马兜铃酸对肾脏管旁毛细血管内皮细胞的毒性作用
发布时间：2018-08-13 18:35
【摘要】：背景和目的： 马兜铃酸(Aristolochic Acid，AA)隶属于马兜铃属，长期服用AA或含AA的药物可引起患者肾小管坏死，肾间质明显纤维化，进而发展为慢性肾功能衰竭，由此，国内外学者将这类肾脏疾病称之为马兜铃酸肾病(Aristolochic Acid Nephropathy，AAN)[1]。与其他肾脏疾病或中毒性肾病不同，AAN患者表现为肾小管上皮细胞变性、坏死、脱落，缺乏明显的上皮细胞再生，基底膜裸露，间质弥漫性增宽、进行性纤维化。目前，这些特殊损伤病理表现机制尚不十分清楚，临床治疗更无有效措施，AAN患者常病情迁延不愈，最终进展为终末期肾衰竭[2]。因此，深入探讨AAN肾小管损伤及修复机制是很有必要的。围绕AA的主要及重要靶点——肾小管，国内外学者进行了大量研究，确立了肾小管上皮细胞凋亡[3]、转分化[4]、胞内AA-DNA加合物形成[5]、泌尿系统肿瘤形成[6]等学说，这些学说能解释AAN肾小管毁损的部分病理特点，然肾小管修复不良的原因仍然需要进一步阐明。近年来，肾小管管旁毛细血管(peritubular capillary，PTC)损伤及丢失或内皮细胞损伤[7]在急慢性肾脏疾病，特别是慢性肾脏疾病进展中的作用引起了广大学者的注意[8]。因此，，有必要观察AAN过程中，PTC损伤及丢失情况，将其与肾小管损伤及增殖修复不良的关系作一系统分析，并深入研究AA对PTC损伤的细胞生物学毒性机制，以期提出治疗马兜铃酸肾病的新机制和保护治疗新策略。 本课题拟通过建立体内动物模型观察PTC损伤及丢失情况，分析该损伤丢失与肾小管损伤、增殖修复的关系，并通过透射电镜进一步观察PTC内皮细胞超微结构改变；体外建立AA对人脐静脉内皮细胞(HUVEC)损伤模型，观察AA毒性下内皮细胞增殖、凋亡及迁移、成管等细胞生物学变化，深入研究AA对内皮细胞的损伤机制，以期为临床治疗AAN提供新的损伤机制和治疗策略。 方法： 本课题拟分为体内、体外两部分进行探讨。 第一部分：体内观察AA对PTC的损伤作用及其与肾小管损伤、修复的相关性分析。 1. AAN小鼠模型建立。C57/BL/6小鼠分为4组：对照2W组，AA2W组，对照4W组，AA4W组。AA组小鼠以5mg/kg剂量每两天腹腔注射一次AA溶液，对照组注射等体积的缓冲液(PBS)。 2.分别于2周、4周时收集小鼠血、尿、肾脏组织，进行血尿生化分析及肾脏病理学观察。 3.分析PTC损伤与小鼠肾小管损伤、增殖和修复的相关关系。 第二部分：体外研究AA损伤血管内皮细胞的机制。 1.采用文献浓度分组[1]，以不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)和不同时间(24h，48h，72h)作用人脐静脉内皮细胞株(HUVEC)，MTT法检测AA对细胞增殖力的影响，筛选出药物最适作用时间。 2. LDH释放：不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)处理HUVEC48h(最佳时间)，检测细胞质LDH释放情况。 3.内皮细胞成管：不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)处理HUVEC48h后，收集各组细胞，转种于基质胶包被的96孔板，每孔103~104个细胞，孵育6h后观察细胞成管情况。 4.内皮细胞迁移：不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)处理HUVEC48h后，收集各组细胞，按105/孔接种于Transwell小室，下室加入含低浓度血清(2.5%FBS)的DMEM高糖培养基刺激细胞迁移，18~24h后，取出小室，固定染色后倒置显微镜下计数小室下层细胞。 5.内皮细胞凋亡：HUVEC经不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)作用48h后，每组收集≥105个细胞，Annexin V-FITC及PI染色后，于流式细胞仪检测细胞凋亡情况。 6.线粒体膜电位变化：HUVEC经不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)作用48h后，JC-1染色，激光共聚焦检测细胞线粒体膜电位变化情况。 7.线粒体介导的凋亡途径： HUVEC经不同浓度AA(0μg/ml，5g/ml，10g/ml，20g/ml)作用48h后，收集细胞进行western blot实验，观察Caspase-3、Caspase-9、Bax、Bcl-2、Apaf-1、Cytochrome C(细胞色素C)表达情况。 结果： 第一部分： 1.经腹腔注射AA的小鼠血Scr、BUN、尿NAG酶较对照组明显升高，其中AA4W较AA2W组更为明显。 2. AA组小鼠肾小管损伤、间质纤维化、PTC损伤及丢失情况较对照组明显，其中AA4W较AA2W组更为明显；与AA4W相比，AA2W的小鼠肾小管上皮细胞增殖修复情况较好。相关分析显示PTC丢失量与肾小管损伤积分高度相关，相关关系为正相关(P＜0.05)；与PCNA阳性表达量高度相关，相关关系为负相关(P＜0.05)。 3. AA组小鼠PTC内皮细胞中可见线粒体肿胀，部分线粒体脊紊乱、消失。 第二部分： 1. AA可诱导HUVEC活力降低、LDH释放增加，并呈浓度依赖性。 2. AA可导致HUVEC体外成管量减少，成管不完整，且内皮迁移能力下降，两者均呈浓度依赖性。 3. AA能诱导HUVEC凋亡，表现为凋亡标记蛋白表达增高(caspase-3、 BAX)、内皮细胞线粒体膜电位下降、线粒体凋亡途径的相关蛋白(Cytochrome C、Apaf-1、Caspase-9)表达上调，而抗凋亡的蛋白Bcl-2表达下调，提示AA可通过线粒体凋亡途径诱导HUVEC凋亡。 结论： 1. AA能导致小鼠肾PTC损伤及丢失，并随AA作用时间延长，损伤逐渐加重，该损伤与肾小管损伤加重及增殖修复不良高度相关； 2. AA能诱导HUVEC增殖抑制、成管抑制、迁移抑制等细胞生物学活动障碍，阻抑内皮细胞损伤时的血管再生，此毒性作用可能影响PTC的修复和功能恢复； 3. AA能诱导HUVEC凋亡，该作用可能与AA激活线粒体凋亡途径有关。
[Abstract]:Background and purpose:
Aristolochic acid (AA) belongs to the genus Aristolochic acid. Long-term use of AA or AA-containing drugs can cause renal tubular necrosis, renal interstitial fibrosis, and then develop into chronic renal failure. Therefore, scholars at home and abroad call this kind of renal disease Aristolochic acid nephropathy (AAN) [1]. Renal diseases or toxic nephropathy are different. AAN patients show renal tubular epithelial cell degeneration, necrosis, exfoliation, lack of obvious epithelial cell regeneration, basement membrane exposure, diffuse interstitial enlargement, progressive fibrosis. At present, the pathological manifestations of these special injuries are not very clear, clinical treatment is even less effective measures, AAN patients often condition. Therefore, it is necessary to explore the mechanism of tubular damage and repair in AAN. A great deal of research has been done around the main and important target of AA, tubules, and established the apoptosis, transdifferentiation, intracellular AA-DNA adduct formation, urinary system. In recent years, peritubular capillary (PTC) injury and loss or endothelial cell injury in acute and chronic kidney diseases, especially in chronic kidneys. Therefore, it is necessary to observe the damage and loss of PTC in the process of AAN, to make a systematic analysis of the relationship between PTC and renal tubular damage and poor proliferation and repair, and to further study the cytotoxic mechanism of AA on PTC injury, in order to propose a new mechanism for the treatment of aristolochic acid nephropathy. New strategies for protection and treatment.
The purpose of this study is to establish an in vivo animal model to observe the damage and loss of PTC, analyze the relationship between the loss of PTC and renal tubular injury, proliferation and repair, and further observe the ultrastructural changes of PTC endothelial cells by transmission electron microscopy; establish an in vitro injury model of human umbilical vein endothelial cells (HUVEC) by AA, and observe the toxicity of AA to endothelial cells. Cell biological changes such as proliferation, apoptosis and migration, tubules and so on, in-depth study of the mechanism of AA damage to endothelial cells, in order to provide a new mechanism and treatment strategy for clinical treatment of AAN.
Method:
This subject is divided into two parts: in vivo and in vitro.
Part one: in vivo observation of AA damage to PTC and its correlation with renal tubular injury and repair.
1. Establishment of AAN mice model. C57/BL/6 mice were divided into four groups: control 2W group, AA2W group, control 4W group, AA4W group. AA mice were injected AA solution intraperitoneally every two days at a dose of 5mg/kg, and control mice were injected with the same volume of buffer solution (PBS).
2. Blood, urine and kidney tissues were collected at 2 and 4 weeks respectively for biochemical analysis and pathological observation.
3. to analyze the relationship between PTC damage and renal tubular injury, proliferation and repair in mice.
The second part: in vitro study of the mechanism of AA damaging vascular endothelial cells.
1. Human umbilical vein endothelial cell line (HUVEC) was treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) and different time (24 h, 48 h, 72 h). MTT assay was used to detect the effect of AA on the proliferation of human umbilical vein endothelial cell line (HUVEC).
