| 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.
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