益气养阴活血方治疗糖尿病肾病的实验研究及远期疗效观察
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摘要
[研究背景]
糖尿病肾病(DN)是糖尿病最常见和主要的慢性微血管并发症,早期表现为尿中微量蛋白,继而出现持续蛋白尿、水肿、高血压、肾功能不全,最终发展至终末期肾病(ESRD)。DN时,肾小球系膜细胞是主要的受累细胞,高血糖作为糖尿病最基本的生化特征,可激活胞外信号调节激酶(ERK)通路并增加其下游产物前列腺素E2(PGE2)、转化生长因子(TGF-β1)及纤维连接蛋白(FN)的表达,引起早期肾小球高滤过、细胞外基质(ECM)合成增多,导致DN的发生、发展。目前关于中药复方对DN时ERK通路的影响多数是动物体内试验,体外实验报道较少,且多数侧重在其非血流动力学效应。中医药在防治DN方面有较大优势,国内关于中医药治疗DN的临床研究一般疗程较短,随访超过2年的报道较少,多数采用随机对照试验,用药及治疗方案单一,临床推广性低,且评价指标侧重在实验室指标及临床症状,缺少对终点事件的评价。本课题乃中国中医科学院优势病种的后续研究,益气养阴活血方是导师治疗DN的经验方,短期疗效观察可以降低DN患者尿蛋白排泄率,改善生存质量,但其作用机制之前并未做相关实验研究,且缺乏远期疗效观察。
[研究目的]
1、实验研究：探讨益气养阴活血方通过抑制肾小球系膜细胞的增殖、ERK通路的活化及其下游产物的表达而治疗糖尿病肾病的作用机制。
2、临床研究：整体评价以益气养阴活血方为主的中医辨证治疗糖尿病肾病的远期疗效,发掘中医治疗本病的特色与优势,为完善中医规范化治疗方案和整体疗效评价体系提供参考。
[研究方法]
1、实验研究
1.1研究对象：大鼠肾小球系膜细胞。
1.2干预措施：以高糖作为刺激因素诱导系膜细胞增殖及ERK通路活化,予益气养阴活血方和福辛普利含药血清加以干预,根据培养条件不同,将系膜细胞分为6组：正常血糖组、高糖组、高糖+福辛普利组和高糖+高、中、低浓度中药组。
1.3观察指标：培养6h、12h、24h、48h、72h后,用Western-blot法检测系膜细胞磷酸化ERK1/2(pERK1/2)蛋白的表达,Real time-PCR法检测系膜细胞TGF-β1基因的表达,ELISA法检测细胞上清液中PGE2和FN的含量。在培养24h、48h、72h时,用4-甲偶氮唑蓝(MTT)法检测系膜细胞的增殖。
2、临床研究
2.1研究对象：2006年12月～2009年12月间在望京医院住院或者门诊治疗的糖尿病肾病患者。
2.2干预措施：以益气养阴活血方为主的中医辨证治疗作为暴露因素,将既往病例划分为两个队列：(1)中西医结合队列(治疗组),接受以益气养阴活血方为主的中医辨证治疗+西医基础治疗；(2)西医队列(对照组),接受单纯西医基础治。对两组病例进行回顾性观察。
2.3观察指标：蛋白尿、肾功能进展,终末期肾病(ESRD)和死亡各类终点事件；实验室指标疗效、临床症状疗效和疾病综合疗效；生存质量及营养状态评估。
[研究结果]
1、实验研究
1.1高糖刺激24h、48h后能显著促进系膜细胞增殖,但至72h,高糖促细胞增殖趋势下降,与正常组相比无显著差异。福辛普利和高浓度中药含药血清组均可明显抑制高糖引起的系膜细胞增殖,两组间未见显著差异。中浓度中药组仅在24h表现出明显的抑制作用,低浓度中药组均未见抑制作用。
1.2高糖刺激6h后系膜细胞中pERK1/2蛋白的表达即明显升高,随时问延长表达逐渐增高,24h达到高峰,后逐渐减弱,至72h恢复至与正常组同等表达水平。福辛普利和高浓度中药组在6h-48h均显著抑制ERK的活化,两组在各时间点均无明显差异。中浓度中药组在6h-24h亦表现出明显的抑制作用。而低浓度中药组仅在12h可见明显的抑制作用。
1.3与正常组相比较,高糖组TGF-β1mRNA的表达从12h开始明显升高,EN的表达则从24h开始明显升高,随着时间延长,至72h升高趋势更为显著。高浓度中药组在各时间点均可显著的抑制TGF-β1mRNA、FN表达的升高。福辛普利组在早期有明显抑制作用,中浓度中药组在后期有明显抑制作用。低浓度中药组均未见抑制作用。
1.4随着ERK通路的激活,高糖组PGE：的表达在6h即明显升高,48h表达量达到峰值,至72h PGE2的表达较正常组仍有明显升高,但表达量较48h有所降低。高浓度中药组在12-48h可明显抑制PGE2的升高,中浓度中药组在24h、48h亦可明显抑制PGE2的升高。福辛普利组和低浓度中药组均未见抑制作用。
2、临床研究
本研究共纳入病例105例,其中治疗组67例,对照组38例,随访时间(从首次纳入观察到末次随访)为2-5年,治疗组中位随访时间为3.6年,对照组中位随访时间为3.8年。两组基线资料经比较具有可比性。
2.1治疗组蛋白尿疗效总有效率52.9%,对照组总有效率28.6%,两组比较差异有统计学意义。治疗组蛋白尿终点事件的总发生率为14.9%,对照组总发生率为34.2%,两组比较差异有统计学意义。其中治疗组早期DN进入临床期DN的发生率为16.1%,对照组发生率为47.4%,两组比较差异有统计学意义。治疗组尿蛋白程度进展的发生率为13.9%,对照组发生率为21.1%,两组比较无统计学差异。
