difference among high,medium,medium and low-dose Tongmai oral solution treatment group too.
3.Post-treatment,compared with the control group,serum t-PA content decreease and serum PAl content increase with varying degrees in other groups. Compared with the model group,serum t-PA content increased and serum PAI content decrease slightly in treatment groups.But there was no significant difference.Post-treatment,compared with the control group,the BUN and Scr have tended to increase in model group and low-dose Tongmai oral soluteion treatment groups without statistical significance.
4.We can see through the light microscope:the model group kidney HE and Masson staining:Most particles tubular-epithelial cells appear granularor vacuolar degeneration.Some tubular epithelial cells necrosis and desquamate,present bare basement membrane.nephric tubule lumen expand,there are some granular cast and cellular cast formed necrotic tissue and cell.Focal tubular atrophy can be seen.Renal interstitial are a widened significantly,inflammatory cell infiltrate,fibrous tissue hyperplasia,appear cable-like,patchy fibrosis. In addition to a part of glomerular renal capsule light-oderate expansion, there are noobvious lesions in glomerulus.All rongmai oral solution treatment group and Lotensin group have analogous changes,But there are different levels of improvement about pathological changes,compared with the model.
5.Post-treatment,CIS increased significantly in other made-modle groups (P＜0.001),compared with the control group.Compared with the model group, CIS decrease with varying degrees in treatment groups,among the total, high dose of Chinese medicine and Lotensin treatment group decreased significantly(P＜0.05).Compared with the lotensin treatment group,Chinese high-dose group has no significant difference(P＞0.05).Compared with the control group,AFS is significantly in other made-modle groups (P＜0.001).Compared with the model group,AFS decrease markedly in treatment groups(P＜0.01).among the total,high dose of Chinese medicine treatment group decreased significantly.Compared with Lotensin and medium and low-dose Tongmai oral solution treatment group,there is significant difference too (P＜0.05).
Experiment 2:
1.No significant expression of TGF-β1 in nephridial tissue of control group. Post-treatment,the expression of TGF-β1 increased significantly in mademodle groups(P＜0.001),compared with the control group.Compared with the model group,the expression of TGF-β1 decrease with varying degrees in treatment groups,among the total,Lotensin treatment group decreased significantly(P＜0.01).High dose of Chinese medicine treatment group decreased obviously(p＜0.05),but worse than treatment group treatment group(p＜0.05); No significant expression of HGF in nephridial tissue of control group, Post-treatmen,the expression of HGF increased significantly in made-modle groups(P＜0.001);Compared with the model group,high-dose of chinese medicine treatment group decreased significantly(P＜0.05).Compared with the model group,the expression of HGF increased significantly in high-dose Tongmai oral solution treatment group(P＜0.05).
2.Post-treatment,compared with the control group,the expression of MMP-9、TIMP-1 increased significantly in made-modle groups(P＜0.001),MMP-9/TIMP-1 decreased significantly.Compared with the model group,MMP-9 and MMP-9/TIMP-1 increase with varying degrees in treatment groups,TIMP-1 decrease signifycantly with varying degrees in treatment groups(P＜0.001),high-dose Tongmai oral solution treatment group is better than Benazepri treatment group (P＜0.001).
3.Post-treatment,compared with the control group,the content of t-PA within obstruction-side kidney of made-modle groups decrease with varying degrees, among the total,the modle group decreased significantly(P＜0.05),the content of PAI within obstruction-side kidney of made-modle groups increase with varying degrees,among the total,the modle group decreased significantly (P＜0.05).Compared with the model group,the content of t-PA within obstructtion-side kidney of made-modle groups increase with varying degrees,but no significant difference(P＞0.05),the content of PAl within obstruction side kidney of made-modle groups decrease with varying degrees,but only high-dose Tongmai oral solution treatment group decrease significantly(P＜0.05).
