高尿酸血症对尿酸氧化酶基因敲除小鼠动脉粥样硬化的促进作用及其相关分子表达机制的研究

Effect of hyperuricemia on promoting atherosclerosis of UOX-gene knock-out mice and its mechanism

目的探讨高尿酸血症(HUA)在促进尿酸氧化酶基因敲除小鼠动脉粥样硬化(AS)的作用以及相关分子表达的机制。方法①入组试验的为雄性SPF级C57BL6J鼠,10周龄,体重为26~28 g,共计24只,其中12只为尿酸氧化酶(UO)基因敲除(UOX-/-)小鼠,即KO小鼠;另12只为野生型小鼠,即WT小鼠。将野生型小鼠采用随机数字表法分为WT组和WT套环组(行颈动脉套环干预的WT小鼠),将KO小鼠采用随机数字表法分为KO组和KO套环组(行颈动脉套环干预的KO小鼠)。四组小鼠均接受8周的高脂肪高胆固醇(HF/HC)西式饮食喂养,其中胆固醇含量0.15%,脂肪含量各为21.00%。②WT套环组和KO套环组于高脂饮食的第4周实施此项干预,完成AS模型的制备。③待实验进行至第4周与第8周,分别由实验动物尾静脉取血,检测血尿酸水平(SUA)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)和C-反应蛋白(hs-CRP)水平。借助酶联免疫吸附法(ELISA)对其单核细胞趋化蛋白-1(MCP-1)、血管细胞黏附分子-1(VCAM-1)以及细胞间黏附因子-1(ICAM-1)的血清含量进行测定;④待实验至第8周,即对部分小鼠行动脉放置套环干预后的第4周,用过量水合氯醛将全部小鼠处死,分别提取各组小鼠的颈动脉组织,同时完成总RNA与蛋白质的提取,借助逆转录多聚酶链反应(RT-PCR)对MCP-1、VCAM-1与ICAM-1在颈动脉内的mRNA表达强度进行测定,用Western blotting对上述3种因子于颈动脉内的蛋白表达强度进行测定。⑤此外,通过苏木精-伊红染色法对四组小鼠颈动脉血管的中膜面积与厚度进行分析。⑥利用免疫组化法(IHC)对巨噬细胞于颈动脉中浸润状况以及细胞核增殖抗原(PCNA)于血管平滑肌细胞(VSMC)内的表达状况进行测定。结果①在SUA、TG与HDL-C水平上,对比KO组和KO套环组,未发现明显不同,然而在这3项指标上,这两组相比WT组和WT套环组均处存在明显提高表现(P<0.05);在hs-CRP水平上,对比KO套环组和WT套环组,未见明显不同,但在此项指标上,这两组相比KO组和WT组均存在显著升高表现(P<0.05)。在LDL-C水平上,四组间未见明显不同。②经ELISA测定发现,在MCP-1含量上,相较于另3组,KO套环组显著较高(P<0.01);且在VCAM-1与ICAM-1含量上,该组相比WT组与WT套环组也明显较高(P<0.05),然而对比KO组未见明显不同。由RT-PCR法对ICAM-1、MCP-1与VCAM-1在颈动脉内mRNA表达强度的测定结果相符于ELISA法。经Western blotting测定发现,在颈动脉VCAM-1与ICAM-1蛋白表达强度上,相较于WT组与WT套环组,KO套环组均存在显著上升表现(P<0.05),然而对比KO组未发现明显不同。在MCP-1蛋白水平上,KO套环组有所上调,且相较于另3组表现为明显偏高(P<0.05)。③经苏木精-伊红染色实验发现,在中膜面积与厚度方面,相较于WT小鼠,UOX-/-小鼠存在明显升高表现(P<0.05),且相较于WT组和KO组,套环组明显偏高(P<0.05)。④经IHC检测发现,KO组与KO套环组,颈总动脉斑块中存在数量可观的F4/80+巨噬细胞浸润表现,但在WT组和WT套环组中,颈总动脉斑块中未见或只见小部分巨噬细胞浸润表现。⑤IHC检测结果表明,UOX-/-小鼠可见PCNA于颈动脉内皮细胞内表达明显,在内膜下可观察到明显增多的棕褐色颗粒。针对切片,借助图像分析软件实施分析计算,结果为,在PCNA表达方面,相较于WT组、WT套环组,另两组显著较高(P<0.01)。结论在HUA小鼠(UOX-/-小鼠)制备的AS模型中,高尿酸血症可使黏附分子表达上调、巨噬细胞浸润加剧、PCNA表达提升等,由此对AS的产生与加剧发挥促进作用。