2.2治疗组肾功能疗效总有效率62.5%,对照组总有效率20%,两组比较差异有统计学意义。治疗组CKD进展的发生率为14.9%,对照组发生率为34.2%,两组比较差异有统计学意义。治疗组Scr由正常进展至异常的发生率为3.9%,对照组发生率为17.9%,两组比较差异有统计学意义。治疗组Scr异常者数值翻倍或升高≥50%的发生率为12.5%,对照组发生率为50%,两组比较差异有统计学意义。
2.3治疗组临床症状疗效总有效率58.2%,对照组总有效率31.6%,两组比较差异有明显统计学意义。
2.4治疗组综合疗效总有效率40.3%,对照组总有效率21.1%,两组比较差异有统计学意义。
2.5治疗组ESRD发生率为3%,对照组发生率为13.2%：两组比较差异有统计学意义。
2.6治疗组死亡率为7.5%,对照组死亡率为10.5%；两组比较无统计学差异。
2.7治疗组和对照组普适性生存质量及糖尿病特异性生存质量积分比较,差异有明显统计学意义。
2.8治疗组营养不良风险的发生率为14.9%,对照组发生率为31.6%,两组比较差异有统计学意义。
[结论]
1、实验研究：益气养阴活血方可抑制早期系膜细胞的增殖、ERK通路的活化及其下游产物PGE2、TGF-β1和FN的表达升高,对ERK通路所引起的血流动力学效应和非血流动力学效应均有干预作用,这可能是其临床作用机制所在。
2、临床研究：以益气养阴活血方为主的中医辨证治疗能较好地改善临床症状；延缓早期蛋白尿的进展；保护肾脏、延缓肾功能的进展及ESRD的进程,改善DN的肾脏预后；提高生存质量、改善营养状况。
[创新性]
1、实验研究
1.1益气养阴活血方为导师临床经验用方,首次对其作用机制做相关实验研究。
1.2从ERK通路血流动力学和非血流动力学两方面效应探讨药物的作用机制。
2、临床研究
2.1观察中医药治疗DN的远期疗效(≥2年)。
2.2从多方面观察指标更全面地反映整体综合疗效。
2.3设定了不同的蛋白尿和肾功能终点事件作为次要结局指标,更早期反映疗效差异。
Background
Diabetic nephropathy(DN) is a common and major chronic microvascular complications of Diabetes Mellitus (DM).The glomerular mesangial cells (GMCs) are mainly affected cells of DN, high blood glucose can activate the extracellular signal-regulated kinase (ERK) pathway of GMCs and increase the expression of prostaglandin E2(PGE2), transforming growth factor-β1(TGF-β1) and fibronectin (FN) and other downstream products. Then, it may cause early glomerular hyperfiltration and increased synthesis of extracellular matrix (ECM) as well as lead to the occurrence and development of DN. At present, the studies on the influence of Chinese herbal compound on ERK pathway in DN are almost vivo animal trials and mostly focus on non-hemodynamic effects, but less vitro experiments were reported. The course of treatment in clinical studies of traditional Chinese medicine (TCM) therapy on DN are short generally, with fewer followed-up for more than2years in domestic. The majority of studies were performed with randomized controlled trial, with single program of medication and therapy as well as low clinical promotion. The evaluation indexes focus on the laboratory indexes and clinical symptoms, lacking of evaluation on endpoint events. Yiqi yangyin huoxue recipe is the empirical recipe for diabetic nephropathy (DN) used by my tutor, reducing urinary albumin excretion rate and improving the quality of life in short-term effect observation. However, its mechanism has not been studied previously and it lacks of long-term efficacy.