Conclusion:
1.To IgA nephropathy(with TIL,24-hour urine protein <3.5 g/d,without hypertension,renal function was normal)patiens,Qi deficiency of Fei and Shen,Qi deficiency of Fei and pi,Qi and Yin deficiency are common to capital syndromes,Yin deficiency of Gan and Shen and Yang deficiency of Pi relatively rare.Blood stasis syndrome and damp-heat syndrome are common to Biao syndrome. Capital syndrome included Qi deficiency syndrome accounted for 94.3%,blood stasis syndrome,blood stasis syndrome accounted for 55.2%among biao syndrome. Qi deficiency syndrome and blood stasis syndrome are common syndromes to the disease.Therefore,it could be inferred that yiqihuoxue herbs have a wider applicability in such diseases。The existence of biao syndromes such as blood stasis,damp-heat and damp-zhuo prompt tubulointerstitial damage to some extent,especially the blood stasis syndrome.The patiens with Yin deficiency of Gan and Shen as capital syndrome cound have more serious tubulo interstitial damage than as capital syndrome.
2.TO made-model group rats,weight and other general condition decreased significantly. Qi deficiency and blood stasis syndrome score increased significantly, the content of t-PA of Serum decreased in varying degrees,the content of t-PA of Serum decreased in varying degrees with typical tubulointerstitial damage pathological changes.YiqihuoxueL compound-Tongmai oral solution have a certain degree of improvement to above-mentioned appearances.This shows that the modle of ombination of disease and syndrome made by aging and swimming fatigue and UUO is relatively successful,It is expected for a similar study of reference.
3.Research shows that:Tongmai oral solution can improve made-modle rats overall performance such as weight,other general condition,Qi deficiency and blood stasis syndrome.At the same time,it can significantly reduce the local micro-pathological changes of tubulointerstitial damage,Improvements about CIS score are similar to benazepril.On the other hand,improvements abo AFS score are better than the benazepril,the results are dose-dependent.
