芪蛭肾综颗粒对难治性肾病综合征患者硫氧还蛋白系统、尿miRNA-192、29c表达的研究
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摘要
原发性肾病综合征(primary nephrotic syndrome,PNS)是以大量蛋白尿、低蛋白血症、水肿等为特征,在激素标准疗法治疗后,出现激素抵抗、依赖或者无效或频繁复发,从而成为难治性肾病综合征(refractory nephropathy syndrome, RNS)。目前RNS的发病机制上不完全清楚,临床治愈率较低,缺乏有效的治疗手段,因此,早期诊断、有效地治疗、判断预后成为RNS医学领域极为重要的问题。
RNS的发生发展与多重因素相关,研究证实,RNS与增强的氧化应激中的活性氧(Reactive Oxygen Species,ROS)反应有关,ROS可能参与了肾小球足细胞、系膜细胞、内皮细胞等肾小球滤过屏障的损伤及后来蛋白尿的发生过程,且与病情的轻重密切相关,所以抗氧化治疗逐渐被人们所重视。硫氧还蛋白系统是一类具有氧化还原活性的小分子蛋白系统,由硫氧还蛋白(thioredoxin, TRX)、还原型辅酶Ⅱ(NADPH)和硫氧还蛋白还原酶(TRX-R)3部分组成,具有调节细胞氧化还原状态、对抗氧化应激、激活转录因子、促进细胞生长以及抑制细胞凋亡等作用。TRX在人体多种组织和细胞中均有表达,它通过调节细胞氧化还原状态来对抗氧化应激。硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)是TRX的结合蛋白之一,通过抑制硫氧还蛋白系统的功能而发挥介导氧化应激作用。因此,TRX和TXNIP在氧化应激中均发挥着重要的作用,是机体维持氧化还原平衡的重要因子。通过对RNS患者血浆TRX、TXNIP表达水平测定,来探讨硫氧还蛋白系统在RNS患者发生发展中的作用。
同时,microRNA(miRNA)具有多种生物学功能,在进化上高度保守,在生长发育、生理功能上起重要作用,尤其对恶性肿瘤的发生发展有重大影响。miRNA与mRNA相比,在体外更稳定,在体内代谢周期更长,它们参与调控基因表达的转录后,通过信使RNA降解或转化抑制蛋白质合成。许多miRNA物种在不同病理条件对肾脏细胞进行调控,可能参与肾脏疾病的发病机制。研究证实niRNA在尿液中大量并稳定存在,因此在肾脏疾病中尿miRNA有潜在的发展前景。许多RNS的病理中存在细胞外基质(extracellular matrix,ECM)蛋白的蓄积、弥漫或结节性肾小球硬化、小管萎缩、间质纤维化等,miR-192参与ECM的发病过程,而miR-192是唯一一种被发现在肾小球系膜细胞表达的miRNA。同时,有研究提示在转化生长因子β1(TGF-β1)诱导大鼠正常肾成纤维细胞(NRK-49F)细胞纤维化过程中miR-29c表达下调,而miR-29c的表达增强可有效抑制TGF-β1刺激NRK-49F细胞株Col Ⅰ的表达,表明miR-29c是抑制纤维化的基因。在肾间质纤维化发生时或发生前,维持细胞的rniR-29c的高表达可抑制或延缓纤维化进展。因此,miR-29c与RNS的肾间质纤维化的相关性尤为重要。miRNA既往的研究表明,miR-29c,miR-192在肾病外周血及组织中有不同表达,在尿沉淀中可以量化,根据不同病理类型尿miRNA水平不同,所以对miRNA的研究可以协助RNS做病理类型的评估,临床治疗的指导,预后的判定等等。
目前西药治疗RNS主要是激素、免疫抑制剂和细胞毒类药物,但副作用大,临床应用受限。因此,发挥中医药特色和优势,探求临床方便有效的中医药治疗RNS有着重要的意义。
RNS属于中医的“水肿”、“腰痛”、“虚劳”、“尿浊”等范畴。“水肿”与RNS最相符。气虚血瘀是RNS发病的基础,湿热蕴结是发展过程中的促进因素,本虚标实是RNS的病机特点。所以以益气健脾益肾、活血化瘀通络、清热利湿消肿等治疗为法,芪蛭肾综颗粒由黄芪、白术、益母草、水蛭、玉米须、石韦组成。根据RNS临床大样本研究,总结出最常见证型：气虚血瘀湿热型,以此证为法提炼出芪蛭肾综颗粒,成为南方医科大学珠江医院和山东省威海市中医院的协定处方。芪蛭肾综颗粒作为院内协定处方,已经应用多年,临床验证表明疗效确切,能显著减少尿蛋白,改善症状,减轻高脂高凝状态,延缓肾小球硬化进展,保护肾功能。但其治疗RNS的作用机制还不清楚,有待进一步研究。本课题通过芪蛭肾综颗粒的治疗,检测临床症状、血生化指标、血清超氧化物歧化酶(SOD)、丙二醛(MDA)、TRX、TXNIP等ROS因子的表达,为临床应用提供依据。通过尿miR-29c,miR-192等基因的表达和病理类型相关性的研究,为RNS的诊疗和预后提供新的靶点。
第一部分芪蛭肾综颗粒对RNS患者的治疗作用及血清TRX、TXNIP表达的影响
目的：观察芪蛭肾综颗粒对气虚血瘀湿热型RNS患者的治疗作用,通过检测RNS患者血清TRX及TXNIP的表达,分析这两种氧化应激因子与RNS的相关性,探讨其在RNS发生发展中可能的作用机制,从而为RNS的治疗提供新的靶点与方向,同时探讨临床分型、病理分型的疗效的关系,指导临床治疗。
方法：选择南方医科大学珠江医院肾病中心和威海市中医院肾病科2010年12月～2012年12月门诊或住院的RNS患者60例,临床分型：常复发22例,激素依赖13例,激素无效2例,激素部分效应23例。其中有46例行肾穿刺活检术,病理分型有：局灶节段性肾小球硬化(Focal segmental glomerulosclerosis, FSGS)23例,微小病变型(Minimal change nephritis,MCD)2例,系膜增生性肾炎(mesangial proliferative nephritis,MsPGN)18例,膜型肾病(Membranous nephropathy,MN)3例。随机分成实验组(芪蛭肾综颗粒加基础治疗)30例,对照组(黄葵胶囊加基础治疗)30例,疗程3个月,各组间在年龄、性别、临床分型、病理分型等方面无统计学差异。分析临床类型和病理类型的关系,及其与疗效的关系,检测两组治疗前后中医证候评分疗效、临床疗效、24h尿蛋白定量、血浆白蛋白(Alb)、血肌酐(Scr)、凝血酶原时间(PT)、部分凝血酶原时间(APTT)、纤维蛋白原(FIB)、SOD、MDA的变化。采用酶联免疫检测技术(ELISA)检测血清TRX及TXNIP的表达水平。
结果：
1.病理类型与临床分型的关系：各病理类型中的临床分型比较,差异无统计学意义(P>0.05)；临床分型与病理类型的关系：FSGS和MsPGN比例较高,与其他类型相比有统计学意义(P<0.05)。
2.两组临床疗效比较：实验组与对照组比较疗效显著(P<0.05)。
3.各个临床分型疗效比较,差异无统计学意义(P>0.05)。
4.各病理类型疗效比较,MsPGN、MCN临床疗效较好,与其他类型相比有统计学意义(P<0.05)。
5.中医证候疗效：两组治疗前后总中医症候评分比较：两组治疗后与治疗前相比中医症候总积分均显著下降,差异均有统计学意义(P<0.01),治疗后实验组在症状改善明显优于对照组(P<0.01)。
