EGCG通過減輕氧化應激與抑制NALP3炎性體對阿霉素腎病有保護作用
發(fā)布時間：2018-09-02 07:21
【摘要】：研究背景和目的 局灶節(jié)段性腎小球硬化(FSGS)是腎病綜合征(NS)常見而嚴重的病理表現，也是導致終末期腎病(ESRD)的重要原因之一。由于其發(fā)病機制尚未完全闡明，目前臨床治療仍是一大難題。近來研究認為，炎癥因子的大量激活以及氧化應激作用的產生對FSGS的發(fā)生發(fā)展起著重要的作用。NALP3(NLR family pyrin domain containing-3protein，NRLP3, NALP3)炎性體是NLRs炎性體家族成員之一，主要表達于巨噬細胞、DCs等抗原遞呈細胞，發(fā)揮啟動炎性體裝配的作用。當危險信號作用于靶細胞，NALP3炎性體被激活，通過募集ASC，激活caspase-1剪切IL-1家族成員前體成為具有活性的IL-1β、IL-18促炎癥細胞因子，從而啟動炎癥反應。研究表明，NALP3炎性體及其下游炎性因子在慢性腎臟疾病中發(fā)揮重要作用，在FSGS腎病患者組織標本檢測發(fā)現NALP3mRNA的表達顯著升高，且與腎功能惡化程度顯著相關。而另一方面，氧化應激的發(fā)生可引起腎組織發(fā)生壞死、凋亡、炎癥以及纖維化已得到廣泛的共識。通過動物模型發(fā)現，與對照相比，FSGS大鼠體內氧化應激相關指標水平明顯上升，抗氧化酶水平降低，并且與病情程度存在明顯相關性。因此，有效減輕炎癥反應及氧化應激作用對減緩FSGS向終末期腎臟疾病發(fā)展具有尤為重要的意義。 新近研究表明，作為綠茶中茶多酚的主要活性物質，表沒食子兒茶素沒食子酸酯(EGCG)具有很強的生物活性。近年來研究發(fā)現，EGCG具有抗炎、抗感染、抗氧化、抑制腫瘤信號通路等作用。而其通過其抗炎、抗氧化,對改善腎組織炎癥、減輕組織損傷、保護殘腎功能方面有積極意義。雖目前未見有關EGCG治療FSGS的相關報道，但我們推測EGCG可以通過減輕炎癥反應及抑制氧化應激作用，減緩FSGS病程進展速度。 本實驗擬觀察FSGS患者腎組織NALP3等炎癥因子的表達，以及EGCG對阿霉素(ADM)誘導的FSGS小鼠腎組織NALP3炎性復合體及氧化應激作用的影響，探討EGCG對阿霉素誘導的腎病小鼠腎組織的保護作用及機制，最終為臨床腎病綜合征的治療方法選擇提供實驗依據。 研究方法 1.臨床標本有關實驗 收集26例我科臨床診斷為腎病綜合征患者經腎活檢確診為FSGS的腎組織石蠟切片，以16例腎錯構瘤患者手術切除的錯構瘤周圍正常腎組織標本為對照組，免疫組化檢測腎組織NALP3/ASC/caspase-1、IL-1β、IL-18、F4/80的表達情況，對腎小管間質損傷程度進行評分，，收集患者治療前24h尿蛋白、血肌酐、血尿素氮、血漿總蛋白、血漿白蛋白等生化指標，計算腎小球濾過率eGFR。將NALP3/ASC/caspase-1和IL-1β、IL-18表達情況分別與腎小管間質損傷程度及各生化指標進行相關性分析。 2.動物實驗 8周齡雄性BALB/C小鼠24只，隨機分為：①正常對照組(N組，6只)，實驗全程每天雙蒸水0.2ml灌胃，5天后生理鹽水(0.1ml)尾靜脈注射，2周后再重復注射生理鹽水，第一次注射后28天開胸腹取血及腎臟標本；②阿霉素腎病模型組(AN組，6只)，實驗全程雙蒸水灌胃，5天后給予10mg/kg阿霉素(生理鹽水配成2mg/ml)經尾靜脈注射，2周后再重復注射同等劑量的阿霉素，第一次注射后28天按對照組方法取血和腎臟標本；③小劑量EGCG處理組(EGCG A組，6只)，實驗全程經50mg/kg.bw EGCG灌胃，后按AN組方法造模，第一次注射后28天按對照組方法取標本；④大劑量EGCG處理組(EGCG B組，6只)，實驗全程經100mg/kg.bw EGCG灌胃，5天后按AN組方法造模，第一次注射后28天按對照組方法取標本。