氯沙坦逆转阿霉素肾病大鼠肾损伤机制的研究
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摘要
目的:通过动物和细胞实验,研究肾内RAS在肾病进展中的作用及氯沙坦保护肾脏作用的机制。
方法:(1)阿霉素肾病大鼠模型制备;(2)人肾系膜细胞(HRMC)培养;(3)组织形态学检测;(4)肾功能检测;(5)RT-PCR;(6)Western-blot;(7)ELISA;(8)MTT;(9)DCFH-DA荧光标记法;(10)流式细胞术;(11)统计学分析。结果:
1.氯沙坦对阿霉素肾病大鼠肾脏功能及病理变化的影响:(1)氯沙坦可以明显降低由阿霉素肾病导致的尿蛋白增高,及血清尿素氮和肌酐水平的增高(。2)HE染色和Masson染色结果显示氯沙坦的治疗可以明显减少由阿霉素作用导致肾小球系膜细胞增生,纤维化增多。(3)免疫组化结果显示,氯沙坦可以降低由阿霉素作用引起的肾小球GRP78/Bip表达增高和NFκB入核增加。(4)ELISA结果显示:氯沙坦可以降低阿霉素引起的肾脏IL-1水平增高。(5)氯沙坦可以降低阿霉素引起肾脏SOD活性和MDA含量增加。(6)氯沙坦影响阿霉素作用下肾内RAS系统表达。
2.氯沙坦对Ang II和/或阿霉素作用下人肾系膜细胞增殖和致纤维化的影响:(1)MTT法选择最佳药物作用浓度和作用时间。(2)Ang II单独或联合ADR可刺激HRMC细胞DNA合成、有丝分裂和细胞增殖;阿霉素可抑制上述刺激作用。(3)DCFH-DA荧光探针标记结果显示氯沙坦可抑制Ang II单独或联合ADR作用引起的细胞内ROS生成增多。(4)Western blot结果显示氯沙坦可抑制Ang II单独或联合ADR作用引起的GRP78/Bip表达上调、Akt活化、NF-κB核表达增加、AT1R表达增加。(5)RT-PCR结果显示氯沙坦可抑制Ang II单独或联合ADR作用引起的TGF-β1和CyclinD1 mRNA表达上调。
结论:
1.氯沙坦主要通过抑制ROS生成、GRP78/Bip表达增加、NFκB激活及促炎性细胞因子分泌增多、细胞外基质异常沉积,延缓或减轻阿霉素导致的大鼠肾损伤。此外氯沙坦还可影响肾内RAS关键成份表达发挥肾脏保护作用。
2.氯沙坦既能抑制Ang II促HRMC细胞增殖、有丝分裂活跃、致纤维化增强的作用,也可有效抑制Ang II和ADR联合刺激作用。该抑制作用主要通过降低ROS生成、下调GRP78/Bip表达、抑制Akt活化介导。氯沙坦可通过阻断AT1R配体依赖和配体非依赖的途径,逆转阿霉素肾病大鼠肾损伤。
Renin-angiotensin system(RAS) is important for the recirculation system and other organs. RAS was included ACE、Ang I、Ang II、ACE、ATR et. According to the traditional theory that RAS was one of the endocrine system, it was mainly adjusted blood pressure and water-electrolyte balance. Now people gradually recognized that RAS was extensively expressed in many kinds of tissues and organs, so it was distinguished into circulation RAS (cRAS) and tissue RAS(tRAS). Last years, the effect of RAS in partly of renal tissue was become to a warm spot. RAS was played very important effect on adjusting body fluid equilibrium and long-term accommodation with heart-blood system. RAS was also played influential effect of nephritis pathophysiology. All of the essential component of RAS, especially Ang II and AT1/AT2 were sure to be expressed in kidney. Ang II was one of the most important biologically active peptide for RAS, it depends on binding with ATR which located in cellular membrance to educe its biological effect. RAS in kidney was not only promoted pantosomatous effect of RAS, but also played very important effects in adjusting kidney function and nephronia. RAS was activated in many kinds of disease, such as hypertension、diabetes、glomerular nephritis(GN) and so on. Ang II was increased in cardiovascular disease and diabetes. In addition, Ang II was found to increased significantly instoma of kidney in pathologic status.