Objective
1.Experimental study rexplore the mechanism of Yiqi yangyin huoxue recipe on diabetic nephropathy from inhibiting mesangial cells multiplication, ERK pathway activation and downstream products expression.
2.Clinical researchcmake overall evaluation of long-term efficacy of Yiqi yangyin huoxue recipe on DN, excavate characteristics and advantages of TCM on the disease and provide references to improve the TCM standardized treatment program and the overall efficacy evaluation system.
Methods
1.Experimental study
1.1Object:rat mesangial cells.
1.2Interventions:induced mesangial cell proliferation and ERK pathway activation, with high glucose as a stimulating factor, serum containing Yiqi yangyin huoxue recipe and Fosinopril to intervene.Rat mesangial cells were divided into six groups, that is normal glucose group, high glucose group, high glucose+Fosinopril group and high glucose+high, medium, low concentration of traditional Chinese medicine group, based on different cultured conditions.
1.3Observation indexes:detected the expression of phospho-ERK1/2(pERK1/2) protein in mesangial cells with Westem-blot assay, the expression of TGF-β1gene with Real time-PCR assay as well as the contents of PGE2and FN in cell supernatant with ELISA assay after6h,12h,24h,48h, and72h culture. Detected mesangial cells multiplication using MTT assay in cultured24h,48h and72h.
2.Clinical research
2.1Object:patients with DN in our hospital or outpatient department between December2006-December2009.
2.2Interventions:with TCM treatment as exposure factors, subjects were divided into integrated traditional Chinese and Western medicine therapy dominating on Yiqi yangyin huoxue recipe (treatment group) and western medicine therapy alone (control group) and were retrospective observed.
2.3Observation indexes:determined the efficacy of laboratory parameters, efficacy of clinical symptoms and synthetic efficacy of disease as well as counted the total efficacy of treatment; observed all kinds of ending indexes, such as progress of albuminuria and renal function, ESRD and death as well as counted the incidence of endpoint events; evaluated the quality of life and nutritional status of patients.
Results
1.Experimental study
1.1Mesangial cell multiplications were promoted significantly after24h and48h high-glucose stimulation, but the multiplications trend declined at72h, there was no significant difference compared with control group. Fosinopril group and high concentration of traditional Chinese medicine group could significantly inhibit high glucose-induced mesangial cell multiplications, but no significant differences were observed between groups. Medium concentration of traditional Chinese medicine group could inhibit mesangial cell multiplications at24h, but there was no inhibition in low concentration of traditional Chinese medicine groups.
1.2The expression of pERK1/2protein in mesangial cells increased obviously after 6h high-glucose stimulation. The expression increased with time goes and reached a peak at24h, then it became weakened gradually and was at the same levels with the control group at the72h point. Fosinopril group and high concentration of traditional Chinese medicine group significantly inhibited the activation of ERK at6h-48h, there was no significant difference in time points between groups. Medium concentration of traditional Chinese medicine group also showed significant inhibition at6h-24h. However, low concentration of traditional Chinese medicine group had obvious inhibition merely at12h.
