EGCG通过减轻氧化应激与抑制NALP3炎性体对阿霉素肾病有保护作用
发布时间：2018-09-02 07:21
【摘要】：研究背景和目的 局灶节段性肾小球硬化(FSGS)是肾病综合征(NS)常见而严重的病理表现，也是导致终末期肾病(ESRD)的重要原因之一。由于其发病机制尚未完全阐明，目前临床治疗仍是一大难题。近来研究认为，炎症因子的大量激活以及氧化应激作用的产生对FSGS的发生发展起着重要的作用。NALP3(NLR family pyrin domain containing-3protein，NRLP3, NALP3)炎性体是NLRs炎性体家族成员之一，主要表达于巨噬细胞、DCs等抗原递呈细胞，发挥启动炎性体装配的作用。当危险信号作用于靶细胞，NALP3炎性体被激活，通过募集ASC，激活caspase-1剪切IL-1家族成员前体成为具有活性的IL-1β、IL-18促炎症细胞因子，从而启动炎症反应。研究表明，NALP3炎性体及其下游炎性因子在慢性肾脏疾病中发挥重要作用，在FSGS肾病患者组织标本检测发现NALP3mRNA的表达显著升高，且与肾功能恶化程度显著相关。而另一方面，氧化应激的发生可引起肾组织发生坏死、凋亡、炎症以及纤维化已得到广泛的共识。通过动物模型发现，与对照相比，FSGS大鼠体内氧化应激相关指标水平明显上升，抗氧化酶水平降低，并且与病情程度存在明显相关性。因此，有效减轻炎症反应及氧化应激作用对减缓FSGS向终末期肾脏疾病发展具有尤为重要的意义。 新近研究表明，作为绿茶中茶多酚的主要活性物质，表没食子儿茶素没食子酸酯(EGCG)具有很强的生物活性。近年来研究发现，EGCG具有抗炎、抗感染、抗氧化、抑制肿瘤信号通路等作用。而其通过其抗炎、抗氧化,对改善肾组织炎症、减轻组织损伤、保护残肾功能方面有积极意义。虽目前未见有关EGCG治疗FSGS的相关报道，但我们推测EGCG可以通过减轻炎症反应及抑制氧化应激作用，减缓FSGS病程进展速度。 本实验拟观察FSGS患者肾组织NALP3等炎症因子的表达，以及EGCG对阿霉素(ADM)诱导的FSGS小鼠肾组织NALP3炎性复合体及氧化应激作用的影响，探讨EGCG对阿霉素诱导的肾病小鼠肾组织的保护作用及机制，最终为临床肾病综合征的治疗方法选择提供实验依据。 研究方法 1.临床标本有关实验 收集26例我科临床诊断为肾病综合征患者经肾活检确诊为FSGS的肾组织石蜡切片，以16例肾错构瘤患者手术切除的错构瘤周围正常肾组织标本为对照组，免疫组化检测肾组织NALP3/ASC/caspase-1、IL-1β、IL-18、F4/80的表达情况，对肾小管间质损伤程度进行评分，，收集患者治疗前24h尿蛋白、血肌酐、血尿素氮、血浆总蛋白、血浆白蛋白等生化指标，计算肾小球滤过率eGFR。将NALP3/ASC/caspase-1和IL-1β、IL-18表达情况分别与肾小管间质损伤程度及各生化指标进行相关性分析。 2.动物实验 8周龄雄性BALB/C小鼠24只，随机分为：①正常对照组(N组，6只)，实验全程每天双蒸水0.2ml灌胃，5天后生理盐水(0.1ml)尾静脉注射，2周后再重复注射生理盐水，第一次注射后28天开胸腹取血及肾脏标本；②阿霉素肾病模型组(AN组，6只)，实验全程双蒸水灌胃，5天后给予10mg/kg阿霉素(生理盐水配成2mg/ml)经尾静脉注射，2周后再重复注射同等剂量的阿霉素，第一次注射后28天按对照组方法取血和肾脏标本；③小剂量EGCG处理组(EGCG A组，6只)，实验全程经50mg/kg.bw EGCG灌胃，后按AN组方法造模，第一次注射后28天按对照组方法取标本；④大剂量EGCG处理组(EGCG B组，6只)，实验全程经100mg/kg.bw EGCG灌胃，5天后按AN组方法造模，第一次注射后28天按对照组方法取标本。PAS染色观察肾组织损伤程度及炎性细胞浸润程度，检测小鼠血肌酐、血尿素氮、血清白蛋白、24h尿蛋白等生化指标，检测肾组织MDA、CAT、GPx、SOD等氧化应激相关指标，免疫荧光检测肾组织NALP3、HO-1表达情况，western-blot法检测肾组织NALP3、ASC、caspase-1、IL-1β、IL-18、Nrf2、HO-1蛋白表达情况，对数据采用SPSS16.0软件，进行统计学分析。 实验结果 1临床研究： 1.1．与对照组相比，FSGS组患者肾组织NALP3/ASC/caspase-1、IL-1β、IL-18、F4/80表达显著增加(P0.01)。 1.2.相关性分析表明，NALP3、ASC、caspase-1的表达与IL-1β、IL-18均呈正相关(P0.01)，NALP3、ASC、caspase-1、IL-1β、IL-18与肾小管间质损伤程度积分、F4/80表达均呈正相关(P0.01)，与24h尿蛋白量、血肌酐浓度呈正相关(P0.05)，与eGFR呈负相关(P0.05)，与尿素氮、血浆总蛋白、血清白蛋白浓度无明显相关性。 2动物实验： 2.1. EGCG对阿霉素小鼠病情的影响。 阿霉素组小鼠较正常对照小鼠24h尿蛋白、血肌酐、尿素氮水平明显升高(P0.05),血浆白蛋白明显降低(P0.05)。经50mg/kg EGCG和100mg/kg EGCG处理后在注射阿霉素的小鼠，其24h尿蛋白、血肌酐、尿素氮的升高程度显著低于AN组(P0.05),血浆白蛋白的降低程度显著低于AN组(P0.05)。肾组织切片PAS染色显示：AN组小鼠出现肾小球局灶节段性硬化(2.10±0.19)，肾小管上皮细胞损伤(2.70±0.34)肾间质炎症细胞浸润(2.70±0.34)等表现；经50mg/kg EGCG和100mg/kg EGCG处理的小鼠，注射阿霉素后，肾小球硬化指数、肾小管间质损伤指数、每高倍视野中性粒细胞数较AN组显著减少(P0.05)。 2.2. EGCG对阿霉素肾病模型小鼠肾组织炎性因子的影响。 