特异性环氧合酶-2抑制剂对实验性梗阻性肾病的保护作用
|
摘要
【背景和目的】肾小管间质性肾炎是仅次于肾小球疾病的第二大类肾脏病,如果病情不能控制,最终将导致肾间质纤维化。在终末期肾衰中约15%～25%的患者是由肾间质纤维化所致,临床上梗阻性肾病是导致肾间质损伤较常见的原因。非甾体类抗炎药(Nonsteroidal anti-inflammatory drugs,NSAIDs)在我国是仅次于抗感染药物的第二大类药物,但它的不良反应限制了它的应用。尤其是对于长期大剂量服药的患者更是如此。人们在寻找不良反应少而疗效更高的NSAIDs的工作中,发现了环氧合酶(Cyclooxygenase,COX)存在两种亚型,即COX-1和COX-2。COX是前列腺素代谢环节中的关键限速酶,通常认为COX-1催化生成的前列腺素主要调节正常组织细胞的生理活动,维持自身稳定,而COX-2作为酶的诱导形式,其表达可以在多种生长因子的作用下急剧增加,主要参与炎症、肿瘤等病理过程。COX-2的发现使人们重新认识了COX的生理及病理作用,提示NSAIDs的治疗作用与不良反应是可以分离的。目前已开发出多种选择性COX-2抑制剂,临床上主要用于治疗风湿性关节炎和骨关节炎。然而,国内外有关选择性COX-2抑制剂对各种肾脏疾病影响的研究报道较少,尤其COX-2与肾小管间质纤维化的相关性研究报道更为罕见。单侧输尿管梗阻(Unilateral ureteral obstruction,UUO)是诱导肾小管间质损伤的常用方法。研究证实,单侧输尿管梗阻后,大量炎性细胞浸润至小管间质,释放转化生长因子—β_1(Transforming
郑州大学2004年硕士毕业论文特异性环氧合酶一2抑制剂对实验性梗阻性肾病的呆护作用
gowth faCtor- pl,TGF一pl)、白介素一l(IL一1)、白介素一6(IL一6)、成纤维细
胞生长因子(FGF)、肿瘤坏死因子(TNF)和血小板起源的生长因子(PDGF)及NO等
活性因子。这些活性因子在UUO发生发展的病理过程中处于十分关键的地位。
研究表明UUO状态下COX一2的基因表达及蛋白合成显著上调,病理性前列腺素生
成增多,证实COX一2参与介导了UUO发生、发展的病理过程,为选择性Cox一2
抑制剂的应用提供了理论依据。近年来,国内外研究结果显示选择性COX一2抑制
剂能有效延缓UUO大鼠的肾小管间质纤维化的进程。有学者认为单侧输尿管梗阻
大鼠的肾脏组织中COX一2的过度表达,可能是肾内炎性细胞浸润和积聚的起始或
促进因素之一。UUO所引发的肾小管间质纤维化的致病过程中存在着刺激COX一2
大量生成的多种因素。传统非幽体类消炎药的较大副作用是导致慢性肾小管间质
性肾炎,此现象可能与其非特异性阻断COX一1而导致肾脏前列腺素的合成障碍、
收缩肾乳头及髓质区血管并最终导致缺血性坏死有关。因此理论上选择性阻断
COX一2的表达在纠正疾病的同时可能并不影响机体的生理状态,从而避免了传统
非幽体类抗炎药物的不良反应。本实验拟采用UUO大鼠模型进行研究,观察
UUO术后2周肾组织中COX一2、NO及TGF一p;的改变,探讨三者在UUO
发生发展中所起的作用,同时应用特异性COX一2抑制剂罗非昔布(万络),观
察其对COX一2、NO以及TGF一p:的影响,了解其肾呆护机制。
[方法124只Wistar大鼠随机分成对照组(C组)、UUO手术组(U组)及万络
治疗组(R组),每组8只,U组和R组行右侧输尿管结扎术。术前24h及术后以
灌胃方式给药,R组给予万络(IOmg/kg/d),C组和U组给予等量生理盐水,每
日一次。术后2周收获动物。24小时尿标本进行尿蛋白定量,放射免疫法测定
尿TXBZ浓度。血标本检测血肌配。肾脏组织进行石蜡切片、HE和MASSON染色、
免疫组织化学(S一法)检测TGF一p,及COX一2的蛋白表达以及硝酸还原酶法测
定肾皮质组织中NO含量。
【结果】UUO术后2周,(1)U组、C组及R组大鼠血压分别为107.60士1.83、
106.80士3.09及106.70士2.94,U组与C组、R组比较均无显著性差异(乃0. 05);
24小时尿蛋白定量分别为0.159士0.03、0.155士0.03及0.148士0.01,U组与C
组、R组比较亦无显著性差异(乃0.05):血肌配分别为25.1士2.48、22.3士2.n
及23.2士2.23,U组与C组、R组比较亦无显著性差异(乃0.05):U组、R组与
郑州大学2004年硕l:毕业论文特异性环氧合酶一2抑制剂对实验性梗阳洲肾病的保护作用
C组比较,尿TxB:浓度变化均有显著性差异(只0.01),R组与U组比较,亦有显
著性差异(只0.01)。(2)UUO术后2周,U组大鼠的右肾肿大,颜色变浅,剖面
示右肾呈缺血状,以肾皮质为重,’肾孟、‘肾瀚明显扩张,充满尿液。’肾实质变薄,
皮髓质分界不清。经Ma 5 Son染色后与C组比较,U组肾间质明显增宽,伴有大
量炎症细胞浸润,明显间质纤维化。与U组比较,万络可显著地改善UUO时的病
理改变,炎症细胞减少,间质纤维化明显减轩。(3)U组、R组与C组比较,肾
皮质匀浆N。:一/N公一含量的变化均有显著性差异(只0.01),其中U组升高尤为明
显:R组与U组比较,亦有显著性变化(只0.01)。表明应用特异性COX一2抑制
剂可以降低NO的合成。(4)U组、R组与C组比较,’肾小管间质TGF一日,的表达
均有显著性差异(只0.01),R组与U组比较,亦有显著性差异(只0.01)。U组、
R组与C组比较,’肾小管间质COX一2的表达均有显著性差异(只0.01),其中U
组升高尤为明显,R组与U组比较,亦有显著性差异(只0.01)。提示COX一2参
与了UUO发生、发展的病理过程。
【结论】本实验证实特?
[Background and Objective Chronic interstitial nephritis is the second kind of kidney disease that is only next to glomerulopathy, if conditions can not be controled , eventually tubulointerstitial fibrosis will appear. In the patients of end-stage ranal stage, about 15%-25% are caused by tubulointerstitial fibrosis. Obstructive nephropathy is the common reason in the clinical diseases of renal stroma damage. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the second kind of medicine that is only next to anti-infective all over the world, but it's side effects have restricted it's application, especially to the patients taking medicine of big dosage for a long period. During the work of seeking NSAIDs with higher curative effect and less side effect, two inferior types of COX had been discovered. Cyc100xygenase(COX) is the key limited enzyme in the metabolism process of prostaglandin, About ten years ago, two COX isoforms(COX-l and-2) were demonstrated: COX-1 expresses stably and COX-2 is expressed by a n
umber of inflammatory stimuli. COX-1 products are involved in regulating physiological functions to maintein the stability of milieu interieur whereas COX-2 products play a pathologic role in many tissues and organs ( such as inflammatory reactions, tumor,et). The discovery of COX-2 make people recognize the physiological and pathologic role of COX again, this indicates the therapy results of NSAIDs can separate from it's side effects. Many selective inhibitors of COX-2 are now available, they are often used to cure rheumatic arthritis ,
ostearthritis and other diseases. However domestic and international researches of selective or specific COX-2 inhibitor about the influences on various kidney disease are scarce, especially the reportes about the correlation of COX-2 and tubulo interstitial fibrosis. Unilateral ureteral obstruction(UUO) is the frequently used method to induce the damage of the renal stroma. It is confirmed that a lot of inflammatory cells infiltrate to tubulointerstitium. The expression of many factors are higher than usual, such as TGF-B1.IL-1, IL-6, FGF, TNF, PDGF, NO. These factors have important role in the occurrence and development of UUO. COX-2 also take part in this pathologic process because many tests confirm that the expression of COX-2 are significant higher in UUO. Domestic and international researches all indicate that selective COX-2 inhibitors can alleviate tubulointerstitial fibrosis of UUO rats. Selective COX-2 inhibitor might exert its salutary effects through down-regulation of TGF-B1. Some scholars repo
rted that the overexpression of COX-2 in the renal cortex of UUO perhaps is the reason of the infiltration and accumulation of inflammatory cells. There are a lot of factors can improve the expression of COX-2 in the pathologic process of tubulointerstitial fibrosis induced by UUO. It is well known that traditional NSAIDs have many bad side effects. One of them is Chronic interstitial nephritis. It might correlate with the blockade of COX-1. In conclusion. Theoretically blocking COX-2 selectively to retify disease does not affect the physiological state of organism at the same time avoid the side effects of traditional nonsteroidal anti-inflammatory drugs. In the present study COX-2, NO and TGF-B1 in the kidney tissue were measured at the 2th week to investigate their effects in the pathogenesis of UUO. Meanwhile rofecoxib was used in the UUO rat model to explore the mechanisms of its renal protective effects on UUO by observing its influence on COX-2, NO and TGF-B1.