1.3Compared with control group, the expression of TGF-β1mRNA obviously increased from12h and FN from24h in high glucose group.With time passing, the increasing trend was more significant at72h. High concentration of traditional Chinese medicine group could inhibit the increase of the expression of TGF-β1mRNA and FN at each time point. Fosinopril group had obviously inhibited effect at early stage, while medium concentration of traditional Chinese medicine group had obviously inhibited effect in the latter stage. Low concentration of traditional Chinese medicine group showed no inhibited effect.
1.4With the activation of ERK pathway, the expression of PGE2increased significantly at6h and reached the peak at48h in high glucose group. The expression was still obviously increased at72h compared to control group but was decreased compared to the expression at48h. The increase of PGE2was inhibited significantly at12-48h in high concentration of traditional Chinese medicine group and at24h as well as48h in medium concentration of traditional Chinese medicine group. Fosinopril group and low concentration of traditional Chinese medicine group showed no inhibited effect.
2.Clinical research
105cases were enrolled in the study, including67cases in treatment group and38cases in control group. Follow-up time (from enrolled for the first time to the last follow-up) was2-5years (the median follow-up time is3.6years in treatment group and3.8years in control group).
2.1Total efficiency of proteinuria decreasing was52.9%in treatment group and28.6%in control group; there was a significant difference between groups. The overall incidence of proteinuria endpoint events was14.9%in treatment group and34.2%in control group; there was a significant difference between groups. The incidence of early DN developed to clinical stage DN was16.1%in treatment group and47.4%in control group; there was a significant difference between groups. The incidence of urinary protein progress was13.9%in treatment group and21.1%in control group; there was no statistical difference between groups.
2.2The total efficacy of renal function was62.5%in treatment group and20%in control group; there was a statistical difference between groups. The incidence of CDK progress was14.9%in treatment group and34.2%in control group; there was a statistical difference between groups. The incidence of Scr progressed from normal to abnormal was3.9%in treatment group and17.9%in control group; there was a statistical difference between groups. The incidence of Scr-value doubled or increased≥50%in patients with abnormal Scr was12.5%in treatment group and50%in control group; there was a statistical difference between groups.
2.3The total efficacy of clinical symptoms was58.2%in treatment group and31.6%in control group; there was a apparent statistical difference between groups.
2.4The total efficiency of synthetic effect was40.3%in treatment group and21.1%in control group; the difference was statistically significant.
2.5The incidence of ESRD was3%in treatment group and13.2%in control group; there was a statistical difference between groups.
2.6The mortality is7.5%in treatment group and10.5%in control group; there was no statistical difference between two groups.
2.7There is a apparent statistical difference in the universal quality of life and the diabetes-specific quality of life between two groups.
2.8The incidence of malnutrition risk was14.9%in treatment group and31.6%in control group; there was a statistical difference between groups.
Conclusions
1.Experimental study:Yiqi yangyin huoxue recipe could inhibit the multiplication of mesangial cells at early stage, the activation of the ERK pathway and the increased expression of PGE2, TGF-β1, FN and other downstream products; it also had intervention effects on hemodynamic effects and non-hemodynamic effects caused by ERK pathway.
2. Clinical research rtraditional Chinese therapy mainly on Yiqi yangyin huoxue recipe could better improve clinical symptoms;delay the progress of early proteinuria; protected the kidneys to slow down the progress of renal function and ESRD proceeding as well as improved the prognosis of kidney in DN; enhanced the quality of life and improved the nutritional status.
Innovations
1.Experimental study
1.1As the empirical recipe for diabetic nephropathy (DN) by my tutor, studied the mechanism of Yiqi yangyin huoxue recipe for the first time.
1.2The study observed ERK pathway activation and downstream products expression; studied the mechanism of the drug from hemodynamics and non-hemodynamic two aspects.
2.Clinical research
2.1Observed the TCM therapy on DN in the long-term efficacy (more than2years).
2.2Evaluate the long-term overall efficacy from endpoint events, efficacy of laboratory parameters, efficacy of clinical symptom, synthetic efficacy of disease, quality of life and nutritional state.
2.3Used the proteinuria and renal endpoint events as the secondary outcomes, earlier to reflect the differences in efficacy.
