托法替布在系统性红斑狼疮小鼠早期动脉粥样硬化中的作用探讨

The role of tofacitinib in early atherosclerosis in mice with systemic lupus erythematosus

目的探讨托法替布对系统性红斑狼疮(SLE)早期动脉粥样硬化的影响;探究狼疮肾炎(LN)与SLE早期动脉粥样硬化可能的关系。方法选择8周龄无特定病原体(SPF)级雌性MRL/lpr小鼠16只,体质量20~25 g,通过完全随机法分为治疗组与安慰剂组,每组8只。治疗组通过0.9%氯化钠溶液稀释托法替布,按照10 mg·kg^(-1)·d^(-1)剂量灌胃,安慰剂组(淀粉片剂)用药方法同治疗组,共给药8周。ELISA方法检测2组小鼠血清抗dsDNA抗体水平;Bradford法蛋白浓度测定小鼠尿蛋白水平;全自动生化分析仪检测血脂、尿素氮、血肌酐、补体C3、补体C4水平;蛋白印迹法测定主动脉及肾组织单核细胞趋化蛋白-1(MCP-1)、非受体型蛋白酪氨酸激酶家族1(JAK1)、信号转导与转录激活因子(STAT)1、STAT2蛋白表达水平;主动脉弓切片后采用油红O染色,使用Image J软件评估血管斑块面积及内中膜厚度;肾脏取病理切片后采用HE染色,使用肾小球活动性评分系统评估LN活动性病变;组间统计学比较采用两独立样本t检验,相关性分析使用Spearman法。结果①治疗组血清抗dsDNA抗体表达水平[(5.2±1.0)U/ml]低于安慰剂组[(6.9±1.2)U/ml](Z=-3.07,P=0.008),补体C3、补体C4水平均高于安慰剂组[(293±10)mg/L与(260±19)mg/L,Z=2.72,P=0.017;(16±6)mg/L与(8±9)mg/L,Z=3.78,P=0.006]。治疗组与安慰剂组血清尿素氮、血肌酐之间差异无统计学意义[(10.6±0.7)mmol/L与(11.5±1.1)mmol/L,Z=-1.96,P=0.071;(17±5)μmol/L与(22±6)μmol/L,Z=-1.79,P=0.095]。②与安慰剂组相比,治疗组小鼠LDL、TC、TG水平均下降[(0.83±0.15)mmol/L与(1.08±1.05)mmol/L,Z=-3.95,P=0.001;(2.90±0.08)mmol/L与(1.81±0.97)mmol/L,Z=-5.17,P=0.001;(1.10±0.08)mmol/L与(1.60±0.42)mmol/L,Z=-3.23,P=0.013],HDL水平升高[(2.02±0.99)mmol/L与(1.81±0.97)mmol/L,Z=4.42,P=0.001]。③治疗组主动脉MCP-1、JAK1、STAT1、STAT2水平较安慰剂组降低(0.17±0.30与0.23±0.05,Z=-3.06,P=0.009;0.83±0.09与1.05±0.19,Z=-3.07,P=0.008;0.77±0.07与0.94±0.13,Z=-2.83,P=0.014;0.70±0.07与0.82±0.09,Z=-2.83,P=0.013),主动脉斑块面积及主动脉内中膜厚度低于安慰剂组[(12±31)μm^(2)与(1242±1101)μm^(2),Z=-3.12,P=0.016;(63±7)μm与(82±8)μm,Z=-5.13,P<0.001]。④与安慰剂组相比,治疗组尿蛋白水平及肾小球肾炎活动性评分均降低[(0.08±0.03)mg/ml与(0.20±0.11)mg/ml,Z=-3.08,P=0.015;(1.79±0.38)分与(2.79±0.14)分,Z=-7.08,P<0.001],肾组织MCP-1、JAK1、STAT1、STAT2水平较安慰剂组降低(0.364±0.040与0.425±0.021,Z=-3.85,P=0.003;0.689±0.074与0.838±0.068,Z=-4.19,P=0.001;0.508±0.070与0.646±0.119,Z=-2.85,P=0.015;0.618±0.062与0.740±0.101,Z=-2.94,P=0.013)。⑤2组小鼠肾小球活动性评分与LDL、TC、TG、主动脉斑块面积、主动脉内中膜厚度均呈正相关(r=0.51,P=0.043;r=0.79,P<0.001;r=0.64,P=0.008;r=0.82,P<0.001;r=0.74,P=0.001),与HDL呈负相关(r=-0.53,P=0.036)。2组小鼠尿蛋白水平与LDL、TC、TG、主动脉斑块面积、主动脉内中膜厚度均呈正相关(r=0.67,P=0.004;r=0.68,P=0.004;r=0.53,P=0.033;r=0.80,P<0.001;r=0.74,P=0.001),与HDL呈负相关(r=-0.57,P=0.021)。结论LN严重程度与SLE早期动脉粥样硬化及血脂异常具有一定相关性;托法替布可能减轻MRL/lpr小鼠早期动脉硬化程度及LN程度,并降低血脂水平,对于改善SLE预后、提高患者生存率可能有效。