[ Methods ] 24 Wistar rats were randomly assigned to following groups: sham-operation group(C) unilateral ureteral obstruction(UUO) group(U) rofecoxib treatment group(R). Each group has 8 rats. A day before operation and after operation, R were treated with rofecoxib(10mg/kg/d) . whereas C and U with same volume N.S. At the 2th week after operation, biochemistry tests of b100d and urine were performed
to measure the quantity of 24h urinary protein and the concentration of urinary TXB2. Kidneys were
郑州大学2004年硕士毕业论文特异性环氧合酶一2抑制剂对实验性梗阻性肾病的呆护作用
gowth faCtor- pl,TGF一pl)、白介素一l(IL一1)、白介素一6(IL一6)、成纤维细
胞生长因子(FGF)、肿瘤坏死因子(TNF)和血小板起源的生长因子(PDGF)及NO等
活性因子。这些活性因子在UUO发生发展的病理过程中处于十分关键的地位。
研究表明UUO状态下COX一2的基因表达及蛋白合成显著上调,病理性前列腺素生
成增多,证实COX一2参与介导了UUO发生、发展的病理过程,为选择性Cox一2
抑制剂的应用提供了理论依据。近年来,国内外研究结果显示选择性COX一2抑制
剂能有效延缓UUO大鼠的肾小管间质纤维化的进程。有学者认为单侧输尿管梗阻
大鼠的肾脏组织中COX一2的过度表达,可能是肾内炎性细胞浸润和积聚的起始或
促进因素之一。UUO所引发的肾小管间质纤维化的致病过程中存在着刺激COX一2
大量生成的多种因素。传统非幽体类消炎药的较大副作用是导致慢性肾小管间质
性肾炎,此现象可能与其非特异性阻断COX一1而导致肾脏前列腺素的合成障碍、
收缩肾乳头及髓质区血管并最终导致缺血性坏死有关。因此理论上选择性阻断
COX一2的表达在纠正疾病的同时可能并不影响机体的生理状态,从而避免了传统
非幽体类抗炎药物的不良反应。本实验拟采用UUO大鼠模型进行研究,观察
UUO术后2周肾组织中COX一2、NO及TGF一p;的改变,探讨三者在UUO
发生发展中所起的作用,同时应用特异性COX一2抑制剂罗非昔布(万络),观
察其对COX一2、NO以及TGF一p:的影响,了解其肾呆护机制。
[方法124只Wistar大鼠随机分成对照组(C组)、UUO手术组(U组)及万络
治疗组(R组),每组8只,U组和R组行右侧输尿管结扎术。术前24h及术后以
灌胃方式给药,R组给予万络(IOmg/kg/d),C组和U组给予等量生理盐水,每
日一次。术后2周收获动物。24小时尿标本进行尿蛋白定量,放射免疫法测定
尿TXBZ浓度。血标本检测血肌配。肾脏组织进行石蜡切片、HE和MASSON染色、
免疫组织化学(S一法)检测TGF一p,及COX一2的蛋白表达以及硝酸还原酶法测
定肾皮质组织中NO含量。
【结果】UUO术后2周,(1)U组、C组及R组大鼠血压分别为107.60士1.83、
106.80士3.09及106.70士2.94,U组与C组、R组比较均无显著性差异(乃0. 05);
24小时尿蛋白定量分别为0.159士0.03、0.155士0.03及0.148士0.01,U组与C
组、R组比较亦无显著性差异(乃0.05):血肌配分别为25.1士2.48、22.3士2.n
及23.2士2.23,U组与C组、R组比较亦无显著性差异(乃0.05):U组、R组与
郑州大学2004年硕l:毕业论文特异性环氧合酶一2抑制剂对实验性梗阳洲肾病的保护作用
C组比较,尿TxB:浓度变化均有显著性差异(只0.01),R组与U组比较,亦有显
著性差异(只0.01)。(2)UUO术后2周,U组大鼠的右肾肿大,颜色变浅,剖面
示右肾呈缺血状,以肾皮质为重,’肾孟、‘肾瀚明显扩张,充满尿液。’肾实质变薄,
皮髓质分界不清。经Ma 5 Son染色后与C组比较,U组肾间质明显增宽,伴有大
量炎症细胞浸润,明显间质纤维化。与U组比较,万络可显著地改善UUO时的病
理改变,炎症细胞减少,间质纤维化明显减轩。(3)U组、R组与C组比较,肾
皮质匀浆N。:一/N公一含量的变化均有显著性差异(只0.01),其中U组升高尤为明
显:R组与U组比较,亦有显著性变化(只0.01)。表明应用特异性COX一2抑制
剂可以降低NO的合成。(4)U组、R组与C组比较,’肾小管间质TGF一日,的表达
均有显著性差异(只0.01),R组与U组比较,亦有显著性差异(只0.01)。U组、
R组与C组比较,’肾小管间质COX一2的表达均有显著性差异(只0.01),其中U
组升高尤为明显,R组与U组比较,亦有显著性差异(只0.01)。提示COX一2参
与了UUO发生、发展的病理过程。
【结论】本实验证实特?