糖尿病肾病(DN)是糖尿病最常见和主要的慢性微血管并发症,早期表现为尿中微量蛋白,继而出现持续蛋白尿、水肿、高血压、肾功能不全,最终发展至终末期肾病(ESRD)。DN时,肾小球系膜细胞是主要的受累细胞,高血糖作为糖尿病最基本的生化特征,可激活胞外信号调节激酶(ERK)通路并增加其下游产物前列腺素E2(PGE2)、转化生长因子(TGF-β1)及纤维连接蛋白(FN)的表达,引起早期肾小球高滤过、细胞外基质(ECM)合成增多,导致DN的发生、发展。目前关于中药复方对DN时ERK通路的影响多数是动物体内试验,体外实验报道较少,且多数侧重在其非血流动力学效应。中医药在防治DN方面有较大优势,国内关于中医药治疗DN的临床研究一般疗程较短,随访超过2年的报道较少,多数采用随机对照试验,用药及治疗方案单一,临床推广性低,且评价指标侧重在实验室指标及临床症状,缺少对终点事件的评价。本课题乃中国中医科学院优势病种的后续研究,益气养阴活血方是导师治疗DN的经验方,短期疗效观察可以降低DN患者尿蛋白排泄率,改善生存质量,但其作用机制之前并未做相关实验研究,且缺乏远期疗效观察。
[研究目的]
1、实验研究：探讨益气养阴活血方通过抑制肾小球系膜细胞的增殖、ERK通路的活化及其下游产物的表达而治疗糖尿病肾病的作用机制。
2、临床研究：整体评价以益气养阴活血方为主的中医辨证治疗糖尿病肾病的远期疗效,发掘中医治疗本病的特色与优势,为完善中医规范化治疗方案和整体疗效评价体系提供参考。
[研究方法]
1、实验研究
1.1研究对象：大鼠肾小球系膜细胞。
1.2干预措施：以高糖作为刺激因素诱导系膜细胞增殖及ERK通路活化,予益气养阴活血方和福辛普利含药血清加以干预,根据培养条件不同,将系膜细胞分为6组：正常血糖组、高糖组、高糖+福辛普利组和高糖+高、中、低浓度中药组。
1.3观察指标：培养6h、12h、24h、48h、72h后,用Western-blot法检测系膜细胞磷酸化ERK1/2(pERK1/2)蛋白的表达,Real time-PCR法检测系膜细胞TGF-β1基因的表达,ELISA法检测细胞上清液中PGE2和FN的含量。在培养24h、48h、72h时,用4-甲偶氮唑蓝(MTT)法检测系膜细胞的增殖。
2、临床研究
2.1研究对象：2006年12月～2009年12月间在望京医院住院或者门诊治疗的糖尿病肾病患者。
2.2干预措施：以益气养阴活血方为主的中医辨证治疗作为暴露因素,将既往病例划分为两个队列：(1)中西医结合队列(治疗组),接受以益气养阴活血方为主的中医辨证治疗+西医基础治疗；(2)西医队列(对照组),接受单纯西医基础治。对两组病例进行回顾性观察。
2.3观察指标：蛋白尿、肾功能进展,终末期肾病(ESRD)和死亡各类终点事件；实验室指标疗效、临床症状疗效和疾病综合疗效；生存质量及营养状态评估。
[研究结果]
1、实验研究
1.1高糖刺激24h、48h后能显著促进系膜细胞增殖,但至72h,高糖促细胞增殖趋势下降,与正常组相比无显著差异。福辛普利和高浓度中药含药血清组均可明显抑制高糖引起的系膜细胞增殖,两组间未见显著差异。中浓度中药组仅在24h表现出明显的抑制作用,低浓度中药组均未见抑制作用。
1.2高糖刺激6h后系膜细胞中pERK1/2蛋白的表达即明显升高,随时问延长表达逐渐增高,24h达到高峰,后逐渐减弱,至72h恢复至与正常组同等表达水平。福辛普利和高浓度中药组在6h-48h均显著抑制ERK的活化,两组在各时间点均无明显差异。中浓度中药组在6h-24h亦表现出明显的抑制作用。而低浓度中药组仅在12h可见明显的抑制作用。
1.3与正常组相比较,高糖组TGF-β1mRNA的表达从12h开始明显升高,EN的表达则从24h开始明显升高,随着时间延长,至72h升高趋势更为显著。高浓度中药组在各时间点均可显著的抑制TGF-β1mRNA、FN表达的升高。福辛普利组在早期有明显抑制作用,中浓度中药组在后期有明显抑制作用。低浓度中药组均未见抑制作用。
1.4随着ERK通路的激活,高糖组PGE：的表达在6h即明显升高,48h表达量达到峰值,至72h PGE2的表达较正常组仍有明显升高,但表达量较48h有所降低。高浓度中药组在12-48h可明显抑制PGE2的升高,中浓度中药组在24h、48h亦可明显抑制PGE2的升高。福辛普利组和低浓度中药组均未见抑制作用。
2、临床研究
本研究共纳入病例105例,其中治疗组67例,对照组38例,随访时间(从首次纳入观察到末次随访)为2-5年,治疗组中位随访时间为3.6年,对照组中位随访时间为3.8年。两组基线资料经比较具有可比性。
2.1治疗组蛋白尿疗效总有效率52.9%,对照组总有效率28.6%,两组比较差异有统计学意义。治疗组蛋白尿终点事件的总发生率为14.9%,对照组总发生率为34.2%,两组比较差异有统计学意义。其中治疗组早期DN进入临床期DN的发生率为16.1%,对照组发生率为47.4%,两组比较差异有统计学意义。治疗组尿蛋白程度进展的发生率为13.9%,对照组发生率为21.1%,两组比较无统计学差异。
2.2治疗组肾功能疗效总有效率62.5%,对照组总有效率20%,两组比较差异有统计学意义。治疗组CKD进展的发生率为14.9%,对照组发生率为34.2%,两组比较差异有统计学意义。治疗组Scr由正常进展至异常的发生率为3.9%,对照组发生率为17.9%,两组比较差异有统计学意义。治疗组Scr异常者数值翻倍或升高≥50%的发生率为12.5%,对照组发生率为50%,两组比较差异有统计学意义。
