大黄复方及其有效成分治疗慢性肾衰竭的临床和机理研究

发布时间：2018-06-13 02:45

本文选题：大黄 + 慢性肾衰竭　；参考：《南京中医药大学》2016年博士论文

【摘要】：大黄治疗慢性肾衰竭的循证医学证据目的：评价大黄治疗慢性肾衰竭的有效性及安全性。方法：计算机检索中国生物医学文献数据库CBM、相关期刊论文CNKI、维普期刊数据库VIP、万方资源数据库、EMBASE、MEDLINE、PUBMED、 Cochrane图书馆中关于大黄治疗慢性肾衰竭的随机和半随机对照试验,同时手工检索相关期刊杂志,并由两位研究者进行独立筛选和资料提取,按照改良Jadad评分量表评价所纳入的文献质量,应用Revman5.3软件进行Meta分析。结果：(一)大黄单方或含大黄的中药复方口服治疗CRF的系统评价纳入35篇文献,共3060例患者,其中治疗组1657例,对照组1403例。Meta分析结果显示,与对照组相比,大黄单方或含大黄的中药复方口服治疗能降低CRF患者Scr[MD=-73.28, 95%CI (-85.96, -60.60) ];降低BUN[MD=-2.47, 95%CI(-3.13, -1.82)];升高CRF患者Ccr[MD=6.66, 95%CI (2.93, 10.40)];升高Hb[MD=9.31, 95%CI (3.81, 14.80)];升高Alb[MD=2.94, 95%CI (1.75, 4.13)];降低24 h尿蛋白定量(24 h UTP) [MD=-0.63, 95%CI (-0.95, -0.30)];降低TG[MD=-0.53, 95%CI (-0.73, -0.32) ]和TC[MD=-1.01, 95%CI (-1.44,-0.58)]；并且可以改善CRF患者症状和体征的总有效率[Peto OR=3.42, 95%CI(2.84,4.12)]；但对于减少ESRD的发生[Peto OR=0.57, 95%CI为(0.19, 1.75)]与对照组相比,差异无统计学意义。(二)含大黄的中药灌肠方治疗CRF的系统评价纳入28篇文献,共2278例患者,其中治疗组1220例,对照组1058例。Meta分析结果显示,与对照组相比,含大黄的中药灌肠方治疗能降低CRF患者Scr[MD=-59.62, 95%CI (-88.58, -30.67) ];降低BUN[MD=-3.27, 95%CI (-4.49, -2.05) ];升高CRF患者Ccr[MD=3.95, 95%CI (-0.03, 7.93) ];改善CRF患者症状和体征的总有效率[Peto OR=4.08, 95%CI (3.25, 5.12) ];并且可以改善中医主要证候纳差[MD=-0.48, 95%CI (-0.77, -0.19) ]和恶心呕吐[MD=- 0.65, 95%CI (- 0.78, - 0.52) ];但对于升高Hb[MD=6.85, 95%CI (-1.82, 15.52) ]、降低尿酸(UA) [MD=-46.84, 95%CI (-102.91, 9.22) ]与对照组相比,差异无统计学意义。结论：与对照组相比,大黄单方或含大黄的中药复方口服和含大黄的中药灌肠方均能明显降低CRF患者Scr、BUN,升高Ccr,同时还能改善患者症状和体征的总有效率,大黄单方或含大黄的中药复方口服能升高Hb、Alb,降低24h UTP、 TG和TC,但无证据显示在减少ESRD的发生方面较对照组更为有效；另外,含大黄的中药灌肠方能改善中医主要证候纳差和恶心呕吐,但对于升高Hb和降低UA无证据显示较对照组更为有效,表明大黄可能是一种相对安全和有效治疗CRF的药物。但由于所纳入的文献存在研究方法学质量低下的问题,因此进一步进行高质量文献研究,对评价其疗效及安全性十分必要。大黄素通过自噬途径对肾小管损伤的保护机制研究背景及目的肾毒性的药物所致的急性肾功能衰竭早已成为临床医生关注的重点,顺铂是一种临床常用于治疗多种恶性肿瘤的广谱、有效的化疗药物,然而,据统计,临床上运用顺铂化疗后,导致的肾损害发生率为25%-35%,且顺铂用药剂量与其引起的肾脏毒性成一定的比例关系,已成为限制其用量及扩大其应用范围的主要考虑因素。西药在治疗肾毒性药物所致肾损伤的同时会带来过多的不良反应,因此,研究人员致力于寻找一种既能降低顺铂肾毒性,又能保留其抗癌活性的中药。自噬(autophagy),即溶酶体依赖性的大分子蛋白质和细胞器的自我降解,通过清除衰老的细胞器和长寿命蛋白质,为细胞修复提供原料与营养,实现细胞器更新。细胞凋亡,是机体为了保持恒定的细胞数目而采取的由基因调控、生物自主的程序化方式。肾毒性的药物损伤肾小管主要表现为肾小管上皮细胞的凋亡,越来越多的实验研究报道,在顺铂导致的肾脏细胞凋亡过程中,自噬发生在凋亡之前,其可能成为其抗凋亡的机制。大黄素是中药大黄的主要的药效成分,大量的研究表明其具有保护肾脏的作用,主要集中在抗炎,抑制增殖,抑制细胞表型转化,以及抗氧化等方面。已有研究报道大黄素能改善顺铂相关性肾毒性,然而,目前还没有研究报道大黄素是否可以通过自噬途径改善顺铂引起的肾损伤。但有研究报道,大黄素是一种有效的单磷酸腺苷(AMP)激活蛋白激酶(AMPK)活化剂,并且可以调节哺乳动物的雷帕霉素靶蛋白(mTOR)途径。由于AMPK/mTOR信号通路在细胞自噬的调节中起着重要的作用,因此,我们推测,在顺铂肾损伤中,大黄素是否有可能通过调节自噬而起到肾保护作用。我们选用NRK-52E细胞作为研究对象,借助顺铂诱导的NRK-52E细胞损伤模型,通过体外实验研究,观察大黄素对顺铂环境下细胞自噬相关蛋白的变化、自噬体形成情况,以及自噬相关信号通路的影响,进一步探讨自噬在肾毒性药物所致的肾损伤中的变化和大黄素的干预作用,试图在体外阐明大黄素通过调节自噬相关信号通路,改善肾毒性药物所致的肾损伤的分子机制,为大黄素的临床应用提供新的途径。方法首先,我们观察了顺铂处理的NRK-52E细胞形态和细胞凋亡相关的标志物表达情况。然后用顺铂加或不加大黄素处理RK-52E细胞,用显微进行观察,并且采用免疫印迹(Western Blot, WB)方法,检测细胞凋亡相关蛋白Caspase-3、cleaved Caspase-3和哺乳动物同源的自噬标记蛋白-微管相关蛋白1轻链3(LC3)Ⅰ/Ⅱ表达的情况,以及采用pmRFP-LC3转染技术,利用荧光显微镜观察了大黄素处理细胞后自噬颗粒在细胞内的表达和分布情况。其次,我们也应用自噬体-溶酶体结合的抑制剂巴弗洛霉素A1,观察其加入前后对大黄素诱导的自噬的影响。此外,雷帕霉素,哺乳动物的雷帕霉素mTOR靶蛋白的抑制剂,在应激条件下可以通过抑制mTOR活性而激活自噬,我们利用免疫印迹,显微镜观察和流式细胞仪检测等方法观察了顺铂和雷帕霉素处理的细胞形态变化和细胞凋亡情况。再次,进一步观察了大黄素通过调节AMPK/mTOR信号通路对自噬活化的影响。