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北京大学学报（医学版） ›› 2024, Vol. 56 ›› Issue (6): 963-971. doi: 10.19723/j.issn.1671-167X.2024.06.004

• 论著 • 上一篇    下一篇

褪黑素调控节律基因表达及其缓解间质性肺纤维化的机制

李炳乐1, 朱凌妍2, 王永福2,*(), 白力3,4,*()   

  1. 1. 内蒙古科技大学包头医学院，内蒙古包头 014010
    2. 包头医学院第一附属医院风湿免疫科，内蒙古包头 014010
    3. 包头医学院第一附属医院中心实验室，内蒙古包头 014010
    4. 内蒙古自体免疫学重点实验室，内蒙古包头 014010
  • 收稿日期:2024-08-02 出版日期:2024-12-18 发布日期:2024-12-18
  • 通讯作者: 王永福,白力 E-mail:wyf18686198868@163.com;182020022@btmc.edu.cn
  • 基金资助:
    内蒙古自治区自然科学基金(2024QN08029);内蒙古自治区自然科学基金(2023MS08052)

Mechanism of melatonin regulating the expression level of rhythm genes to alleviate interstitial pulmonary fibrosis

Bingle LI1, Lingyan ZHU2, Yongfu WANG2,*(), Li BAI3,4,*()   

  1. 1. Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014010, Inner Mongolia, China
    2. Department of Rheumatology and Immunology, the First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia, China
    3. The Central Lab, the First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia, China
    4. Inner Mongolia Autoimmune Key Laboratory, Baotou 014010, Inner Mongolia, China
  • Received:2024-08-02 Online:2024-12-18 Published:2024-12-18
  • Contact: Yongfu WANG, Li BAI E-mail:wyf18686198868@163.com;182020022@btmc.edu.cn
  • Supported by:
    Natural Science Foundation of Inner Mongolia Autonomous Region(2024QN08029);Natural Science Foundation of Inner Mongolia Autonomous Region(2023MS08052)

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摘要：

目的: 研究褪黑素(melatonin，MT)干预肺纤维化患者节律基因表达，并分析其缓解肺纤维化疾病进展的机制。方法: 通过高通量基因表达数据库(Gene Expression Omnibus，GEO)筛选肺纤维化患者与健康对照组之间差异表达的生物钟基因，分析节律基因与肺功能及肺纤维化相关基因之间的相关性。构建博来霉素(bleomycin，BLM)诱导小鼠肺纤维化模型，通过测序及免疫组织化学染色观察BLM组以及使用MT干预后(BLM+MT组)肺组织PER2、CRY2表达的差异，通过HE染色及Masson染色观察MT对肺纤维化的影响。用Western blot检测转化生长因子β(transforming growth factor β，TGF-β)诱导肺上皮细胞P-smad2/3的表达，采用实时荧光定量逆转录PCR技术，探究对照组、TGF-β组、TGF-β+MT组生物钟基因的节律表达变化，最后通过MT受体阻滞剂luzindole干预TGF-β+MT组的肺上皮细胞，通过Western blot探究MT减轻TGF-β诱导上皮-间质转化的受体依赖性。结果: (1) 通过对GEO数据集的分析发现，节律基因PER2和CRY2与TGF-β的表达存在负相关性，且与患者肺功能指标存在正相关性。(2)小鼠肺组织转录组测序分析发现BLM组PER2和CRY2的表达较正常组明显减少。同时，病理染色结果显示正常组小鼠肺组织结构完整清晰，肺泡间隔薄；BLM组中肺组织出现大量胶原纤维增生，肺泡结构紊乱；与BLM组相比，BLM+MT组胶原纤维增生及炎性细胞浸润减少；免疫组化染色结果提示BLM组PER2和CRY2的表达量比正常组降低，BLM+MT组比BLM组增加。(3)肺上皮细胞体外TGF-β干预实验结果表明，相较于对照组，TGF-β组P-smad2/3表达量增加，MT干预抑制了TGF-β对P-smad2/3的诱导作用，而MT受体阻滞剂干预又逆转了这一现象，说明MT可以抑制TGF-β通路激活，且该过程存在MT受体依赖性。(4)肺上皮细胞48 h节律实验结果显示，TGF-β+MT组中PER2和CRY2的mRNA节律接近24 h，且有向对照组节律恢复的趋势，而在加入MT受体阻断剂后，其节律的时程和振幅均趋向于TGF-β组。结论: MT通过与其受体结合，可以恢复正常生物钟基因PER2和CRY2的周期性表达，进而抑制TGF-β经典通路的激活，抑制肺纤维化上皮-间质转化病理进程。这一发现为肺纤维化的治疗提供了新的分子靶点和潜在的治疗策略。

关键词: 褪黑素, 节律基因, 肺纤维化

Abstract:

Objective: To investigate the intervention of melatonin (MT) in the expression of circadian genes in patients with pulmonary fibrosis and to analyze the mechanism by which it alleviates the progression of pulmonary fibrosis. Methods: By utilizing the Gene Expression Omnibus (GEO) database, we identified differentially expressed circadian genes between patients with pulmonary fibrosis and controls. We analyzed the correlation between circadian genes and pulmonary function as well as genes related to pulmonary fibrosis. A bleomycin-induced mouse model of pulmonary fibrosis (BLM group) was constructed to observe the expression differences of PER2 and CRY2 by sequencing and immunohistochemical staining in the BLM group and after MT intervention (BLM+MT group). Hematoxylin and eosin (HE) staining and Masson staining were used to observe the effects of MT on fibrosis. We used Western blot to detect the expression of P-smad2/3 in lung epithelial cells induced by transforming growth factor β (TGF-β). Reverse transcription quantitative real-time PCR technology was employed to investigate the rhythmic expression changes of circadian genes in the control group, TGF-β group, and TGF-β+MT group. Finally, luzindole, a MT receptor antagonist, was used to intervene in TGF-β+MT group, and Western blot was used to explore the receptor dependence of MT in alleviating TGF-β-induced epithelial-mesenchymal transition. Results: (1) Analysis of the GEO dataset (GSE) revealed a negative correlation between circadian genes PER2 and CRY2 and the expression of TGF-β, and a positive correlation with pulmonary function indicators in patients. (2) Transcriptome sequencing analysis of lung tissue in BLM group found that the expression of PER2 and CRY2 was significantly reduced compared with the normal group. Histopathological staining results showed that the lung tissue structure of the normal group was intact and clear, with thin alveolar septa; in the BLM group, there was a large increase in collagen fibers and disordered alveolar structure; compared with the BLM group, the BLM+MT group had reduced collagen fiber proliferation and inflammatory cell infiltration; the expression of PER2 and CRY2 in the BLM group was lower than in the normal group, and the expression in the BLM+MT group was increased compared with the BLM group. (3) In vitro lung epithelial cell experiments with TGF-β intervention showed that compared with the control group, the expression of P-smad2/3 increased in the TGF-β group, and MT intervention inhibited the inducing effect of TGF-β on P-smad2/3, while intervention with the MT receptor antagonist reversed this phenomenon. The results indicated that MT could inhibit the activation of the TGF-β pathway, and this process was dependent on MT receptors. (4) The 48-hour rhythm experiment in lung epithelial cells showed that the mRNA rhythm of PER2 and CRY2 in the TGF-β+MT group was close to 24 hours and showed a trend towards restoring the rhythm of the control group, while the addition of the MT receptor blocker tended to make the rhythm duration and amplitude of both groups approach that of the TGF-β group. Conclusion: MT, by binding to its receptors, can restore the periodic expression of the circadian genes PER2 and CRY2, thereby inhibiting the activation of the TGF-β classical pathway and suppressing the pathological process of epithelial-mesenchymal transition in pulmonary fibrosis. This finding provides new molecular targets and potential therapeutic strategies for the treatment of pulmonary fibrosis.

Key words: Melatonin, Rhythm genes, Pulmonary fibrosis

中图分类号: 

  • R563

表1

RT-qPCR扩增引物列表"

Gene Primer sequence (5′-3′)
GAPDH Forward: GGAGCGAGATCCCTCCAAAAT
Reverse: GGCTGTTGTCATACTTCTCATGG
PER2 Forward: GACATGAGACCAACGAAAACTGC
Reverse: AGGCTAAAGGTATCTGGACTCTG
CRY2 Forward: GCTAGAGTGACGGAGATGCC
Reverse: TCAGGAGTCCTTGCTTGCTG

图1

GSE47460数据集中对照组与普通型间质性肺炎组节律基因的表达差异及与肺纤维化基因的相关性分析"

图2

节律基因PER2 (A~C)和CRY2 (D~F)与肺功能指标的相关性"

图3

正常组和BLM组小鼠测序基因差异分析的热图及火山图"

图4

正常组、BLM组、BLM+MT组小鼠肺组织的HE及Masson染色"

图5

各组小鼠肺组织PER2及CRY2的免疫组织化学结果"

图6

肺上皮细胞对照组、TGF-β组以TGF-β+MT组中smad2/3及P-smad2/3的表达差异"

图7

肺上皮细胞系各组标志物变化(A)及PER2 (B)、CRY2 (C)mRNA的48 h节律变化"

图8

肺上皮细胞系中TGF-β组、TGF-β+MT组和MT受体阻断剂组PER2和CRY2 mRNA的48 h节律拟合曲线"

图9

Western blot检测对照组、TGF-β组、TGF-β+MT组和MT受体阻断剂组肺上皮细胞系中Col-1及E-cad蛋白表达差异"

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ISSN 1671-167X
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