Objective To investigate the effect of tofacitinib on early atherosclerosis of patients with systemic lupus erythematosus and explore the possible relationship between lupus nephritis and early atherosclerosis of systemic lupus erythematosus.Methods Sixteen 8-week-old female MRL/lpr mice with a body weight of 20~25 g were selected and randomly divided into the treatment group and placebo group,with 8 mice in each group.The treatment group diluted tofacitinib by normal saline,and given at a dose of 10 mg·kg^(-1)·d^(-1),and the placebo group(starch tablets)administered the medication in the same way as the treatment group for a total of 8 weeks.The ELISA method was applied to detect serum anti-dsDNA antibody levels in the two groups of mice.Bradford method protein concentration was used to determine the level of urine protein in mice.Automatic biochemical analyzer was used to detect blood lipids,urea nitrogen,serum creatinine,complement C3,complement C4 levels.Western blotting was used to determine the protein expression levels of monocyte chemoattractant protein-1(MCP-1),non-receptor protein tyrosine kinase family 1(JAK1),signal transducer and activator of transcription 1(STAT1)and signal transducer and activator of transcription 2(STAT2)in aortic and kidney tissues.After the aortic arch section were prepared,oil red O was used to stain the sections,and the vascular plaque area and intimal thickness were evaluated by ImageJ software.The kidneys were dissected and stained with HE,and the active lesions of lupus nephritis were evaluated using the glomerular activity scoring system.SPSS 23.0 software was used for statistical analysis,in which the between-group comparison was performed using two independent samples t-test,and the correlation analysis was performed using the Spearman method.Results①The serum anti-dsDNA antibody expression level in the treatment group[(5.2±1.0)U/ml]was lower than that in the placebo group[(6.9±1.2)U/ml],(Z=-3.07,P=0.008),and the levels of complement C3 and complement C4 were higher than those in the placebo group[(293±10)mg/L vs.(260±19)mg/L,Z=2.72,P=0.017];(16±6)mg/L vs.(8±9)mg/L,Z=3.78,P=0.006].There was no significant difference in serum BUN and Scr between the treatment group and the placebo group[(10.6±0.7)mmol/L vs.(11.5±1.1)mmol/L,Z=-1.96,P=0.071;(17±5)μmol/L vs.(22±6)μmol/L,Z=-1.79,P=0.095].②Compared with the placebo group,the levels of LDL,TC and TG in the treatment group decreased[(0.83±0.15)mmol/L vs.(1.08±1.05)mmol/L,Z=-3.95,P=0.001;(2.90±0.08)mmol/L vs.(1.81±0.97)mmol/L,Z=-5.17,P=0.001;(1.10±0.08)mmol/L vs.(1.60±0.42)mmol/L,Z=-3.23,P=0.013],and HDL level increased[(2.02±0.99)mmol/L vs.(1.81±0.97)mmol/L,Z=4.42,P=0.001].③Compared with the placebo group,the levels of aortic MCP-1,JAK1,STAT1 and STAT2 in the treatment group were reduced[(0.17±0.30)vs.(0.23±0.05),Z=-3.06,P=0.009;(0.83±0.09)vs.(1.05±0.19),Z=-3.07,P=0.008;(0.77±0.07)vs.(0.94±0.13),Z=-2.83,P=0.014;(0.70±0.07)vs.(0.82±0.09),Z=-2.83,P=0.013],the aortic plaque area and aortic intimal thickness were lower than those in the placebo group[(12±31)μm^(2)vs.(1242±1101)μm^(2),Z=-3.12,P=0.016;(63±7)μm vs.(82.10±8.06)μm,Z=-5.13,P<0.001].④Compared with the placebo group,the urine protein level and glomerulonephritis activity score in the treatment group were decreased[(0.08±0.03)mg/mL vs.(0.20±0.11)mg/mL,Z=-3.08,P=0.015;(1.79±0.38)vs.(2.79±0.14)points,Z=-7.08,P<0.001],and renal tissue MCP-1,JAK1,STAT1.Compared with the placebo group,STAT2 levels were reduced[(0.364±0.040)vs.(0.425±0.021),Z=-3.85,P=0.003;(0.689±0.074)vs.(0.838±0.068),Z=-4.19,P=0.001;(0.508±0.070)vs.(0.646±0.019),Z=-2.85,P=0.015;(0.618±0.062)vs.(0.740±0.101),Z=-2.94,P=0.013]...⑤The glomerular mobility scores of the two groups were positively correlated with LDL,TCHO,TG,aortic plaque area and aortic intimal thickness(r=0.51,P=0.043;r=0.79,P<0.001;r=0.64,P=0.008;r=0.82,P<0.001;r=0.74,P=0.001),and negatively correlated with HDL(r=-0.53,P=0.036).The urine protein levels in the two groups were positively correlated with LDL,TC,TG,aortic plaque area and aortic intimal thickness(r=0.67,P=0.004;r=0.68,P=0.004;r=0.53,P=0.033;r=0.80,P<0.001;r=0.74,P=0.001),and negatively correlated with HDL(r=-0.57,P=0.021).Conclusion The severity of lupus nephritis is correlated with atherosclerosis and dyslipidemia in the early stage of systemic lupus erythematosus.Tofacitinib may reduce the degree of early arteriosclerosis and lupus nephritis in MRL/LPR mice,and reduce blood lipid levels,which may be effective in improving the prognosis of SLE and improving the survival rate of patients.