2.3治疗组临床症状疗效总有效率58.2%,对照组总有效率31.6%,两组比较差异有明显统计学意义。
2.4治疗组综合疗效总有效率40.3%,对照组总有效率21.1%,两组比较差异有统计学意义。
2.5治疗组ESRD发生率为3%,对照组发生率为13.2%：两组比较差异有统计学意义。
2.6治疗组死亡率为7.5%,对照组死亡率为10.5%；两组比较无统计学差异。
2.7治疗组和对照组普适性生存质量及糖尿病特异性生存质量积分比较,差异有明显统计学意义。
2.8治疗组营养不良风险的发生率为14.9%,对照组发生率为31.6%,两组比较差异有统计学意义。
[结论]
1、实验研究：益气养阴活血方可抑制早期系膜细胞的增殖、ERK通路的活化及其下游产物PGE2、TGF-β1和FN的表达升高,对ERK通路所引起的血流动力学效应和非血流动力学效应均有干预作用,这可能是其临床作用机制所在。
2、临床研究：以益气养阴活血方为主的中医辨证治疗能较好地改善临床症状；延缓早期蛋白尿的进展；保护肾脏、延缓肾功能的进展及ESRD的进程,改善DN的肾脏预后；提高生存质量、改善营养状况。
[创新性]
1、实验研究
1.1益气养阴活血方为导师临床经验用方,首次对其作用机制做相关实验研究。
1.2从ERK通路血流动力学和非血流动力学两方面效应探讨药物的作用机制。
2、临床研究
2.1观察中医药治疗DN的远期疗效(≥2年)。
2.2从多方面观察指标更全面地反映整体综合疗效。
2.3设定了不同的蛋白尿和肾功能终点事件作为次要结局指标,更早期反映疗效差异。
Background
Diabetic nephropathy(DN) is a common and major chronic microvascular complications of Diabetes Mellitus (DM).The glomerular mesangial cells (GMCs) are mainly affected cells of DN, high blood glucose can activate the extracellular signal-regulated kinase (ERK) pathway of GMCs and increase the expression of prostaglandin E2(PGE2), transforming growth factor-β1(TGF-β1) and fibronectin (FN) and other downstream products. Then, it may cause early glomerular hyperfiltration and increased synthesis of extracellular matrix (ECM) as well as lead to the occurrence and development of DN. At present, the studies on the influence of Chinese herbal compound on ERK pathway in DN are almost vivo animal trials and mostly focus on non-hemodynamic effects, but less vitro experiments were reported. The course of treatment in clinical studies of traditional Chinese medicine (TCM) therapy on DN are short generally, with fewer followed-up for more than2years in domestic. The majority of studies were performed with randomized controlled trial, with single program of medication and therapy as well as low clinical promotion. The evaluation indexes focus on the laboratory indexes and clinical symptoms, lacking of evaluation on endpoint events. Yiqi yangyin huoxue recipe is the empirical recipe for diabetic nephropathy (DN) used by my tutor, reducing urinary albumin excretion rate and improving the quality of life in short-term effect observation. However, its mechanism has not been studied previously and it lacks of long-term efficacy.
