【作者】 张耀全；

【导师】 袁发焕；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 背景与目的:心血管疾病是终末期肾病(end stage renal disease, ESRD)患者最常见的并发症和最主要的死亡原因。其中尿毒症动脉粥样硬化(atherosclerosis, AS)倍受关注。慢性肾衰竭患者发生AS与普通人群不同,具有发病率高、发病年龄早、病变累及广泛等自身特点,其发病机制尚不十分清楚。传统的危险因素如:高血压、糖尿病、血脂代谢紊乱等虽在CRF病人中也较为普遍,但并不能满意解释慢性肾衰竭(chronic renal failure, CRF)病人心血管疾病如此高的发病率和死亡率,提示慢性肾衰竭患者体内可能有其它因素加速了动脉粥样硬化的发生发展。已明确AS实质是血管壁细胞的慢性损伤性炎症反应,但目前仍缺乏有效的防治手段。ESRD患者具有严重的内环境紊乱,体内存在“微炎症状态”,这种微炎症状态可能和其动脉血管细胞慢性损伤性炎症反应有关,加速了动脉粥样硬化的进展。核因子-κB(NF-κB)是一种具有多向调控作用的转录因子,存在于多种不同的细胞类型中,处于炎症反应网络的中心位置,研究其生物学特性并通过药理学作用适当地进行调控,对于防治组织细胞炎症反应的发生发展具有重要的临床意义。近来的研究发现,泛素-蛋白酶体途径(ubiquitin-proteasome pathway, UPP)是一种广泛存在于真核细胞胞浆中非常重要的、依赖ATP的非溶酶体蛋白降解通路,可从多个环节参与调控NF-κB信号通路。且UPP组分具有明确的组织特异性,为靶向性地针对血管细胞NF-κB活化提供了可能。本课题通过建立慢性肾衰兔模型和体外细胞培养两层次观察UPP—NF-κB信号组份在慢性肾衰加速性动脉粥样硬化发生发展过程中的表达和活化规律,分析阻断泛素-蛋白酶体信号途径对慢性肾衰主动脉血管细胞炎症病理变化的影响,为临床有效防治慢性肾衰加速性动脉粥样硬化提供理论依据。方法:1.采用结扎左肾动脉分支并切除右肾的方法建立慢性肾衰竭兔模型,将动物随机分为假手术组(Sham)、慢性肾衰组(CRF)、假手术+MG132组(Sham +MG132)、慢性肾衰+MG132组(CRF+MG132)、假手术组+PDTC组(Sham+PDTC)、慢性肾衰+PDTC组(CRF+PDTC)。各组设术后4w、8w、12w三时相点,动态观察血压、肾功能、血脂、肾脏及主动脉病理变化情况。2.采用慢性肾衰模型12 w兔血清刺激体外培养的兔主动脉内皮细胞和平滑肌细胞,MTT、流式细胞仪及Hoechst33342染色检测内皮细胞和平滑肌细胞的形态增殖、凋亡的变化。并采用MG132和PDTC进行干预,观察其对细胞增殖的量效和时效情况。3.免疫组化检测主动脉中增殖细胞核抗原(PCNA)的表达变化。4.应用RT-PCR方法检测各组兔主动脉及体外培养细胞泛素(Ub)、泛素激活酶(E1)、NF-κBp65及IκBαmRNA表达变化.5.Western blot检测主动脉及体外培养细胞泛素化蛋白、E1、NF-κB、IκBα、PCNA及TNFα蛋白表达变化。6.免疫荧光检测培养细胞NF-κBp65核转位情况。7.EMSA检测NF-κB—DNA结合活性。8.特异性荧光底物反应检测蛋白酶体活性20s亚基活性。9.ELISA方法检测各组兔血清和细胞培养上清中炎症因子IL-6、TNFα浓度。结果:1.结扎新西兰白兔左肾动脉分支并切除右肾的方法建立了慢性肾衰竭动脉粥样硬化的模型:模型组兔术后出现明显的蛋白尿和血尿,并逐渐出现贫血;出现明显的高血压;血清BUN、SCr逐渐升高;肾脏病理出现明显的肾小球硬化和肾间质纤维化。兔主动脉逐渐出现脂纹、粥样斑块等动脉粥样硬化早中期的变化。使用MG132和PDTC干预后能明显减轻主动脉动脉粥样硬化的病理变化。2.慢性肾衰竭兔主动脉NF-κB信号通路组分表达及MG132和PDTC干预的效果:与Sham相比,NF-κBmRNA和蛋白水平表达均增加,NF-κB-DNA结合活性明显增强,随病程延长更为明显。其抑制因子IκBαmRNA和蛋白水平表达和NF-κB呈现相反的变化。MG132干预后能使NF-κBmRNA和蛋白水平表达降低;NF-κB-DNA结合活性明显下降;IκBαmRNA和蛋白水平表达上调。PDTC干预后和MG132作用类似,证实慢性肾衰竭兔主动脉内存在NF-κB信号通路的活化。3.慢性肾衰竭兔主动脉泛素-蛋白酶体信号通路组分表达及MG132和PDTC的干预作用:与Sham组相比,慢性肾衰竭兔主动脉中Ub mRNA表达逐渐上调、泛素化蛋白表达未见明显变化;E1 mRNA和蛋白表达逐渐增加;蛋白酶体活性明显增强,且随病程进展更为明显。MG132干预后,Ub mRNA表达下调,泛素化蛋白表达增加,E1 mRNA和蛋白表达下降;蛋白酶体活性下降。证实慢性肾衰竭兔主动脉内存在泛素蛋白酶体通路的活化。与MG132相比,PDTC干预后对Ub mRNA和E1mRNA和蛋白表达与MG132相仿,但对蛋白酶体活性影响较小,对泛素化蛋白表达无影响。4.慢性肾衰兔血清中的炎症因子TNFα、IL-6浓度和兔主动脉中TNFα的表达变化及MG132和PDTC的干预作用:与对照组相比,血清中的炎症因子TNFα、IL-6浓度逐渐升高,主动脉中TNFα的表达逐渐增加。MG132和PDTC干预后均能降低血清中的炎症因子TNFα、IL-6浓度和主动脉中TNFα的表达。证实慢性肾衰竭兔存在全身和主动脉局部的微炎症反应。5.慢性肾衰血清在较低浓度时(3%~10%)时呈浓度和时间依赖性促进内皮细胞和平滑肌细胞的增殖,较高浓度时(>10%),促增殖作用减弱,促细胞凋亡增强。MG132和PDTC均能抑制10%慢性肾衰血清诱导的内皮细胞和平滑肌细胞增殖。6.与正常血清相比,10%慢性肾衰血清能使内皮细胞和平滑肌细胞NF-κB mRNA和蛋白表达增加,IκBαmRNA和蛋白表达降低;10%慢性肾衰血清能使NF-κBp65发生核转位;不同浓度慢性肾衰血清(3%~10%)使内皮细胞和平滑肌细胞NF-κB DNA结合活性呈浓度依赖性增加,10%慢性肾衰血清使内皮细胞和平滑肌细胞NF-κB DNA结合活性24h持续增加。MG132和PDTC干预后,能抑制10%慢性肾衰血清刺激的内皮细胞和平滑肌细胞NF-κBmRNA和蛋白的表达,增加IκBαmRNA和蛋白表达,抑制NF-κB DNA结合活性。7.10%慢性肾衰血清使主动脉内皮细胞和平滑肌细胞培养上清IL-6和TNFα含量明显增加,刺激6小时后升高幅度最为明显,以后仍缓慢上升。同时内皮细胞和平滑肌细胞内TNFα蛋白表达也较对照明显增加。MG132和PDTC干预后,均能降低细胞炎症因子TNFα、IL-6的分泌和主动脉中TNFα的表达。8.10%慢性肾衰血清刺激后,主动脉内皮细胞和平滑肌细胞Ub和E1mRNA的表达上调,E1蛋白的表达增加,蛋白酶体活性较对照组明显升高。MG132干预后,能抑制主动脉内皮细胞和平滑肌细胞Ub和E1mRNA的表达,降低E1蛋白的表达,抑制蛋白酶体活性。与MG132相比,PDTC干预后对细胞Ub mRNA和E1mRNA和蛋白表达与MG132相仿,但对蛋白酶体活性影响较小。结论:1、慢性肾衰竭兔随病程的进展主动脉逐渐出现动脉粥样硬化的病理改变。2、慢性肾衰兔主动脉内存在NF-κB信号通路的活化和炎症细胞因子的表达,并随慢性肾衰病程进展呈现出加重的趋势。3、慢性肾衰兔主动脉内存在泛素-蛋白酶体信号通路的活化,阻断泛素-蛋白酶体途径可显著抑制慢性肾衰兔主动脉NF-κB信号通路的活化和炎症细胞因子的表达;明显减轻兔主动脉粥样硬化的病理改变。4、较低浓度的慢性肾衰兔血清(≤10%)能促进兔主动脉内皮细胞和平滑肌细胞异常增殖,呈一定浓度和时间依赖性;而高浓度的慢性肾衰兔血清对兔主动脉内皮细胞和平滑肌细胞增殖作用减弱,促凋亡作用增强。5、较低浓度的慢性肾衰兔血清(≤10%)能使兔主动脉内皮细胞和平滑肌细胞NF-κB信号通路的活化和炎症细胞因子的表达,呈一定的浓度和时间依赖性。6、较低浓度的慢性肾衰兔血清(≤10%)能使兔主动脉内皮细胞和平滑肌细胞泛素-蛋白酶体途径明显活化;阻断泛素-蛋白酶体途径可显著抑制慢性肾衰兔主动脉内皮细胞和平滑肌细胞NF-κB信号通路的活化和炎症细胞因子的表达,抑制细胞的炎症增殖反应。更多还原

