乳脂球表皮生长因子8通过调控缺氧诱导生长因子-1α/血管内皮细胞生长因子/Notch信号通路减轻移植肺缺血再灌注损伤

Protective effects of milk fat globule epidermal growth factor 8 on ischemic reperfusion injury of pulmonary grafts by modulating hypoxia inducible factor-1α/vascular endothelial growth factor/Notch signaling pathway

目的探讨乳脂球表皮生长因子8(MFG-E8)对移植肺缺血再灌注损伤的保护作用及缺氧诱导生长因子-1α(HIF-1α)/血管内皮细胞生长因子(VEGF)/Notch信号转导通路的影响。方法雄性SD大鼠60只,随机分为移植组、MFG-E8组、MFG-E8+HIF-1α抑制剂组,改良袖套法建立大鼠左肺移植模型,MFG-E8组受体大鼠术前30 min尾静脉注射rhMFG-E820μg/kg,MFG-E8+HIF-1α抑制剂组在MFG-E8组基础上,术前经腹腔注射4 mg/kg HIF-1α抑制剂YC-1。移植组受体大鼠尾静脉注射等量生理盐水。移植肺再灌注2 h后阻断右肺门5 min,行动脉血气分析,并测量移植肺组织测髓过氧化物酶(MPO)活性、肺组织湿/干重量比率(W/D);苏木精-伊红(HE)染色观察移植肺组织病理学变化;原位缺口末端标记法(TUNEL)法检测移植肺组织细胞凋亡指数。蛋白免疫印迹(Western blot)法检测HIF-1α/VEGF/Notch通路蛋白及细胞凋亡蛋白B细胞淋巴瘤/白血病-2(bcl-2)和bcl-2相关X蛋白(bax)表达,酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)表达水平。结果移植肺再灌注2 h后,MFG-E8组动脉血氧分压/吸氧浓度(PaO_(2)/FiO_(2))较移植组明显提升[(307.23±29.17)mmHg比(215.48±20.92)mmHg,t=8.08,P<0.05]。与移植组比较,MFG-E8组肺组织MPO活性和W/D均明显降低[(1.76±0.21)U/g tissue比(2.51±0.46)U/g tissue,(6.03±1.07)mg/dl比(8.41±1.36)mg/dl,t=4.69、4.35,P<0.05]。移植组病理检测可见肺泡壁水肿、肺泡结构破坏、肺泡腔内明显炎性细胞浸润,与移植组比较,MFG-E8组肺泡结构保持完整、肺泡腔和间质内浸润的炎性细胞明显减少,炎性反应显著减轻。与移植组比较,MFG-E8组肺组织细胞凋亡指数(AI)、bax、TNF-α、IL-6蛋白表达显著降低[(11.47±1.23)%比(27.09±2.57)%、(1.34±0.12)比(1.92±0.13)、(250.1±11.76)pg/ml比(392.7±28.14)pg/ml、(134.6±11.25)pg/ml比(251.3±14.33)pg/ml,t=17.34、10.37、14.79、20.26,P<0.05],bcl-2、HIF-1α、VEGF、Notch1、HES1蛋白表达明显升高(1.75±0.16比1.36±0.12、0.87±0.14比0.36±0.09、0.79±0.08比0.39±0.03、0.58±0.05比0.24±0.03、0.59±0.05比0.28±0.03,t=6.17、9.69、14.80、18.44、16.81,P<0.05)。与MFG-E8组比较,MFG-E8+HIF-1α抑制剂组PaO_(2)/FiO_(2)、bcl-2、HIF-1α、VEGF、Notch1、HES1蛋白表达显著降低[(237.25±21.34)mmHg比(307.23±29.17)mmHg、1.40±0.13比1.75±0.16、0.41±0.08比0.87±0.14、0.41±0.04比0.79±0.08、0.26±0.03比0.58±0.05、0.27±0.03比0.59±0.05,t=6.12、5.37、9.02、13.43、17.35、17.34,P<0.05],肺组织MPO活性、W/D、细胞凋亡指数、bax、TNF-α、IL-6蛋白表达显著升高[(2.47±0.37)U/g tissue比(1.76±0.21)U/g tissue、(7.85±1.32)mg/dl比(6.03±1.07)mg/dl、(25.76±2.63)%比(11.47±1.23)%、1.87±0.14比1.34±0.12、(385.4±27.93)pg/ml比(250.1±11.76)pg/ml、(247.5±14.21)pg/ml比(134.6±11.25)pg/ml,t=5.28、3.39、15.56、9.09、14.12、19.70,P<0.05]。结论MFG-E8能减轻大鼠移植肺缺血再灌注损伤,其作用机制可能与其激活HIF-1α/VEGF/Notch信号转导通路,抑制细胞凋亡,降低炎性细胞因子表达,减轻脂质过氧化反应有关。