6.实验组治疗后24h尿蛋白、Scr、BUN较治疗前显著降低,Alb显著升高,差异有统计学意义(P<0.01)；对照组治疗后与治疗前比较24h尿蛋白、Alb显著升高,差异有统计学意义(P<0.01)；两组治疗后比较,实验组在Alb显著上升,BUN显著下降,实验组均优于对照组,差异有统计学意义(P<0.05,P<0.01)。
7.实验组治疗后与治疗前比较FIB显著降低,PT、APTT显著升高,差异有统计学意义(P<0.01),对照组治疗后与治疗前比较PT、APTT显著升高,差异有统计学意义(P<0.01)；两组治疗后比较,实验组FIB显著降低,差异有统计学意义(P<0.05)。
8.实验组治疗后与治疗前比较,SOD显著升高,MDA显著降低,差异有统计学意义(P<0.01)；两组治疗后比较,实验组较对照组SOD明显升高,MDA明显下降,差异有统计学意义(P<0.01)。
9.实验组治疗后与治疗前比较,TRX显著升高,TXNIP显著降低,差异有统计学意义(P<0.05)；对照组治疗后与治疗前比较TRX、TXNIP变化不明显,差异无统计学意义(P>0.05),治疗后两组比较,治疗组与对照组相比TRX显著上升,TXNIP显著降低明显,差异有统计学意义(P<0.05)。
结论：
1.在各临床分型中FSGS和MsPGN比例较高,在病理类型中MCD、MsPGN疗效较高。
2.芪蛭肾综颗粒治疗RNS患者疗效评价：能明显的降尿蛋白,优于对照组。
3.临床分型与疗效关系证实：激素依赖和常复发疗效稍好,但与激素无效和部分效应的对比无显著性差异。即临床分型和疗效无明显相关性。
4.病理分型与疗效关系证实：治疗后MCD、MsPGN治疗效果明显,优于与其他病理分型。
5.芪蛭肾综颗粒在中医症候疗效比较,实验组优于对照组：治疗后中医症候总积分明显下降,实验组优于对照组。即芪蛭肾综颗粒在中医症候改善优势明显。
6.芪蛭肾综颗粒有更好的升高血浆白蛋白,保护肾功的作用。
7.芪蛭肾综颗粒能够改善RNS患者血液高凝状态,升高PT、APTT,在降低FIB上显著优于对照组。
8.芪蛭肾综颗粒能够升高RNS患者SOD水平,降低其MDA水平,改善机体氧化应激状态。
9.芪蛭肾综颗粒能够升高RNS患者TRX水平,降低TXNIP水平。TRX、TXNIP作为氧化应激的相关因子,在RNS中发挥作用。
10.芪蛭肾综颗粒可能通过抗氧化应激机制,在RNS的治疗中发挥着重要的作用。
第二部分尿miRNA-192,29c的表达与RNS芪蛭肾综颗粒的疗效和
RNS病理关系的初步研究
目的：通过检测尿miR-192、miR-29c在RNS不同病理类型的患者和健康对照者尿中的表达,探讨尿miR-192、miR-29c作为RNS辨别肾组织学病变和反映疾病严重程度的可靠的非侵入性的生物标记物的可能性。检测芪蛭肾综颗粒治疗前后尿miR-192和miR-29c的表达,研究与24h尿蛋白、Alb、TC、脂蛋白a (Lp-a)等临床指标的相关性。
方法：选择南方医科大学珠江医院肾病中心和威海市中医院肾病科2010年12月～2012年12月门诊或住院的经肾活检确诊的44例RNS患者,其中包括18例MsPGN,3例MN和23例FSGS,随机分成实验组和对照组,随机分成实验组(芪蛭肾综颗粒加基础治疗)23例,对照组(黄葵胶囊加基础治疗)21例,疗程3个月,各组间在年龄、性别、病理分型等方面无差异。设立健康对照组15人,为威海市中医院肾病科医护人员中的健康志愿者。RNS患者取治疗前和治疗3个月后的清晨空腹尿液标本100ml和血标本,健康人清晨空腹收集尿液标本100ml,尿标本先在3000rmp离心30分钟后,再在13000rmp离心5分钟,丢弃上清液,将尿沉渣分装于1.5ml EP管中,每管0.25m1。每只EP管中加入0.75ml Trizol LS,剧烈震荡1min充分裂解,室温静置5min后-80℃冰箱保存待用。Trizol提取总RNA,进行RNA质量检测合格后,即可构建miR-192、miR-29c的RT-PCR反应体系,分别检测各样本miR-192、miR-29c的相对表达量并做统计分析。治疗前后检测24小时尿蛋白,血标本用生化仪做治疗前后TC、Lp-a、Alb等指标的检测,测试尿miR-192、miR-29c与RNS患者症状指标的相关性。
结果：
1与健康对照组比较,miR-29c在FSGS中的表达明显下降,差异有统计学意义(P<0.05),在MsPGN、MN中的表达下降不明显,差异无统计学意义(P>0.05)。miR-192在MsPGN表达明显上升(P<0.05),在FSGS、MN中的表达上升不明显(P>0.05)。
2.尿miR-29c表达与Lp-a表达呈负相关,与Alb表达呈正相关,均有统计学意义(P<0.01)。尿miR-192表达与24h尿蛋白和TC表达正相关,均有统计学意义(P<0.05,P<0.01)。
3.实验组治疗后与治疗前比较尿miR-29c显著升高,miR-192表达显著降低,有统计学意义(P<0.01)；对照组治疗前后比较,尿miR-29c显著升高,miR-192表达显著降低,有统计学意义(P<0.05)；治疗后实验组与对照组比较,治疗组miR-29c显著升高,有统计学意(P<0.05)；
结论：
1.尿miR-192表达在MsPGN患者中显著升高；
2.MsPGN患者可能存在较多的ECM分泌和沉积,可以判断肾小球ECM的程度。指导临床治疗和预后的判断；
3.尿miR-29c表达在FSGS患者显著降低；
4.FSGS患者可能存在肾间质纤维化明显,可以判断肾间质纤维化的程度,上调尿miR-29c水平可能有助于肾纤维化的治疗；
5.尿miR-192,29c的差异表达显示了RNS各病理类型之间的差异,提供RNS分型的新方法,更好地理解RNS的发病机理；
6.尿miR-192,29c的表达与24h尿蛋白、TC、Lp-a、Alb的相关性显示,多方面因素均可加重肾纤维化和ECM沉积；
7.芪蛭肾综颗粒能上调尿miR-29c水平表达,下调尿miR-192水平表达,对ECM的清除、抗纤维化有较好的作用；可能通过双向作用来抑制ECM的增生,延缓肾小球硬化。
Primary nephrotic syndrome (PNS) is characterized by a massive proteinuria, hypoalbuminemia, edema, etc of a kind of clinical common kidney disease, after standard therapy of the hormone to treat disease, can't effectively relieve and accur hormone resistance, dependency or invalid or frequent recurrence, which in turn become refractory nephrotic syndrome (RNS). At present, the pathogenesis of RNS are not entirely clear, clinical cure rate is low and lack of effective treatment, therefore, early diagnosis, effective treatment and judge prognosis for RNS become extremely important medicine field problems.