[Background and Objective Chronic interstitial nephritis is the second kind of kidney disease that is only next to glomerulopathy, if conditions can not be controled , eventually tubulointerstitial fibrosis will appear. In the patients of end-stage ranal stage, about 15%-25% are caused by tubulointerstitial fibrosis. Obstructive nephropathy is the common reason in the clinical diseases of renal stroma damage. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the second kind of medicine that is only next to anti-infective all over the world, but it's side effects have restricted it's application, especially to the patients taking medicine of big dosage for a long period. During the work of seeking NSAIDs with higher curative effect and less side effect, two inferior types of COX had been discovered. Cyc100xygenase(COX) is the key limited enzyme in the metabolism process of prostaglandin, About ten years ago, two COX isoforms(COX-l and-2) were demonstrated: COX-1 expresses stably and COX-2 is expressed by a n
umber of inflammatory stimuli. COX-1 products are involved in regulating physiological functions to maintein the stability of milieu interieur whereas COX-2 products play a pathologic role in many tissues and organs ( such as inflammatory reactions, tumor,et). The discovery of COX-2 make people recognize the physiological and pathologic role of COX again, this indicates the therapy results of NSAIDs can separate from it's side effects. Many selective inhibitors of COX-2 are now available, they are often used to cure rheumatic arthritis ,
ostearthritis and other diseases. However domestic and international researches of selective or specific COX-2 inhibitor about the influences on various kidney disease are scarce, especially the reportes about the correlation of COX-2 and tubulo interstitial fibrosis. Unilateral ureteral obstruction(UUO) is the frequently used method to induce the damage of the renal stroma. It is confirmed that a lot of inflammatory cells infiltrate to tubulointerstitium. The expression of many factors are higher than usual, such as TGF-B1.IL-1, IL-6, FGF, TNF, PDGF, NO. These factors have important role in the occurrence and development of UUO. COX-2 also take part in this pathologic process because many tests confirm that the expression of COX-2 are significant higher in UUO. Domestic and international researches all indicate that selective COX-2 inhibitors can alleviate tubulointerstitial fibrosis of UUO rats. Selective COX-2 inhibitor might exert its salutary effects through down-regulation of TGF-B1. Some scholars repo
rted that the overexpression of COX-2 in the renal cortex of UUO perhaps is the reason of the infiltration and accumulation of inflammatory cells. There are a lot of factors can improve the expression of COX-2 in the pathologic process of tubulointerstitial fibrosis induced by UUO. It is well known that traditional NSAIDs have many bad side effects. One of them is Chronic interstitial nephritis. It might correlate with the blockade of COX-1. In conclusion. Theoretically blocking COX-2 selectively to retify disease does not affect the physiological state of organism at the same time avoid the side effects of traditional nonsteroidal anti-inflammatory drugs. In the present study COX-2, NO and TGF-B1 in the kidney tissue were measured at the 2th week to investigate their effects in the pathogenesis of UUO. Meanwhile rofecoxib was used in the UUO rat model to explore the mechanisms of its renal protective effects on UUO by observing its influence on COX-2, NO and TGF-B1.
[ Methods ] 24 Wistar rats were randomly assigned to following groups: sham-operation group(C) unilateral ureteral obstruction(UUO) group(U) rofecoxib treatment group(R). Each group has 8 rats. A day before operation and after operation, R were treated with rofecoxib(10mg/kg/d) . whereas C and U with same volume N.S. At the 2th week after operation, biochemistry tests of b100d and urine were performed
to measure the quantity of 24h urinary protein and the concentration of urinary TXB2. Kidneys were
引文