摘要
肾缺血再灌注损伤(renal ischemia/ reperfusion injury , RI/ RI)是临床上常见病理生理现象,在缺血性急性肾损伤的病理过程中起重要作用,是多因素参与的复杂的病理过程。其中主要的损伤因素有自由基损伤、白细胞-内皮细胞相互作用、胞内钙超载、NO等舒血管物质减少、内皮素等缩血管物质产生过多等。
他汀类药物是三羟基三甲基戊二酰辅酶A (HMG-COA)还原酶抑制剂,通过对该酶特异性抑制作用,使HMG-CoA不能转变为甲羟戊酸(Mevalonate,MVA),从而阻断胆固醇的合成,降低血胆固醇的水平,这一作用已普遍用于临床治疗高脂血症。他汀类药物作用的多效性除降脂作用外,近年研究还发现,其对肾脏也有一定的保护功能。辛伐他汀及洛伐他汀可通过诱导培养人内皮细胞eNOS基因的转录激活,促进一氧化氮(NO)合成,而NO可扩张肾小球入球和出球动脉,降低肾脏血管阻力,使肾脏血浆流量和肾小球滤过率增加。此外也有报道,他汀类药物可减少烟酰胺腺嘌呤二核苷酸磷酸(Nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶亚基表达,增加抗氧化酶活性,发挥其细胞内抗氧化作用,从而减少氧自由基产生或直接清除机体产生的氧自由基,增强机体的抗氧化能力。
但他汀化学等方法观察辛伐他汀对RI/RI的作用,并探讨其可能的作用机制。类药物是否对缺血再灌注损伤肾脏有保护作用,目前少有报道。因此探讨他汀类药物对缺血再灌注损伤大鼠肾脏的保护作用及其作用机制,对RI/ RI的防治具有十分重要的意义。本实验旨在应用生理、生化、分子生物学及免疫组织
目的:观察辛伐他汀对RI/RI的作用,并探讨其可能的作用机制。方法:采用夹闭双侧肾动、静脉45分钟后松夹再灌的方法制作RI/RI模型,观察辛伐他汀对大鼠RI/RI损伤的影响。成年健康雄性Sprague-Dawley(SD)大鼠60只,体重150～180g,在18-22℃实验室安静饲养两日后动物随机分为5组,即:(1)假手术组(Sham) (2)缺血再灌注组(I/R) (3)缺血再灌注+辛伐他汀低剂量组5mg/kg/d(Sim-L) (4)缺血再灌注+辛伐他汀中剂量组20mg/kg/d(Sim-M) (5)缺血再灌注+辛伐他汀高剂量组40mg/kg/d(Sim-H)动物分组后,Sim-L组每日给予辛伐他汀5mg/kg灌胃,Sim-M组每日20mg/kg灌胃,Sim-H组每日40 mg/kg灌胃,持续2周。假手术组和缺血再灌组每日予等量生理盐水灌胃。
2周后复制动物模型。
动物经1%戊巴比妥钠腹腔麻醉,开腹,暴露双侧肾动、静脉。除假手术组只开腹不夹闭肾动、静脉外,其余各组均夹闭双侧肾动、静脉45分钟,然后去夹再灌。再灌后逐层缝合腹膜,腹壁肌肉和皮肤。分别于再灌后6小时和24小时分批取血及双侧肾脏测定血清肌酐(Scr)、尿素氮(BUN)、肾组织丙二醛(MDA)、一氧化氮(NO)、诱生型一氧化氮合酶(iNOS)的含量及超氧化物歧化酶(SOD)的活性。免疫组化法观察肾组织内皮型一氧化氮合酶(eNOS)、P47phox的表达。Western blot法检测肾组织eNOS和P47phox蛋白表达水平。
结果:
1.大鼠肾脏缺血再灌注损伤后,光镜所见:Sham组肾组织无明显异常;I/R组肾组织损伤随缺血再灌时间延长逐渐加重,表现为肾小管上皮细胞混浊肿胀、坏死、管腔明显扩张,管内可见管型和脱落细胞、间质水肿、核染色质边集以及明显的胞浆空泡样变性。Sim-M和Sim-H组各时点肾组织损伤程度较I/R组明显减轻,Sim-L组各时点肾组织损伤程度与I/R组相似。
2.大鼠肾脏缺血再灌注损伤后,I/R组肾功能明显下降,BUN、Scr均明显高于Sham组(P<0.01),且随缺血时间延长呈加重趋势;与I/R组相比,Sim-L、Sim-M和Sim-H组各时点BUN、Scr均显著降低(P<0.05～0.01)。
3.大鼠肾脏缺血再灌注损伤后,I/R组各时点SOD活力较Sham组显著降低(P<0.01),MDA含量显著升高(P<0.01);与I/R组相比,Sim-M组和Sim-H组各时点SOD活力均明显增加(P<0.01), MDA含量均显著下降(P<0.01),Sim-L组的各项指标与I/R组相比没有明显的变化。
4.大鼠肾脏缺血再灌注损伤后,I/R组各时点NO及iNOS含量均明显高于Sham组(P<0.01);与I/R组相比,Sim-M组和Sim-H组各时点NO含量显著增加(P<0.01),Sim-M组和Sim-H组iNOS含量降低(P<0.01);Sham组肾皮质eNOS无明显表达,I/R组肾皮质eNOS表达均较Sham组增加,与I/R组相比,Sim-M组和Sim-H组各时点eNOS表达显著增加; Western blot法检测肾组织eNOS蛋白含量,I/R组肾皮质eNOS蛋白含量较Sham组增加,与I/R组相比,Sim-M组和Sim-H组各时点eNOS蛋白含量均明显增加,Sim-L组与I/R组相比没有明显的变化。
5.大鼠肾脏缺血再灌注损伤后,肾皮质P47phox表达较Sham组明显增加,Western blot法检测肾组织P47phox蛋白含量, I/R组肾皮质P47phox蛋白含量较Sham组明显增加;与I/R组相比,Sim-M组和Sim-H组各时点P47phox表达及蛋白含量均明显降低。Sim-L组与I/R组相比无明显变化。
结论:
1. RI/RI后,肾功能明显受损。肾小管上皮细胞肿胀、坏死、间质水肿。RI/RI大鼠BUN、Scr、MDA和iNOS含量均较sham组明显增加,SOD活力及eNOS表达均较sham组明显降低,肾皮质P47phox免疫反应阳性颗粒和蛋白表达均明显增加。表明P47Phox参与了肾缺血/再灌注损伤,并在其中发挥了重要作用。