应用western blot检测各组小鼠肾组织炎性因子的表达发现，与正常对照小鼠相比，AN组小鼠肾组织NALP3、ASC、caspase-1、IL-1β、IL-18蛋白表达明显升高。经EGCG处理的小鼠肾组织NALP3炎性体及其下游炎性因子的蛋白表达较AN组显著减弱(P0.05)，并且100mg/kg EGCG处理组较50mg/kg EGCG处理组小鼠肾组织炎性因子表达的减弱程度更加明显(P0.05)。 2.3EGCG对阿霉素肾病模型小鼠肾组织氧化应激作用的影响 AN组小鼠较正常对照小鼠肾组织抗氧化酶SOD、CAT、CPx活性显著降低、脂质过氧化标志物MDA含量显著升高(P0.05)。经100mg/kg EGCG处理的小鼠注射阿霉素后肾组织抗氧化酶SOD、CAT、CPx活性的降低程度和脂质过氧化标志物MDA含量的升高程度显著低于AN组(P0.05)。采用western blot检测各组小鼠肾组织具有抗氧化及保护作用的Nrf2、HO-1蛋白表达水平发现，经EGCG处理的小鼠Nrf2、HO-1的蛋白表达水平较正常对照和单纯阿霉素处理的小鼠肾组织均明显升高(P0.05)。 结论 1.NALP3炎性复合体及氧化应激参与了FSGS的发生发展。 2.口服EGCG可改善阿霉素肾病模型小鼠的肾功能损伤程度、有效降低24h尿蛋白排泄量、缓解肾小球硬化和肾小管损伤程度，可以抑制肾组织NALP3炎性复合体的表达，减轻氧化应激作用，增强Nrf2/HO-1对肾脏的保护作用。
[Abstract]:Research background and purpose
Focal segmental glomerulosclerosis (FSGS) is a common and serious pathological manifestation of nephrotic syndrome (NS) and one of the important causes leading to end-stage renal disease (ESRD). Clinical treatment is still a difficult problem because of its pathogenesis has not been fully elucidated. NALP3 (NLR family pyrin domain containing-3 protein, NRLP3, NALP3) inflammatory body is one of the members of the NLRs inflammatory body family, mainly expressed in macrophages, DCs and other antigen presenting cells, plays an important role in initiating inflammatory body assembly. When dangerous signals act on target cells, NALP3 inflammatory body is stimulated. Activate caspase-1 by recruiting ASC and splicing precursors of IL-1 family members into active IL-1 beta and IL-18 pro-inflammatory cytokines to initiate inflammation. Studies have shown that NALP-3 inflammatory bodies and their downstream inflammatory factors play an important role in chronic kidney disease, and NALP-3 mRNA expression is detected in tissue specimens of patients with FSGS nephropathy. On the other hand, oxidative stress can cause renal tissue necrosis, apoptosis, inflammation and fibrosis, which has been widely recognized. Therefore, it is very important to reduce the inflammation and oxidative stress to slow down the progression of FSGS to end-stage renal disease.
Recent studies have shown that epigallocatechin gallate (EGCG), as the main active substance of tea polyphenols in green tea, has strong biological activity. In recent years, it has been found that EGCG has anti-inflammatory, anti-infective, anti-oxidant, anti-tumor signaling pathways and other effects. Although there are no reports about EGCG in treating FSGS, we speculate that EGCG can slow down the progression of FSGS by reducing inflammation and inhibiting oxidative stress.