4.The mechanism of Tongmai oral solution to TIL may be related to the following: increasing HGF expression,decreasing TGF-β1 expression;increasing MMP-9 expression,decreasing TIMP-1 expression,increasing MMP-9/ TIMP-1 ratio, Reducing the content of the PAI of kidney mechanisms and so on.
慢性肾脏病(CKD)是世界范围的医疗和公共卫生问题,美国肾脏数据系统(USRDS)的数据提示,CKD导致的终末期肾衰的发病率呈逐年上升趋势。CKD是一组临床综合征,可以由多种疾病引起,在我国主要由慢性肾小球肾炎引起,近年来,糖尿病肾病和高血压肾病所引起的CKD也呈上升趋势,是当今社会威胁人类健康和长寿的重要新流行疾病。因此,积极探讨和研究预防本病的有效措施及治疗方法具有非常重要的现实意义。
CKD属于中医学的“水肿”、“腰痛”等疾病范畴,后期可演变为“虚劳”“关格”等病证,导师杨霓芝教授根据对中西医理论的认识,结合多年的临床证候调查及治疗研究,认为CKD的病性属本虚标实,其中以气虚血瘀为关键病机之一,由此开展了以益气活血为主的方药治疗多种CKD的临床研究,取得了较好临床疗效。在此基础上,研制了具有益气活血功效的中药复方制剂“通脉口服液”用于治疗具有气虚血瘀证的慢性肾小球疾病等多种CKD,通过10余年的临床观察,证实有助于改善临床表现及相关实验室指标,延缓CKD的进展。进一步针对通脉口服液的配伍规律、有效部位、作用机制等科学问题申报了国家自然科学基金面上项目、国家中医药管理局新药开发项目、广东省中医药管理局项目、广东省科技攻关项目等一系列科研项目,研究明确了通脉口服液的有效部位、组分的最佳配伍比,而且体内、体外实验证明肾小球系膜细胞增生、防治肾小球硬化可能为其治疗CKD的作用机制之一。
长期以来,针对慢性肾小球肾炎、糖尿病肾病等CKD主要原发病的研究主要集中于其肾小球损害、肾小球硬化,肾小管间质损害(TIL)被认为是继发于肾小球损害的结果而未得到重视。随着对人类肾脏疾病病理改变和肾损害动物模型研究的深入,提示慢性肾小球疾病等CKD的发展和预后不仅与肾小球的损害有关,更取决于肾小管间质病变的有无及程度的轻重,TIL是决定十年肾脏存活率的最重要影响因素。肾小管间质损害发病机理十分复杂,早期表现可为肾间质局部炎症,随后出现细胞因子分泌,继而肾小管上皮细胞等表型转化、细胞凋亡或细胞过度增殖,细胞外基质(ECM)合成增多、降解减少,造成ECM过度沉积,最终导致肾间质纤维化(RIF),而后者几乎是所有CKD发展到后期的病理学特征。因此,通过早期防治TIL以阻止RIF的进展进而改善CKD的预后具有重大意义。
目的:
1.IgA肾病(存在不同程度TIL,尿蛋白定量＜3.5g/d,无高血压,肾功能正常)是我国CKD中的常见临床类型,其TIL对预后起着非常关键的作用。因此,本人在导师杨霓芝教授的指导下,选择上述病人为代表,开展中医证候调查,观察其中医证候特点及微观病理表现及宏观证候表现的关系,以探讨CKD肾小管间质病变的中医证候规律,指导临床辨证治疗和进一步的中医药治疗研究。
2.建立气虚血瘀证肾小管间质损害动物模型,从动物实验角度探讨通脉口服液对其宏观气虚血瘀证、病理形态学等微观表现的干预作用,进一步完善对通脉口服液防治CKD的治疗靶点及可能作用机制的研究,为将其开发为防治气虚血瘀证CKD的中药新药奠定基础。
方法:
第一部分临床研究
收集2002.9～2007.12在广东省中医院肾内科住院肾穿刺活检确诊IgA肾病(存在肾小管间质损害,尿蛋白定量＜3.5g/d,无高血压,肾功能正常)患者,总计87例;通过对中医证候学及病理学、血压、血肌酐、24小时尿蛋白定量等实验室检查资料调查分析,探索其中医证候的分布特点及与证候与肾小管间质病理改变的关系。
第二部分实验研究
实验一:采用洛汀新为的对照药物,选SD大鼠60只,分为空白对照组、模型对照组、洛汀新治疗组、通脉口服液高、中、低剂量治疗组组;造模组采用衰老+游泳疲劳+单侧输尿管结扎致气虚血瘀证肾小管间质损害病证结合模型:空白组予假手术处理;模型成功后予相应药物治疗4周。观测大鼠体重等一般状况;进行中医气虚证、血瘀证评分;检测血清中BUN、Scr、t-PA、PAI等指标水平;对梗阻侧肾组织病理切片进行HE、Masson染色,光镜下进行间质细胞浸润情况评分(CIS)及肾小管间质慢性病变(包括肾小管萎缩和间质纤维化评分(AFS),综合评价通脉口服液的疗效。
实验二:在实验一证明通脉口服液高剂量治疗组疗效显著的前提下,取空白对照组,模型对照组,通脉口服液大剂量治疗组,洛汀新治疗组梗阻肾组织,应用免疫组织化学染色法检测其HGF、TGF-β1、MMP-9、TIMP-1等指标表达;应用ELISA法检测梗阻肾匀浆PAI、t-PA等指标;以探讨其可能作用机制。
结果:
第一部分临床研究
1.研究发现此类病人中医本证:肺肾气虚证占39.1%,脾肺气虚证占35.6%,气阴两虚证占19.5%,肝肾阴虚证占5.7%,脾肾阳虚证占2.3%;主证中包含气虚证者占94.3%;标证:血瘀证占55.2%,湿热证占50.1%,湿浊证占17.2%;标证在各本证之间分布情况比较无显著性差异;
2.各证型间24h尿蛋白水平、血压、血肌酐无明显差异;