2. LDH release: Different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) were used to treat HUVEC for 48h (the best time) to detect the release of LDH in cytoplasm.
3. Endothelial cell tube formation: After treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, HUVEC cells were collected and transferred to 96-well plate coated with matrix gel. 103-104 cells per hole were incubated for 6 hours.
4. Endothelial cell migration: After 48 hours of treatment with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), the cells in each group were collected and inoculated into the Transwell chamber according to 105/hole. The cells were stimulated by DMEM high glucose medium containing low concentration of serum (2.5% FBS) in the lower chamber. After 18-24 hours, the cells were removed from the chamber and stained and counted under the inverted microscope.
5. Endothelial cell apoptosis: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), more than 105 cells were collected from each group. After staining with Annexin V-FITC and PI, the apoptosis was detected by flow cytometry.
6. Mitochondrial membrane potential changes: HUVEC treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, JC-1 staining, laser confocal detection of cell mitochondrial membrane potential changes.
7. Mitochondrial-mediated apoptosis pathway: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), HUVEC cells were collected for Western blot assay to observe the expression of Caspase-3, Caspase-9, Bax, Bcl-2, Apaf-1, Cytochrome C.
Result:
Part one:
1. The levels of serum Scr, BUN and urinary NAG enzymes in AA-injected mice were significantly higher than those in the control group, especially in AA4W group.
2. The renal tubular injury, interstitial fibrosis, PTC injury and loss in AA group were more obvious than those in control group, especially in AA4W group. Compared with AA4W group, the proliferation and repair of renal tubular epithelial cells in AA2W group were better. Correlation analysis showed that the loss of PTC was highly correlated with the renal tubular injury score, and the correlation was positive (P < 0.05). It was highly correlated with the positive expression level of PCNA, and the correlation was negatively correlated (P < 0.05).
3. mitochondria of PTC endothelial cells in group AA showed swelling of mitochondria and partial mitochondrial ridge disappearance.
The second part:
1. AA could induce the decrease of HUVEC activity and the increase of LDH release in a concentration dependent manner.
2. AA can decrease the volume of tube formation, incomplete tube formation and the ability of endothelial migration of HUVEC in vitro, both of which are concentration-dependent.
3. AA can induce apoptosis of HUVEC, which is manifested by increased expression of apoptosis marker protein (caspase-3, BAX), decreased mitochondrial membrane potential, up-regulated expression of mitochondrial apoptosis pathway-related proteins (Cytochrome C, Apaf-1, Caspase-9), and down-regulated expression of anti-apoptotic proteins Bcl-2, suggesting that AA can induce apoptosis of HUVEC through mitochondrial apoptosis pathway.
Conclusion:
1. AA can cause the damage and loss of PTC in mice kidney, and with the prolongation of AA action time, the damage is gradually aggravated, which is highly related to the aggravation of renal tubular injury and poor proliferation and repair.
2. AA can induce proliferation inhibition, tubular inhibition and migration inhibition of HUVEC, and inhibit vascular regeneration during endothelial cell injury, which may affect the repair and functional recovery of PTC.
3. AA can induce HUVEC apoptosis, which may be related to AA activation of mitochondrial apoptotic pathway.
【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：2181833
[Abstract]:Background and purpose:
Aristolochic acid (AA) belongs to the genus Aristolochic acid. Long-term use of AA or AA-containing drugs can cause renal tubular necrosis, renal interstitial fibrosis, and then develop into chronic renal failure. Therefore, scholars at home and abroad call this kind of renal disease Aristolochic acid nephropathy (AAN) [1]. Renal diseases or toxic nephropathy are different. AAN patients show renal tubular epithelial cell degeneration, necrosis, exfoliation, lack of obvious epithelial cell regeneration, basement membrane exposure, diffuse interstitial enlargement, progressive fibrosis. At present, the pathological manifestations of these special injuries are not very clear, clinical treatment is even less effective measures, AAN patients often condition. Therefore, it is necessary to explore the mechanism of tubular damage and repair in AAN. A great deal of research has been done around the main and important target of AA, tubules, and established the apoptosis, transdifferentiation, intracellular AA-DNA adduct formation, urinary system. In recent years, peritubular capillary (PTC) injury and loss or endothelial cell injury in acute and chronic kidney diseases, especially in chronic kidneys. Therefore, it is necessary to observe the damage and loss of PTC in the process of AAN, to make a systematic analysis of the relationship between PTC and renal tubular damage and poor proliferation and repair, and to further study the cytotoxic mechanism of AA on PTC injury, in order to propose a new mechanism for the treatment of aristolochic acid nephropathy. New strategies for protection and treatment.