Objective
1.Experimental study rexplore the mechanism of Yiqi yangyin huoxue recipe on diabetic nephropathy from inhibiting mesangial cells multiplication, ERK pathway activation and downstream products expression.
2.Clinical researchcmake overall evaluation of long-term efficacy of Yiqi yangyin huoxue recipe on DN, excavate characteristics and advantages of TCM on the disease and provide references to improve the TCM standardized treatment program and the overall efficacy evaluation system.
Methods
1.Experimental study
1.1Object:rat mesangial cells.
1.2Interventions:induced mesangial cell proliferation and ERK pathway activation, with high glucose as a stimulating factor, serum containing Yiqi yangyin huoxue recipe and Fosinopril to intervene.Rat mesangial cells were divided into six groups, that is normal glucose group, high glucose group, high glucose+Fosinopril group and high glucose+high, medium, low concentration of traditional Chinese medicine group, based on different cultured conditions.
1.3Observation indexes:detected the expression of phospho-ERK1/2(pERK1/2) protein in mesangial cells with Westem-blot assay, the expression of TGF-β1gene with Real time-PCR assay as well as the contents of PGE2and FN in cell supernatant with ELISA assay after6h,12h,24h,48h, and72h culture. Detected mesangial cells multiplication using MTT assay in cultured24h,48h and72h.
2.Clinical research
2.1Object:patients with DN in our hospital or outpatient department between December2006-December2009.
2.2Interventions:with TCM treatment as exposure factors, subjects were divided into integrated traditional Chinese and Western medicine therapy dominating on Yiqi yangyin huoxue recipe (treatment group) and western medicine therapy alone (control group) and were retrospective observed.
2.3Observation indexes:determined the efficacy of laboratory parameters, efficacy of clinical symptoms and synthetic efficacy of disease as well as counted the total efficacy of treatment; observed all kinds of ending indexes, such as progress of albuminuria and renal function, ESRD and death as well as counted the incidence of endpoint events; evaluated the quality of life and nutritional status of patients.
Results
1.Experimental study
1.1Mesangial cell multiplications were promoted significantly after24h and48h high-glucose stimulation, but the multiplications trend declined at72h, there was no significant difference compared with control group. Fosinopril group and high concentration of traditional Chinese medicine group could significantly inhibit high glucose-induced mesangial cell multiplications, but no significant differences were observed between groups. Medium concentration of traditional Chinese medicine group could inhibit mesangial cell multiplications at24h, but there was no inhibition in low concentration of traditional Chinese medicine groups.
1.2The expression of pERK1/2protein in mesangial cells increased obviously after 6h high-glucose stimulation. The expression increased with time goes and reached a peak at24h, then it became weakened gradually and was at the same levels with the control group at the72h point. Fosinopril group and high concentration of traditional Chinese medicine group significantly inhibited the activation of ERK at6h-48h, there was no significant difference in time points between groups. Medium concentration of traditional Chinese medicine group also showed significant inhibition at6h-24h. However, low concentration of traditional Chinese medicine group had obvious inhibition merely at12h.