Objective To investigate the effects of milk fat globule epidermal growth factor 8(MFG-E8)on hypoxia inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)/Notch signaling pathway in pulmonary grafts with ischemic reperfusion injury.Methods Totally,60 male Sprague Dawley(SD)rats were randomly divided into transplant group,MFG-E8 group,and MFG-E8+HIF-1α inhibitor group.The rat left lung transplantation model was established by a"cuff-like"technique.Rats in MFG-E8 group were given rhMFG-E820μg/kg via the tail vein 30 min before transplantation,and in the MFG-E8+HIF-1α inhibitor group,rats were given rhMFG-E820μg/kg via the tail vein and peritoneal injection of YC-1(HIF-1α inhibitor)4 mg/kg 30 min before transplantation,while the transplant group was injected with the same amount of normal saline.The gas exchange and oxygenation of the grafts were assessed under occlusion of the right pulmonary artery and main bronchus for 5 min after 2-h reperfusion,as well as measurement of Wet/Dry lung weight ratio and myeloperoxidase(MPO)activity.The pathological changes of grafts were observed by hematoxylin-eosin(HE)staining.Apoptosis index of grafts was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)assay.The expression levels of HIF-1α,VEGF,Notch1,HES1,B cell lymphoma/leukemia-2(bcl-2)and bcl-2 associated X protein(bax)were detected by Western blotting,and those of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by enzyme linked immunosorbent assay(ELISA).Results After 2-h reperfusion,compared to the transplant group,the PaO_(2)/FiO_(2) was improved significantly in the MFG-E8 group[(307.23±29.17)vs.(215.48±20.92)mmHg,t=8.08,P<0.05].The activity of MPO and Wet/Dry lung weight ratio in the MFG-E8 group were(1.76±0.21)U/g tissue and(6.03±1.07)mg/dL,respectively,which were significantly lower than those in transplant group[(2.51±0.46)U/g tissue and(8.41±1.36)mg/dL,respectively,t=4.69,4.35,P<0.05].Extensive alveolar hemorrhage,interstitial thickening and neutrophil infiltration were observed in the transplant group.By contrast,alveolar exudates,neutrophil infiltration and edema were markedly attenuated in the MFG-E8 group.Compared to the transplant group,the apoptosis index and expression of bax,TNF-α and IL-6 were decreased significantly in the MFG-E8 group[(11.47±1.23)%vs.(27.09±2.57)%,1.34±0.12 vs.1.92±0.13,(250.1±11.76)pg/ml vs.(392.7±28.14)pg/ml,(134.6±11.25)pg/ml vs.(251.3±14.33)pg/ml,t=17.34,10.37,14.79,20.26,P<0.05],and the expression levels of bcl-2,HIF-1α,VEGF,Notch1 and HES1 were improved significantly in the MFG-E8 group(1.75±0.16 vs.1.36±0.12,0.87±0.14 vs.0.36±0.09,0.79±0.08 vs.0.39±0.03,0.58±0.05 vs.0.24±0.03,0.59±0.05 vs.0.28±0.03,t=6.17,9.69,14.80,18.44,16.81,P<0.05).Compared to the MFG-E8 group,the PaO_(2)/FiO_(2) and expression of bcl-2,HIF-1α,VEGF,Notch1 and HES1 were decreased significantly in the MFG-E8+HIF-1αinhibitor group[(237.25±21.34)mmHg vs.(307.23±29.17)mmHg,1.40±0.13 vs.1.75±0.16,0.41±0.08 vs.0.87±0.14,0.41±0.04 vs.0.79±0.08,0.26±0.03 vs.0.58±0.05,0.27±0.03 vs.0.59±0.05,t=6.12,5.37,9.02,13.43,17.35,17.34,P<0.05],and the activity of MPO,Wet/Dry lung weight ratio,apoptosis index and the expression of bax,TNF-αand IL-6 were improved significantly in the MFG-E8+HIF-1α inhibitor group[(2.47±0.37)U/g tissue vs.(1.76±0.21)U/g tissue,(7.85±1.32)mg/dL vs.(6.03±1.07)mg/dL,(25.76±2.63)%vs.(11.47±1.23)%,1.87±0.14 vs.1.34±0.12,(385.4±27.93)pg/ml vs.(250.1±11.76)pg/ml,(247.5±14.21)pg/ml vs.(134.6±11.25)pg/ml,t=5.28,3.39,15.56,9.09,14.12,19.70,P<0.05].Conclusion MFG-E8 can ameliorate ischemic reperfusion injury of pulmonary grafts,which may be related to the regulation of apoptosis and inflammatory lipid peroxidation by inducing HIF-1α/VEGF/Notch signaling pathway activation.