采用WB的方法检测了大黄素处理细胞后AMPK/mTOF信号通路相关蛋白的表达情况,与此同时,我们也在大黄素处理的细胞中加或不加compound C,个公认的AMPK印制剂,观察NRK-52E细胞形态和凋亡相关蛋白的表达。最后,除了上述通路的研究外,我们还观察了MAPKs家族,包括ERK、P38和JNK所介导的信号通路,是否也参与了大黄素诱导的自噬活化。采用WB方法,检测了大黄素不同时间点处理细胞后p-ERK、p-P38和p-JNK蛋白的表达情况,以及LC3-I和LC3-II的表达情况。同时,我们还分别选用了ERK和p38的抑制剂PD098059和SB203580,用大黄素加或不加PD098059和SB203580处理细胞,观察p-ERK蛋白、p-p38蛋白的表达情况。结果(1)随着顺铂用药时间和剂量的增加,观察到细胞出现凋亡,WB结果显示,早期自噬标志性蛋白LC3-I币LC3-Ⅱ的表达增加,LC3-Ⅱ/LC3-Ⅰ的比值增加,晚期凋亡相关蛋白cleaved Caspase-3的表达增加,CCK-8法和流式细胞仪检测结果与WB的结果一致,晚期凋亡增加。(2)顺铂处理组存活细胞数目减少,经大黄素干预后,存活细胞数目明显增多。免疫印迹处理后,表明大黄素与顺铂共同处理细胞,能够明显逆转单独顺铂处理细胞所致的cleaved Caspase-3蛋白的上调。同时,大黄素处理细胞,能够明显增加LC3-Ⅱ/Ⅰ的比值和自噬颗粒RFP-LC3点状结构的表达。我们选用雷帕霉素处理细胞,同样观察到其可以增加LC3-I和LC3-Ⅱ的表达,增加]LC3-Ⅱ/LC3-Ⅰ的比值,倒置显微镜下观察到在顺铂和大黄素共同处理细胞时加入巴佛洛霉素A1,凋亡细胞数反而明显增加。此外,大黄素还能以时间依赖性的方式下调p-p70S6K蛋白和p-mTOR蛋白的表达,同时上调p-AMPK蛋白的表达。WB结果表明,AMPK的抑制齐compoundC能够明显的抑制大黄素诱导的MPK的磷酸化,LC3-I向LC3-Ⅱ的转化也被明显的抑制,荧光显微镜观察的结果与WB结果相同,加入了compound C之后,大黄素诱导的红色荧光自噬颗粒明显减少。倒置显微镜下观察也发现compound C能明显的抑制大黄素的抗凋亡作用,WB结果同样表明,大黄素能明显下调顺铂诱导的cleaved Caspase-3蛋白的表达,但当加入compound C共处理之后,又上调了cleaved Caspase-3蛋白的表达。(3)WB处理结果表明,大黄素处理细胞,p-ERK、p-P38和p-JNK蛋白的表达水平随着时间的变化先增加后降低；当加入ERK的抑制剂PD098059和p38的抑制剂SB203580后,大黄素引起的p-ERK蛋白、p-p38蛋白的增加被抑制。结论我们的研究结果发现顺铂处理NRK-52E细胞早期可以出现自噬,晚期则出现凋亡。大黄素能诱导自噬,进而达到改善顺铂导致的NRK-52E细胞凋亡的作用。我们的机制研究结果提示,AMPK/mTOR信号通路和MAPKs家族成员介导的信号通路可能参与了大黄素诱导自噬的过程。“益肾清利、和络泄浊法加减大黄”辨证方延缓慢性肾衰竭进展的临床疗效观察目的：评价“益肾清利、和络泄浊法加减大黄”辨证方延缓CRF患者肾功能进展的有效性。方法：采用回顾性的研究方法,选择2013年9月-2015年9月就诊于江苏省中医院肾内科导师孙伟教授门诊,诊断为慢性肾衰竭的51例患者,观察“益肾清利、和络泄浊法加减大黄”辨证方治疗后,临床症状和疗效指标的变化,总疗程为2年,其中每位患者大黄连续或间断用药疗程≥1个月。结果：51例患者经“益肾清利、和络泄浊法加减大黄”辨证方治疗24个月后,中医临床症状积分较初诊时相比显著下降(P0.01)。经治疗患者的主要疗效指标Scr、BUN、UA较治疗前相比,均有所下降,具有显著统计学差异(P0.01)。治疗前后相比较,RBC有所上升,具有显著统计学差异(P0.01),治疗后Hb轻微上升,但无统计学差异(P0.05)；治疗前后的TC比较,具有显著统计学差异(P0.01)；治疗后TG有轻微下降,但无统计学差异(P0.05)。结论：“益肾清利、和络泄浊法加减大黄”辨证方可以明显减轻患者临床症状,稳定并改善患者的肾功能,不同程度降低Scr、BUN、UA、血脂水平,贫血也一定程度上得到了改善,说明该法很大程度上能延缓肾功能进展,改善患者生活质量。
[Abstract]:Evidence based evidence-based medicine for Rhubarb for chronic renal failure: To evaluate the effectiveness and safety of Rhubarb for chronic renal failure. Methods: computer retrieval of Chinese biomedical literature database CBM, Chinese journal full text database CNKI, VP journal database VIP, data base of Wanfang resource, EMBASE, MEDLINE, PUBMED, Cochrane Library A randomized and semi randomized controlled trial of Rhubarb for chronic renal failure was conducted by manual retrieval of periodicals in related journals and two researchers were selected for independent screening and data extraction. The quality of the literature was evaluated according to the modified Jadad scale. The Revman5.3 software was used for Meta analysis. Results: (1) rhubarb single or rhubarb The systematic evaluation of oral Chinese medicine for oral treatment of CRF included 35 articles, including 3060 cases, of which 1657 cases were in the treatment group and 1403 cases in the control group showed that, compared with the control group, the oral administration of rhubarb single or rhubarb compound oral Chinese