This study was designed to investigate the expression of NALP3 and other inflammatory factors in kidney tissue of FSGS patients and the effect of EGCG on NALP3 inflammatory complex and oxidative stress in kidney tissue of FSGS mice induced by adriamycin (ADM). Provide experimental evidence.
research method
1. clinical specimen related experiments
The expression of NALP3/ASC/caspase-1, IL-1beta, IL-18, F4/80 in renal tissue and tubulointerstitial injury were detected by immunohistochemistry in 26 patients with renal biopsy-confirmed FSGS and 16 normal renal tissues around renal hamartoma as control group. The levels of urinary protein, serum creatinine, blood urea nitrogen, plasma total protein, plasma albumin and other biochemical indicators were collected 24 hours before treatment, and the glomerular filtration rate (eGFR) was calculated.
2. animal experiments
Twenty-four eight-week-old male BALB/C mice were randomly divided into two groups: (1) normal control group (N group, 6 mice). The rats in the experimental group were given 0.2 ml of double-steamed water daily, followed by tail vein injection of 0.1 ml of normal saline 5 days later, followed by repeated injection of normal saline 2 weeks later. Blood and kidney samples were taken from thoracotomy and abdomen 28 days after the first injection; and (2) adriamycin nephropathy model group (AN group, 6 mice). After 5 days, 10 mg/kg of adriamycin (2 mg/ml of normal saline) was injected into the tail vein. After 2 weeks, the same dose of adriamycin was injected again. Blood and kidney samples were taken from the control group 28 days after the first injection. 3. The small dose of EGCG treatment group (EGCG A group, 6 rats) was given 50 mg/kg. BW EGCG through the stomach, and then AN group was used. (4) High dose EGCG treatment group (EGCG B group, 6 mice) was given 100 mg/kg. BW EGCG by gastric lavage. The model was made by AN group 5 days later, and the samples were taken by control group 28 days after the first injection. The degree of renal tissue injury and inflammatory cell infiltration were observed by PAS staining, and the mice were detected. Serum creatinine, blood urea nitrogen, serum albumin, 24h urine protein, MDA, CAT, GPx, SOD and other indicators of oxidative stress in kidney tissue, immunofluorescence detection of NALP3, HO-1 expression in kidney tissue, Western-blot detection of NALP3, ASC, caspase-1, IL-1beta, IL-18, Nrf2, HO-1 protein expression in kidney tissue, data using SPSS16.0 software, radical. Statistical analysis was performed.