Development of RNS is associated with multiple factors, studies confirm that RNS is associated with enhanced oxidative stress (ROS) reaction, ROS may participate in the glomerular podocytes, mesangial cells, endothelial cells, such as glomerular filtration barrier damage and subsequent process of the occurrence of proteinuria, and closely related to the severity of illness, so antioxidant treatment gradually attention by people. Thus, thioredoxin (TRX) and TXNIP play an important role in oxidative stress, is an important factor for the body to maintain redox balance. TRX are expressed in various body tissues and cells, it is by regulating cellular redox status against oxidative stress, thioredoxin-interacting protein (TXNIP) is one of the binding protein of TRX, by inhibiting the function of thioredoxin system and play a role of mediated oxidative stress. Therefore, TRX and TXNIP play an important role in oxidative stress, is an important factor of the body to maintain redox balance. RNS patients measure plasma TRX, TXNIP expression level, to explore the thioredoxin system in its role in the development of RNS patients. At the same time, micrornas (miRNA) has a variety of biological functions, highly conserved in evolution, plays an important role in the growth, physiological function, especially have significant effects on the occurrence of malignant tumor development. MiRNA compared with mRNA, more stable in vitro and in vivo metabolic cycle longer, they are involved in regulation of gene expression after transcription, via messenger RNA degradation or inhibition of protein synthesis.Many miRNA species control kidney cells in different pathological conditions, may be involved in the pathogenesis of kidney diseases. Study of miRNS in urine, large and stable, so in kidney disease, urinary micrornas have potential development prospects. Many RNS pathology exists in the extracellular matrix (ECM) protein accumulation, diffuse or nodular, glomerular sclerosis, tubular atrophy and interstitial fibrosis, miR-192is involved in the onset of the ECM process, and miR-192is the only one was found in glomerular mesangial cell expression of miRNA. At the same time, research hints in the transforming growth factor beta1(TGF-β1) induction of normal rat renal fibroblasts (NRK-49F) cell miR-29c expression by fibrosis process, while the expression of miR-29c enhancement can effectively inhibit TGF-β1and stimulate express of NRK-49cell lin Col Ⅰ, showed that miR-29c is inhibiting fibrosis genes. When or before in renal interstitial fibrosis, miR-29c high expression of cell can inhibit or delay the fibrosis progression. Therefore, correlation of miR-29c and RNS of renal interstitial fibrosis is particularly important. MiRNA always studies have shown that miR-29c, miR-192in kidney disease have different expression in peripheral blood and tissue, can be quantified in the urinary sediment, according to different pathological type urinary miRNA level is different, So the study of the miRNA can assist assessment of RNS pathological types, and judgement of the guidance of the clinical treatment and prognosis, and so on.
At present, western medicine treatment of RNS is mainly hormone and immune inhibitors and cytotoxic drugs, but the side effects,limite clinical application. Therefore, developing the features and advantages of TCM, searching convenient and effective treatment of traditional Chinese medicine in RNS has important significance.
RNS belongs to "edema","back pain","consumptive disease","urine turbid" and etc. in TCM."Edema" consistent with the RNS. Qi deficiency and blood stasis is the basis of RNS onset. Damp and hot accumulate knot is the catalyst in the process of development. The asthenia in origin and pathogenic factors in superficiality is the pathogenesis characteristics of RNS. So treatment with replenishing qi to invigorate the spleen and kidney and promoting blood circulation to remove blood stasis and clearing heat and promoting diuresisbeen detumescence, By astragalus, atractylodes, motherwort, leech, corn stigma, pyrrosia leaf of QZSZ particle comprise Huangqi,Baizhu,Yimucao,Shuizhi,Yumixu,Shiwei. According to the large sample study of RNS clinical, summed up the most often type of syndrome:qi deficiency and blood stasis and damp and hot, QZSZ particle become prescription for agreement of zhujiang hospital, southern medical university and TCM hospital in weihai city shandong province. QZSZ particle as an agreement prescription in hospital, has been used for many years, the clinical curative effect is verified, and can significantly reduce urinary protein, improve symptoms, reduce the high fat high coagulation state, delay the progress of glomerular sclerosis and protect renal function. But therapeutical mechanism of it in RNS is not clear, and needs further research. This topic through the treatment of QZSZ particle Detection clinical symptoms, blood biochemical indicators, serum superoxide dismutase (SOD), malondialdehyde (MDA), TRX and TXNIP ROS factor expression, provide the basis for clinical application. Through correlation study of gene expression of the urine miR-29c and miR-192and pathological types, it provide new targets for the diagnosis and treatment and prognosis of RNS.
Chart Ⅰ QZSZ particle in the treatment of patients with RNS function and serum TRX, TXNIP expression
Objective:To observe the treatment of the QZSZ particle on qi deficiency blood stasis damp and hot syndrome in RNS patients, through testing the patient's serum TRX and TXNIP expression, analysis of the two kinds of oxidative stress factor and RNS severity and prognosis of relevance, to explore its possible role in RNS development mechanism, which provide new targets for the treatment of RNS, and to study the relation of different clinical and pathological classification in curative effect.
Methods:Choice of kidney disease center of zhujiang hospital, and TCM hospital in weihai in December2010to December2012outpatient or hospitalization RNS,60patients with clinical classification:22cases of often relapse,13cases of hormone dependence,2cases of hormone is invalid in,23cases of hormone some effects.46cases of whom underwent renal biopsy and pathological classification are:23cases of focal segmental glomerular sclerosis (FSGS),2cases of minimal change disease small lesion type (MCD),18cases of mesangial proliferative glomerulonephritis (MsPGN),3patients of membrane nephropathy (MN). randomly divided into30cases in experimental group (QZSZ particle plus foundation treatment) and30cases in control group (ambrette capsule plus and foundation treatment), treatment for3months, there is no difference between groups in age, sex, clinical classification, pathological classification, etc. Detection of two groups of curative effect of TCM syndrome score before and after the treatment, clinical efficacy,24h urinary protein, Alb, serum creatinine (Scr), prothrombin time (PT), partial prothrombin time (APTT), fibrinogen (FIB), the change of SOD and MDA. Using enzyme-linked immune detection technology (ELISA) to detect serum TRX and TXNIP expression level.
