细胞焦亡介导慢性间歇缺氧大鼠肾脏损伤及依达拉奉的干预作用

目的：探究依达拉奉对慢性间歇缺氧导致大鼠肾脏损伤的保护作用及其对Caspase-1介导的细胞焦亡信号通路的影响。方法：24只SPF级雄性SD大鼠随机分为正常对照（NC）组、间歇缺氧（IH）组、间歇缺氧+生理盐水（IH+NS）组、间歇缺氧+依达拉奉（IH+EDA）组，每组6只。将4组大鼠放置在密闭式饲养舱内造模，NC组舱内氧气浓度维持在21%左右，IH组、IH+NS组、IH+EDA组定时输入纯氧气、纯氮气、压缩空气，使舱内形成缺氧-复氧循环（60 s低氧期+60 s复氧期），低氧期舱内氧浓度降至6%～7%，每日造模8 h（10∶00-18∶00），同时IH+EDA组大鼠每天造模前按照5 mg/kg剂量标准予以腹腔注射依达拉奉，IH+NS组大鼠按照同等剂量标准腹腔注射生理盐水。造模8周后采集大鼠血液标本及肾脏组织标本，测定各组大鼠血肌酐（Crea）、尿素（Urea）水平；HE、Masson染色后光镜下观察肾脏病理形态变化、纤维化程度；化学法测定丙二醛（MDA）含量、超氧化物歧化酶（SOD）活力；免疫组织化学染色法测定肾组织NLRP3、Caspase-1、IL-1β蛋白表达水平；Western blot法测定肾组织Caspase-1、IL-1β蛋白表达水平；RT-PCR法测定消皮素D（gasdermin D, GSDMD）、IL-18 mRNA扩增水平。结果：间歇缺氧暴露后，大鼠血清Crea、Urea明显升高（P<0.01），肾小管发生病理损害、肾单位球囊间隙出现胶原纤维沉积，MDA含量增加、SOD活力降低（P<0.01），Caspase-1、NLRP3、IL-1β蛋白表达增加（P<0.01或P<0.05）、GSDMD mRNA、IL-18 mRNA扩增增加（P<0.01）；而经过依达拉奉干预后，上述指标呈现出与间歇低氧暴露后相反的变化趋势，肾脏病理损害减轻（P<0.01或P<0.05）。 结论：慢性间歇缺氧可能通过氧化应激活化Caspase-1参与的细胞焦亡信号通路介导肾脏损伤过程，而依达拉奉可能通过清除氧自由基、下调机体氧化应激水平，抑制焦亡通路的活化，起到保护肾脏的作用。

Pyroptosis mediated renal injury caused by chronic intermittent hypoxia and the intervention effect of edaravone in rats

AIM: To study the protective effect of edaravone on renal injury induced by chronic intermittent hypoxia and its effect on Caspase-1 mediated pyroptosis signaling pathway in rats. METHODS: Twenty four SPF male SD rats were randomly divided into normal control group, intermittent hypoxia group, intermittent hypoxia + normal saline group and intermittent hypoxia + edaravone group, with 6 rats in each group. The four groups of rats were placed in the closed feeding chamber for modeling. The oxygen concentration in the NC group was maintained at about 21%; the IH group, IH+NS group and IH+EDA group were given regular input of pure oxygen, pure nitrogen and compressed air to form anoxic-reoxygenation cycle (60 s hypoxic period + 60 s reoxygenation period). During the hypoxic period, the oxygen concentration in the chamber was reduced to 6%-7%, and the rats in the IH+EDA group were intraperitoneally injected with edaravone at a dose of 5 mg/kg per day before modeling, while the rats in the IH+NS group were intraperitoneally injected with normal saline at the same dose per day. After 8 weeks of modeling, blood and kidney tissue samples were collected to measure the levels of Crea and Urea in each group. The pathological changes and fibrosis degree of kidney were observed under light microscope after HE and Masson staining. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were determined by chemical method. The expression levels of NLRP3, Caspase-1 and IL-1β in renal tissues were determined by immunohistochemical staining. The expression levels of caspase-1 and IL-1β in renal tissues were determined by Western blot. GSDMD and IL-18 mRNA were detected by RT-PCR.RESULTS: After intermittent hypoxia exposure, serum Crea and Urea were increased significantly (P<0.01), renal tubules were damaged by pathology, collagen fiber deposition occurred in balloon space of renal units, MDA content was increased and SOD activity was decreased (P<0.01). Caspase-1, NLRP3, IL-1β protein expression increased (P<0.01 or P<0.05), GSDMD mRNA and IL-18 mRNA amplification increased (P<0.01); After Edaravone intervention, the above indexes showed a reverse trend compared with that after intermittent hypoxia exposure, and the pathological damage of kidney was reduced (P<0.01 or P<0.05). CONCLUSION: Chronic intermittent hypoxia may mediate kidney injury through oxidative stress activation of caspase-1 involved in the cell pyroptosis signaling pathway, while edaravone may inhibit the activation of pyroptosis signaling pathway by scavenging oxygen free radicals and down-regulating the level of oxidative stress in the body, thus playing a protective role in kidney.