3.各证型肾小管间质损害平均积分排序:血瘀＞湿热＞湿浊＞肝肾阴虚＞肺肾气虚＞气阴两虚＞脾肾气虚＞脾肾阳虚。
第二部分实验研究
实验一:
1治疗后,与空白组相比,各组大鼠体重明显下降(P＜0.001);与模型组比较,通脉口服液高、中剂量组均可显著改善UUO大鼠体重(P＜0.05)。治疗前造模各组与空白组相比气虚证、血瘀证积分值明显升高(P＜0.001);治疗后中药高、中、低剂量组气虚证、血瘀证积分值较模型组、西药组显著降低(P＜0.01),西药组和模型组比较差异无显著差异,中药高、中、低剂量组之间无显著性差异。
3治疗后与空白组相比,各造模组大鼠血清t-PA含量均有不同程度的降低趋势,血清PAI有不同程度的升高趋势;与模型组比较,各用药组血清t-PA有所升高,血清PAI有所降低,但均无显著性差异。治疗后模型组、通脉低剂量组BUN、Scr与空白组有升高趋势,但无统计学意义。
4光镜下,模型组大鼠HE、Masson染色可见:大部分肾小管上皮细胞颗粒、空泡变性,部分肾小管上皮细胞坏死脱落,呈裸基底膜,管腔扩张,部分管腔内可见脱落坏死组织及细胞形成颗粒管型及细胞管型,可见灶性肾小管萎缩;间质区明显增宽,炎性细胞浸润,纤维组织增生,呈条索状、片状纤维化;肾小球除部分有肾小囊轻-中度扩张外,无其他明显病变。中药各剂量组和西药组也有以上类似的改变,但病变与模型组相比有不同程度的减轻。
5治疗后与空白组相比,各造模组炎症细胞浸润积分均明显增高(P＜0.001);与模型组比较,各用药组炎症细胞浸润积分均有不同程度的降低,其中中药高剂量组与西药组下降显著(P＜0.05);与西药组比较,中药高剂量组均无显著性差异(P＞0.05)。与空白组相比,各造模组肾小管萎缩和间质纤维化积分均明显增高(P＜0.001);与模型组比较,各用药组肾小管萎缩和间质纤维化均有显著降低(P＜0.01),其中中药高剂量组降低最明显,且与西药组比较具有显著性差异(P＜0.05),与中药中、低剂量组相比也具有显著性差异(P＜0.05)。
实验二:
1空白组肾组织无明显TGF-β1表达,治疗结束后与空白组相比,各造模组肾小管及间质TGF-β1的表达明显增高(P＜0.001);与模型组比较,各用药组TGF-β1表达均有不同程度的降低,洛汀新组下降显著(P＜0.01),通脉大剂量组下降明显(p＜0.05),但不如西药组(p＜0.05);空白组肾组织无明显HGF表达,治疗后与空白组相比,各组HGF的表达都明显增高(P＜0.001);与模型组比较,通脉高剂量组表达明显增高(P＜0.05):与西药组比较,通脉高剂量组表达明显增高(P＜0.05)。
2治疗后与空白组相比,各造模组MMP-9、TIMP-1的表达均明显增高(P＜0.001),MMP-9/TIMP-1比例明显下降(P＜0.001);与模型组比较,各用药组MMP-9表达均有不同程度的升高,TIMP-1表达均有不同程度的降低,MMP-9/TIMP-1均有不同程度的升高,且具有显著性差异(P＜0.001),与西药组比较,中药高剂量组在升高MMP-9、降低TIMP-1及MMP-9/TIMP-1方面优势明显(P＜0.001)。
3治疗后与空白组相比,各造模组梗阻侧肾组织内t-PA含量均有不同程度的降低,,PAI含量均有不同程度的升高,但无统计学差异;与模型组比较,各用药组梗阻侧肾组织内t-PA有所升高,但无显著性差异(P＞0.05),PAI含量均有不同程度的下降,但仅有中药高剂量组同模型组比较具有显著性差异(P＜0.05)。
结论:
1 IgA肾病(存在TIL,尿蛋白定量＜3.5g/d,无高血压,肾功能正常)患者本证以肺肾气虚证,脾肺气虚证,气阴两虚证多见,肝肾阴虚证,脾肾阳虚所占比例较少;标证以血瘀证、湿热证为主。本证中包含气虚证者占94.3%,标证中血瘀证占55.2%;因此可以推论:气虚证、血瘀证是此类疾病的常见临床证候,血瘀、湿热、湿浊等标实证特别是血瘀证可能与肾小管间质损害存在较直接的相关性,提示益气活血方药在此类疾病有较广泛适用性和针对性。同时血瘀、湿热、湿浊等标证及本证中肝肾阴虚证的存在提示肾小管间质损害可能较其他证为重的结果,可为根据临床表现推测病理损害所借鉴。
2衰老+游泳疲劳+单侧输尿管结扎致气虚血瘀证肾小管间质损害病证结合模型大鼠与空白对照组比较,体重等一般状况明显下降;气虚证、血瘀证积分值明显升高;大鼠血清t-PA含量均有不同程度的降低趋势,PAI含量均有不同程度的升高趋势;具有典型的肾小管间质损害病理改变;益气活血方药对上述表现都一定程度的改善作用;可见衰老+游泳疲劳+单侧输尿管结扎致气虚血瘀证肾小管间质纤维化病证结合模型是相对成功的,可为类似研究参考应用。
3通脉口服液可改善改善模型大鼠体重等一般情况;改善气虚证、血瘀证积分等宏观整体表现;明显减轻其肾小管间质损害的微观局部病理改变,其中降低肾小管间质炎症细胞浸润积分与洛汀新相当,降低肾小管萎缩和间质纤维化积分优于洛汀新,而且具有量效依赖性,其中以通脉高剂量最佳。
4通脉口服液治疗肾小管间质损害可能与:上调梗阻侧肾组织HGF表达;下调梗阻侧肾组织TGF-β1表达;上调梗阻侧MMP-9表达,下调梗阻侧肾组织TIMP-1表达,上调MMP-9/TIMP-1比值;降低肾组织PAI含量等机制有关,特别在上调作为主要的抑制纤维化因子HGF及调节细胞外基质酶降解系统MMP-9/TIMP-1,t-PA/PAI比例失衡方面较洛汀新有优势,值得进一步深入研究。
