HMGB1中和抗体抑制细胞焦亡改善系统性红斑狼疮小鼠肺损伤的机制研究

Mechanism of HMGB1 Neutralizing Antibody Inhibiting Pyroptosis and Improving Lung Injury in Mice with Systemic Lupus Erythematosus

目的探究高迁移率族蛋白1(high-mobility group box 1,HMGB1)中和抗体对系统性红斑狼疮(systemic lupus erythematosus,SLE)小鼠肺损伤的影响及机制。方法30只MRL/lpr小鼠随机分为MRL/lpr组、MRL/lpr+HMGB1中和抗体(anti-HMGB1)组、MRL/lpr+MCC950组,每组10只,另取10只野生型C57BL/6小鼠作为对照组,给药4周。苏木精-伊红(HE)染色和马松三色(Masson)染色观察各组小鼠肺组织病理学变化及胶原纤维沉积情况,酶联免疫吸附法(ELISA)检测各组小鼠肺泡灌洗液中白细胞介素-1β(IL-1β)、IL-6、IL-18及肿瘤坏死因子-α(TNF-α)含量,免疫荧光染色观察各组小鼠肺组织内核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)荧光表达,蛋白质免疫印记(Western blotting)检测各组小鼠肺组织中HMGB1及NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶-1(Caspase-1)、gasdermin D(GSDMD)的蛋白表达水平。结果与对照组比较,MRL/lpr组小鼠肺组织呈现严重病理损伤症状,胶原纤维沉积面积显著增加(P<0.05),肺泡灌洗液中IL-1β、IL-6、IL-18、TNF-α含量显著升高(P<0.05),肺组织内NLRP3平均荧光强度显著增加(P<0.05),HMGB1蛋白相对表达量及NLRP3、ASC、Caspase-1、GSDMD蛋白相对表达量均显著上调(P<0.05);与MRL/lpr组比较,MRL/lpr+anti-HMGB1组和MRL/lpr+MCC950组小鼠肺组织损伤得到明显改善,胶原纤维沉积面积显著减少(P<0.05),肺泡灌洗液中IL-1β、IL-6、IL-18、TNF-α含量显著降低(P<0.05),肺组织内NLRP3平均荧光强度显著减小(P<0.05),同时,肺组织中HMGB1蛋白相对表达量及NLRP3、ASC、Caspase-1、GSDMD蛋白相对表达量均显著下调(P<0.05)。结论HMGB1中和抗体能够改善SLE模型小鼠肺损伤,该作用可能是通过抑制细胞焦亡实现的。

Objective To investigate the effect of neutralizing antibody of high mobility group protein 1(HMGB1)on lung injury in systemic lupus erythematosus(SLE)mice and the underlying mechanism.Methods Thirty MRL/lpr mice were randomly divided into 3 groups:MRL/lpr group,MRL/lpr+anti-HMGB1 group and MRL/lpr+MCC950 group,with 10 mice in each group,and another 10 wild-type C57BL/6 mice were taken as the control group.Mice in each group were administered for 4 weeks.Hematoxylin-eosin(HE)staining and Masson staining were used to observe the lung histopathological changes and collagen fiber deposition in each group.The levels of interleukin-1β(IL-1β),IL-6,IL-18 and tumor necrosis factor-α(TNF-α)in alveolar lavage fluid of mice in each group were detected by enzyme-linked immunosorbent assay(ELISA).The fluorescence expression of nucleotide-binding oligomerized domain-like receptor protein 3(NLRP3)in lung tissues of mice in each group was observed by immunofluorescence staining.The expression levels of HMGB1 and NLRP3,apoptosis-related spect-like protein containing a CARD(ASC)protein,Caspase-1,gasdermin D(GSDMD)in mice lung tissues were detected by Western blotting.Results The lung tissues in the MRL/lpr group showed serious pathological injury symptoms,the collagen fiber deposition area was significantly increased(P<0.05),and the levels of IL-1β,IL-6,IL-18 and TNF-αin alveolar lavage fluid were significantly increased,when compared with those in the control group(P<0.05).In addition,the mean fluorescence intensity of NLRP3 in lung tissues in the MRL/lpr group was significantly increased(P<0.05),and the relative protein expression levels of HMGB1 and NLRP3,ASC,Caspase-1,GSDMD were significantly up-regulated when compared with those in the control group(P<0.05).The lung tissue injuries in the MRL/lpr+anti-HMGB1 group and the MRL/lpr+MCC950 group were significantly improved,and the collagen fiber deposition area was significantly reduced,when compared with those in the MRL/lpr group(P<0.05).In addition,the levels of IL-1β,IL-6,IL-18 and TNF-αin alveolar lavage fluid in the MRL/lpr+anti-HMGB1 group and the MRL/lpr+MCC950 group were significantly decreased(P<0.05),the mean fluorescence intensity of NLRP3 in lung tissues was significantly decreased(P<0.05),and the relative protein expression levels of HMGB1 and NLRP3,ASC,Caspase-1,GSDMD in lung tissues were significantly down-regulated,when compared with those in the control group(P<0.05).Conclusion HMGB1 neutralizing antibody can ameliorate lung injury in mice with SLE,which may be achieved by inhibiting pyroptosis.