中性粒细胞膜纳米囊泡对小鼠脓毒症致急性肾损伤的保护作用研究

Protective effect of neutrophil membrane nanovesicles against sepsis-associated acute kidney injury in mice

目的基于小鼠脓毒症性急性肾损伤(acute kidney injury,AKI)模型,探究中性粒细胞膜纳米囊泡对脓毒症致AKI的保护作用。方法选用雄性8周龄BALB/c小鼠,腹腔注射10 mg/kg脂多糖(lipopolysaccharide,LPS)构建脓毒症模型后,随机分成3组,脓毒症模型组(LPS组):尾静脉注射0.2 ml PBS溶液;红细胞膜纳米囊泡治疗组(LRM组):尾静脉注射0.2 ml浓度为40 mg/ml红细胞膜来源的纳米囊泡溶液;中性粒细胞膜纳米囊泡治疗组(LNM组):尾静脉注射0.2 ml浓度为40 mg/ml的中性粒细胞膜来源的纳米囊泡溶液。正常小鼠作为空白对照(CK组)。8 h后,麻醉小鼠,取血浆和肾组织。用全自动生化分析仪检测血浆中肌酐(creatinine,CREA)和尿素(UREA)含量以评价肾功能改变,生化分析方法分别检测肾组织中丙二醛(malondialdehyde,MDA)含量和髓过氧化物酶(myeloperoxidase,MPO)活性变化,采用ELISA方法检测小鼠细胞外组蛋白H4(H4)、白细胞介素(IL)-1β以及巨噬细胞炎症蛋白-2(macrophage inflammatory protein 2,MIP-2)水平变化,采用免疫组织化学法检测肾组织中促凋亡蛋白Bax的表达水平。结果与CK组相比,LPS组小鼠血浆中尿素氮和肌酐水平显著升高(P<0.05),而LNM和LRM组均显著降低(P<0.05)。与LPS组相比,LNM组小鼠肾组织MDA含量(P<0.01)和MPO活性均显著降低(P<0.05);肾组织中细胞外组蛋白H4和IL-1β的表达水平显著降低(P<0.05)。LPS处理能显著增强LPS组肾组织Bax的表达,而中性粒细胞膜纳米囊泡能显著降低LNM组肾组织Bax的表达(P<0.01)。结论中性粒细胞膜纳米囊泡可抑制脓毒症小鼠肾MDA含量和MPO活性,降低肾细胞外组蛋白H4、IL-1β和MIP-2含量并降低促凋亡蛋白Bax表达,改善脓毒症所致AKI。

Objective To explore the protective effect of neutrophil membrane nanovesicles against lipopolysaccharide(LPS)-induced sepsis-associated acute kidney injury(AKI)in mice.Methods Eight-week-old male BABL/c mice were selected and randomly divided into three groups to establish animal models for sepsis-associated AKI via the injection of LPS at 10 mg/kg.The model group(LPS group)was administered with 0.2 ml of PBS solution by tail vein injection,while the erythrocyte membrane nanovesicle treatment group(LRM group)and the neutrophil membrane nanovesicle treatment group(LNM group)were administered with 0.2 ml of erythrocyte membrane nanovesicle solution and neutrophil membrane nanovesicle solution at 40 mg/ml,respectively.Normal mice were used as a control(CK group).The mice were anesthetized after 8 h,and the plasma and kidney tissues were stored.The CREA and UREA levels in the plasma were assessed using an automatic biochemical analyzer.The renal malondialdehyde(MDA)content and myeloperoxidase(MPO)activity were detected using a biochemical method.The contents of extracellular histones 4(H4),interleukin(IL)-1β,and macrophage inflammatory protein 2 in the kidneys were determined via an enzyme-linked immunosorbent assay.The expression of Bax was evaluated using an immunohistochemical method.Results Compared with the CK group,the plasma CREA and UREA contents in the LPS group were significantly increased(P<0.05),while neutrophil membrane nanovesicle and erythrocyte membrane nanovesicle treatment both significantly decreased the CREA and UREA(P<0.05)contents.Compared with the LPS group,the renal MDA content(P<0.01)and MPO activity in the LNM group were significantly reduced(P<0.05).Neutrophil membrane-derived nanovesicle administration substantially decreased the expression levels of H4 and IL-1βin the kidneys(P<0.05).Immunohistochemical staining showed a higher Bax expression in the LPS group than in the LNM group(P<0.01).Conclusion Neutrophil membrane-derived nanovesicles can alleviate LPS induced sepsis-associated AKI in mice by inhibiting renal MDA content and MPO activity,decreasing the contents of H4,IL1β,and MIP-2,and reducing Bax protein expression.