Objective

Idiopathic pulmonary fibrosis (IPF) is considered as a chronic interstitial lung disease with underlying mechanism of IPF remaining unclear, while there are no definitive treatment options. In recent years, scientists have gradually paid attention to the influence of angiogenesis on IPF. Because IPF is a progressive with microvascular remodeling disorder, scientists have postulated that angiogenesis may also be one of the initiating and contributing factors of the disease. Bupleurum is a common natural Chinese herbal medicine with antibacterial, anti-inflammatory, anti-tumor and other pharmacological effects. As the most important active monomer of Bupleurum, Saikosaponin-d (SSd) is a new discovery with anti-pulmonary fibrosis (PF) activity. This study attempts to investigate the role of SSd in the interference of PF through regulation of angiogenesis in IPF through Angiopoietin (Angpt) /Tie receptor 2 (Tie2) pathway.

Methods

Randomly, we allocated C57BL/6 mice into four groups (n = 20 in each group). Afterwards, establishment of IPF model was accomplished via intratracheal administration of bleomycin (BLM, 5 mg/kg), while corresponding drug intervention was given accordingly. On 3rd, 7th, 14th and 28th days after modeling, we performed histopathological examination through staining. Meanwhile, immunohistochemistry (IHC) of PF and the expression of related factors were observed, while Ang/Tie2 pathway was assessed by ELISA with the effect of SSd on angiogenesis related proteins in IPF being explored with IHC and Western Blot technique.

Results

Our results showed that SSd could reduce inflammation and PF levels in lung tissue of experimental mice, while levels of angiogenesis-related factors, namely Tie-2, Ang-1 and ANGPT2 (Ang-2), fibrosis- associated factors like Alpha-smooth muscle actin (α-SMA), collagen-I and hydroxyproline in SSd and dexamethasone (DXM) mice were significantly reduced at each time point compared to BLM (p < 0.01). Additionally, we discovered substantial decreased expressions of Ang-1, Ang-2, Tie-2, α-SMA and collagen-I at protein level in SSd and DXM mice at each time point compared to BLM (p < 0.05). Besides, insignificant differences were observed between SSd and DXM groups (p > 0.05).

Conclusion

This study has demonstrated that SSd could down-regulate the expression of ANG-1, Ang-2 and Tie2 in the Ang/Tie2 pathway, and may reduce lung inflammation and PF in BLM-induced mice via inhibition of angiogenesis.

中文翻译：

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柴胡皂苷-d 通过血管生成素/Tie-2 途径调节特发性肺纤维化的血管生成

客观的

特发性肺纤维化（IPF）被认为是一种慢性间质性肺疾病，其潜在机制尚不清楚，同时也没有明确的治疗方案。近年来，科学家逐渐关注血管生成对IPF的影响。由于 IPF 是一种进行性微血管重塑疾病，科学家推测血管生成也可能是该疾病的引发和促成因素之一。柴胡是一种常见的天然中草药，具有抗菌、抗炎、抗肿瘤等药理作用。作为柴胡最重要的活性单体，柴胡皂苷-d（SSd）是具有抗肺纤维化（PF）活性的新发现。本研究试图通过血管生成素（Angpt）/Tie受体2（Tie2）途径调节IPF中的血管生成，探讨SSd在干扰PF中的作用。

方法

我们随机将 C57BL/6 小鼠分为四组（每组 n = 20）。随后气管内给予博莱霉素（BLM，5 mg/kg）建立IPF模型，并给予相应的药物干预。造模后第3、7、14、28天，通过染色进行组织病理学检查。同时观察PF的免疫组织化学(IHC)及相关因子的表达情况，并通过ELISA评估Ang/Tie2通路，并通过IHC和Western Blot技术探讨SSd对IPF中血管生成相关蛋白的影响。

结果

我们的结果表明，SSd 可以降低实验小鼠肺组织中的炎症和 PF 水平，同时降低血管生成相关因子（即 Tie-2、Ang-1 和 ANGPT2 (Ang-2)）以及纤维化相关因子（如 Alpha-smooth）的水平与 BLM 相比，SSd 和地塞米松 (DXM) 小鼠的肌肉肌动蛋白 (α-SMA)、胶原蛋白 I 和羟脯氨酸在每个时间点均显着减少 (p < 0.01)。此外，我们发现与 BLM 相比，SSd 和 DXM 小鼠在每个时间点的蛋白水平上 Ang-1、Ang-2、Tie-2、α-SMA 和胶原蛋白表达显着降低 (p < 0.05)。此外，SSd 组和 DXM 组之间观察到不显着差异 (p > 0.05)。

结论

这项研究表明，SSd 可以下调 Ang/Tie2 通路中 ANG-1、Ang-2 和 Tie2 的表达，并可能通过抑制血管生成来减轻 BLM 诱导的小鼠的肺部炎症和 PF。