【Abstract】 Background and objective:Cardiovascular diseases are the most common complications and the main causes of death seen in patients with end-stage renal disease(ESRD), whose death rate were 10-20 fold as high as the general population. The uremic accelerated atherosclerosis is paid more attention to in recent years. The Characteristics of uremic accelerated atherosclerosis, which has high morbidity, small age and widely lesions, is different from that of the general population. The pathogenesis of which is not yet understood sufficiently. Most of the traditional CVD risk factors, such as older age, diabetes mellitus, systolic hypertension, metabolism disorder of lipid, left ventricular hypertrophy(LVH), are highly prevalent in ESRD, but which can not explain why such a high morbidity and mortality of CVD, suggesting that there are other factors in the patients of chronic renal failure accelerating the course of atherosclerosis. It is very important to further find what causes lead to promote the CVD development in uremia. It is definitely sure that atherosclerosis is a kind of chronic inflammatory response process of artery wall to the traumatic factors. However, the effective prevention is still the main problem to be solved. Persistent special pathophysiological state and various toxin form a serious internal milieu disorder, which triggers prolonged microinflammatory state in ESRD patients. The microinflammatory might be related to accelerated atherosclerosis of chronic renal failure.Nuclear factor of kappa B (NF-κB) is a kind of pleiotropic transcription factors, widely found in multiple cell types, regulating the transcriptions of multiple genes. involved in inflammatory response, cells proliferation and apoptosis. Investigating NF-κB biological characteristics and controlling it’s activity has important clinical significance for prevention of cell tissue inflammatory response. Currently intensive researches have founed that the ubiquitin-proteasome pathway(UPP) was an important protein regulation system in eukaryotic cells, which was involved in various cell processes such as the regulation of cell cycle, proliferation and differentiation of cells, and signal transduction. It has been demonstrated that the UPP are closely related to the regulation of activation and inactivation of NF-κB by different links. Especially, it has been found that the UPP components expression such as ubiquitin ligases enzymes(E3) displayed the characteristic of tissue and organ specialty. Then ,it was proposed that the inflammation of artery could be inhibited through blocking UPP directly using the artery -specialized UPP inhibitor.In the present