Losartan is one of competitive antagonist of AT1R, it was not only used to be treat with hypertension, but also be used to alleviated and to deteriortate diabetic nepropathy, it can help undepended-diabetic to heal renal disease、hypertension and others disease. In addition many clinical and animal researches evidence certify that long-term used high dose, even ultrahigh dose Losartan can further more postponed chronic nephritic, but the curative effect was not depended on haemodynamics. Even though Losartan had been confirmed to curative chronic kidney disease, but the exactly mechanism was not completely clear. Our research copy the model of ADR nephritis to study the mainly mechanism of Losartan deteriorate renal injury; Based on cultural human renal mesagial cell to study HRMC proliferation, fibrotic influence. To deep reseach the exactly effect of RAS and the mainly mechanism of AT1R antagon Losartan.
Objective:
1. Through copy the model of ADR nephritis to study the effect of RAS and Losartan in the development of nephritis; 2.Through the cell experiment, to study the effect of AngⅡand ADR for HRMC cells, further confirmed the protection mechanism of RAS in ADR nephritis.
Method:
1. The influence of Losartan on renal functional and pathological changes in ADR nephritis:(1)Choose animals、breeding and multiply;(2)Made ADR animal model,divided into groups and administration;(3)Convention fixed tissues with neutral-fomalin, routine paraffin imbedding and slicing, observed the changes of morphology by HE staining;(4)Used immunohistochemistry to detect the expression changes of GRP78/Bip;(5)Observe the changes of collagen deposit by Masson staining;(6)Used metabolic cage to collect 24h urine to detect urinary volume、urine protein and biochemical indicator;(7)Blood-serum biochemical indicator detection;(8)Detect the SOD and MDA levels by chemiluminescence method;(9)Detect AT1R、AT2R、AGT、ACE mRNA expression by RT-PCR;(10)Detect the expression of IL-1 in renal tissue by ELISA; (11) Datas were analysised by the SPSS 11.0 software.
2. The influence of fibrotic on Losartan when affected on AngⅡand/or ADR;(1)Cell culture;(2)Used MTT to detect the cell multiplication;(3)Used RT-PCR to detect the expression of TGF-β1 and CyclinD1; (4)Detect the expression of EGFR and NF-κB by western-blot;(5)Detect intracellel Ros by DCFH-DA staining;(6)Detective cell cycle by flow cytometry;(7) Used ELISA to detect the expression of type 1 collogen;(8) Used SPSS software to analysis the datas.
Results:
1. The influence of Losartan on renal functional and pathological changes when ADR nephritis:
(1)Losartan can significantly decreased urine protein that increase by ADR, so dose blood serum urea nitrogen.
(2)HE staining and Masson staining show that losartan can reduce mesangial cell proliferation and fibrous degeneration in ADR nephritis.
(3)Immunohistochemistry results show that GRP78/Bip expression was obviously decreased in ADR nephritis group.
(4) Losartan can reduce the expression level of IL-1 in ADR nephritis groups, it was possible reduce glomerulitis, to diminish injury of glomerulum.
(5)Losartan can reduce SOD and MDA that increased by ADR in kidney, it can protect kidney to avoid oxidation injury.
(6)Losartan can change some mainly gene of RAS system expression in kidney to avoid the injury of ADR.
2. The influence of fibrotic on Losartan when affected on AngⅡand/or ADR
(1)Cell proliferation was increased when it was affected by AngⅡ, 1×10-6mol/L AngⅡwas significant promote cells proliferation; Low dose ADR(0.2μg/ml)has no cytotoxicity for cells; every density losartan had no influence on cell proliferation, and all density losartan can suppressed HRMC cell proliferation; 5×10-5mol/L losartan was one of the most efficency dose. The inhibitory action of losartan was concentration dependent; The cell proliferation of combination AngII and low dose ADR for HRMC was stronger than AngII, and this kind of proliferation can also be inhibited by losartan.
(2)HRMC cell DNA was promoted to synthesis when it was affected by AngⅡor association with low dose ADR, cell caryocinesia activity, cell cycle changed. Losartan can utility suppressed the caryocinesia by AngⅡor association with ADR, the ratio of S+G2/M were obviously decreased, even lower than control groups.