Results:
1. The relationships of pathological types and clinical classification:it was no significant difference (P>0.05); Relationships of clinical classification and pathological types of:FSGS and MsPGN ratio is higher, compared with other types have statistical significance (P<0.05).
2. Two groups of clinical curative effect comparison:the total effective rate of experimental group compared with the control group, have statistical significance (P<0.05).
3. Comparison of the curative effect of each clinical classification:there was no statistically significant difference (P>0.05).
4. Comparison of the curative effect of each pathological types:curative effect of MN, MsPGN is obvious, it was statistically differences (P<0.05).
5. The main syndromes of traditional Chinese medicine:the symptoms were improved after treatment, compared with before in two groups, treatment were statistically significant difference (P<0.01); The symptoms of comparative differences of two groups after treatment are statistically significant (P<0.01). the experimental group is better than the control group.
6. Experimental group after treatment,24h urinary protein, Scr, BUN was significantly reduced, significantly reduced the difference was statistically significant (P<0.01); The control group after treatment compared with before treatment,24h urinary protein significantly reduced, significantly reduced,the difference was statistically significant (P<0.01); Compare the two groups after treatment, the experimental group in Alb to rise significantly, BUN dropped significantly, the experimental group were better than control group, the difference was statistically significant (P<0.05, P<0.01).
7. Comparison of the experimental group before and after treatment,FIB decreased significantly, PT, APTT significantly increased (P<0.01), the control group before and after treatment comparison of PT,APTT significantly higher (P<0.01); Compare the two groups after treatment, the experimental group FIB decreased significantly, the difference was statistically significant (P<0.05).
8. Comparison of the experimental group before and after treatment, a significant rise in SOD, MDA decreased significantly (P<0.01).Comparing the two groups after treatment, the experimental group than the control group SOD significantly increased, MDA decreased obviously (P<0.01).
9. Comparison of the experimental group before and after treatment, TRX was significantly higher, TXNIP decreased significantly (P<0.05). Control group TRX, TXNIP comparison before and after the treatment, there was no statistically significant difference (P>0.05). Comparing the two groups after treatment, the experimental group than the control group TRX significantly increased, TXNIP decreased obviously (P<0.01).
Conclusion:
1. Proportion of FSGS and MsPGN in various clinical classification is higher. Efficacy of MCD, MsPGN in the pathological types of is higher.
2.The QZSZ particle curative effect evaluation:can obviously drop of urine protein, is better than the control group.
3. The relationship of the clinical classification and effect has proved that the curative effect of hormone dependence and often relapse is better, but there is no significant difference compared with hormone is invalid and some effects.The clinical has no obvious correlation with curative effect.
4. The pathological classification relationship has proved that the effect of MsPGN and MCD is obvious, is better than with other pathological types.
5. Comparitting curative effect of of QZSZ particle in TCM syndrome, the experimental group is better than the control group, after treatment the total integral of TCM symptoms decreased obviously, the experimental group is better than the control group. QZSZ particle can heal RNS better in TCM symptoms.
6. QZSZ particle significantly elevated plasma Albumin, protect the renal function.
7. QZSZ particle can improve high blood coagulation state in patients with RNS, rise in PT and APTT, reduce FIB was significantly better than the control group.
8. QZSZ particle can increases SOD level and reduce the MDA level of the RNS patients, improve the body state of oxidative stress.
9. QZSZ particle can increase TRX levels, lower levels of TXNIP. TRX, TXNIP as oxidative stress related factors, play a role in the RNS.
10. QZSZ particle may play an important role in the treatment of RNS through the mechanism of resistance to oxidative stress.
Chart II Preliminary study of expression of the urinary miRNA-192,29c with curative effect of QZSZ particle inRNS and pathology of RNS
Objective:Study internal relation of urine miR-192and miR-29c in different clinical pathological types, explore the urine miR-192and miR-29c to distinguish the severity of disease and renal histological lesions of the possibility as a non-invasive biomarkers. Urinary miR-192and miR-29c expression of before and after QZSZ particle treatment are associated with24h urinary protein, Alb, TC, Lp-a and so on.
Methods:Selection of kidney disease center of zhujiang hospital, southern medical university and nephrology department in TCM hospital in weihai in December2010to December2012outpatient or hospitalization of the renal biopsy diagnosed44RNS patients, including18cases of MsPGN,3cases of MN and23cases of FSGS, randomly divided into the experimental group(QZSZ particle plus foundation treatment) and the control group,21cases of control group (ambrette capsule plus foundation treatment), treatment for3months, each group in age, gender, pathological classification, no difference. Set up15healthy volunteers as healthy controls in medical staff weihai hospital. Before and after3months of treatment RNS patient t collect specimens of blood and urine samples of100ml early in the morning on an empty stomach, healthy people to collect urine samples of100ml early in the morning on an empty stomach, urine specimen in3000RMP centrifugal before30minutes, again in13000RMP centrifuge for5minutes, discarding the supernatant fluid, the urine sediment repackaging in1.5ml EP tube,0.25ml per tube. Add0.75ml per EP tube Trizol LS, volatility1min cracking fully, let stand at room temperature after5min-80℃refrigerator. Trizol to extract total RNA, RNA quality testing after qualified, can build miR-192and miR-29c RT-PCR reaction system, each sample was detected in miR-192and miR-29c. The relative expression quantity and do statistical analysis. Test24hours urinary protein, Lp-a,TC, Alb before and after the treatment, test correlation of the urine miR-192and miR-29c and the above indexes.
Results:
1. Compared with healthy controls, miR-29c expression in the FSGS decreased obviously (P<0.05), the expression of MsPGN, MN decline is not obvious (P>0.05). miR-192in MsPGN expression increased obviously (P<0.05), the expression of MN,FSGS is not obvious (P>0.05).
2. The expression of urinary miR-29c and Lp-a showed a negative correlation, It was positively correlated with Alb, had statistical significance (P<0.05). The expression of urinary miR-192express positive correlation with the expression of urinary24h urinary protein and TC, had statistical significance (P<0.05).
3. Comparison of the experimental group before and after the treatment, the expression of miR-192significantly reduced and the expression of miR-29c significantly rised (P<0.01). Comparison of the control group before and after the treatment, the expression of miR-192significantly reduced and the expression of miR-29c significantly rised (P<0.05).Comparison of two groups after treatment, the expression of miR-29c in the experimental group significantly rised (P<0.05)
Conclusion:
1. Urine miR-192expression significantly increased in MsPGN patients.
2. MsPGN patients may have more ECM secretion and deposition, can determine glomerular ECM. It can guide clinical treatment and prognosis.
3. The urine miR-29c expression in patients with FSGS decreased significantly.
4. Patients with FSGS may be renal interstitial fibrosis significantly. It can judge the degree of renal fibrosis, increase urine levels of miR-29, help in treatment of renal fibrosis.
5. Differences in expression of urine miR-192,29c shows the differences between the various pathological types in RNS, and provides new ways to better understand the pathogenesis of RNS.
6. The expression of urine miR-192,29c with24h urinary protein, TC, Lp-a shows that several factors can increase renal fibrosis and ECM deposition.