2.辛伐他汀可显著降低RI/RI大鼠BUN、Scr和MDA含量,提高肾组织SOD活力。降低肾组织中iNOS含量,增加eNOS免疫反应阳性颗粒和蛋白的表达,增加NO含量。表明辛伐他汀可减轻RI/RI大鼠肾组织损伤,改善肾功能。
3.辛伐他汀能明显减少缺血再灌损伤大鼠肾皮质P47phox免疫反应阳性颗粒和蛋白的表达,减轻肾缺血再灌注损伤。表明辛伐他汀减轻RI/RI大鼠肾组织损伤的机制可能与其降低p47phox的表达水平、干扰NAD(P)H氧化酶生成通路的某些环节、抗自由基损伤有关。
Renal ischemia-reperfusion injury (RI/RI) is acommon pathophysiologic phenomenon,and it is a complex and important patho- process with many factors in acute renal ischemia injury. The main damage factors are free radical damage, leukocyte-endothelial cell interaction, intracellular calcium overload, NO and other vasodilator substances decrease, Endothelin and other vasoconstrictor substances increase.
Statins, a group of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are widely used in clinical practice for theI/R efficacy in producing significant reductions in plasma cholesterol and LDL cholesterol via inhibited mevalonic acid (MVA) pathway of cholesterol synthesis. A number of recent reports have shown that statins may also have important kidney protection effects, in addition to theI/R lipid-lowering effects on plasma lipids. Simvastatin and lovastatin could induce eNOS gene transcription in the cultured human endothelial cells, promote NO synthesis. NO can dilate efferent and afferent glomerular arterioles, lower renal vascular resistance while increase renal plasma flow and glomerular filtration rate. The molecular basis of the observed effects of statins may relate to theI/R ability block the production and/or activity of ROS. The inhibition of ROS generation by statins, through interference with NAD(P)H oxidase expression and activity, and the actions of statins that serve to blunt the damaging effects of these radicals, including effects on antioxidant enzymes, lipid peroxidation and nitric oxide synthase. These antioxidant effects of statins likely contribute to theI/R clinical efficacy in treating cardiovascular disease as well as other chronic conditions associated with increased oxidative stress in humans.
However, whether statins has a protective effect on renal ischemia-reperfusion injury is still unknown. The present study observed the protective effects and explored mechanisms of Simvastatin on renal ischemia-reperfusion injury through use of physiological, biochemical, molecular biological and immunohistological methods.
Objective: This study aimed to investigate the role of simvastatin on RI/RI and to explore its possible mechanism.
