Abstract：
This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.
摘要：
本研究旨在探讨淫羊藿水提物对博莱霉素(BLM)诱导小鼠肺纤维化模型的干预作用,从而为肺纤维化的临床治疗提供数据支持。90只雄性C57BL/6N小鼠随机分为正常小鼠（n=10），模型(BLM，n=20),吡非尼酮(PFD,270mg·kg~(-1),n=15),和低，medium-,和大剂量龙骨提取物(1.67g·kg~(-1)，n=15;3.33g·kg~(-1),n=15;6.67g·kg~(-1),n=15)组。除正常组外,其余5组均采用气管内滴注BLM(5mg·kg~(-1))建立肺纤维化模型,用等量的生理盐水处理。在建模后的第二天，每组用相应药物灌胃治疗21天.在此期间,计算小鼠的存活率。灌胃后,计算肺指数，苏木精-伊红(HE)和Masson染色观察肺组织的形态和胶原沉积,分别。通过流式细胞术测量肺细胞悬浮液中的活性氧（ROS）水平。谷胱甘肽过氧化物酶（GSH-Px）的水平，总超氧化物歧化酶（T-SOD），测定肺组织中丙二醛(MDA)含量。末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）用于检查肺组织细胞的凋亡。白细胞介素-6（IL-6）的含量，趋化因子C-C基序配体2（CCL-2），基质金属蛋白酶-8(MMP-8),转化生长因子-β1（TGF-β1），α-平滑肌肌动蛋白(α-SMA)，E-cadherin,胶原蛋白Ⅰ,采用酶联免疫吸附试验(ELISA)检测肺组织中的纤连蛋白。F4/80、Ly-6G的表达水平,免疫组化法检测肺组织中TGF-β1和Ⅰ型胶原的表达。通过qRT-PCR检测肺组织中CCL-2、IL-6和MMP-7的mRNA水平。碱性水解法测定肺组织中羟脯氨酸(HYP)的含量。免疫荧光法检测α-SMA和E-cadherin的表达,和α-SMA的蛋白质水平，波形蛋白,通过Western印迹测定肺组织中的E-钙黏着蛋白。结果表明，龙骨水提物提高了成活率，降低肺指数，减轻了病理损伤，胶原蛋白沉积，和肺组织中的氧化应激，并减少凋亡细胞。此外，矢状芽孢杆菌的水提取物下调了肺组织中F4/80和Ly-6G的蛋白质水平以及CCL-2，IL-6和MMP-7的mRNA水平，降低了肺泡灌洗液中IL-6，CCL-2和MMP-8的含量。此外,它降低了HYP的水平,TGF-β1，α-SMA，胶原蛋白Ⅰ,纤连蛋白,还有波形蛋白,并提高了肺组织中E-cadherin的水平。龙骨水提物可抑制胶原沉积,缓解氧化应激，并通过调节与上皮间质转化相关的分子的表达来减少炎症反应，从而减轻博来霉素诱导的小鼠肺纤维化的症状。