experimental result
1 clinical research:
Compared with the control group, the expression of NALP3/ASC/caspase-1, IL-1 beta, IL-18 and F4/80 in renal tissue of FSGS group increased significantly (P 0.01).
1.2 Correlation analysis showed that the expression of NALP3, ASC, caspase-1 was positively correlated with IL-1 beta, IL-18 (P 0.01), NALP3, ASC, caspase-1, IL-1 beta, IL-18 and renal tubulointerstitial injury score, F4/80 expression were positively correlated (P 0.01), 24 h urinary protein content, serum creatinine concentration was positively correlated (P 0.05), and eGFR was negatively correlated (P 0.05), and urea nitrogen, plasma total levels of IL-1 beta, IL-18 were positively correlated (P 0.05). There was no significant correlation between protein and serum albumin concentration.
2 animal experiments:
Effect of 2.1. EGCG on the condition of adriamycin in mice.
The levels of urinary protein, serum creatinine, urea nitrogen and plasma albumin were significantly increased (P 0.05) and decreased (P 0.05) in the adriamycin treated mice compared with the normal control mice at 24 h. The levels of urinary protein, serum creatinine and urea nitrogen in the adriamycin treated mice at 50 mg/kg EGCG and 100 mg/kg EGCG were significantly lower than those in the AN group (P 0.05). PAS staining showed focal segmental glomerulosclerosis (2.10 (+ 0.19)) and renal tubular epithelial cell injury (2.70 (+ 0.34)) in AN mice, renal interstitial inflammatory cell infiltration (2.70 (+ 0.34)) in 50 mg / kg EGCG and 100 mg / kg EGCG treated mice, and glomerulosclerosis finger after adriamycin injection. The number of neutrophils in each high field of vision was significantly lower than that in group AN (P0.05).
Effect of 2.2. EGCG on inflammatory factors in kidney tissue of adriamycin nephropathy mice.
Western blot showed that the expression of NALP3, ASC, caspase-1, IL-1beta and IL-18 in kidney tissues of AN mice was significantly higher than that of normal control mice. Compared with 50 mg/kg EGCG treatment group, the expression of inflammatory factors in kidney tissue of mice treated with mg/kg EGCG decreased more significantly (P 0.05).
Effect of 2.3EGCG on oxidative stress in kidney tissue of adriamycin nephropathy mice
The activities of SOD, CAT and CPx in kidney tissue of AN group mice were significantly lower than those of normal control mice, and the content of MDA, a marker of lipid peroxidation, was significantly increased (P 0.05). The activity of SOD, CAT and CPx and the content of MDA, a marker of lipid peroxidation, were significantly decreased in kidney tissue of mice treated with 100mg/kg EGCG. The expression levels of Nrf2 and HO-1 proteins in the kidneys of mice treated with EGCG were significantly higher than those of normal control and adriamycin-treated mice (P 0.05).
conclusion
1.NALP3 inflammatory complex and oxidative stress are involved in the development of FSGS.