Background:
Chronic kidney disease(CKD)is a worldwide medical and public health issues, The data of U.S.Renal Data System(USRDS)prompted that the incidence of end-stage renal failure caused by the CKD rised year by year.Chronic kidney disease is a clinical syndrome which can be caused by various diseases,but in China mainly caused by chronic glomerulonephritis.In recent years,chronic kidney disease caused by diabetes,hypertension and other kidney disease also showed an upward trend,which is a new an important new epidemic threat to human health and longevity in today' s society which lead to cardiovascular disease that is a threat to human health and an important reason 1 to death. Therefore,actively exploring and studying the prevention and effective treatment of the disease has very important and practical significance.
Chronic kidney disease belongs to the TCM medicine edema,back pain and other disease areas in the latter part of which can be evolve into Xulao,guange and other related diseases.According to the theory of knowledge of Chinese and Western Medicine with many years of clinical investigation and syndrome study Mentor Professor Yang Nizhi considers that the chronic kidney disease is a disease of deficiency in origin and excess in superficiality,in which qi deficiency and blood stasis is one of the critical machines.Professor Yang carried out a variety of clinical research related to chronic kidney disease with the treatment of supplementing Qi and activating blood circulation method and achieved good effect.On this basis,"Tongmai oral solution" had been developed which has the efficacy of supplementing Qi and activating blood circulation.Through more than 10 years of clinical research,"Tongmai oral solution" could interfere with the progress of chronic kidney disease and improve their prognosis through various mechanisms.To further adopt various science funding,and in the in vivo/in vitro studies,we found that Tongmai oral solution through a variety of mechanisms could inhibit mesangial cell proliferation which indicated that Tongmai oral solution can improve the prognosis of chronic kidney disease through control glomerulosclerosis.
