蛋白酪氨酸激酶2/信号转导和转录活化因子3信号通路在局灶节段性肾小球硬化小鼠模型中的机制研究

Mechanism of Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway in model mice with focal segmental glomerulosclerosis

目的通过精准化5/6肾切除术建立局灶节段性肾小球硬化(FSGS)小鼠模型,探讨蛋白酪氨酸激酶2/信号转导和转录活化因子3(JAK2/STAT3)信号通路在FSGS小鼠模型中的发病机制。方法选取6周龄雄性C57BL/6小鼠60只,并随机分为对照组(n=30)和FSGS组(n=30)。FSGS组小鼠接受精准化5/6肾切除术,应用体积公式计算肾脏切除体积。对照组为假手术组,仅暴露双侧肾脏,剥离肾周脂肪组织后复位缝合。比较2组小鼠24 h尿蛋白、血肌酐、尿素氮等指标水平;应用苏木精-伊红(HE)染色观察肾脏组织的病理学变化;应用实时荧光定量PCR(RT-qPCR)测定肾组织JAK2/STAT3信号通路的目标基因;应用Western Blot测定JAK2/STAT3信号通路的目标蛋白;应用酶联免疫吸附试验(ELISA)检测JAK2/STAT3信号通路下游炎症因子及纤维化因子的水平。结果造模过程中,FSGS组有1只小鼠死于麻醉,予以排除;精准化5/6肾切除术建立FSGS小鼠模型的造模成功率为89.7%(26/29)。与对照组相比,FSGS组小鼠24 h尿蛋白、血肌酐、尿素氮水平较高,差异有统计学意义(P<0.01)。FSGS组小鼠HE染色可见大量肾小管上皮细胞凋亡、坏死,肾小囊腔扩张,肾小球代偿性肥大及局灶性硬化,系膜细胞和系膜基质增生,伴炎细胞浸润。术后10周,FSGS组小鼠肾组织JAK2 mRNA、STAT3 mRNA表达水平,磷酸化酪氨酸激酶2(p-JAK2)、JAK2、磷酸化信号转导和转录活化因子3(p-STAT3)、STAT3蛋白表达水平,以及白细胞介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)、α-平滑肌肌动蛋白(α-SMA)、转化生长因子-β(TGF-β)水平均升高,差异有统计学意义(P<0.01)。结论通过精准化5/6肾切除术可成功建立小鼠FSGS模型,而JAK2/STAT3信号通路活化可通过促进炎症反应、肾组织纤维化等多种途径参与FSGS的发生及发展过程。

Objective To establish the model mice with focal segmental glomerulosclerosis(FSGS) by precise 5/6 nephrectomy, and to explore the pathogenesis of Januskinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway in model mice with FSGS. Methods A total of 60 male C57 BL/6 mice aged 6 weeks were selected and randomly divided into control group(n=30) and FSGS group(n=30). The mice in the FSGS group received precise 5/6 nephrectomy, and the volume of nephrectomy was calculated by volume formula. The control group was a sham operation group, the bilateral kidneys of mice were exposed only, and the adipose tissue around the kidney was stripped before restoration and suturing. The 24 h urinary protein, serum creatinine and urea nitrogen were compared between the two groups;hematoxylin-eosin(HE) staining was used to observe the pathological change of kidney tissue;the target genes of JAK2/STAT3 signal pathway in renal tissues were detected by real-time fluorescent quantitative PCR( RT-qPCR);the target proteins of JAK2/STAT3 signal pathway were detected by western blot;the enzyme-linked immunosorbent assay( ELISA) was used to detect the levels of inflammatory factors and fibrosis factors in the downstream of JAK2/STAT3 signal pathway. Results During the modeling process,one mouse in the FSGS group died of anesthesia and was excluded;the success rate of establishing model mice with FSGS by precise 5/6 nephrectomy was 89. 7%( 26/29). Compared with the control group,the 24 h urine protein,blood creatinine and urea nitrogen levels in the mice of the FSGS group were significantly higher( P < 0. 01). In the FSGS group,HE staining showed a large number of apoptosis and necrosis of renal tubular epithelial cells,expansion of renal vesicles,compensatory glomerular hypertrophy and focal sclerosis,proliferation of mesangial cells and mesangial matrix,and infiltration of inflammatory cells. At 10 weeks after operation,the expression levels of JAK2 mRNA and STAT3 mRNA,the protein expression levels of phosphorylated Janus kinase 2( p-JAK2),JAK2,phosphorylated signal transducer and activator of transcription 3( p-STAT3) and STAT3,and levels of interleukin-6( IL-6),monocyte chemoattractant protein-1( MCP-1),α-smooth muscle actin( α-SMA) and transforming growth factor-β1( TGF-β1) were increased significantly( P <0. 01). Conclusion Precise 5/6 nephrectomy is helpful in successfully establishing model mice with FSGS,and the activation of JAK2/STAT3 signaling pathway can participate in the occurrence and development of FSGS by promoting inflammatory response,renal fibrosis and other ways.