2. Oral administration of EGCG can improve the degree of renal function damage in adriamycin nephropathy model mice, effectively reduce 24-hour urinary protein excretion, alleviate glomerulosclerosis and renal tubular damage, inhibit the expression of NALP3 inflammatory complex in renal tissue, reduce oxidative stress, and enhance the protective effect of Nrf2/HO-1 on kidney.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692
本文编号：2218663
[Abstract]:Research background and purpose
Focal segmental glomerulosclerosis (FSGS) is a common and serious pathological manifestation of nephrotic syndrome (NS) and one of the important causes leading to end-stage renal disease (ESRD). Clinical treatment is still a difficult problem because of its pathogenesis has not been fully elucidated. NALP3 (NLR family pyrin domain containing-3 protein, NRLP3, NALP3) inflammatory body is one of the members of the NLRs inflammatory body family, mainly expressed in macrophages, DCs and other antigen presenting cells, plays an important role in initiating inflammatory body assembly. When dangerous signals act on target cells, NALP3 inflammatory body is stimulated. Activate caspase-1 by recruiting ASC and splicing precursors of IL-1 family members into active IL-1 beta and IL-18 pro-inflammatory cytokines to initiate inflammation. Studies have shown that NALP-3 inflammatory bodies and their downstream inflammatory factors play an important role in chronic kidney disease, and NALP-3 mRNA expression is detected in tissue specimens of patients with FSGS nephropathy. On the other hand, oxidative stress can cause renal tissue necrosis, apoptosis, inflammation and fibrosis, which has been widely recognized. Therefore, it is very important to reduce the inflammation and oxidative stress to slow down the progression of FSGS to end-stage renal disease.
Recent studies have shown that epigallocatechin gallate (EGCG), as the main active substance of tea polyphenols in green tea, has strong biological activity. In recent years, it has been found that EGCG has anti-inflammatory, anti-infective, anti-oxidant, anti-tumor signaling pathways and other effects. Although there are no reports about EGCG in treating FSGS, we speculate that EGCG can slow down the progression of FSGS by reducing inflammation and inhibiting oxidative stress.
This study was designed to investigate the expression of NALP3 and other inflammatory factors in kidney tissue of FSGS patients and the effect of EGCG on NALP3 inflammatory complex and oxidative stress in kidney tissue of FSGS mice induced by adriamycin (ADM). Provide experimental evidence.
research method
1. clinical specimen related experiments
The expression of NALP3/ASC/caspase-1, IL-1beta, IL-18, F4/80 in renal tissue and tubulointerstitial injury were detected by immunohistochemistry in 26 patients with renal biopsy-confirmed FSGS and 16 normal renal tissues around renal hamartoma as control group. The levels of urinary protein, serum creatinine, blood urea nitrogen, plasma total protein, plasma albumin and other biochemical indicators were collected 24 hours before treatment, and the glomerular filtration rate (eGFR) was calculated.
2. animal experiments
Twenty-four eight-week-old male BALB/C mice were randomly divided into two groups: (1) normal control group (N group, 6 mice). The rats in the experimental group were given 0.2 ml of double-steamed water daily, followed by tail vein injection of 0.1 ml of normal saline 5 days later, followed by repeated injection of normal saline 2 weeks later. Blood and kidney samples were taken from thoracotomy and abdomen 28 days after the first injection; and (2) adriamycin nephropathy model group (AN group, 6 mice). After 5 days, 10 mg/kg of adriamycin (2 mg/ml of normal saline) was injected into the tail vein. After 2 weeks, the same dose of adriamycin was injected again. Blood and kidney samples were taken from the control group 28 days after the first injection. 3. The small dose of EGCG treatment group (EGCG A group, 6 rats) was given 50 mg/kg. BW EGCG through the stomach, and then AN group was used. (4) High dose EGCG treatment group (EGCG B group, 6 mice) was given 100 mg/kg. BW EGCG by gastric lavage. The model was made by AN group 5 days later, and the samples were taken by control group 28 days after the first injection. The degree of renal tissue injury and inflammatory cell infiltration were observed by PAS staining, and the mice were detected. Serum creatinine, blood urea nitrogen, serum albumin, 24h urine protein, MDA, CAT, GPx, SOD and other indicators of oxidative stress in kidney tissue, immunofluorescence detection of NALP3, HO-1 expression in kidney tissue, Western-blot detection of NALP3, ASC, caspase-1, IL-1beta, IL-18, Nrf2, HO-1 protein expression in kidney tissue, data using SPSS16.0 software, radical. Statistical analysis was performed.
