Abstract：
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a persistent and advanced pulmonary ailment. The roles of innate immunity and adaptive immunity are pivotal in the evolution of IPF. An ill-adjusted interaction between epithelial cells and immune cells is responsible for initiating the epithelial-mesenchymal transition (EMT) process and sustaining chronic inflammation, thereby fostering fibrosis progression. The intricacy of IPF pathogenesis has hindered the availability of efficacious agents. Elephantopus scaber Linn. (ESL) is a canonical Chinese medicine with significant immunoregulatory effects, and its aqueous extract has been proven to attenuate IPF symptoms in bleomycin (BLM)-induced mice. However, the underlying mechanism through which ESL relieves IPF remains unclear.
OBJECTIVE: To validate whether ESL reverses IPF by mediating the immune response and EMT.
METHODS: Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and UPLC were used to identify the components and determine the concentrations of the specific compounds in the ESL. Network pharmacology and molecular docking were applied to predict the potential mechanism underlying the anti-IPF effect of ESL. BLM-induced IPF mice were used to validate the anti-IPF effect of ESL, and lung tissue was collected to test putative pathways involved in inflammation and EMT via immunohistochemistry (ICH), real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.
RESULTS: Sixty-one compounds were identified, and thirteen main ingredients were quantified in the ESL. In silico experiments predicted that the IPF-mediated reversal of adverse effects by ESL would be related to interruption of the Toll-like receptor 4 (TLR4)/nuclear factor-k-gene binding (NF-ĸB) inflammatory pathway and the transforming growth factor-beta l (TGF-β1)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O3 (FOXO3a) fibrosis pathway. In vivo experiments showed that ESL alleviates BLM-induced lung inflammation and fibrosis by reducing neutrophil aggregation and fibroblast foci, similar to the effects of the positive control drug pirfenidone (PFD). ESL markedly inhibited the transcription of TNF-α, IL-1β, and IL-6, which are downstream genes of the NF-κB signaling pathway. Furthermore, the protein levels of TLR4 and p-NF-κB were correspondingly inhibited in response to ESL treatment. Additionally, ESL reverses BLM-induced changes in the expression of EMT-related biological characteristic indicators (collagen I [COLIA1], E-cadherin, and alpha smooth muscle actin [α-SMA]) at the messenger ribonucleic acid (mRNA) level and markedly inhibits the expression of EMT-related upstream proteins (TGF-β1, p-PI3K, p-Akt, and p-FOXO3a).
CONCLUSIONS: Our research suggested that ESL attenuates BLM-induced IPF through mediating the EMT process via the TGF-β1/PI3K/Akt/FOXO3a signaling pathway and inhibiting inflammation through the TLR4/NF-κB signaling pathway, highlighting that ESL can serve as an immunoregulator for relieving the abnormal immune response and reversing the EMT in IPF.
OBJECTIVE: To validate whether ESL reverses IPF by mediating the immune response and EMT.
METHODS: Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and UPLC were used to identify the components and determine the concentrations of the specific compounds in the ESL. Network pharmacology and molecular docking were applied to predict the potential mechanism underlying the anti-IPF effect of ESL. BLM-induced IPF mice were used to validate the anti-IPF effect of ESL, and lung tissue was collected to test putative pathways involved in inflammation and EMT via immunohistochemistry (ICH), real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.
RESULTS: Sixty-one compounds were identified, and thirteen main ingredients were quantified in the ESL. In silico experiments predicted that the IPF-mediated reversal of adverse effects by ESL would be related to interruption of the Toll-like receptor 4 (TLR4)/nuclear factor-k-gene binding (NF-ĸB) inflammatory pathway and the transforming growth factor-beta l (TGF-β1)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O3 (FOXO3a) fibrosis pathway. In vivo experiments showed that ESL alleviates BLM-induced lung inflammation and fibrosis by reducing neutrophil aggregation and fibroblast foci, similar to the effects of the positive control drug pirfenidone (PFD). ESL markedly inhibited the transcription of TNF-α, IL-1β, and IL-6, which are downstream genes of the NF-κB signaling pathway. Furthermore, the protein levels of TLR4 and p-NF-κB were correspondingly inhibited in response to ESL treatment. Additionally, ESL reverses BLM-induced changes in the expression of EMT-related biological characteristic indicators (collagen I [COLIA1], E-cadherin, and alpha smooth muscle actin [α-SMA]) at the messenger ribonucleic acid (mRNA) level and markedly inhibits the expression of EMT-related upstream proteins (TGF-β1, p-PI3K, p-Akt, and p-FOXO3a).
CONCLUSIONS: Our research suggested that ESL attenuates BLM-induced IPF through mediating the EMT process via the TGF-β1/PI3K/Akt/FOXO3a signaling pathway and inhibiting inflammation through the TLR4/NF-κB signaling pathway, highlighting that ESL can serve as an immunoregulator for relieving the abnormal immune response and reversing the EMT in IPF.