study, we investigate the dynamic pathological changes and NF-κB activity of the aorta of rabbits with chronc renal failure in vivo and the proliferation, apoptosis and NF-κB activity of the rabbit aortic endothelial cells and smooth muscle cells induced by uremic serum in vitro and the regulatory role of ubiquitin-proteasome pathway.Methods:1. Rabbit models with chronic renal failure(CRF)were established by partial ligation of renal pedical artery and side nephroectomy. The rabbits were randomly divided into six groups:Sham group, CRF group, Sham+MG132 treatment group(Sham+MG132), CRF+ MG132 treatment group(CRF+MG132), Sham+PDTC treatment group(Sham+PDTC), CRF+ PDTC treatment group(CRF+PDTC). The time of all observation index was set at postorperation 4wk, 8wk and12wk. The life signs, renal function, blood lipid and the pathological changes of renal and aorta2. The proliferation and apoptosis of the rabbit aortic endothelial cells(AECs) and aortic smooth muscle cells(ASMCs) induced by uremic serum in vitro and the effect of MG132 treatment was measured by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis and Hoechst33342 staining3. The distribution of proliferating cell nuclear antigen (PCNA)in the rabbits aorta was measured by immunohistochemistry.4. The mRNA expression of ubiquitin(Ub), ubiquitin activating enzyme(E1), NF-κBp65 and IκBαin the rabbits’aortic and the cells in vitro was surveyed by using RT-PCR.5. The protein expression of ubiquitinative proteins, E1, NF-κB, IκBα, PCNA and TNFαdetected by western blot.6. NF-κB p65 nuclear translocation of the cells in vitro were analyzed by immunofluorescence.7. The activity of NF-κB were measured by EMSA.8. The three enzyme activities of 20S proteasomes in the total protein was examined using three special fluorogenic peptide substrate.9. The level of inflammatory factors IL-6, TNFαin the rabbit serum and the cellular supernatant were examined by ELISA method.Result:1.The establishment of rabbits model with atherosclerosis of chronic renal failure: Compared with Sham group , the rabbits of CRF group developed heavy proteinuria , high level of creatinine, anaemia and heavy hypertension. The remnant kidneys showed glomerular hypertrophy and extensive glomerular damage consisting of global or segmental sclerosis, tubular dilation, cast formation, tubular atrophy, interstitial fibrosis, as well as lymphocyte infiltration. The typical changes of artherosclerosis in rabbits aortic, such as foam cells, fatty streak and atheromatous plaque were gradually oberserved in CRF group. The pathological changes of the aortic tissues in CRF+MG132 group and CRF+PDTC group were improved significantly, It was different from CRF group2. The expression of NF-κB constituents and the effects of MG132 and PDTC tretment in the aorta of rabbits with chronic renal failure. Compared with Sham group, the mRNA and protein expression of NF-κB and the activity of NF-κB in the aorta of rabbits with chronic renal failure were significantly increased. It was more obvious followed the development of CRF. the mRNA and protein expression