7. QZSZ particle can increase the expression of urine miR-29c levels, and lower the expression of urinary miR-192levels in patients, play a regulatory role of the removal of the ECM, anti fibrosis, may through the two-way function to inhibit proliferation of ECM, delay the glomerular sclerosis.
RNS的发生发展与多重因素相关,研究证实,RNS与增强的氧化应激中的活性氧(Reactive Oxygen Species,ROS)反应有关,ROS可能参与了肾小球足细胞、系膜细胞、内皮细胞等肾小球滤过屏障的损伤及后来蛋白尿的发生过程,且与病情的轻重密切相关,所以抗氧化治疗逐渐被人们所重视。硫氧还蛋白系统是一类具有氧化还原活性的小分子蛋白系统,由硫氧还蛋白(thioredoxin, TRX)、还原型辅酶Ⅱ(NADPH)和硫氧还蛋白还原酶(TRX-R)3部分组成,具有调节细胞氧化还原状态、对抗氧化应激、激活转录因子、促进细胞生长以及抑制细胞凋亡等作用。TRX在人体多种组织和细胞中均有表达,它通过调节细胞氧化还原状态来对抗氧化应激。硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)是TRX的结合蛋白之一,通过抑制硫氧还蛋白系统的功能而发挥介导氧化应激作用。因此,TRX和TXNIP在氧化应激中均发挥着重要的作用,是机体维持氧化还原平衡的重要因子。通过对RNS患者血浆TRX、TXNIP表达水平测定,来探讨硫氧还蛋白系统在RNS患者发生发展中的作用。
同时,microRNA(miRNA)具有多种生物学功能,在进化上高度保守,在生长发育、生理功能上起重要作用,尤其对恶性肿瘤的发生发展有重大影响。miRNA与mRNA相比,在体外更稳定,在体内代谢周期更长,它们参与调控基因表达的转录后,通过信使RNA降解或转化抑制蛋白质合成。许多miRNA物种在不同病理条件对肾脏细胞进行调控,可能参与肾脏疾病的发病机制。研究证实niRNA在尿液中大量并稳定存在,因此在肾脏疾病中尿miRNA有潜在的发展前景。许多RNS的病理中存在细胞外基质(extracellular matrix,ECM)蛋白的蓄积、弥漫或结节性肾小球硬化、小管萎缩、间质纤维化等,miR-192参与ECM的发病过程,而miR-192是唯一一种被发现在肾小球系膜细胞表达的miRNA。同时,有研究提示在转化生长因子β1(TGF-β1)诱导大鼠正常肾成纤维细胞(NRK-49F)细胞纤维化过程中miR-29c表达下调,而miR-29c的表达增强可有效抑制TGF-β1刺激NRK-49F细胞株Col Ⅰ的表达,表明miR-29c是抑制纤维化的基因。在肾间质纤维化发生时或发生前,维持细胞的rniR-29c的高表达可抑制或延缓纤维化进展。因此,miR-29c与RNS的肾间质纤维化的相关性尤为重要。miRNA既往的研究表明,miR-29c,miR-192在肾病外周血及组织中有不同表达,在尿沉淀中可以量化,根据不同病理类型尿miRNA水平不同,所以对miRNA的研究可以协助RNS做病理类型的评估,临床治疗的指导,预后的判定等等。
目前西药治疗RNS主要是激素、免疫抑制剂和细胞毒类药物,但副作用大,临床应用受限。因此,发挥中医药特色和优势,探求临床方便有效的中医药治疗RNS有着重要的意义。
RNS属于中医的“水肿”、“腰痛”、“虚劳”、“尿浊”等范畴。“水肿”与RNS最相符。气虚血瘀是RNS发病的基础,湿热蕴结是发展过程中的促进因素,本虚标实是RNS的病机特点。所以以益气健脾益肾、活血化瘀通络、清热利湿消肿等治疗为法,芪蛭肾综颗粒由黄芪、白术、益母草、水蛭、玉米须、石韦组成。根据RNS临床大样本研究,总结出最常见证型：气虚血瘀湿热型,以此证为法提炼出芪蛭肾综颗粒,成为南方医科大学珠江医院和山东省威海市中医院的协定处方。芪蛭肾综颗粒作为院内协定处方,已经应用多年,临床验证表明疗效确切,能显著减少尿蛋白,改善症状,减轻高脂高凝状态,延缓肾小球硬化进展,保护肾功能。但其治疗RNS的作用机制还不清楚,有待进一步研究。本课题通过芪蛭肾综颗粒的治疗,检测临床症状、血生化指标、血清超氧化物歧化酶(SOD)、丙二醛(MDA)、TRX、TXNIP等ROS因子的表达,为临床应用提供依据。通过尿miR-29c,miR-192等基因的表达和病理类型相关性的研究,为RNS的诊疗和预后提供新的靶点。
第一部分芪蛭肾综颗粒对RNS患者的治疗作用及血清TRX、TXNIP表达的影响
目的：观察芪蛭肾综颗粒对气虚血瘀湿热型RNS患者的治疗作用,通过检测RNS患者血清TRX及TXNIP的表达,分析这两种氧化应激因子与RNS的相关性,探讨其在RNS发生发展中可能的作用机制,从而为RNS的治疗提供新的靶点与方向,同时探讨临床分型、病理分型的疗效的关系,指导临床治疗。
方法：选择南方医科大学珠江医院肾病中心和威海市中医院肾病科2010年12月～2012年12月门诊或住院的RNS患者60例,临床分型：常复发22例,激素依赖13例,激素无效2例,激素部分效应23例。其中有46例行肾穿刺活检术,病理分型有：局灶节段性肾小球硬化(Focal segmental glomerulosclerosis, FSGS)23例,微小病变型(Minimal change nephritis,MCD)2例,系膜增生性肾炎(mesangial proliferative nephritis,MsPGN)18例,膜型肾病(Membranous nephropathy,MN)3例。随机分成实验组(芪蛭肾综颗粒加基础治疗)30例,对照组(黄葵胶囊加基础治疗)30例,疗程3个月,各组间在年龄、性别、临床分型、病理分型等方面无统计学差异。分析临床类型和病理类型的关系,及其与疗效的关系,检测两组治疗前后中医证候评分疗效、临床疗效、24h尿蛋白定量、血浆白蛋白(Alb)、血肌酐(Scr)、凝血酶原时间(PT)、部分凝血酶原时间(APTT)、纤维蛋白原(FIB)、SOD、MDA的变化。采用酶联免疫检测技术(ELISA)检测血清TRX及TXNIP的表达水平。
结果：
1.病理类型与临床分型的关系：各病理类型中的临床分型比较,差异无统计学意义(P>0.05)；临床分型与病理类型的关系：FSGS和MsPGN比例较高,与其他类型相比有统计学意义(P<0.05)。
2.两组临床疗效比较：实验组与对照组比较疗效显著(P<0.05)。
3.各个临床分型疗效比较,差异无统计学意义(P>0.05)。
4.各病理类型疗效比较,MsPGN、MCN临床疗效较好,与其他类型相比有统计学意义(P<0.05)。
5.中医证候疗效：两组治疗前后总中医症候评分比较：两组治疗后与治疗前相比中医症候总积分均显著下降,差异均有统计学意义(P<0.01),治疗后实验组在症状改善明显优于对照组(P<0.01)。
6.实验组治疗后24h尿蛋白、Scr、BUN较治疗前显著降低,Alb显著升高,差异有统计学意义(P<0.01)；对照组治疗后与治疗前比较24h尿蛋白、Alb显著升高,差异有统计学意义(P<0.01)；两组治疗后比较,实验组在Alb显著上升,BUN显著下降,实验组均优于对照组,差异有统计学意义(P<0.05,P<0.01)。