摘要：
背景：特发性肺纤维化（IPF）是一种持续性和晚期的肺部疾病。先天免疫和适应性免疫的作用在IPF的进化中至关重要。上皮细胞和免疫细胞之间失调的相互作用负责启动上皮-间质转化（EMT）过程并维持慢性炎症，从而促进纤维化进展。IPF发病机制的复杂性阻碍了有效药物的可用性。象形龙疮Linn.(ESL)是一种具有显著免疫调节作用的中药,并且其水性提取物已被证明可减轻博来霉素（BLM）诱导的小鼠的IPF症状。然而,ESL缓解IPF的潜在机制尚不清楚.
目的：验证ESL是否通过介导免疫应答和EMT逆转IPF。
方法：超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF-MS/MS）和UPLC用于鉴定成分并确定ESL中特定化合物的浓度。网络药理学和分子对接被用来预测ESL抗IPF作用的潜在机制。BLM诱导的IPF小鼠用于验证ESL的抗IPF作用。收集肺组织以通过免疫组织化学（ICH）测试参与炎症和EMT的推定途径，实时定量聚合酶链反应（RT-qPCR）和蛋白质印迹。
结果：鉴定出61种化合物，在ESL中定量了13种主要成分。在计算机模拟实验中预测，IPF介导的ESL逆转不良反应将与Toll样受体4（TLR4）/核因子-k基因结合（NF-κB）炎症途径的中断和转化生长因子-β1（TGF-β1）/磷酸肌醇3激酶（PI3K）/蛋白激酶B（Akt）/叉头O3盒（FOXO3a）纤维化途径。体内实验表明，ESL通过减少中性粒细胞聚集和成纤维细胞病灶来减轻BLM诱导的肺部炎症和纤维化，与阳性对照药物吡非尼酮（PFD）的效果相似。ESL显著抑制TNF-α的转录,IL-1β,和IL-6，它们是NF-κB信号通路的下游基因。此外，对ESL治疗的反应,TLR4和p-NF-κB的蛋白水平受到相应抑制.此外,ESL逆转了BLM诱导的EMT相关生物学特性指标表达的变化（胶原蛋白I[COLIA1]，E-cadherin,和α平滑肌肌动蛋白[α-SMA]）在信使核糖核酸（mRNA）水平，并显着抑制EMT相关上游蛋白的表达（TGF-β1，p-PI3K，p-Akt,和p-FOXO3a）。
结论：我们的研究表明，ESL通过TGF-β1/PI3K/Akt/FOXO3a信号通路介导EMT过程，通过TLR4/NF-κB信号通路抑制炎症，减轻BLM诱导的IPF，强调ESL可以作为缓解IPF中异常免疫反应和逆转EMT的免疫调节剂。
目的：验证ESL是否通过介导免疫应答和EMT逆转IPF。
方法：超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF-MS/MS）和UPLC用于鉴定成分并确定ESL中特定化合物的浓度。网络药理学和分子对接被用来预测ESL抗IPF作用的潜在机制。BLM诱导的IPF小鼠用于验证ESL的抗IPF作用。收集肺组织以通过免疫组织化学（ICH）测试参与炎症和EMT的推定途径，实时定量聚合酶链反应（RT-qPCR）和蛋白质印迹。
结果：鉴定出61种化合物，在ESL中定量了13种主要成分。在计算机模拟实验中预测，IPF介导的ESL逆转不良反应将与Toll样受体4（TLR4）/核因子-k基因结合（NF-κB）炎症途径的中断和转化生长因子-β1（TGF-β1）/磷酸肌醇3激酶（PI3K）/蛋白激酶B（Akt）/叉头O3盒（FOXO3a）纤维化途径。体内实验表明，ESL通过减少中性粒细胞聚集和成纤维细胞病灶来减轻BLM诱导的肺部炎症和纤维化，与阳性对照药物吡非尼酮（PFD）的效果相似。ESL显著抑制TNF-α的转录,IL-1β,和IL-6，它们是NF-κB信号通路的下游基因。此外，对ESL治疗的反应,TLR4和p-NF-κB的蛋白水平受到相应抑制.此外,ESL逆转了BLM诱导的EMT相关生物学特性指标表达的变化（胶原蛋白I[COLIA1]，E-cadherin,和α平滑肌肌动蛋白[α-SMA]）在信使核糖核酸（mRNA）水平，并显着抑制EMT相关上游蛋白的表达（TGF-β1，p-PI3K，p-Akt,和p-FOXO3a）。
结论：我们的研究表明，ESL通过TGF-β1/PI3K/Akt/FOXO3a信号通路介导EMT过程，通过TLR4/NF-κB信号通路抑制炎症，减轻BLM诱导的IPF，强调ESL可以作为缓解IPF中异常免疫反应和逆转EMT的免疫调节剂。