The purpose of this study is to establish an in vivo animal model to observe the damage and loss of PTC, analyze the relationship between the loss of PTC and renal tubular injury, proliferation and repair, and further observe the ultrastructural changes of PTC endothelial cells by transmission electron microscopy; establish an in vitro injury model of human umbilical vein endothelial cells (HUVEC) by AA, and observe the toxicity of AA to endothelial cells. Cell biological changes such as proliferation, apoptosis and migration, tubules and so on, in-depth study of the mechanism of AA damage to endothelial cells, in order to provide a new mechanism and treatment strategy for clinical treatment of AAN.
Method:
This subject is divided into two parts: in vivo and in vitro.
Part one: in vivo observation of AA damage to PTC and its correlation with renal tubular injury and repair.
1. Establishment of AAN mice model. C57/BL/6 mice were divided into four groups: control 2W group, AA2W group, control 4W group, AA4W group. AA mice were injected AA solution intraperitoneally every two days at a dose of 5mg/kg, and control mice were injected with the same volume of buffer solution (PBS).
2. Blood, urine and kidney tissues were collected at 2 and 4 weeks respectively for biochemical analysis and pathological observation.
3. to analyze the relationship between PTC damage and renal tubular injury, proliferation and repair in mice.
The second part: in vitro study of the mechanism of AA damaging vascular endothelial cells.
1. Human umbilical vein endothelial cell line (HUVEC) was treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) and different time (24 h, 48 h, 72 h). MTT assay was used to detect the effect of AA on the proliferation of human umbilical vein endothelial cell line (HUVEC).
2. LDH release: Different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) were used to treat HUVEC for 48h (the best time) to detect the release of LDH in cytoplasm.
3. Endothelial cell tube formation: After treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, HUVEC cells were collected and transferred to 96-well plate coated with matrix gel. 103-104 cells per hole were incubated for 6 hours.
4. Endothelial cell migration: After 48 hours of treatment with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), the cells in each group were collected and inoculated into the Transwell chamber according to 105/hole. The cells were stimulated by DMEM high glucose medium containing low concentration of serum (2.5% FBS) in the lower chamber. After 18-24 hours, the cells were removed from the chamber and stained and counted under the inverted microscope.
5. Endothelial cell apoptosis: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), more than 105 cells were collected from each group. After staining with Annexin V-FITC and PI, the apoptosis was detected by flow cytometry.
6. Mitochondrial membrane potential changes: HUVEC treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, JC-1 staining, laser confocal detection of cell mitochondrial membrane potential changes.
7. Mitochondrial-mediated apoptosis pathway: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), HUVEC cells were collected for Western blot assay to observe the expression of Caspase-3, Caspase-9, Bax, Bcl-2, Apaf-1, Cytochrome C.
Result:
Part one:
1. The levels of serum Scr, BUN and urinary NAG enzymes in AA-injected mice were significantly higher than those in the control group, especially in AA4W group.
2. The renal tubular injury, interstitial fibrosis, PTC injury and loss in AA group were more obvious than those in control group, especially in AA4W group. Compared with AA4W group, the proliferation and repair of renal tubular epithelial cells in AA2W group were better. Correlation analysis showed that the loss of PTC was highly correlated with the renal tubular injury score, and the correlation was positive (P < 0.05). It was highly correlated with the positive expression level of PCNA, and the correlation was negatively correlated (P < 0.05).
3. mitochondria of PTC endothelial cells in group AA showed swelling of mitochondria and partial mitochondrial ridge disappearance.
The second part:
1. AA could induce the decrease of HUVEC activity and the increase of LDH release in a concentration dependent manner.
2. AA can decrease the volume of tube formation, incomplete tube formation and the ability of endothelial migration of HUVEC in vitro, both of which are concentration-dependent.
3. AA can induce apoptosis of HUVEC, which is manifested by increased expression of apoptosis marker protein (caspase-3, BAX), decreased mitochondrial membrane potential, up-regulated expression of mitochondrial apoptosis pathway-related proteins (Cytochrome C, Apaf-1, Caspase-9), and down-regulated expression of anti-apoptotic proteins Bcl-2, suggesting that AA can induce apoptosis of HUVEC through mitochondrial apoptosis pathway.
Conclusion:
1. AA can cause the damage and loss of PTC in mice kidney, and with the prolongation of AA action time, the damage is gradually aggravated, which is highly related to the aggravation of renal tubular injury and poor proliferation and repair.
2. AA can induce proliferation inhibition, tubular inhibition and migration inhibition of HUVEC, and inhibit vascular regeneration during endothelial cell injury, which may affect the repair and functional recovery of PTC.
3. AA can induce HUVEC apoptosis, which may be related to AA activation of mitochondrial apoptotic pathway.
【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
【参考文献】
相关期刊论文 前1条
1 刘圆圆;李艺;王帅;官涛;郑科;黄云剑;;马兜铃酸致肾小管周围毛细血管丢失的体内实验研究[J];第三军医大学学报;2014年06期
本文编号：2181833
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