15-甲基-脂氧素A_4对大鼠系膜增殖性肾炎干预作用的研究

Protective effects of 15-methy-lipoxin A_4 on mesangioproliferative nephritis in rats

目的已发现脂氧素A4（LXA4）可抑制肾小球系膜细胞的体外增殖，该研究旨在了解LXA4同系物15-甲基-LXA4是否抑制大鼠系膜增殖性肾炎的病理进展，并探讨其作用的信号转导分子机制。方法使用小鼠抗大鼠Thy1．1单克隆抗体静脉内1次性注射制备大鼠系膜增殖性肾炎。使用15-甲基-LXA4静脉内注射干预大鼠系膜增殖性肾炎。检测尿蛋白、肾小球白细胞浸润、系膜细胞增生评分、增殖性细胞核抗原（PCNA）表达。应用RT—PCR方法检测肾小球白介素（IL）-1β，IL-6的mRNA表达，应用放免法测定肾小球IL-1β，IL-6水平。应用Western Blot测定肾小球磷酸化的磷脂酰肌醇-3-激酶（PI3-K）、Akt1与p27^kip1表达，应用凝胶电泳迁移率试验（EMSA）测定肾小球核因子-κB（NF—κB）及信号转导及转录活化子-3（STAT3）活性。结果大鼠系膜增殖性肾炎发病后第1～4天，肾小球白细胞计数、IL-1β、IL-6的mRNA与蛋白表达、NF—κB活性升高；第4天尿蛋白、肾小球系膜细胞增生评分、PCNA表达、肾小球PI3-K、Akt1与STAT3活性升高，p27^kip1表达减低。应用15-甲基-LXA4干预，可减少肾炎大鼠尿蛋白、肾小球白细胞计数、系膜细胞增生评分、PCNA表达、IL-1β、IL-6的mRNA与蛋白表达（均P〈0．05），减低PI3-K、Akt1、NF—κB、STAT3活性，阻止p27^kip1表达减低。结论15-甲基-LXA4可有效地抑制大鼠系膜增殖性肾炎的尿蛋白、肾小球炎性反应，系膜细胞增殖，其机制与抑制PI3-K／Akt1／p27^kip1／cyclin途径、STAT3、NF—κB活性有关。

Objective To investigate the protective effects of 15-methy-lipoxin A4 （LXA4 ） on mesangioproliferative nephritis in rats and the possible mechanisms. Methods Mesangioproliferative nephritis was induced by a single intravenous injection of the mouse monoclonal anti-Thy1. 1 antibodies （ ER4 ） in 20 rats. Ten nephritic rats were injected with 15-methy-LXA4 at 10 minutes before ER4 antibody injection and then 8-hourly until the rats were sacrificed on day 4 after nephritis induction. The nephritis was evidenced by presence of proteinuria, histologic examination with light microscopy, infiltrating leukocyte assessed by immunofluorescence microscopy, and mesangial cell proliferation assessed by proliferation scoring and by immunohistochemical staining of proliferating cell nuclear antigen （PCNA）. Expressions of interleukin （IL）-1β and IL-6 protein or mRNA in glomeruli were determined by radioimmunoassay or RT-PCR, respectively. Phosphorylated phosphoinositide 3-kinase （PI3-K）, Akt1 and p27^kip1 in glomeruli were analyzed by Western Blot. Activities of nuclear factor-κB （NF-κB） and signal transducer and activator of transcription 3 （STAT3 ） in glomeruli were assessed by electrophroretic mobility shift assay （EMSA）. Results There were increases in glomerular infiltration of leukocyte, expressions of IL-1β and IL-6 protein and mRNA, and activities of NF-κB in nephritic rats between days 1 and 4 after nephritis induction. The enhanced proteinuria, score of mesangial proliferation, glomerular PCNA positive cells, activities of phosphorylated PI3-K, Akt1 and STAT3, and reduced p27^kip1 expression were found on day 4 after nephritis induction. 15-methy-LXA4 treatment significantly reduced the proteinuria, glomerular infiltration of leukocyte, expressions of IL-1β and IL-6 protein and mRNA, score of mesangial proliferation, glomerular PCNA positive cells, activities of phosphorylated PI3-K, Akt1, NF-κB and STAT3 , and increased the p27^kip1 expression. Conclusions 15-methy-LXA4 can markedly inhibit the proteinuria, glomerular inflammation, and mesangial cell proliferation induced by anti-Thy1. 1 antibodies. The inhibition effects are related to PI3-K/Akt1/p27^kip1/cyclin pathway, STAT3 and NF-κB pathway-dependent signal transduction.