哮喘小鼠HMGB1/TLR4/NF-κB信号通路及维生素D的作用

HMGB1/TLR4/NF-κB signaling pathway and role of vitamin D in asthmatic mice

摘要:

目的 观察哮喘小鼠肺组织高迁移率族蛋白B1（HMGB1）、Toll样受体4（TLR4）和核因子κB（NF-κB）mRNA和蛋白表达变化，以及维生素D的干预作用。方法 将48只BALB/c小鼠随机分为对照组、哮喘组和1,25- (OH)2D3干预组，每组16只。建立哮喘动物模型，干预组在每次雾化激发前给予腹腔注射1,25- (OH)2D3（4 μg/kg）。分别在雾化激发1周和2周采集各组标本，常规苏木精-伊红（HE）染色测定气道壁厚度；免疫组化染色观察HMGB1、TLR4和NF-κB在肺组织中的表达；采用实时荧光定量PCR和Western blot分别从mRNA和蛋白质水平检测HMGB1、TLR4和NF-κB表达变化。结果 雾化激发1周和2周，哮喘组小鼠气道壁厚度较对照组明显增加，干预组较哮喘组明显减少（P < 0.05）；哮喘组肺组织中HMGB1、TLR4和NF-κB的mRNA表达量均明显高于对照组，干预组TLR4和NF-κB 的mRNA均明显低于哮喘组（P < 0.05）。雾化激发1周时干预组HMGB1 mRNA表达量与哮喘组比较差异无统计学意义（P > 0.05），但在雾化激发2周时则明显下降（P < 0.05）。雾化激发1周和2周，肺组织中HMGB1、TLR4和NF-κB的蛋白表达量均明显高于对照组，干预组均明显低于哮喘组（P < 0.05）。小鼠气道壁厚度与肺组织HMGB1、TLR4和NF-κB的mRNA的表达量均呈正相关性（r分别为0.804、0.895、0.834；P < 0.05）。结论 HMGB1/TLR4/NF-κB信号通路在哮喘发病中起重要作用，适量1,25- (OH)₂D₃可能通过对其的调控作用阻止哮喘的进展，有望成为哮喘治疗的新选择。

Abstract:

Objective To investigate the changes in the mRNA and protein expression of high-mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), and nuclear factor-kappa B (NF-κB) in lung tissues of asthmatic mice and the interventional effect of vitamin D. Methods A total of 48 BALB/c mice were randomly divided into control group, asthma group, and 1,25-(OH)₂D₃ intervention group, with 16 mice in each group. An animal model of asthma was established, and lung tissue samples were taken in each group at weeks 1 and 2 of ovalbumin challenging. Conventional hematoxylin-eosin staining was used to measure airway wall thickness. Immunohistochemical staining was used to observe the expression of HMGB1, TLR4, and NF-κB in lung tissues. Quantitative real-time PCR and Western blot were used to investigate the changes in the mRNA and protein expression of HMGB1, TLR4, and NF-κB. Results At weeks 1 and 2 of ovalbumin challenging, compared with the control group, the asthma group had a significant increase in airway wall thickness and the intervention group had a significant reduction compared with the asthma group (P < 0.05). The asthma group had significantly higher mRNA expression of HMGB1, TLR4, and NF-κB in lung tissues than the control group, and the intervention group had significantly lower mRNA expression of TLR4 and NF-κB than the asthma group (P < 0.05). At week 1 of ovalbumin challenging, there was no significant difference in the mRNA expression of HMGB1 between the intervention group and the asthma group (P > 0.05). At week 2, the intervention group had a significant reduction in the mRNA expression of HMGB1 compared with the asthma group (P < 0.05). At weeks 1 and 2 of ovalbumin challenging, the asthma group had significantly higher protein expression of HMGB1, TLR4, and NF-κB in lung tissues than the control group, and the intervention group had significantly lower expression than the asthma group (P < 0.05). Airway wall thickness was positively correlated with the mRNA expression of HMGB1, TLR4, and NF-κB in lung tissues (r=0.804, 0.895, and 0.834; P < 0.05). Conclusions The HMGB1/TLR4/NF-κB signaling pathway plays an important role in the pathogenesis of asthma, and an appropriate amount of 1,25-(OH)₂D₃ has a regulatory effect on this pathway and may prevent the progression of asthma. Therefore, 1,25-(OH)₂D₃ is expected to become a new choice for the treatment of asthma.