Method: The model of RI/RI was induced by bilateral clamping the renal artery and vein for 45 minutes followed by reperfusion and observing the effect of simvastatin on RI/RI in rats. After two days breeding in 18-22℃cI/Rcumstance, Sixty male Sprague-Dawley rats weighing between 150g to 180g were divided into five groups randomly (n=12 on each group): (1) sham-operation group (Sham); (2) ischemia-reperfusion group (I/R); (3) low-dose Simvastatin group (Sim-L, 5mg/kg/d); (4) middle-dose Simvastatin (Sim-M, 20mg/kg/d); (5) high-dose simvastatin group (Sim-H , 40mg/kg/d).
Sim-L, M and H group rats were started on oral Simvastatin 50, 20 and 40 mg/kg/d treatment respectively for 2 weeks. Sham and I/R group rats were given the same doses solution in the same way. The model of RI/RI was made after two weeks later.
The animals were anesthetized with 1% pentobarbital sodium,the bilateral renal artery and vein were clamped for 45 minutes followed by reflow in I/R, Sim-L, M and H group rats. After reperfusion, each layer opened was closed with suture, including the peritoneum, abdominal wall muscle and skin. The rats in the sham group were treated identically except for the clamping. After 6 and 24 hours of reperfusion, the blood samples were taken for detecting contents of serum creatinine (Scr), serum urea nitrogen (BUN). After blood was taken, both side of kidney were excised for observing renal histological examination, content of Nitric Oxide (NO), activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), inducible nitric oxide synthase (iNOS), the expression of endothelial nitric oxide synthase (eNOS) and P47phox.
Results:
1. After RI/RI, the renal tubule epithelial cells showed signs of damage in I/R group rats, especially proximal convoluted tubule in I/R group rats, in which the lumen of tubule was enlarged; also there were some cast in many of the renal tubules. Chromatin was localized in the cell nucleus periphery. The degree of renal tissue injury significantly mitigate in Sim-M and Sim-H group rats compared with I/R group rats at different time point, but no any improvement of renal tissue injury in Sim-L group rats compared with I/R group rats.
2. After RI/RI, the contents of Scr and BUN were significantly increased in I/R group rats than that of sham group rats at different time point (P﹤0.01); Compared with the I/R group rats, contents of Scr and BUN were significantly lower in Sim-L , Sim-M and Sim-H group rats at different time point (P﹤0.05～0.01).
3. After RI/RI, activities of SOD were significantly decreased (P﹤0.01) and the contents of MDA were significantly increased (P﹤0.01) in I/R group rats at different time point; Compared with I/R group rats, the activities of SOD were significantly increased (P﹤0.01) and the contents of MDA were significantly decreased (P﹤0.01) in Sim-L and Sim-H group rats, but not in Sim-L group rats.
4. After RI/RI, NO and iNOS were significantly increased in I/R group rats at different time point (P <0.01); compared with the I/R group rats, NO contents were significantly increased (P <0.01) and iNOS were decreased (P <0.01) in Sim-M and Sim-H group rats; there were no significant eNOS expression of renal cortex in Sham group rats, Positive immunoreactive particles of eNOS in renal proximal convoluted tubular epithelial cells were significantly increased in I/R group rats. Compared with I/R group rats, Sim-M and Sim-H group rats were showed more positive immunoreactive particles expression; eNOS protein expression were up-regulated in I/R group rats than that of Sham group rats, Compared with the I/R group, eNOS protein expression were significantly increased in Sim-M and Sim-H group rats, but not in Sim-L group rats.
5. The expression of positive immunoreactive particles and protein of P47 phox were increased in I/R group rats than that of in Sham group rats. Compared with I/R group rats, both of positive immunoreactive particles and protein expression of P47phox were decreased in Sim-M and Sim-H group rats, but not in Sim-L group rats.
Conclusions:
1. After RI/RI, the renal function was markedly impaI/Red and renal tubule epithelial cells showed signs of damage. It was found that Scr,MDA and iNOS were increased, the activity of SOD and expression of eNOS were decreased in RI/RI rats. The positive immunoreactive particles and protein of P47phox in renal proximal convoluted tubular epithelial cells were markedly increased in RI/RI rats. These results indicated that P47phox is involved in the pathogenesis of RI/RI in rats.
2. Simvastatin could decreased the Scr, BUN, MDA and iNOS, increased the activity of SOD and content of NO, upregulated the expression of eNOS in the renal tissue. It is indicated that Simvastatin could reduced renal tissue injury and improved renal function in RI/RI rats.
3. Simvastatin could down-regulated the expression of P47phox of renal tissue in RI/RI rats and mitigated renal ischemia-repufution injury. It is indicated that the protective effects of Simvastatin to the RI/RI may be related to block the NAD (P) H oxidate pathway and anti-free radical damage.
引文
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