medicine can reduce Scr[MD=-73.28, 95%CI (-85.96, -60.60)) in CRF patients and reduce BUN[MD=-2.47, 95%CI (-3.13). Ccr[MD=6.66, 95%CI (2.93, 10.40)]; elevated Hb[MD=9.31, 95%CI (3.81, 14.80)]; elevated Alb[MD=2.94, 95%CI (1.75, 4.13)]; reduced 24 h proteinuria (24 h UTP). The total effective rate to improve the symptoms and signs of CRF patients was [Peto OR=3.42, 95%CI (2.84,4.12)]; but there was no significant difference between the control group and the control group for the occurrence of [Peto OR=0.57 in the reduction of ESRD, and (0.19, 1.75)]. (two) the system evaluation of the traditional Chinese medicine enema containing Rhubarb in the treatment of CRF was included in 28 literature, of which the treatment group was 1220. In the control group, 1058 cases of.Meta analysis showed that compared with the control group, Chinese medicine enema containing rhubarb could reduce Scr[MD=-59.62, 95%CI (-88.58, -30.67)) and BUN[MD=-3.27, 95%CI (-4.49, -2.05)] and CRF patient Ccr[MD=3.95, 95 (7.93)), and improve the total effective rate of symptoms and signs of patients. R=4.08, 95%CI (3.25, 5.12)]; and can improve the main syndromes of TCM [MD=-0.48, 95%CI (-0.77, -0.19)] and nausea and vomiting [MD=- 0.65, 95%CI (- 0.78, - 0.52)]; but for the increase of Hb[MD=6.85, 95%CI (-1.82, 15.52)], lower uric acid (UA), compared to the control group, there is no statistical difference. Conclusion: compared with the control group, the Chinese medicine compound of rhubarb single or rhubarb containing oral Chinese medicine and Chinese herbal enema containing rhubarb can significantly reduce the Scr, BUN, Ccr, and improve the total effective rate of the symptoms and signs of CRF patients. The oral administration of rhubarb alone or with rhubarb can increase Hb, Alb, 24h UTP, TG and TC, but it is no more. The evidence shows that the reduction of ESRD is more effective than the control group; in addition, the traditional Chinese medicine enema containing rhubarb can improve the main syndromes and nausea and vomiting of traditional Chinese medicine, but it is more effective for raising Hb and reducing UA than in the control group, suggesting that rhubarb may be a safe and effective drug for the treatment of CRF. There is a problem of low quality of research methodology in the literature included, so it is necessary to further study the high quality literature to evaluate its efficacy and safety. The research background of the protection mechanism of renal tubule injury by autophagy and the acute renal failure caused by the drugs for the purpose of renal toxicity have already become a clinician. The focus of attention, cisplatin is a broad spectrum, effective chemotherapeutic drug commonly used in the treatment of various malignant tumors. However, according to the statistics, the incidence of renal damage caused by cisplatin chemotherapy is 25%-35%, and the dosage of cisplatin is proportional to the renal