7.实验组治疗后与治疗前比较FIB显著降低,PT、APTT显著升高,差异有统计学意义(P<0.01),对照组治疗后与治疗前比较PT、APTT显著升高,差异有统计学意义(P<0.01)；两组治疗后比较,实验组FIB显著降低,差异有统计学意义(P<0.05)。
8.实验组治疗后与治疗前比较,SOD显著升高,MDA显著降低,差异有统计学意义(P<0.01)；两组治疗后比较,实验组较对照组SOD明显升高,MDA明显下降,差异有统计学意义(P<0.01)。
9.实验组治疗后与治疗前比较,TRX显著升高,TXNIP显著降低,差异有统计学意义(P<0.05)；对照组治疗后与治疗前比较TRX、TXNIP变化不明显,差异无统计学意义(P>0.05),治疗后两组比较,治疗组与对照组相比TRX显著上升,TXNIP显著降低明显,差异有统计学意义(P<0.05)。
结论：
1.在各临床分型中FSGS和MsPGN比例较高,在病理类型中MCD、MsPGN疗效较高。
2.芪蛭肾综颗粒治疗RNS患者疗效评价：能明显的降尿蛋白,优于对照组。
3.临床分型与疗效关系证实：激素依赖和常复发疗效稍好,但与激素无效和部分效应的对比无显著性差异。即临床分型和疗效无明显相关性。
4.病理分型与疗效关系证实：治疗后MCD、MsPGN治疗效果明显,优于与其他病理分型。
5.芪蛭肾综颗粒在中医症候疗效比较,实验组优于对照组：治疗后中医症候总积分明显下降,实验组优于对照组。即芪蛭肾综颗粒在中医症候改善优势明显。
6.芪蛭肾综颗粒有更好的升高血浆白蛋白,保护肾功的作用。
7.芪蛭肾综颗粒能够改善RNS患者血液高凝状态,升高PT、APTT,在降低FIB上显著优于对照组。
8.芪蛭肾综颗粒能够升高RNS患者SOD水平,降低其MDA水平,改善机体氧化应激状态。
9.芪蛭肾综颗粒能够升高RNS患者TRX水平,降低TXNIP水平。TRX、TXNIP作为氧化应激的相关因子,在RNS中发挥作用。
10.芪蛭肾综颗粒可能通过抗氧化应激机制,在RNS的治疗中发挥着重要的作用。
第二部分尿miRNA-192,29c的表达与RNS芪蛭肾综颗粒的疗效和
RNS病理关系的初步研究
目的：通过检测尿miR-192、miR-29c在RNS不同病理类型的患者和健康对照者尿中的表达,探讨尿miR-192、miR-29c作为RNS辨别肾组织学病变和反映疾病严重程度的可靠的非侵入性的生物标记物的可能性。检测芪蛭肾综颗粒治疗前后尿miR-192和miR-29c的表达,研究与24h尿蛋白、Alb、TC、脂蛋白a (Lp-a)等临床指标的相关性。
方法：选择南方医科大学珠江医院肾病中心和威海市中医院肾病科2010年12月～2012年12月门诊或住院的经肾活检确诊的44例RNS患者,其中包括18例MsPGN,3例MN和23例FSGS,随机分成实验组和对照组,随机分成实验组(芪蛭肾综颗粒加基础治疗)23例,对照组(黄葵胶囊加基础治疗)21例,疗程3个月,各组间在年龄、性别、病理分型等方面无差异。设立健康对照组15人,为威海市中医院肾病科医护人员中的健康志愿者。RNS患者取治疗前和治疗3个月后的清晨空腹尿液标本100ml和血标本,健康人清晨空腹收集尿液标本100ml,尿标本先在3000rmp离心30分钟后,再在13000rmp离心5分钟,丢弃上清液,将尿沉渣分装于1.5ml EP管中,每管0.25m1。每只EP管中加入0.75ml Trizol LS,剧烈震荡1min充分裂解,室温静置5min后-80℃冰箱保存待用。Trizol提取总RNA,进行RNA质量检测合格后,即可构建miR-192、miR-29c的RT-PCR反应体系,分别检测各样本miR-192、miR-29c的相对表达量并做统计分析。治疗前后检测24小时尿蛋白,血标本用生化仪做治疗前后TC、Lp-a、Alb等指标的检测,测试尿miR-192、miR-29c与RNS患者症状指标的相关性。
结果：
1与健康对照组比较,miR-29c在FSGS中的表达明显下降,差异有统计学意义(P<0.05),在MsPGN、MN中的表达下降不明显,差异无统计学意义(P>0.05)。miR-192在MsPGN表达明显上升(P<0.05),在FSGS、MN中的表达上升不明显(P>0.05)。
2.尿miR-29c表达与Lp-a表达呈负相关,与Alb表达呈正相关,均有统计学意义(P<0.01)。尿miR-192表达与24h尿蛋白和TC表达正相关,均有统计学意义(P<0.05,P<0.01)。
3.实验组治疗后与治疗前比较尿miR-29c显著升高,miR-192表达显著降低,有统计学意义(P<0.01)；对照组治疗前后比较,尿miR-29c显著升高,miR-192表达显著降低,有统计学意义(P<0.05)；治疗后实验组与对照组比较,治疗组miR-29c显著升高,有统计学意(P<0.05)；
结论：
1.尿miR-192表达在MsPGN患者中显著升高；
2.MsPGN患者可能存在较多的ECM分泌和沉积,可以判断肾小球ECM的程度。指导临床治疗和预后的判断；
3.尿miR-29c表达在FSGS患者显著降低；
4.FSGS患者可能存在肾间质纤维化明显,可以判断肾间质纤维化的程度,上调尿miR-29c水平可能有助于肾纤维化的治疗；
5.尿miR-192,29c的差异表达显示了RNS各病理类型之间的差异,提供RNS分型的新方法,更好地理解RNS的发病机理；
6.尿miR-192,29c的表达与24h尿蛋白、TC、Lp-a、Alb的相关性显示,多方面因素均可加重肾纤维化和ECM沉积；
7.芪蛭肾综颗粒能上调尿miR-29c水平表达,下调尿miR-192水平表达,对ECM的清除、抗纤维化有较好的作用；可能通过双向作用来抑制ECM的增生,延缓肾小球硬化。
Primary nephrotic syndrome (PNS) is characterized by a massive proteinuria, hypoalbuminemia, edema, etc of a kind of clinical common kidney disease, after standard therapy of the hormone to treat disease, can't effectively relieve and accur hormone resistance, dependency or invalid or frequent recurrence, which in turn become refractory nephrotic syndrome (RNS). At present, the pathogenesis of RNS are not entirely clear, clinical cure rate is low and lack of effective treatment, therefore, early diagnosis, effective treatment and judge prognosis for RNS become extremely important medicine field problems.