(3)Many ROS were induced to produce when AngⅡaffect alone or association with ADR, losartan can suppressed this kind of effect, and make it close to nomal level.
(4) Ang II or/and associated with ADR can increase GRP78/Bip expression in HRMC cells, Losartan can suppressed this kind of effect.
(5) NFκB can be promote to come into nuclear by Ang II or/and associated with ADR, the effect of NFκB into nuclear induced by Ang I can be obviously suppressed by losartan.
(6) AngII and ADR can up-regulate the expression of TGF-β1、CyclinD1 mRNA in HRMC cell, collogen type I expression up regulation , AT1R protein expression increased, and losartan can inhibit these kinds of effects.
Conclusion:
1. ADR nephronia rats kidney show functional and structure lesion. Losartan can reduced and/or delayed the injury of kidney that cause of ADR. ADR induced ROS increased, activated NFκB and promoted phlegmasia cytokine to secrete increased, and to lead extracellular matrix to abnormality deposition, all of these kinds of effects can be inhibited by losartan. Endocytoplasmic reticulum stress was happened in ADR nephronia rats, GRP78/Bip expressed increase, the important component of kidney were changed. Through affected the endocytoplasmic reticulum stress and the level of Ang to delay the kidney injury.
2. Used Ang II alone or associated with lower dose ADR can stimulate DNA synthesis of HRMC cell. TGF-β1 expressed increased, cllogen secreted increased, ADR can enhance the promotion of proliferation by Ang II in HRMC cell. All of these kind of effects mainly caused by ROS product increase, to up regulate GRP78/Bip expression, Akt activation and activated some downstream signals, include Akt-mTOR pathway, TGF-β1 pathway, NF-κB pathway, all of these kind of pathway can affect cell proliferation, differentiated, inflammatory reaction and ECM synthesis. Losartan not only can inhibit the effect of cell proliferation by AngII, but also can utility inhibit the effect of association of AngII and ADR. This inhibition was mainly through decrease ROS production and down-regulate CRP78/Bip and inhibit Akt activation. Losartan can also retroconversion the renal injury by intercept the AT1R ligand dependent pathway and undependent pathway.
方法:(1)阿霉素肾病大鼠模型制备;(2)人肾系膜细胞(HRMC)培养;(3)组织形态学检测;(4)肾功能检测;(5)RT-PCR;(6)Western-blot;(7)ELISA;(8)MTT;(9)DCFH-DA荧光标记法;(10)流式细胞术;(11)统计学分析。结果:
1.氯沙坦对阿霉素肾病大鼠肾脏功能及病理变化的影响:(1)氯沙坦可以明显降低由阿霉素肾病导致的尿蛋白增高,及血清尿素氮和肌酐水平的增高(。2)HE染色和Masson染色结果显示氯沙坦的治疗可以明显减少由阿霉素作用导致肾小球系膜细胞增生,纤维化增多。(3)免疫组化结果显示,氯沙坦可以降低由阿霉素作用引起的肾小球GRP78/Bip表达增高和NFκB入核增加。(4)ELISA结果显示:氯沙坦可以降低阿霉素引起的肾脏IL-1水平增高。(5)氯沙坦可以降低阿霉素引起肾脏SOD活性和MDA含量增加。(6)氯沙坦影响阿霉素作用下肾内RAS系统表达。
2.氯沙坦对Ang II和/或阿霉素作用下人肾系膜细胞增殖和致纤维化的影响:(1)MTT法选择最佳药物作用浓度和作用时间。(2)Ang II单独或联合ADR可刺激HRMC细胞DNA合成、有丝分裂和细胞增殖;阿霉素可抑制上述刺激作用。(3)DCFH-DA荧光探针标记结果显示氯沙坦可抑制Ang II单独或联合ADR作用引起的细胞内ROS生成增多。(4)Western blot结果显示氯沙坦可抑制Ang II单独或联合ADR作用引起的GRP78/Bip表达上调、Akt活化、NF-κB核表达增加、AT1R表达增加。(5)RT-PCR结果显示氯沙坦可抑制Ang II单独或联合ADR作用引起的TGF-β1和CyclinD1 mRNA表达上调。
结论:
1.氯沙坦主要通过抑制ROS生成、GRP78/Bip表达增加、NFκB激活及促炎性细胞因子分泌增多、细胞外基质异常沉积,延缓或减轻阿霉素导致的大鼠肾损伤。此外氯沙坦还可影响肾内RAS关键成份表达发挥肾脏保护作用。
2.氯沙坦既能抑制Ang II促HRMC细胞增殖、有丝分裂活跃、致纤维化增强的作用,也可有效抑制Ang II和ADR联合刺激作用。该抑制作用主要通过降低ROS生成、下调GRP78/Bip表达、抑制Akt活化介导。氯沙坦可通过阻断AT1R配体依赖和配体非依赖的途径,逆转阿霉素肾病大鼠肾损伤。
