12/15-脂氧化酶基因敲除对肥胖相关性肾小球疾病模型小鼠肾组织的保护作用及其机制

Protective effect of 12/15-lipoxygenase gene knockout on kidney tissue of obesity-related glomerulopathy model mice and its mechanism

目的:探讨12/15-脂氧化酶(12/15-LO)基因敲除(12/15-LOKO)对高脂饮食(HFD)诱导的肥胖小鼠肾组织的保护作用,阐明其在肾脏疾病进展中的作用。方法:将16只12/15-LOKO小鼠和16只WT小鼠各自随机分为HFD组和标准饮食喂养(SFD)组,每组8只。HFD组小鼠给予HFD喂养,SFD组小鼠给予SFD喂养(即WT+HFD、WT+SFD、LOKO+HFD和LOKO+SFD组)。分别于喂养1、8和14周收集小鼠24 h尿液,14周后采血并处死小鼠。检测各组小鼠体质量、双肾质量和后腿胫骨长,分别在第1、8和14周时代谢笼收集各组小鼠24 h尿液,检测尿蛋白水平、尿蛋白肌酐比(PCR)和尿微量白蛋白肌酐比(ACR)。葡萄糖耐量试验(GTT)检测各组小鼠空腹血糖水平,实时定量逆转录PCR(RT-qPCR)法检测各组小鼠肾皮质组织中adiponectin、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、单核细胞趋化蛋白1(MCP-1)、纤溶酶原激活物抑制剂1(PAI-1)和转化生长因子β1(TGF-β1)mRNA表达水平,ELASA法检则各组小鼠血清中adiponectin、TNF-α、IL-6、血管紧张素Ⅱ(AngⅡ)和胰岛素水平。PAS染色观察小鼠肾组织病理形态表现,Westernblotting法检测各组小鼠肾皮质组织中TGF-β1和PAI-1蛋白表达情况。结果:HFD组小鼠体质量明显高于SFD组(P<0.01),肾脏质量和肾小球面积明显大于SFD组(P<0.01);WT+HFD组小鼠ACR和PCR高于WT+SFD组(P<0.05);与LOKO+HFD组比较,WT+HFD组小鼠空腹血糖水平升高(P<0.05),WT+HFD组小鼠肾皮质组织中TNF-α、IL-6和MCP-1 mRNA表达水平明显高于WT+SFD组(P<0.05);与LOKO+HFD组比较,WT+HFD组小鼠肾皮质组织中IL-6和MCP-1 mRNA表达水平明显升高(P<0.05),adiponectinmRNA表达水平降低(P<0.05)。WT+HFD组小鼠肾皮质组织中PAI-1和TGF-β1 mRNA表达水平高于WT+SFD组(P<0.05),LOKO+HFD组小鼠肾皮质中PAI-1和TGF-β1 mRNA表达水平明显低于WT+HFD组(P<0.01)。结论:12/15-LOKO有利于改善肥胖小鼠胰岛素抵抗、蛋白尿水平和肾小球肥大程度,其机制可能与抑制TNF-α和IL-6等炎症因子在肾组织中的表达、影响PAI-1和TGF-β1 mRNA的表达、从而发挥肾脏保护作用有关。

Objective:To explore the protective effect of 12/15-lipoxygenase(12/15-LO)gene knockout(12/15-LOKO)on the kidney tissue of the obese mice induced by high-fat diet(HFD),and to clarify its role in the progression of kidney disease.Methods:A total of 1612/15-LOKO mice and 16 WT mice were randomly divided into HFD group and standard diet(SFD)group,respectively,and there were 8 mice in each group.The mice in HFD group were fed with HFD,and the mice in SFD were fed with SFD,namely WT+HFD group,WT+SFD group,LOKO+HFD group,and LOKO+SFD group.The 24 h urine of mice in various groups was collected after feeding for 1,8,and 14 weeks.The body weights,double kidney weights and tibia lengths of hind legs of mice in various groups were detected.The 24 h urine of the mice in various groups was collected after 1,8,and 14 week,the urinary protein level,urinary perotein creatinine ratios(PCR),and urinary microalbumin creartinine ratio(ACR)of the mice were detected.The blood glucose levels of the mice in various groups were detected by glucose tolerance test(GTT),and the mRNA levels of adiponectin,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1),plasminogen activator inhibitor-1(PAI-1),and transforming growth factor-β1(TGF-β1)in kindey cortex tissue of the mice in various groups were detected by Real-time fluorescence quantitative PCR(RT-qPCR)method.ELISA method was used to detect the levels of adiponectin,TNF-α,IL-6,angioteinsinⅡ(AngⅡ)and insulin in serum of the mice in various groups.PAS staining was used to observe the pathomorphology of kidney tissue and glomerularareas of the mice in various groups,and Western blotting method was used to detect the expressions of TGF-β1 and PAI-1βproteins in kidney tissue of the mice in various groups.Results:The body weight,kidney weight and glomerular area of the mice in HFD group were significantly higher than those in SFD group(P<0.01),and the ACR and PCR of the mice in WT+HFD group were higher than those in SFD group(P<0.05);compared with LOKO+HFD group,the fasting blood glucose level of the mice in WT+HFD group was increased(P<0.05).The expression levels of TNF-α,IL-6,and MCP-1 mRNA in kidney cortex tissue of the mice in WT+HFD group were significantly higher than those in WT+SFD group(P<0.05).Compared with LOKO+HFD group,the expression levels of IL-6 and MCP-1 mRNA in kidney cortex tissue of the mice in WT+HFD group were increased(P<0.05),while the expression level of adiponectin mRNA was decreased significantly(P<0.05).The expression levels of PAI-1 and TGF-β1 mRNA in kidney cortex tissue of the mice in WT+HFD group were higher than those of the mice in WT+SFD group(P<0.05).The expression levels of PAI-1 and TGF-β1 mRNA in kidney cortex tissue of the mice in LOKO+HFD group were lower than those of the mice in WT+HFD group(P<0.01).Conclusion:12/15-LOKO can improve insulin resistance,proteinuria level and degree of glomerular hypertrophy of the obese mice,and its mechanism may be related to the inhibition the expressions of inflammatory factors such as TNF-αand IL-6 in kidney tissue and affecting the expressions of PAI-1 and TGF-β1 mRNA,so as to play a protective role in kidney.