toxicity caused by cisplatin, which has become a restriction of its dosage and enlargement. The main consideration of the scope of application. Western medicine will bring too many adverse reactions to kidney damage caused by nephrotoxic drugs. Therefore, researchers are committed to finding a traditional Chinese medicine that can reduce the renal toxicity of cisplatin and retain its anticancer activity. Autophagy (autophagy), a lysosomal dependent macromolecule protein and organelle Self degradation, by removing aging organelles and long life proteins to provide material and nutrition for cell repair, and to realize organelle renewal. Cell apoptosis is regulated by genes to maintain a constant number of cells and is bioautonomic programming. Nephrotoxic drug damage renal tubules are mainly shown on renal tubules. A growing number of experimental studies have reported that in the process of renal cell apoptosis induced by cisplatin, autophagy occurs before apoptosis, and it may become a mechanism for its anti apoptosis. Emodin is the main pharmacological component of rhubarb. A large number of studies have shown that it has the role of protecting the kidney, mainly focusing on anti-inflammatory and inhibiting proliferation. It has been reported that emodin can improve cisplatin induced renal damage. However, emodin is an effective monophosphate (AMP) activation protein stimulated by emodin, however, however, it is not reported that emodin can improve cisplatin induced renal damage. Enzyme (AMPK) activator and can regulate the mammalian rapamycin target protein (mTOR) pathway. As the AMPK/mTOR signaling pathway plays an important role in the regulation of autophagy, we speculate whether emodin may play a role in renal protection by autophagy in cisplatin. We choose NRK-52E cells as a cell. With the aid of cisplatin induced NRK-52E cell damage model, the changes in autophagy related proteins, the formation of autophagic bodies and the influence of autophagy related signaling pathways in cisplatin were investigated by cisplatin induced damage model in vitro. The changes of autophagy in renal injury induced by nephrotoxic drugs and emodin were further explored. The effect of the intervention is to elucidate in vitro the molecular mechanism of emodin by regulating autophagy related signaling pathways and improving renal injury induced by nephrotoxic drugs, providing a new approach for the clinical application of emodin. First, we observed the expression of NRK-52E cell morphology and apoptosis related markers in cisplatin treatment. RK-52E cells were treated with cisplatin plus or without enlarging flavin, and the expression of apoptosis related protein Caspase-3, cleaved Caspase-3 and autophagy related protein microtubule related protein 1 light chain 3 (LC3) I / II were detected by Western Blot (WB). The expression and distribution of autophagic particles in cells were observed by