桔梗皂苷D对支原体肺炎小鼠肺组织转化生长因子-β1、表皮生长因子表达的影响

Effect of Platycodin D on the expression of transforming growth factor-β1 and epidermal growth factor in lung tissue of mice with mycoplasma pneumonia

摘要:

目的 通过抑制纤维因子的表达，探究桔梗皂苷D对支原体肺炎的治疗机制。方法 将桔梗皂苷D与转化生长因子-β₁（TGF-β₁）进行结合能力测试，采用Biacore软件进行桔梗皂苷D与TGF-β₁的亲和力分析，并进行分子对接模拟实验。将雄性SPF级BALB/c小鼠60只分为对照组、模型组、阿奇霉素组、桔梗皂苷D高、中、低剂量组，每组10只。采用滴鼻法复制小鼠支原体肺炎模型，小鼠麻醉后经鼻滴入20 μL含1×10⁶ ccu/mL肺炎支原体的悬液，对照组采用等体积生理盐水滴鼻，1次/d，持续3 d。桔梗皂苷D高、中、低剂量组小鼠分别给予桔梗皂苷D 10.0、5.0、2.5 g/（kg·d），阿奇霉素组小鼠给予32.0 mg/（kg·d）的阿奇霉素，对照组和模型组小鼠则给予等体积的生理盐水，连续给药7 d。制备小鼠肺组织病理切片，采用苏木精-伊红（HE）染色和Masson染色观察肺组织的病理学变化。采用免疫组织化学染色（IHC）检测小鼠肺组织TGF-β₁和表皮生长因子（EGF）的蛋白阳性表达。采用实时荧光定量聚合酶链反应（qRT-PCR）检测TGF-β₁、EGF mRNA的表达。结果 Biacore分子互作和分子对接结果显示，桔梗皂苷D在体外能够与TGF-β₁蛋白进行特异性结合。各组小鼠肺组织的病理学分析显示，与模型组相比，桔梗皂苷D各剂量组小鼠肺组织中仅见少量肺泡壁充血，炎症细胞浸润显著减少，其组织结构与对照组和阿奇霉素组相似，且桔梗皂苷D高剂量组的炎症细胞浸润最少、肺泡壁充血范围最小。各组小鼠肺组织纤维化病理结果显示，与模型组相比，桔梗皂苷D各剂量组的胶原纤维均减少，与对照组和阿奇霉素组的肺组织相似，且桔梗皂苷D高、中剂量组的胶原纤维显著减少、仅有少量肺泡壁增厚。与对照组比较，模型组TGF-β₁阳性表达升高（P < 0.05）；与模型组比较，桔梗皂苷D高、中、低剂量组TGF-β₁阳性表达均减少（P < 0.05）。与对照组比较，模型组EGF蛋白阳性表达降低（P < 0.05）；与模型组比较，桔梗皂苷D高、中剂量组EGF蛋白阳性表达均增多（P < 0.05）。与对照组比较，模型组TGF-β₁ mRNA相对表达量升高（P < 0.05）；与模型组相比，桔梗皂苷D高、中剂量组的TGF-β₁ mRNA相对表达量降低（P < 0.05）。与对照组比较，模型组EGF mRNA相对表达量降低（P < 0.05）；与模型组相比，桔梗皂苷D各给药组的EGF mRNA相对表达量均有不同程度升高（P < 0.05）。结论 桔梗皂苷D能够有效改善肺炎支原体肺炎的肺间质纤维化，其作用机制可能是通过降低TGF-β₁表达、提高EGF表达来抑制上皮间质转化的发生。

Abstract:

Objective To investigate the therapeutic mechanism of Platycodin D in mycoplasma pneumonia by inhibiting the expression of fibrosis-related factors.Methods The binding affinity between Platycodin D and transforming growth factor-β₁ (TGF-β₁) was analyzed by surface plasmon resonance (SPR) using a Biacore system, and molecular docking simulations were performed. Sixty male SPF-grade BALB/c mice were divided into six groups (10/group): control, model, azithromycin, and high/medium/low-dose Platycodin D. The mycoplasma pneumonia model was established via intranasal instillation of 20 μL suspension containing 10⁶ ccu/mL Mycoplasma pneumoniae. Treatments were administered for 7 days. Lung tissues were subjected to histopathological analysis (HE and Masson staining), immunohistochemical staining for TGF-β₁ and EGF protein, and qRT-PCR for mRNA expression.Results The results of Biacore molecular interaction and molecular docking showed that Platycodin D could specifically bind to TGF-β₁ protein in vitro. The pathological analysis of the lung tissues of mice in each group showed that, compared with the model group, only a small amount of congestion in the alveolar walls was observed in the lung tissues of mice in each dose group of Platycodin D, and the infiltration of inflammatory cells was significantly reduced. The tissue structure was similar to that of the control group and the azithromycin group, and the high-dose group of Platycodin D had the least infiltration of inflammatory cells and the smallest range of alveolar wall congestion. The pathological results of pulmonary fibrosis in the lung tissues of mice in each group showed that, compared with the model group, the collagen fibers in each dose group of Platycodin D were reduced, which was similar to the lung tissues of the control group and the azithromycin group. Moreover, the collagen fibers in the high- and medium-dose groups of Platycodin D were significantly reduced, and only a small amount of alveolar wall thickening was observed. Compared with the control group, the positive expression of TGF-β₁ in the model group was increased (P < 0.05); compared with the model group, the positive expressions of TGF-β₁ in the high-, medium-, and low-dose groups of Platycodin D were all decreased (P < 0.05). Compared with the control group, the positive expression of EGF protein in the model group was decreased (P < 0.05); compared with the model group, the positive expressions of EGF protein in the high- and medium-dose groups of Platycodin D were both increased (P < 0.05). Compared with the control group, the relative expression level of TGF-β₁ mRNA in the model group was increased (P < 0.05); compared with the model group, the relative expression levels of TGF-β₁ mRNA in the high- and medium-dose groups of Platycodin D were decreased (P < 0.05). Compared with the control group, the relative expression level of EGF mRNA in the model group was decreased (P < 0.05); compared with the model group, the relative expression levels of EGF mRNA in each administration group of Platycodin D were all increased to varying degrees (P < 0.05).Conclusion Platycodin D alleviates pulmonary interstitial fibrosis in mycoplasma pneumonia, potentially by suppressing EMT through TGF-β₁ inhibition and EGF promotion.