Renin-angiotensin system(RAS) is important for the recirculation system and other organs. RAS was included ACE、Ang I、Ang II、ACE、ATR et. According to the traditional theory that RAS was one of the endocrine system, it was mainly adjusted blood pressure and water-electrolyte balance. Now people gradually recognized that RAS was extensively expressed in many kinds of tissues and organs, so it was distinguished into circulation RAS (cRAS) and tissue RAS(tRAS). Last years, the effect of RAS in partly of renal tissue was become to a warm spot. RAS was played very important effect on adjusting body fluid equilibrium and long-term accommodation with heart-blood system. RAS was also played influential effect of nephritis pathophysiology. All of the essential component of RAS, especially Ang II and AT1/AT2 were sure to be expressed in kidney. Ang II was one of the most important biologically active peptide for RAS, it depends on binding with ATR which located in cellular membrance to educe its biological effect. RAS in kidney was not only promoted pantosomatous effect of RAS, but also played very important effects in adjusting kidney function and nephronia. RAS was activated in many kinds of disease, such as hypertension、diabetes、glomerular nephritis(GN) and so on. Ang II was increased in cardiovascular disease and diabetes. In addition, Ang II was found to increased significantly instoma of kidney in pathologic status.
Losartan is one of competitive antagonist of AT1R, it was not only used to be treat with hypertension, but also be used to alleviated and to deteriortate diabetic nepropathy, it can help undepended-diabetic to heal renal disease、hypertension and others disease. In addition many clinical and animal researches evidence certify that long-term used high dose, even ultrahigh dose Losartan can further more postponed chronic nephritic, but the curative effect was not depended on haemodynamics. Even though Losartan had been confirmed to curative chronic kidney disease, but the exactly mechanism was not completely clear. Our research copy the model of ADR nephritis to study the mainly mechanism of Losartan deteriorate renal injury; Based on cultural human renal mesagial cell to study HRMC proliferation, fibrotic influence. To deep reseach the exactly effect of RAS and the mainly mechanism of AT1R antagon Losartan.
Objective:
1. Through copy the model of ADR nephritis to study the effect of RAS and Losartan in the development of nephritis; 2.Through the cell experiment, to study the effect of AngⅡand ADR for HRMC cells, further confirmed the protection mechanism of RAS in ADR nephritis.
Method:
1. The influence of Losartan on renal functional and pathological changes in ADR nephritis:(1)Choose animals、breeding and multiply;(2)Made ADR animal model,divided into groups and administration;(3)Convention fixed tissues with neutral-fomalin, routine paraffin imbedding and slicing, observed the changes of morphology by HE staining;(4)Used immunohistochemistry to detect the expression changes of GRP78/Bip;(5)Observe the changes of collagen deposit by Masson staining;(6)Used metabolic cage to collect 24h urine to detect urinary volume、urine protein and biochemical indicator;(7)Blood-serum biochemical indicator detection;(8)Detect the SOD and MDA levels by chemiluminescence method;(9)Detect AT1R、AT2R、AGT、ACE mRNA expression by RT-PCR;(10)Detect the expression of IL-1 in renal tissue by ELISA; (11) Datas were analysised by the SPSS 11.0 software.
