隐丹参酮减轻LPS诱导的ARDS小鼠早期肺纤维化及机制研究

Effect and mechanism of cryptotanshinone on early pulmonary fibrosis in LPS-induced acute respiratory distress syndrome in mice

目的 探讨隐丹参酮(cryptotanshinone, CTS)对脂多糖(lipopolysaccharide, LPS)诱导的急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)小鼠的早期肺纤维化的影响及其可能的作用机制。方法 将40只8周龄雄性C57BL/6J小鼠随机分为正常组、模型组、处理组、CTS组(n=10)。模型组采用气管滴注LPS(5 mg/kg)制备ARDS模型。处理组在ARDS建模前腹腔注射CTS(60 mg/kg)预处理3 d,建模后再连续予以CTS 7 d。正常组和CTS组分别注射等体积的无菌磷酸盐缓冲液(phosphate-buffered saline, PBS)或CTS。末次给药24 h后处死小鼠,取支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)及肺组织。通过HE染色、肺组织湿/干质量(wet-to-dry, W/D)比值、BALF中蛋白浓度和总细胞计数评估肺组织损伤情况;采用酶联免疫吸附试验(ELISA)检测BALF中炎症因子水平;通过Masson染色及羟脯氨酸(hydroxyproline,HYP)含量检测评估肺组织纤维化程度;利用免疫组化染色或Western blot检测VE-cadherin、CD31、波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的蛋白表达。结果 与正常组比较,模型组的肺组织W/D、BALF中总细胞数胞计数和蛋白浓度升高,BALF中的TGF-β1、TNF-α、IL-1β、IL-6升高,IL-10降低(P<0.05),小鼠肺组织出现肺损伤和早期肺纤维化;HYP含量升高(P<0.05);Vimentin、α-SMA的表达升高,而VE-cadherin、CD31的表达隆低(P<0.05)。与模型组比较,处理组的肺组织W/D、 BALF中总细胞计数和蛋白浓度降低,BALF中的TGF-β1、TNF-α、IL-1β、IL-6降低,IL-10升高(P<0.05);肺损伤和早期肺纤维化程度明显减轻,肺损伤评分和肺纤维化程度评分降低(P<0.05);HYP含量显著降低(P<0.05);Vimentin、α-SMA的表达降低,但VE-cadherin、CD31的表达增强(P<0.05)。结论 CTS可能通过抑制LPS刺激后肺组织中的内皮间质转化(endothelial-to-mesenchymal transition, EndMT)过程从而减轻ARDS小鼠早期肺纤维化。

Objective To investigate the effects and underlying mechanism of cryptotanshinone(CTS) on early pulmonary fibrosis in mice with lipopolysaccharide(LPS)-induced acute respiratory distress syndrome(ARDS). Methods Forty male C57 BL/6 J mice(8 weeks old) were randomly divided into control group, model group, treatment group, and CTS group, with 10 mice in each group. A ARDS model was established by intratracheal instillation of LPS(5 mg/kg). The mice in the treatment group was intraperitoneally pretreated with CTS(60 mg/kg) for 3 d before modeling, followed by CTS administion for 7 consecutive days. Similarly, the mice of the control and CTS groups were injected with an equivalent volume of sterile phosphate-buffered saline(PBS) or CTS, respectively. The mice were sacrificed in 24 h after the last challenge, and bronchoalveolar lavage fluid(BALF) and lung tissues were collected. The injury of lung was observed with HE staining, lung wet-to-dry(W/D) ratio, total protein concentration and total cell number in BALF. The levels of inflammatory cytokines in BALF were determined by enzyme-linked immunosorbent assay(ELISA). The fibrosis of lung was characterized by Masson staining and content of hydroxyproline(HYP). The protein levels of VE-cadherin, CD31, Vimentin and α-smooth muscle actin(α-SMA) were detected by immunohistochemistry or Western blotting. Results Obviously increased lung W/D ratio and total protein concentration and total cell number in BALF, elevated contents of TNF-α, TGF-β1, IL-1β and IL-6 while reduced content of IL-10 in BALF were seen in the model group when compared with the control group(P<0.05). Compared with the model group, the treatment group had significantly decreased lung W/D ratio and total protein concentration and total cell number in BALF, down-regulated levels of IL-1β, IL-6, TNF-α and TGF-β1 and up-regulated level of IL-10 in BALF(P<0.05). In addition, CTS also ameliorated lung injury and early fibrosis, decreased lung injury and fibrosis score, and reduced HYP content in LPS-induced ARDS(P<0.05), up-regulated the VE-cadherin and CD31 levels, and suppressed the Vimentin and α-SMA levels(P<0.05). Conclusion CTS alleviates LPS-induced early pulmonary fibrosis in ARDS mice, and its underlying mechanism may be related to the inhibition of endothelial-to-mesenchymal transition(EndMT).