OBJECTIVE To investigate the therapeutic effect and side effect of ibrutinib long-term treatment in systemic lupus erythematosus (SLE) model mice induced by pristane. METHODS Female 6-week old BALB/c mice were ip given pristane 0.5 mL once for SLE induction. Four weeks later, the SLE model mice were divided into three groups based on the body mass and serum level of anti-double-strand DNA (DS-DNA) antibody and treated with 1%methylcellulose (model control group, ig), ibrutinib (30 mg·kg-1, ig) or prednisone (10 mg·kg-1, ig), respectively, once daily for 28 weeks. Every 4 weeks, body mass of each mouse was measured. Autoantibodies against DS-DNA, single-strand DNA (SS-DAN), and histone were tested by ELISA. The incidence of lupus arthritis and clinical score of inflammation and edema were recorded and graded. Biochemical analysis of urea protein, serum urea nitrogen and creatinine was used to evaluate kidney function. Mice were euthanized post 28 weeks of dosing. Inter-leukin-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were tested by ELISA. The heart, liver, spleen, lung, and kidney were collected and weighed for organ relative mass calculation. Biochemical analysis of serum glutamic-pyruvic transaminase (GPT), glutamic-oxal(o)acetic transaminase (GOT) and alkaline phosphatase (ALP) was used to evaluate liver function. Hind feet were collected for HE staining and pathological scoring to observe renal injury and inflammation. Immunohistochemical staining was used to study IgG immune complexes deposition in kidneys of lupus. RESULTS Compared with normal control group, autoantibodies (anti-DS-DNA, anti-SS-DNA and anti-histone antibodies), renal function indexes (serum creatinine, urea nitrogen and urine protein), and cytokines (IL-6, IFN-γ and TNF-α) levels in model group were significantly increased (P<0.01). The model group mice had obvious clinical symptoms of arthritis (P<0.01), serious inflammation cell invasion (P<0.01), and the mass of kidneys and spleen increased significantly (P<0.01). Compared with model group, after 28 weeks of treatment, ibrutinib decreased the level of anti-DS-DNA, anti-SS-DNA and anti-histone antibodies (P<0.01), decreased the lupus arthritis score (P<0.01) and the morbidity of arthritis, reduced the level of cyto-kines IL-6, IFN-γand TNF-α(P<0.01), reduced the level of serum creatinine, serum urea nitrogen and urine protein (P<0.01), improved pathological symptoms of hind feet such as inflammation, cartilage destruction, bone resorption and pannus (P<0.01), alleviated renal tissue inflammatory cell invasion and the immune-complex precipitation, and reduced the mass of organs (spleen and kidneys, P<0.01) and the level of liver function (GPT and ALP, P<0.01). CONCLUSION Long-term treatment with ibrutinib has therapeutic effect on the model mice of SLE, and has no obvious side effect.%目的 评估依鲁替尼长期给药对姥鲛烷诱导的系统性红斑狼疮(SLE)模型小鼠的治疗作用及副作用.方法 6周龄雌性BALB/c小鼠ip给予姥鲛烷0.5 mL制备SLE小鼠模型.4周后,根据抗双链DNA(DS-DNA)抗体滴度和体质量均匀分为模型组、依鲁替尼30 mg·kg-1治疗组和泼尼松10 mg·kg-1治疗组,每天ig给药1次,连续28周.每4周测定1次体质量,ELISA法测定血清中抗DS-DNA抗体、抗单链DNA(SS-DNA)抗体和抗组蛋白抗体水平,评估关节炎红肿等症状的评分及发病率;生物化学法检测血清肌酐、尿素氮和尿液尿蛋白水平评估肾功能.给药28周后处死小鼠,ELISA检测白细胞介素6(IL-6)、干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)水平;采集心、肝、脾、肺和肾,称重并计算脏器系数;生物化学法检测血清谷丙转氨酶、谷草转氨酶和碱性磷酸酶活性评估肝功能;HE染色观察后足和肾组织病理变化,并用免疫组化法检测肾组织免疫复合物IgG沉积.结果 与正常对照组比较,模型组抗DS-DNA,SS-DNA和组蛋白抗体及IL-6,IFN-γ和TNF-α水平均明显升高(P<0.01),血清肌酐、尿素氮和尿蛋白升高,关节炎症状严重(P<0.01),后足组织炎症细胞浸润严重(P<0.01),肾和脾显著增大(P<0.01).与模型组相比,依鲁替尼治疗28周可减缓SLE模型小鼠体质量下降,降低抗DS-DNA,SS-DNA和组蛋白抗体水平(P<0.01),减轻小鼠关节红肿等症状(P<0.01),并降低关节炎发病率,降低血清细胞因子IL-6,IFN-γ和TNF-α水平(P<0.01);后足关节病理观察显示,炎症细胞浸润、血管翳形成、软骨破坏和骨吸收减轻(P<0.01);肾组织病理观察显示,炎症细胞浸润和IgG免疫复合物沉积减少,血清肌酐、尿素氮和尿蛋白水平降低(P<0.01),血清GPT和ALP活性亦降低(P<0.01).结论 依鲁替尼给药28周对SLE模型小鼠有一定的治疗作用,未见明显副作用.
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