of IκBαwere just contrary to that of NF-κB. the mRNA and protein expression of NF-κB and the activity of NF-κB in the aorta with CRF rabbits treated with MG132 or PDTC were significantly decreased compared with CRF group, but the mRNA and protein expression of IκBαwas significantly increased at the same time. These data showed that the NF-κB signal pathway was activated in the aorta of rabbits with CRF and its activation was regulated by the ubiquitin-proteasome.3. The expression of UPP constituents and the effects of MG132 and PDTC tretment in the aorta of rabbits with chronic renal failure: Compared with Sham group, the mRNA expression of Ub and E1 and the protein expression of E1 in the aortic of rabbits with CRF were significantly increased. The activity of 20S proteasomes in the aorta of rabbits with CRF were significantly increased. It was more obvious followed the development of CRF. The ubiquitinative proteins of CRF group did not change obviously. The mRNA expression of Ub and E1 and the protein expression of E1 and the activity of 20S proteasomes in the aorta of rabbits with CRF treated with MG132 were significantly decreased compared with CRF group, but the expression of ubiquitinative proteins was significantly increased at the same time. The mRNA expression of Ub and E1 and the protein expression of E1 in the rabbits aorta with CRF treated with PDTC were similar to these in CRF+MG132 group. PDTC influenced the activity of 20S proteasomes lighty and did not changed the expressed of ubiquitinative proteins obviously compared with MG132.4. Compared with Sham group, the concentration of serum IL-6 and TNFαin the rabbits with CRF was significantly increased followed the development of CRF. The protein expression of TNFαin the aorta of rabbits with CRF was also increased markedly than that in Sham rabbits. Contrasted to CRF group, the level of serum IL-6 and TNFαwas markedly decreased in the CRF rabbits treated with MG132 or PDTC. The protein expression of TNFαin the aorta of CRF rabbits treated with MG132 or PDTC was also decreased significantly than that in CRF rabbits.5. Various lower concentrations serum of chronic renal failure(≤10%) could significantly promot the proliferation of AECs and ASMCs in a dose and time dependent manner. Higher concentrations of serum of chronic renal failure (>10%) could significantly inhibit the proliferation and induce apoptosis of AECs and ASMCs in dose dependent manne. MG132 and PDTC all could inhibit the proliferation of AECs and ASMCs induced by 10% serum of chronic renal failure.6. Under the stimulation of 10% serum of chronic renal failure, the mRNA and protein expression of NF-κB in AECs and ASMCs were increased significantly compared with 10% control serum, but the mRNA and protein expression of IκBαwere just contrary to that of NF-κB. The nuclear translocation of NF-κBp65 in AECs and ASMCs was taken place. The activity of NF-κB in AECs and ASMCs increased significantly in a dose dependent manner induced by various lower concentrations serum of chronic renal failure(3%~10%). The activity of NF-κB in AECs