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.
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 progres-sion 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 construc-ted 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.Fi-nally,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-mesen-chymal transition.Results:(1)Analysis of the GEO dataset(GSE)revealed a negative correlation be-tween circadian genes PER2 and CRY2 and the expression of TGF-β,and a positive correlation with pul-monary 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 in-tact 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 col-lagen 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 com-pared 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 re-ceptor 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 experi-ment 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 ad-dition of the MT receptor blocker tended to make the rhythm duration and amplitude of both groups ap-proach 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 classi-cal pathway and suppressing the pathological process of epithelial-mesenchymal transition in pulmonary fi-brosis.This finding provides new molecular targets and potential therapeutic strategies for the treatment of pulmonary fibrosis.
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