All along,for the primary diseases of CKD such as chronic glomerulonephritis, diabetes,kidney disease,and other diseases,the related research main focus on glomerular damage,glomerulosclerosis.The previous view considered that tubulointerstitial lesions(TIL)was not so important because it was only in the results of glomerular lesions.But in recent years,with the In-depth study of kidney pathological changes of human and animal model of kidney damage,the new view prompted that the development of chronic glomerular diseases and the prognosis was not only with the glomerular damage on its own, but also with the existence and extent Of the severity of the tubulointerstitial lesions.The latest point of view considered that tubulointerstitial damage is a most important factor which determined the the kidney survival rate in 10 years.The mechanism of TIL is very complicated,in the early stage the kidney mesenchymal became local inflammatory,subsequently cytokines secreted,Then the tubular epithelial cells and other forms transformed, the cell apoptosised or excessive proliferated,extracellular matrix (ECM)synthesis increased,and degradation reduced,resulted in excessive sedimentation of ECM,eventually led to renal fibrosis(RIF),the latter was the pathological features which performanced by almost all chronic kidney disease in the end stage.Therefore,early prevention TIL to stop the progress of RIF could improve the prognosis of CKD that was of great significance.
Objective:
Therefore,under the guidance of Professor Yang Nizhi,to further conduct the investigation of TCM Clinical Syndrome of IgA nephropathy-the most common chronic kidney disease in china,and investigate the TCM Syndrome characteristic of the tubulointerstitial lesions in order to illustrate the chinese medical pathogenesis of the tubulointerstitial lesions to guide clinical research and treatment,and to further conduct the the study of Tongmai oral solution intervention on experimental Qi blood stasis tubulointerstitial damage of animal,to discuss the possible treatment target and the mechanism of the Tongmai oral solution to chronic kidney disease for further development of chinese medicine to the prevention and treatment of chronic kidney disease.
Method:
The first part clinical research
The patients confirmed IgA nephropathy by renal biopsy in Chinese medicine hospitals of Guangdong Province from 2002.9 to 2007.12 who had tubulointerstitial damage,urine protein <3.5 g/d,without hypertension,renal function was normal,ther toatle numbe of the patients were 87.Through investigations of Chinese medicine and pathology Syndrome,blood pressure,serum creatinine,a 24-hour urine protein quantitative information etc we explored the relationship of distribution of TCM syndrome with tubuloin- terstitial pathological changes.
The second part experimental study
Experiment 1:
Lotensin for the use of as control medicine,the election of SD 60,into the control group,model,Lotensin group,Tongmai Solution high,medium and low-dose group.The models were blood stasis Qi Syndrome combining tubulointerstitial fibrosis model caused by swimming fatigue +aging + unilateral ureteral ligation.The rats in the control group were treated with sham treatment.After the model successed drug was treated for four weeks. To Observe the general body weight;Chinese Qi Deficiency Syndrome,blood stasis score;serum BUN,Scr,t-PA,PAI,and other indicators;Obstruction side kidney HE,Masson stain,light microscope to stromal cells infiltration score(CIS)and chronic tubulointerstitial change(including tubular atrophy and fibrosis)score(AFS),and other indicators on the base of which Evaluate the effect of Tongmai oral solution.
Experiment 2:
On the basis of experiment one proving that high-dose oral solution Tongmai was effective,Applicating immunohistochemistry to detected HGF,TGF-β1, MMP-9,TIMP-1 and other targets by ELISA serum PAI,t-PA,and other indicators, and to explore its possible mechanism.