2. The influence of fibrotic on Losartan when affected on AngⅡand/or ADR;(1)Cell culture;(2)Used MTT to detect the cell multiplication;(3)Used RT-PCR to detect the expression of TGF-β1 and CyclinD1; (4)Detect the expression of EGFR and NF-κB by western-blot;(5)Detect intracellel Ros by DCFH-DA staining;(6)Detective cell cycle by flow cytometry;(7) Used ELISA to detect the expression of type 1 collogen;(8) Used SPSS software to analysis the datas.
Results:
1. The influence of Losartan on renal functional and pathological changes when ADR nephritis:
(1)Losartan can significantly decreased urine protein that increase by ADR, so dose blood serum urea nitrogen.
(2)HE staining and Masson staining show that losartan can reduce mesangial cell proliferation and fibrous degeneration in ADR nephritis.
(3)Immunohistochemistry results show that GRP78/Bip expression was obviously decreased in ADR nephritis group.
(4) Losartan can reduce the expression level of IL-1 in ADR nephritis groups, it was possible reduce glomerulitis, to diminish injury of glomerulum.
(5)Losartan can reduce SOD and MDA that increased by ADR in kidney, it can protect kidney to avoid oxidation injury.
(6)Losartan can change some mainly gene of RAS system expression in kidney to avoid the injury of ADR.
2. The influence of fibrotic on Losartan when affected on AngⅡand/or ADR
(1)Cell proliferation was increased when it was affected by AngⅡ, 1×10-6mol/L AngⅡwas significant promote cells proliferation; Low dose ADR(0.2μg/ml)has no cytotoxicity for cells; every density losartan had no influence on cell proliferation, and all density losartan can suppressed HRMC cell proliferation; 5×10-5mol/L losartan was one of the most efficency dose. The inhibitory action of losartan was concentration dependent; The cell proliferation of combination AngII and low dose ADR for HRMC was stronger than AngII, and this kind of proliferation can also be inhibited by losartan.
(2)HRMC cell DNA was promoted to synthesis when it was affected by AngⅡor association with low dose ADR, cell caryocinesia activity, cell cycle changed. Losartan can utility suppressed the caryocinesia by AngⅡor association with ADR, the ratio of S+G2/M were obviously decreased, even lower than control groups.
(3)Many ROS were induced to produce when AngⅡaffect alone or association with ADR, losartan can suppressed this kind of effect, and make it close to nomal level.
(4) Ang II or/and associated with ADR can increase GRP78/Bip expression in HRMC cells, Losartan can suppressed this kind of effect.
(5) NFκB can be promote to come into nuclear by Ang II or/and associated with ADR, the effect of NFκB into nuclear induced by Ang I can be obviously suppressed by losartan.
(6) AngII and ADR can up-regulate the expression of TGF-β1、CyclinD1 mRNA in HRMC cell, collogen type I expression up regulation , AT1R protein expression increased, and losartan can inhibit these kinds of effects.
Conclusion:
1. ADR nephronia rats kidney show functional and structure lesion. Losartan can reduced and/or delayed the injury of kidney that cause of ADR. ADR induced ROS increased, activated NFκB and promoted phlegmasia cytokine to secrete increased, and to lead extracellular matrix to abnormality deposition, all of these kinds of effects can be inhibited by losartan. Endocytoplasmic reticulum stress was happened in ADR nephronia rats, GRP78/Bip expressed increase, the important component of kidney were changed. Through affected the endocytoplasmic reticulum stress and the level of Ang to delay the kidney injury.
2. Used Ang II alone or associated with lower dose ADR can stimulate DNA synthesis of HRMC cell. TGF-β1 expressed increased, cllogen secreted increased, ADR can enhance the promotion of proliferation by Ang II in HRMC cell. All of these kind of effects mainly caused by ROS product increase, to up regulate GRP78/Bip expression, Akt activation and activated some downstream signals, include Akt-mTOR pathway, TGF-β1 pathway, NF-κB pathway, all of these kind of pathway can affect cell proliferation, differentiated, inflammatory reaction and ECM synthesis. Losartan not only can inhibit the effect of cell proliferation by AngII, but also can utility inhibit the effect of association of AngII and ADR. This inhibition was mainly through decrease ROS production and down-regulate CRP78/Bip and inhibit Akt activation. Losartan can also retroconversion the renal injury by intercept the AT1R ligand dependent pathway and undependent pathway.
引文
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