fluorescence microscopy. Secondly, we also applied the inhibitor of autophagosome - lysosome binding inhibitor, buffalamycin A1, to observe the effect of emodin on autophagy induced by emodin. In addition, rapamycin, rapamycin mTOR in mammals The inhibitor of target protein can activate autophagy by inhibiting the activity of mTOR under stress conditions. We observed the morphological changes and apoptosis of cisplatin and rapamycin by immunoblotting, microscope observation and flow cytometry. Again, the emodin was observed by adjusting the AMPK/mTOR signal. The effect of pathway on autophagy activation. WB method was used to detect the expression of AMPK/mTOF signaling pathway related proteins after emodin treated cells. At the same time, we also added or without compound C in emodin treated cells, a recognized AMPK printing agent, to observe the expression of NRK-52E cell morphology and apoptosis related proteins. Finally, the expression of apoptosis related proteins was observed. In addition to the above pathway, we also observed whether the MAPKs family, including ERK, P38 and JNK, also participated in emodin induced autophagy activation. The expression of p-ERK, p-P38 and p-JNK protein, as well as the expression of LC3-I and LC3-II, were detected by the WB method. We also selected ERK and p38 inhibitors PD098059 and SB203580 respectively. The expression of p-ERK protein and p-p38 protein was observed with emodin plus or without PD098059 and SB203580 cells. Results (1) the apoptosis of cells was observed with the increase of time and dosage of cisplatin. WB results showed that the early autophagic marker protein LC3-I currency LC3 was found. The expression of - II increased, the ratio of LC3- II /LC3- I increased, and the expression of advanced apoptosis related protein cleaved Caspase-3 increased. The results of CCK-8 and flow cytometry were consistent with the results of WB, and advanced apoptosis increased. (2) the number of surviving cells in cisplatin treatment group decreased, and the number of surviving cells increased significantly after the prognosis of emodin. Immunoblotting The results showed that the CO treatment of emodin and cisplatin could obviously reverse the up regulation of cleaved Caspase-3 protein caused by cisplatin treated cells. At the same time, emodin treated cells could significantly increase the ratio of LC3- II / I and the expression of RFP-LC3 dot structure of autophagy particles. It could increase the expression of LC3-I and LC3- II and increase the ratio of]LC3- II /LC3- I. Under the inverted microscope, it was observed that the number of apoptotic cells increased obviously when cisplatin and emodin co treated cells, and the number of apoptotic cells increased significantly. In addition, emodin could also reduce the expression of p-p70S6K and p-mTOR protein in a time dependent manner. The expression of p-AMPK protein expression.WB showed that AMPK