Result:
The first part clinical research
1.Study found that Qi deficiency of Fei and Shen accounted for 39.1%,Qi deficiency of Fei and pi accounted for 35.6%,Qi and Yin deficiency accounted for 19.5%,Yin deficiency of Gan and Shen accounted for 5.7%,Yang deficiency of Pi and Shen accounted for about capital syndroms in selected patients. This shows that Qi deficiency accounted for 94.3%in capital syndromes. Among biao syndrom,blood stasis syndrome accounted for 55.2%,damp-heat syndrome accounted for 50.1%,damp-zhuo syndrome accounted for 17.2%.There is no significant difference about distribution of biao syndrom of no significant difference among every capital syndrom.
2.There was no significant difference about urine protein,blood pressure, serum creatinine among TCM syndrome types.
3.The sorting of tubulointerstitial damage average points of every TCM syndrome type:blood stasis> damp-heat> damp-zhuo> Yin deficiency of Gan and Shen> Qi deficiency of Fei and pi> Qi and Yin deficiency> Qi deficiency of Fei and Shen> Yang deficiency of Pi and Shen.
The second part empirical study
Experiment 1:
1.Post-treatment,compared with the control group,body weight decreased significantly in other groups(P＜0.001).Compared with the model group,high, medium-dose Tongmai Oral Solution treatment group rats can be signif- icantly improved in weight(P＜0.05).Before treatment,each made-modle group Qi deficiency and blood stasis syndrome' s score increased significantly compared with the control group(P＜0.001).Post-treatment,Qi deficiency and blood stasis syndrome' s score of high,medium,medium and low-dose Tongmai oral solution treatment group significantly decrease compared with the model group and lotensin treatment group(P＜0.01),there are no significant differentce between the model group and lotensin treatment group,no significant difference among high,medium,medium and low-dose Tongmai oral solution treatment group too.
3.Post-treatment,compared with the control group,serum t-PA content decreease and serum PAl content increase with varying degrees in other groups. Compared with the model group,serum t-PA content increased and serum PAI content decrease slightly in treatment groups.But there was no significant difference.Post-treatment,compared with the control group,the BUN and Scr have tended to increase in model group and low-dose Tongmai oral soluteion treatment groups without statistical significance.
4.We can see through the light microscope:the model group kidney HE and Masson staining:Most particles tubular-epithelial cells appear granularor vacuolar degeneration.Some tubular epithelial cells necrosis and desquamate,present bare basement membrane.nephric tubule lumen expand,there are some granular cast and cellular cast formed necrotic tissue and cell.Focal tubular atrophy can be seen.Renal interstitial are a widened significantly,inflammatory cell infiltrate,fibrous tissue hyperplasia,appear cable-like,patchy fibrosis. In addition to a part of glomerular renal capsule light-oderate expansion, there are noobvious lesions in glomerulus.All rongmai oral solution treatment group and Lotensin group have analogous changes,But there are different levels of improvement about pathological changes,compared with the model.
5.Post-treatment,CIS increased significantly in other made-modle groups (P＜0.001),compared with the control group.Compared with the model group, CIS decrease with varying degrees in treatment groups,among the total, high dose of Chinese medicine and Lotensin treatment group decreased significantly(P＜0.05).Compared with the lotensin treatment group,Chinese high-dose group has no significant difference(P＞0.05).Compared with the control group,AFS is significantly in other made-modle groups (P＜0.001).Compared with the model group,AFS decrease markedly in treatment groups(P＜0.01).among the total,high dose of Chinese medicine treatment group decreased significantly.Compared with Lotensin and medium and low-dose Tongmai oral solution treatment group,there is significant difference too (P＜0.05).