experimental result
1 clinical research:
Compared with the control group, the expression of NALP3/ASC/caspase-1, IL-1 beta, IL-18 and F4/80 in renal tissue of FSGS group increased significantly (P 0.01).
1.2 Correlation analysis showed that the expression of NALP3, ASC, caspase-1 was positively correlated with IL-1 beta, IL-18 (P 0.01), NALP3, ASC, caspase-1, IL-1 beta, IL-18 and renal tubulointerstitial injury score, F4/80 expression were positively correlated (P 0.01), 24 h urinary protein content, serum creatinine concentration was positively correlated (P 0.05), and eGFR was negatively correlated (P 0.05), and urea nitrogen, plasma total levels of IL-1 beta, IL-18 were positively correlated (P 0.05). There was no significant correlation between protein and serum albumin concentration.
2 animal experiments:
Effect of 2.1. EGCG on the condition of adriamycin in mice.
The levels of urinary protein, serum creatinine, urea nitrogen and plasma albumin were significantly increased (P 0.05) and decreased (P 0.05) in the adriamycin treated mice compared with the normal control mice at 24 h. The levels of urinary protein, serum creatinine and urea nitrogen in the adriamycin treated mice at 50 mg/kg EGCG and 100 mg/kg EGCG were significantly lower than those in the AN group (P 0.05). PAS staining showed focal segmental glomerulosclerosis (2.10 (+ 0.19)) and renal tubular epithelial cell injury (2.70 (+ 0.34)) in AN mice, renal interstitial inflammatory cell infiltration (2.70 (+ 0.34)) in 50 mg / kg EGCG and 100 mg / kg EGCG treated mice, and glomerulosclerosis finger after adriamycin injection. The number of neutrophils in each high field of vision was significantly lower than that in group AN (P0.05).
Effect of 2.2. EGCG on inflammatory factors in kidney tissue of adriamycin nephropathy mice.
Western blot showed that the expression of NALP3, ASC, caspase-1, IL-1beta and IL-18 in kidney tissues of AN mice was significantly higher than that of normal control mice. Compared with 50 mg/kg EGCG treatment group, the expression of inflammatory factors in kidney tissue of mice treated with mg/kg EGCG decreased more significantly (P 0.05).
Effect of 2.3EGCG on oxidative stress in kidney tissue of adriamycin nephropathy mice
The activities of SOD, CAT and CPx in kidney tissue of AN group mice were significantly lower than those of normal control mice, and the content of MDA, a marker of lipid peroxidation, was significantly increased (P 0.05). The activity of SOD, CAT and CPx and the content of MDA, a marker of lipid peroxidation, were significantly decreased in kidney tissue of mice treated with 100mg/kg EGCG. The expression levels of Nrf2 and HO-1 proteins in the kidneys of mice treated with EGCG were significantly higher than those of normal control and adriamycin-treated mice (P 0.05).
conclusion
1.NALP3 inflammatory complex and oxidative stress are involved in the development of FSGS.
2. Oral administration of EGCG can improve the degree of renal function damage in adriamycin nephropathy model mice, effectively reduce 24-hour urinary protein excretion, alleviate glomerulosclerosis and renal tubular damage, inhibit the expression of NALP3 inflammatory complex in renal tissue, reduce oxidative stress, and enhance the protective effect of Nrf2/HO-1 on kidney.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692
【引证文献】
相关期刊论文 前1条
1 王璐;李璐;陈光亮;;NALP3炎性体在痛风发病中的作用与药物治疗研究进展[J];生命科学;2016年03期
本文编号：2218663
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