【摘要】 目的 探讨高迁移率族蛋白1(HMGB1)介导细胞焦亡参与肺动脉高压(PAH)小鼠肺血管重构的机制。方法 将40只SD小鼠分为对照组、PAH组、PAH+HMGB1中和抗体(HMGB1 Ab)组、PAH+焦亡抑制剂(NSA)组,除对照组外的其余3组均采用低氧处理建立PAH模型,PAH+HMGB1 Ab组和PAH+NSA组再分别给予HMGB1 Ab、NSA处理,结束后,检测各组小鼠平均肺动脉压(m PAP)、右心室肥厚指数(RVHI),苏木精-伊红染色观察各组小鼠肺组织病理学变化情况并计算肺动脉血管壁厚度百分比(WT)及肺动脉管壁面积百分比(WA)。酶联免疫吸附试验检测各组小鼠血清中白细胞介素(IL)-1β、IL-18水平。免疫组化染色观察各组小鼠肺组织中消皮素D(GSDMD)表达。实时荧光定量PCR和蛋白质免疫印迹检测各组小鼠肺组织中HMGB1及核苷酸结合寡聚化结构域样受体蛋白3(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)、GSDMD表达。结果 与对照组[(1.81±0.19)k Pa、(0.27±0.03)]比较,PAH组小鼠m PAP和RVHI[(3.97±0.41)k Pa、(0.41±0.04)]显著升高,差异有统计学意义(P<0.05)。与对照组比较,PAH组肺动脉血管壁明显增厚,血管平滑肌细胞增殖肥大;PAH+HMGB1 Ab组和PAH+NSA组肺动脉血管壁增厚程度较PAH组明显减轻。PAH组小鼠WT和WA[(42.06±4.38)%、(50.56±5.24)%]显著高于对照组[(23.64±2.46)%、(25.12±2.63)%],差异有统计学意义(P<0.05)。与对照组[(23.56±2.48)pg/m L、(22.68±2.32)pg/m L]比较,PAH组小鼠血清中IL-1β、IL-18水平[(94.51±9.62)pg/m L、(58.21±5.97)pg/m L]显著增加,差异有统计学意义(P<0.05)。与PAH组[(48.57±5.02)%]比较,PAH+HMGB1 Ab组和PAH+NSA组肺组织中GSDMD阳性率[(16.52±1.76)%、(14.62±1.59)%]显著降低,差异有统计学意义(P<0.05)。PAH组小鼠肺组织中HMGB1、NLRP3、ASC、Caspase-1、GSDMD蛋白表达显著高于对照组(P<0.05)。结论 HMGB1中和抗体能够抑制细胞焦亡从而降低PAH小鼠肺动脉压力,改善肺血管重构。更多还原

【Abstract】 Objective To investigate the mechanism of high mobility group protein 1(HMGB1)-mediated pyroptosis involved in pulmonary vascular remodeling in mice with pulmonary arterial hypertension(PAH).Methods Forty SD mice were divided into control group,PAH group,PAH+HMGB1 neutralizing antibody(HMGB1 Ab) group and PAH+ necronecroamide(NSA) group.Except the control group,the remaining 3 groups were treated with hypoxia to establish PAH model.PAH+HMGB1 Ab group and PAH+NSA group were treated with HMGB1 Ab and NSA respectively.After the treatment,the mean pulmonary artery pressure(m PAP) and right ventricular hypertrophy index(RVHI) of mice in each group were detected.Hematoxylin-eosin staining was used to observe the pulmonary histopathological changes and calculate the percentage of pulmonary artery wall thickness(WT) and the percentage of pulmonary artery wall area(WA).The serum levels of interleukin(IL)-1β and IL-18 were detected by enzyme-linked immunosorbent assay.The expression of gasdermin D(GSDMD) in lung tissues of mice in each group was observed by immunohistochemical staining.Real-time fluorescence quantitative PCR and western blot were used to detect the expression of HMGB1,nucleotide-binding oligomerized domain-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),Cysteinyl aspartate-specific proteinase-1(Caspase-1),and GSDMD in lung tissues of mice in each group.Results Compared with control group[(1.81±0.19)k Pa,(0.27±0.03)],m PAP and RVHI in PAH group [(3.97±0.41)k Pa,(0.41±0.04)]were significantly increased,and the difference was statistically significant(P<0.05).Compared with the control group,the pulmonary artery wall of PAH group was significantly thickened,and the vascular smooth muscle cells proliferated and hypertrophy.The degree of pulmonary artery wall thickening in PAH+ HMGB1Ab and PAH+NSA groups was significantly reduced compared with PAH group.WT and WA in PAH group[(42.06±4.38)%,(50.56±5.24)%] were significantly higher than those in control group[(23.64±2.46)%,(25.12±2.63)%],the difference was statistically significant(P<0.05).Compared with control group[(23.56±2.48)pg/m L,(22.68±2.32)pg/m L],serum levels of IL-1β and IL-18 in PAH group [(94.51±9.62)pg/m L,(58.21±5.97)pg/m L] were significantly increased,and the difference was statistically significant(P<0.05).Compared with PAH group[(48.57±5.02)%],the positive rate of GSDMD in PAH+ HMGB1Ab and PAH+NSA groups [(16.52±1.76)%,(14.62±1.59)% ]was significantly decreased,and the difference was statistically significant(P<0.05).The protein expression of HMGB1,NLRP3,ASC,Caspase-1 and GSDMD in lung tissues of PAH group were significantly higher than those of control group(P<0.05).Conclusion HMGB1 neutralizing antibody can inhibit pyroptosis of PAH mice,thereby reducing pulmonary artery pressure and improving pulmonary vascular remodeling.更多还原