Development of RNS is associated with multiple factors, studies confirm that RNS is associated with enhanced oxidative stress (ROS) reaction, ROS may participate in the glomerular podocytes, mesangial cells, endothelial cells, such as glomerular filtration barrier damage and subsequent process of the occurrence of proteinuria, and closely related to the severity of illness, so antioxidant treatment gradually attention by people. Thus, thioredoxin (TRX) and TXNIP play an important role in oxidative stress, is an important factor for the body to maintain redox balance. TRX are expressed in various body tissues and cells, it is by regulating cellular redox status against oxidative stress, thioredoxin-interacting protein (TXNIP) is one of the binding protein of TRX, by inhibiting the function of thioredoxin system and play a role of mediated oxidative stress. Therefore, TRX and TXNIP play an important role in oxidative stress, is an important factor of the body to maintain redox balance. RNS patients measure plasma TRX, TXNIP expression level, to explore the thioredoxin system in its role in the development of RNS patients. At the same time, micrornas (miRNA) has a variety of biological functions, highly conserved in evolution, plays an important role in the growth, physiological function, especially have significant effects on the occurrence of malignant tumor development. MiRNA compared with mRNA, more stable in vitro and in vivo metabolic cycle longer, they are involved in regulation of gene expression after transcription, via messenger RNA degradation or inhibition of protein synthesis.Many miRNA species control kidney cells in different pathological conditions, may be involved in the pathogenesis of kidney diseases. Study of miRNS in urine, large and stable, so in kidney disease, urinary micrornas have potential development prospects. Many RNS pathology exists in the extracellular matrix (ECM) protein accumulation, diffuse or nodular, glomerular sclerosis, tubular atrophy and interstitial fibrosis, miR-192is involved in the onset of the ECM process, and miR-192is the only one was found in glomerular mesangial cell expression of miRNA. At the same time, research hints in the transforming growth factor beta1(TGF-β1) induction of normal rat renal fibroblasts (NRK-49F) cell miR-29c expression by fibrosis process, while the expression of miR-29c enhancement can effectively inhibit TGF-β1and stimulate express of NRK-49cell lin Col Ⅰ, showed that miR-29c is inhibiting fibrosis genes. When or before in renal interstitial fibrosis, miR-29c high expression of cell can inhibit or delay the fibrosis progression. Therefore, correlation of miR-29c and RNS of renal interstitial fibrosis is particularly important. MiRNA always studies have shown that miR-29c, miR-192in kidney disease have different expression in peripheral blood and tissue, can be quantified in the urinary sediment, according to different pathological type urinary miRNA level is different, So the study of the miRNA can assist assessment of RNS pathological types, and judgement of the guidance of the clinical treatment and prognosis, and so on.
At present, western medicine treatment of RNS is mainly hormone and immune inhibitors and cytotoxic drugs, but the side effects,limite clinical application. Therefore, developing the features and advantages of TCM, searching convenient and effective treatment of traditional Chinese medicine in RNS has important significance.
RNS belongs to "edema","back pain","consumptive disease","urine turbid" and etc. in TCM."Edema" consistent with the RNS. Qi deficiency and blood stasis is the basis of RNS onset. Damp and hot accumulate knot is the catalyst in the process of development. The asthenia in origin and pathogenic factors in superficiality is the pathogenesis characteristics of RNS. So treatment with replenishing qi to invigorate the spleen and kidney and promoting blood circulation to remove blood stasis and clearing heat and promoting diuresisbeen detumescence, By astragalus, atractylodes, motherwort, leech, corn stigma, pyrrosia leaf of QZSZ particle comprise Huangqi,Baizhu,Yimucao,Shuizhi,Yumixu,Shiwei. According to the large sample study of RNS clinical, summed up the most often type of syndrome:qi deficiency and blood stasis and damp and hot, QZSZ particle become prescription for agreement of zhujiang hospital, southern medical university and TCM hospital in weihai city shandong province. QZSZ particle as an agreement prescription in hospital, has been used for many years, the clinical curative effect is verified, and can significantly reduce urinary protein, improve symptoms, reduce the high fat high coagulation state, delay the progress of glomerular sclerosis and protect renal function. But therapeutical mechanism of it in RNS is not clear, and needs further research. This topic through the treatment of QZSZ particle Detection clinical symptoms, blood biochemical indicators, serum superoxide dismutase (SOD), malondialdehyde (MDA), TRX and TXNIP ROS factor expression, provide the basis for clinical application. Through correlation study of gene expression of the urine miR-29c and miR-192and pathological types, it provide new targets for the diagnosis and treatment and prognosis of RNS.
Chart Ⅰ QZSZ particle in the treatment of patients with RNS function and serum TRX, TXNIP expression
Objective:To observe the treatment of the QZSZ particle on qi deficiency blood stasis damp and hot syndrome in RNS patients, through testing the patient's serum TRX and TXNIP expression, analysis of the two kinds of oxidative stress factor and RNS severity and prognosis of relevance, to explore its possible role in RNS development mechanism, which provide new targets for the treatment of RNS, and to study the relation of different clinical and pathological classification in curative effect.
Methods:Choice of kidney disease center of zhujiang hospital, and TCM hospital in weihai in December2010to December2012outpatient or hospitalization RNS,60patients with clinical classification:22cases of often relapse,13cases of hormone dependence,2cases of hormone is invalid in,23cases of hormone some effects.46cases of whom underwent renal biopsy and pathological classification are:23cases of focal segmental glomerular sclerosis (FSGS),2cases of minimal change disease small lesion type (MCD),18cases of mesangial proliferative glomerulonephritis (MsPGN),3patients of membrane nephropathy (MN). randomly divided into30cases in experimental group (QZSZ particle plus foundation treatment) and30cases in control group (ambrette capsule plus and foundation treatment), treatment for3months, there is no difference between groups in age, sex, clinical classification, pathological classification, etc. Detection of two groups of curative effect of TCM syndrome score before and after the treatment, clinical efficacy,24h urinary protein, Alb, serum creatinine (Scr), prothrombin time (PT), partial prothrombin time (APTT), fibrinogen (FIB), the change of SOD and MDA. Using enzyme-linked immune detection technology (ELISA) to detect serum TRX and TXNIP expression level.
Results:
1. The relationships of pathological types and clinical classification:it was no significant difference (P>0.05); Relationships of clinical classification and pathological types of:FSGS and MsPGN ratio is higher, compared with other types have statistical significance (P<0.05).
2. Two groups of clinical curative effect comparison:the total effective rate of experimental group compared with the control group, have statistical significance (P<0.05).
