毛蕊异黄酮对大鼠呼吸机相关性肺损伤的影响及其与HB-EGF的关系

Effects of calycosin on ventilator-induced lung injury and the relationship with heparin-binding EGF-like growth factor in rats

目的评价毛蕊异黄酮对大鼠呼吸机相关性肺损伤(VILI)的影响及其与肝素结合性表皮生长因子(HB-EGF)的关系。方法清洁级雄性Wistar大鼠25只,6周龄,体重250~300 g,采用随机数字表法分为5组(n=5):假手术组(Sham组)、VILI组、毛蕊异黄酮5 mg/kg组(M1组)、毛蕊异黄酮10 mg/kg组(M2组)、毛蕊异黄酮20 mg/kg组(M3组)。于机械通气前7 d开始口饲药物,Sham组和VILI组口饲玉米油1 ml/d;M1组-M3组分别口饲毛蕊异黄酮溶液5、10、30 mg/kg,共7 d。之后禁食8 h,制备VILI模型。机械通气4 h后,行左肺肺泡支气管灌洗术,回收支气管肺泡灌洗液(BALF),采用BCA法检测总蛋白浓度,采用ELISA法测定IL-1β、TNF-α和IL-10浓度,瑞氏染色后计算中性粒细胞比例;随后处死大鼠,取肺组织,计算湿重/干重(W/D)比值,采用Western blot法检测Bax、Bcl-2及HB-EGF表达,免疫组化法检测HB-EGF染色面积,HE染色法观察肺病理学改变,TUNEL法检测细胞凋亡情况,计算TUNEL阳性细胞密度。结果与Sham组比较,VILI组W/D比值、BALF中性粒细胞比例、总蛋白、TNF-α和IL-10浓度升高,HB-EGF表达下调,免疫组化染色面积增加,Bax和Bcl-2表达上调,TUNEL阳性细胞密度升高(P<0.05),肺组织病理学损伤加重。与VILI组比较,M1组BALF总蛋白、TNF-α和IL-1β浓度降低,HB-EGF表达上调,TUNEL阳性细胞密度降低;M2组W/D比值、BALF总蛋白、TNF-α和IL-1β浓度降低,M3组W/D比值、BALF中性粒细胞比例、总蛋白、TNF-α、IL-1β和IL-10浓度降低,M2组和M3组HB-EGF表达上调,免疫组化染色面积增加,Bax表达下调,Bcl-2表达上调,TUNEL阳性细胞密度降低(P<0.05),肺组织病理学损伤减轻。结论毛蕊异黄酮可减轻大鼠VILI,机制可能与促进肺组织HB-EGF的表达,减轻炎症反应及细胞凋亡有关。

Objective To evaluate the effects of calycosin on ventilator-induced lung injury(VILI)and the relationship with heparin-binding EGF-like growth factor(HB-EGF)in the rats.Methods Twenty-five clean-grade male Wistar rats,aged 6 weeks,weighing 250-300 g,were divided into 5 groups(n=5 each)using a random number table method:sham operation group(Sham group),VILI group,calycosin 5 mg/kg group(M1 group),calycosin 10 mg/kg group(M2 group)and calycosin 20 mg/kg group(M3 group).The oral administration was started at 7 days before mechanical ventilation.Corn oil 1 ml/d was orally administered once a day for 7 days in Sham group and VILI group.The calycosin solution(dissolved in 1 ml of corn oil)5,10 and 30 mg/kg was orally administered once a day for 7 days in M1-3 groups,respectivley.The rats were then fasted for 8 h,and VILI model was established.The left main bronchus was lavaged and broncho-alveolar lavage fluid(BALF)was collected for determination of total protein(by BCA assay),interleukin-1beta(IL-1β),tumor necrosis factor-alpha(TNF-α)and IL-10 concentrations(by enzyme-linked immunosorbent assay),and the proportion of neutrophils was calculated after Wright staining.The rats were then sacrificed,and the lungs were harvested to calculate wet/dry weight(W/D)ratio and to determine the expression of Bax,Bcl-2 and HB-EGF(by Western blot),stained area of HB-EGF(using immunohistochemistry),pathological changes of lung tissues(using HE staining),and cell apoptosis(using TUNEL assay).The density of TUNEL-positive cells was calculated.Results Compared with Sham group,the W/D ratio,proportion of neutrophils and concentrations of total protein,TNF-αand IL-10 in BALF were significantly increased,the expression of HB-EGF was down-regulated,the stained area of HB-EGF was increased,the expression of Bax and Bcl-2 was up-regulated,and the density of TUNEL-positive cells was increased(P<0.05),and the pathological changes of lung tissues were accentuated in VILI group.Compared with VILI group,the concentrations of total protein,TNF-αand IL-10 in BALF were significantly decreased,the expression of HB-EGF was up-regulated,and the density of TUNEL-positive cells was decreased in M1 group,the W/D ratio and concentrations of total protein and TNF-αand IL-10 in BALF were significantly decreased in M2 group,the W/D ratio,proportion of neutrophils and concentrations of total protein,TNF-α,IL-1βand IL-10 in BALF were significantly decreased in M3 group,and the expression of HB-EGF was significantly up-regulated,the stained area of HB-EGF was increased,and the Bax protein expression was down-regulated,the Bcl-2 expression was up-regulated,and the density of TUNEL-positive cells was decreased in M2 and M3 groups(P<0.05).Conclusion Calycosin can attenuate VILI,and the mechanism may be related to promoting HB-EGF expression and reducing inflammatory responses and cell apoptosis in rats.