Experiment 2:
1.No significant expression of TGF-β1 in nephridial tissue of control group. Post-treatment,the expression of TGF-β1 increased significantly in mademodle groups(P＜0.001),compared with the control group.Compared with the model group,the expression of TGF-β1 decrease with varying degrees in treatment groups,among the total,Lotensin treatment group decreased significantly(P＜0.01).High dose of Chinese medicine treatment group decreased obviously(p＜0.05),but worse than treatment group treatment group(p＜0.05); No significant expression of HGF in nephridial tissue of control group, Post-treatmen,the expression of HGF increased significantly in made-modle groups(P＜0.001);Compared with the model group,high-dose of chinese medicine treatment group decreased significantly(P＜0.05).Compared with the model group,the expression of HGF increased significantly in high-dose Tongmai oral solution treatment group(P＜0.05).
2.Post-treatment,compared with the control group,the expression of MMP-9、TIMP-1 increased significantly in made-modle groups(P＜0.001),MMP-9/TIMP-1 decreased significantly.Compared with the model group,MMP-9 and MMP-9/TIMP-1 increase with varying degrees in treatment groups,TIMP-1 decrease signifycantly with varying degrees in treatment groups(P＜0.001),high-dose Tongmai oral solution treatment group is better than Benazepri treatment group (P＜0.001).
3.Post-treatment,compared with the control group,the content of t-PA within obstruction-side kidney of made-modle groups decrease with varying degrees, among the total,the modle group decreased significantly(P＜0.05),the content of PAI within obstruction-side kidney of made-modle groups increase with varying degrees,among the total,the modle group decreased significantly (P＜0.05).Compared with the model group,the content of t-PA within obstructtion-side kidney of made-modle groups increase with varying degrees,but no significant difference(P＞0.05),the content of PAl within obstruction side kidney of made-modle groups decrease with varying degrees,but only high-dose Tongmai oral solution treatment group decrease significantly(P＜0.05).
Conclusion:
1.To IgA nephropathy(with TIL,24-hour urine protein <3.5 g/d,without hypertension,renal function was normal)patiens,Qi deficiency of Fei and Shen,Qi deficiency of Fei and pi,Qi and Yin deficiency are common to capital syndromes,Yin deficiency of Gan and Shen and Yang deficiency of Pi relatively rare.Blood stasis syndrome and damp-heat syndrome are common to Biao syndrome. Capital syndrome included Qi deficiency syndrome accounted for 94.3%,blood stasis syndrome,blood stasis syndrome accounted for 55.2%among biao syndrome. Qi deficiency syndrome and blood stasis syndrome are common syndromes to the disease.Therefore,it could be inferred that yiqihuoxue herbs have a wider applicability in such diseases。The existence of biao syndromes such as blood stasis,damp-heat and damp-zhuo prompt tubulointerstitial damage to some extent,especially the blood stasis syndrome.The patiens with Yin deficiency of Gan and Shen as capital syndrome cound have more serious tubulo interstitial damage than as capital syndrome.
2.TO made-model group rats,weight and other general condition decreased significantly. Qi deficiency and blood stasis syndrome score increased significantly, the content of t-PA of Serum decreased in varying degrees,the content of t-PA of Serum decreased in varying degrees with typical tubulointerstitial damage pathological changes.YiqihuoxueL compound-Tongmai oral solution have a certain degree of improvement to above-mentioned appearances.This shows that the modle of ombination of disease and syndrome made by aging and swimming fatigue and UUO is relatively successful,It is expected for a similar study of reference.
3.Research shows that:Tongmai oral solution can improve made-modle rats overall performance such as weight,other general condition,Qi deficiency and blood stasis syndrome.At the same time,it can significantly reduce the local micro-pathological changes of tubulointerstitial damage,Improvements about CIS score are similar to benazepril.On the other hand,improvements abo AFS score are better than the benazepril,the results are dose-dependent.
4.The mechanism of Tongmai oral solution to TIL may be related to the following: increasing HGF expression,decreasing TGF-β1 expression;increasing MMP-9 expression,decreasing TIMP-1 expression,increasing MMP-9/ TIMP-1 ratio, Reducing the content of the PAI of kidney mechanisms and so on.
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