3. Comparison of the curative effect of each clinical classification:there was no statistically significant difference (P>0.05).
4. Comparison of the curative effect of each pathological types:curative effect of MN, MsPGN is obvious, it was statistically differences (P<0.05).
5. The main syndromes of traditional Chinese medicine:the symptoms were improved after treatment, compared with before in two groups, treatment were statistically significant difference (P<0.01); The symptoms of comparative differences of two groups after treatment are statistically significant (P<0.01). the experimental group is better than the control group.
6. Experimental group after treatment,24h urinary protein, Scr, BUN was significantly reduced, significantly reduced the difference was statistically significant (P<0.01); The control group after treatment compared with before treatment,24h urinary protein significantly reduced, significantly reduced,the difference was statistically significant (P<0.01); Compare the two groups after treatment, the experimental group in Alb to rise significantly, BUN dropped significantly, the experimental group were better than control group, the difference was statistically significant (P<0.05, P<0.01).
7. Comparison of the experimental group before and after treatment,FIB decreased significantly, PT, APTT significantly increased (P<0.01), the control group before and after treatment comparison of PT,APTT significantly higher (P<0.01); Compare the two groups after treatment, the experimental group FIB decreased significantly, the difference was statistically significant (P<0.05).
8. Comparison of the experimental group before and after treatment, a significant rise in SOD, MDA decreased significantly (P<0.01).Comparing the two groups after treatment, the experimental group than the control group SOD significantly increased, MDA decreased obviously (P<0.01).
9. Comparison of the experimental group before and after treatment, TRX was significantly higher, TXNIP decreased significantly (P<0.05). Control group TRX, TXNIP comparison before and after the treatment, there was no statistically significant difference (P>0.05). Comparing the two groups after treatment, the experimental group than the control group TRX significantly increased, TXNIP decreased obviously (P<0.01).
Conclusion:
1. Proportion of FSGS and MsPGN in various clinical classification is higher. Efficacy of MCD, MsPGN in the pathological types of is higher.
2.The QZSZ particle curative effect evaluation:can obviously drop of urine protein, is better than the control group.
3. The relationship of the clinical classification and effect has proved that the curative effect of hormone dependence and often relapse is better, but there is no significant difference compared with hormone is invalid and some effects.The clinical has no obvious correlation with curative effect.
4. The pathological classification relationship has proved that the effect of MsPGN and MCD is obvious, is better than with other pathological types.
5. Comparitting curative effect of of QZSZ particle in TCM syndrome, the experimental group is better than the control group, after treatment the total integral of TCM symptoms decreased obviously, the experimental group is better than the control group. QZSZ particle can heal RNS better in TCM symptoms.
6. QZSZ particle significantly elevated plasma Albumin, protect the renal function.
7. QZSZ particle can improve high blood coagulation state in patients with RNS, rise in PT and APTT, reduce FIB was significantly better than the control group.
8. QZSZ particle can increases SOD level and reduce the MDA level of the RNS patients, improve the body state of oxidative stress.
9. QZSZ particle can increase TRX levels, lower levels of TXNIP. TRX, TXNIP as oxidative stress related factors, play a role in the RNS.
10. QZSZ particle may play an important role in the treatment of RNS through the mechanism of resistance to oxidative stress.
Chart II Preliminary study of expression of the urinary miRNA-192,29c with curative effect of QZSZ particle inRNS and pathology of RNS
Objective:Study internal relation of urine miR-192and miR-29c in different clinical pathological types, explore the urine miR-192and miR-29c to distinguish the severity of disease and renal histological lesions of the possibility as a non-invasive biomarkers. Urinary miR-192and miR-29c expression of before and after QZSZ particle treatment are associated with24h urinary protein, Alb, TC, Lp-a and so on.
Methods:Selection of kidney disease center of zhujiang hospital, southern medical university and nephrology department in TCM hospital in weihai in December2010to December2012outpatient or hospitalization of the renal biopsy diagnosed44RNS patients, including18cases of MsPGN,3cases of MN and23cases of FSGS, randomly divided into the experimental group(QZSZ particle plus foundation treatment) and the control group,21cases of control group (ambrette capsule plus foundation treatment), treatment for3months, each group in age, gender, pathological classification, no difference. Set up15healthy volunteers as healthy controls in medical staff weihai hospital. Before and after3months of treatment RNS patient t collect specimens of blood and urine samples of100ml early in the morning on an empty stomach, healthy people to collect urine samples of100ml early in the morning on an empty stomach, urine specimen in3000RMP centrifugal before30minutes, again in13000RMP centrifuge for5minutes, discarding the supernatant fluid, the urine sediment repackaging in1.5ml EP tube,0.25ml per tube. Add0.75ml per EP tube Trizol LS, volatility1min cracking fully, let stand at room temperature after5min-80℃refrigerator. Trizol to extract total RNA, RNA quality testing after qualified, can build miR-192and miR-29c RT-PCR reaction system, each sample was detected in miR-192and miR-29c. The relative expression quantity and do statistical analysis. Test24hours urinary protein, Lp-a,TC, Alb before and after the treatment, test correlation of the urine miR-192and miR-29c and the above indexes.
Results:
1. Compared with healthy controls, miR-29c expression in the FSGS decreased obviously (P<0.05), the expression of MsPGN, MN decline is not obvious (P>0.05). miR-192in MsPGN expression increased obviously (P<0.05), the expression of MN,FSGS is not obvious (P>0.05).
2. The expression of urinary miR-29c and Lp-a showed a negative correlation, It was positively correlated with Alb, had statistical significance (P<0.05). The expression of urinary miR-192express positive correlation with the expression of urinary24h urinary protein and TC, had statistical significance (P<0.05).
3. Comparison of the experimental group before and after the treatment, the expression of miR-192significantly reduced and the expression of miR-29c significantly rised (P<0.01). Comparison of the control group before and after the treatment, the expression of miR-192significantly reduced and the expression of miR-29c significantly rised (P<0.05).Comparison of two groups after treatment, the expression of miR-29c in the experimental group significantly rised (P<0.05)
Conclusion:
1. Urine miR-192expression significantly increased in MsPGN patients.
2. MsPGN patients may have more ECM secretion and deposition, can determine glomerular ECM. It can guide clinical treatment and prognosis.
3. The urine miR-29c expression in patients with FSGS decreased significantly.
4. Patients with FSGS may be renal interstitial fibrosis significantly. It can judge the degree of renal fibrosis, increase urine levels of miR-29, help in treatment of renal fibrosis.
5. Differences in expression of urine miR-192,29c shows the differences between the various pathological types in RNS, and provides new ways to better understand the pathogenesis of RNS.
6. The expression of urine miR-192,29c with24h urinary protein, TC, Lp-a shows that several factors can increase renal fibrosis and ECM deposition.
7. QZSZ particle can increase the expression of urine miR-29c levels, and lower the expression of urinary miR-192levels in patients, play a regulatory role of the removal of the ECM, anti fibrosis, may through the two-way function to inhibit proliferation of ECM, delay the glomerular sclerosis.
引文
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