PAS染色觀察腎組織損傷程度及炎性細胞浸潤程度，檢測小鼠血肌酐、血尿素氮、血清白蛋白、24h尿蛋白等生化指標，檢測腎組織MDA、CAT、GPx、SOD等氧化應激相關指標，免疫熒光檢測腎組織NALP3、HO-1表達情況，western-blot法檢測腎組織NALP3、ASC、caspase-1、IL-1β、IL-18、Nrf2、HO-1蛋白表達情況，對數據采用SPSS16.0軟件，進行統(tǒng)計學分析。 實驗結果 1臨床研究： 1.1．與對照組相比，FSGS組患者腎組織NALP3/ASC/caspase-1、IL-1β、IL-18、F4/80表達顯著增加(P0.01)。 1.2.相關性分析表明，NALP3、ASC、caspase-1的表達與IL-1β、IL-18均呈正相關(P0.01)，NALP3、ASC、caspase-1、IL-1β、IL-18與腎小管間質損傷程度積分、F4/80表達均呈正相關(P0.01)，與24h尿蛋白量、血肌酐濃度呈正相關(P0.05)，與eGFR呈負相關(P0.05)，與尿素氮、血漿總蛋白、血清白蛋白濃度無明顯相關性。 2動物實驗： 2.1. EGCG對阿霉素小鼠病情的影響。 阿霉素組小鼠較正常對照小鼠24h尿蛋白、血肌酐、尿素氮水平明顯升高(P0.05),血漿白蛋白明顯降低(P0.05)。經50mg/kg EGCG和100mg/kg EGCG處理后在注射阿霉素的小鼠，其24h尿蛋白、血肌酐、尿素氮的升高程度顯著低于AN組(P0.05),血漿白蛋白的降低程度顯著低于AN組(P0.05)。腎組織切片PAS染色顯示：AN組小鼠出現腎小球局灶節(jié)段性硬化(2.10±0.19)，腎小管上皮細胞損傷(2.70±0.34)腎間質炎癥細胞浸潤(2.70±0.34)等表現；經50mg/kg EGCG和100mg/kg EGCG處理的小鼠，注射阿霉素后，腎小球硬化指數、腎小管間質損傷指數、每高倍視野中性粒細胞數較AN組顯著減少(P0.05)。 2.2. EGCG對阿霉素腎病模型小鼠腎組織炎性因子的影響。 應用western blot檢測各組小鼠腎組織炎性因子的表達發(fā)現，與正常對照小鼠相比，AN組小鼠腎組織NALP3、ASC、caspase-1、IL-1β、IL-18蛋白表達明顯升高。經EGCG處理的小鼠腎組織NALP3炎性體及其下游炎性因子的蛋白表達較AN組顯著減弱(P0.05)，并且100mg/kg EGCG處理組較50mg/kg EGCG處理組小鼠腎組織炎性因子表達的減弱程度更加明顯(P0.05)。 2.3EGCG對阿霉素腎病模型小鼠腎組織氧化應激作用的影響 AN組小鼠較正常對照小鼠腎組織抗氧化酶SOD、CAT、CPx活性顯著降低、脂質過氧化標志物MDA含量顯著升高(P0.05)。經100mg/kg EGCG處理的小鼠注射阿霉素后腎組織抗氧化酶SOD、CAT、CPx活性的降低程度和脂質過氧化標志物MDA含量的升高程度顯著低于AN組(P0.05)。采用western blot檢測各組小鼠腎組織具有抗氧化及保護作用的Nrf2、HO-1蛋白表達水平發(fā)現，經EGCG處理的小鼠Nrf2、HO-1的蛋白表達水平較正常對照和單純阿霉素處理的小鼠腎組織均明顯升高(P0.05)。 結論 1.NALP3炎性復合體及氧化應激參與了FSGS的發(fā)生發(fā)展。 2.口服EGCG可改善阿霉素腎病模型小鼠的腎功能損傷程度、有效降低24h尿蛋白排泄量、緩解腎小球硬化和腎小管損傷程度，可以抑制腎組織NALP3炎性復合體的表達，減輕氧化應激作用，增強Nrf2/HO-1對腎臟的保護作用。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692
【引證文獻】
相關期刊論文 前1條
1 王璐;李璐;陳光亮;;NALP3炎性體在痛風發(fā)病中的作用與藥物治療研究進展[J];生命科學;2016年03期
本文編號：2218663
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