1.3Compared with control group, the expression of TGF-β1mRNA obviously increased from12h and FN from24h in high glucose group.With time passing, the increasing trend was more significant at72h. High concentration of traditional Chinese medicine group could inhibit the increase of the expression of TGF-β1mRNA and FN at each time point. Fosinopril group had obviously inhibited effect at early stage, while medium concentration of traditional Chinese medicine group had obviously inhibited effect in the latter stage. Low concentration of traditional Chinese medicine group showed no inhibited effect.
1.4With the activation of ERK pathway, the expression of PGE2increased significantly at6h and reached the peak at48h in high glucose group. The expression was still obviously increased at72h compared to control group but was decreased compared to the expression at48h. The increase of PGE2was inhibited significantly at12-48h in high concentration of traditional Chinese medicine group and at24h as well as48h in medium concentration of traditional Chinese medicine group. Fosinopril group and low concentration of traditional Chinese medicine group showed no inhibited effect.
2.Clinical research
105cases were enrolled in the study, including67cases in treatment group and38cases in control group. Follow-up time (from enrolled for the first time to the last follow-up) was2-5years (the median follow-up time is3.6years in treatment group and3.8years in control group).
2.1Total efficiency of proteinuria decreasing was52.9%in treatment group and28.6%in control group; there was a significant difference between groups. The overall incidence of proteinuria endpoint events was14.9%in treatment group and34.2%in control group; there was a significant difference between groups. The incidence of early DN developed to clinical stage DN was16.1%in treatment group and47.4%in control group; there was a significant difference between groups. The incidence of urinary protein progress was13.9%in treatment group and21.1%in control group; there was no statistical difference between groups.
2.2The total efficacy of renal function was62.5%in treatment group and20%in control group; there was a statistical difference between groups. The incidence of CDK progress was14.9%in treatment group and34.2%in control group; there was a statistical difference between groups. The incidence of Scr progressed from normal to abnormal was3.9%in treatment group and17.9%in control group; there was a statistical difference between groups. The incidence of Scr-value doubled or increased≥50%in patients with abnormal Scr was12.5%in treatment group and50%in control group; there was a statistical difference between groups.
2.3The total efficacy of clinical symptoms was58.2%in treatment group and31.6%in control group; there was a apparent statistical difference between groups.
2.4The total efficiency of synthetic effect was40.3%in treatment group and21.1%in control group; the difference was statistically significant.
2.5The incidence of ESRD was3%in treatment group and13.2%in control group; there was a statistical difference between groups.
2.6The mortality is7.5%in treatment group and10.5%in control group; there was no statistical difference between two groups.
2.7There is a apparent statistical difference in the universal quality of life and the diabetes-specific quality of life between two groups.
2.8The incidence of malnutrition risk was14.9%in treatment group and31.6%in control group; there was a statistical difference between groups.
Conclusions
1.Experimental study:Yiqi yangyin huoxue recipe could inhibit the multiplication of mesangial cells at early stage, the activation of the ERK pathway and the increased expression of PGE2, TGF-β1, FN and other downstream products; it also had intervention effects on hemodynamic effects and non-hemodynamic effects caused by ERK pathway.
2. Clinical research rtraditional Chinese therapy mainly on Yiqi yangyin huoxue recipe could better improve clinical symptoms;delay the progress of early proteinuria; protected the kidneys to slow down the progress of renal function and ESRD proceeding as well as improved the prognosis of kidney in DN; enhanced the quality of life and improved the nutritional status.
Innovations
1.Experimental study
1.1As the empirical recipe for diabetic nephropathy (DN) by my tutor, studied the mechanism of Yiqi yangyin huoxue recipe for the first time.
1.2The study observed ERK pathway activation and downstream products expression; studied the mechanism of the drug from hemodynamics and non-hemodynamic two aspects.
2.Clinical research
2.1Observed the TCM therapy on DN in the long-term efficacy (more than2years).
2.2Evaluate the long-term overall efficacy from endpoint events, efficacy of laboratory parameters, efficacy of clinical symptom, synthetic efficacy of disease, quality of life and nutritional state.
2.3Used the proteinuria and renal endpoint events as the secondary outcomes, earlier to reflect the differences in efficacy.
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