Objective To investigate the effect of hyperuricemia on promoting atherosclerosis of UOX-gene knock-out mice and the related molecular expression mechanism.Methods ①A total of 24 male SPF C57 BL6 J mice,aged 10 weeks and weighing 26 ~ 28 g,were included in the study,including 12 urate oxidase(UO) gene knockout(UOX-/-) mice,namely KO mice;the other 12 were wild-type,namely WT mice.The wild-type mice were randomly divided into the WT group and the WT thimble group(WT mice received carotid thimble intervention),and the KO mice were randomly divided into the KO group and the KO thimble group(KO mice received carotid thimble intervention).All four groups received a high-fat,high-cholesterol(HF/HC) western diet(with 0.15% cholesterol and 21% fat) for eight weeks. ②The intervention was performed in the WT thimble group and KO thimble group at the 4 th week of high-fat diet to make the AS model. ③At the 4 th and 8 th weeks of the experiment,blood sample was taken from the tail vein of each experimental animal to detect the level of serum uric acid(SUA),triglyceride(TG),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C) and C-reactive protein(hs-CRP).Serum of monocyte chemoattractant protein-1(MCP-1),vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1(ICAM-1) were detected by enzyme-linked immunosorbent assay(ELISA) . ④At the 8 th week of the experiment,all mice were sacrificed with excess chloral hydrate,and the carotid artery tissue of each group of mice was extracted.The mRNA expression of MCP-1,VCAM-1 and ICAM-1 in the carotid artery was measured by reverse transcription polymerase chain reaction(RT-PCR),and the protein expression of MCP-1,VCAM-1 and ICAM-1 were detected by Western blotting. ⑤In addition,hematoxylin-eosin staining was used to analyze the media area and thickness of carotid arteries in four groups of mice. ⑥Immunohistochemistry(IHC) was used to measure the infiltration of macrophages in carotid arteries and the expression of proliferation cell nuclear antigen(PCNA) in vascular smooth muscle cells(VSMC).Results ①There was no significant difference between the KO group and the KO thimble group in the level of SUA,TG and HDL-C,but the level of SUA,TG and HDL-C in the KO and KO thimble group were significantly higher than those of the WT and WT thimble group(P< 0.05).The C-reactive protein level(hs-CRP) of the KO thimble group was not statistically different from WT thimble group,but significantly higher than that of the KO group and WT group(P< 0.05).There was no significant difference in LDL-C level between the four groups. ②It was found by ELISA that the serum MCP-1 in the KO thimble group was significantly higher than that in the other three groups(P< 0.01).The serum ICAM-1 and VCAM-1 in the KO thimble group were significantly higher than those of the WT group and WT thimble group(P< 0.05),but was not significantly different from the KO group.The mRNA expression of ICAM-1,VCAM-1 and MCP-1 were detected by RT-PCR,and the results were consistent with the results of ELISA.Western blotting results showed that the levels of ICAM-1 and VCAM-1 in the KO thimble group were significantly increased compared with those of the WT and WT thimble group(P< 0.05),but there was no significant difference with the KO group.The level of MCP-1 in the KO group was significantly higher the that in the other three groups(P< 0.05) . ③Hematoxylin-eosin stain results showed that the media area and thickness of carotid arteries of the KO group were significantly higher than those of the WT group;the KO and WT thimble group were significantly higher than the KO and WT group(P< 0.05) . ④Immunohistochemistry results showed that there were considerable F4/80+macrophage infiltration in carotid plaques in KO group and KO thimble group,but in WT group and WT thimble group,no or only a small amount of macrophage infiltration was seen. ⑤Immunohistochemistry showed that the KO group had a significant increase in PCNA expression in carotid endothelial cells,and an increase of brown granules could be observed under the endometrium.For the slices,the analysis and calculation were carried out with the help of image analysis software.The results showed that the expression of PCNA was significantly higher in the other two groups compared with the WT group and the WT thimble group(P< 0.01).Conclusion In the AS model prepared by HUA mice(UOX-/-mice),hyperuricemia can increase the expression of adhesion molecules,intensify macrophage infiltration,and increase the expression of PCNA,thereby promoting the production and exacerbation of AS.