and ASMCs was resulted in a continuous increase within 24h induced by 10% serum of chronic renal failure. MG132 and PDTC all could inhibit the mRNA and protein expression and the activity of NF-κB and increase the mRNA and protein expression of IκBαof AECs and ASMCs induced by 10% serum of chronic renal failure.7. Under the stimulation of 10% serum of chronic renal failure, The level of inflammatory factors IL-6 and TNFαin the cellular supernatant of AECs and ASMCs were increased significantly compared with 10% control serum. After stimulated 6 hours, The increasing amplitude of the level of IL-6 and TNFαin the cellular supernatant of AECs and ASMCs was highest. At the same time, the protein expression of TNFαin AECs and ASMCs induced by 10% serum of chronic renal failure increased significantly compared with 10% control serum. MG132 and PDTC all could inhibit the IL-6 and TNFαSecretion and the TNFαprotein expression of AECs and ASMCs induced by 10% serum of chronic renal failure.8. Under the stimulation of 10% serum of chronic renal failure, the mRNA expression of Ub and E1, the protein expression of E1 and the activity of 20S proteasomes in AECs and ASMCs were increased significantly compared with 10% control serum. MG132 could inhibit the mRNA expression of Ub and E1, the protein expression of E1 and the activity of 20S proteasomes in AECs and ASMCs induced by 10% serum of CRF. PDTC could also inhibit the mRNA expression of Ub and E1 and the protein expression of E1 in AECs and ASMCs stimulated by 10% serum of CRF, but it influenced the activity of 20S proteasomes slightly.Conclusion:1. The pathological changes of artherosclerosis were gradually oberserved in the aortic of rabbits followed the development of CRF.2. The NF-κB signaling pathway and its downstream inflammatory cytokine expression was acticated in the rabbits with CRF, and the inflammatory reaction was aggrevated gradually with the progress of CRF.3. The ubiquitin-proteasome pathway was activated in the chronic renal failure rabbits. The activation of NF-κB and its downstream inflammatory cytokine expression was inhibited markedly by the inhibition of proteasome with MG132, and the pathological changes of the aortic tissues were improved significantly.4. Lower concentrations of serum of chronic renal failure(≤10%) could significantly promot the proliferation of AECs and ASMCs in a dose and time dependent manner. Higher concentrations of serum of chronic renal failure (>10%) could significantly inhibit the proliferation and induce apoptosis of AECs and ASMCs in dose dependent manne.5. The NF-κB signaling pathway and its downstream inflammatory cytokine expression of the AECs and ASMCs induced by 10% serum of CRF was activated in a dose and time dependent manner.6. The ubiquitin-proteasome pathway of the AECs and ASMCs induced by 10% serum of CRF was activated. The proliferation of AECs and ASMCs induced by 10% serum of chronic renal failure and the activation of NF-κB and its downstream inflammatory cytokine expression were inhibited markedly by the inhibition of proteasome with MG132.更多还原