inhibition of homogeneous compoundC could obviously inhibit the phosphorylation of MPK induced by emodin, and the transformation of LC3-I to LC3- II was obviously suppressed. The results of fluorescence microscopy were the same as WB. After adding compound C, the red fluorescent autophagic particles induced by emodin was significantly reduced. Compound C could obviously inhibit the anti apoptotic effect of emodin. WB results also showed that rhubarb could obviously reduce the expression of cleaved Caspase-3 protein induced by cisplatin, but when compound C was added, the expression of cleaved Caspase-3 protein was up-regulated. (3) the result of WB treatment showed emodin. The expression level of p-ERK, p-P38 and p-JNK protein increased first and then decreased with the change of time; the increase of p-ERK protein and p-p38 protein caused by emodin was inhibited when the inhibitor of ERK was added to PD098059 and p38, the inhibitor of SB2035 80. Conclusion our results showed that autophagy could occur at the early stage of cisplatin treatment for NRK-52E cells. There is apoptosis in late stage. Rhein can induce autophagy to improve the apoptosis of NRK-52E cells induced by cisplatin. Our mechanism study suggests that the signal pathway mediated by AMPK/mTOR signaling pathway and MAPKs family members may be involved in the process of emodin induced autophagy. The clinical effect of syndrome differentiation in delaying the progress of chronic renal failure: To evaluate the effectiveness of "Yishen Qingli, harmony and turbidity method plus subtraction of rhubarb" to delay the progression of renal function in CRF patients. Methods: a retrospective study was adopted to select professor Sun Wei's clinic in Jiangsu Province Traditional Chinese Medicine Hospital, the nephrology department of Jiangsu Province Traditional Chinese Medicine Hospital in September September 2013. 51 cases of chronic renal failure were diagnosed and observed the changes of clinical symptoms and curative effects for 2 years after the treatment of "Yishen Qingli, rhubarb and rhubarb", and the course of treatment for each patient was more than 1 months. Results: 51 patients were identified by "Yishen Qingli, harmony and turbidity method adding rhubarb" After 24 months of treatment, the clinical symptom score of TCM was significantly lower than that of the first diagnosis (P0.01). The main curative effect indexes of the patients were Scr, BUN, UA compared with before treatment, and there were significant differences (P0.01). After treatment, RBC had a significant difference (P0.01), and Hb slightly increased after treatment. But there was no statistical difference (P0.05). There was a significant difference in TC between before and after treatment (P0.01), but there was a slight decrease in TG after treatment.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R277.5

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