乳脂球表皮生长因子8通过抑制活性氧簇/核苷酸结合寡聚化结构域样受体蛋白3炎症小体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 lung grafts via reactive oxygen species/nucleotide-binding oligomerization domain-like receptor protein 3 signaling pathway and mechanisms

目的从活性氧簇(ROS)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体信号通路探讨乳脂球表皮生长因子8(MFG-E8)抗移植肺缺血再灌注损伤的作用机制。方法健康雄性SD大鼠50只,随机分为对照组、移植组和MFG-E8组,袖套改良法建立大鼠左肺移植模型。MFG-E8组受体鼠术前30min经尾静脉注射rhMFG-E820μg/kg,对照组和移植组受体大鼠尾静脉注射同等量生理盐水。肺移植再灌注2 h后阻断右肺门5min,全自动血气分析检测仪检测动脉血中氧分压(PaO_(2))和二氧化碳分压(PaCO_(2))。苏木精-伊红(HE)染色观察移植肺组织显微病理改变,原位缺口末端标记法(TUNEL)染色检测移植肺组织细胞凋亡指数。组织活性氧检测试剂盒(DHE)检测肺组织中ROS水平,干/湿法测量肺湿质量/干质量(W/D)比。蛋白免疫印迹(Western blot)法检测肺组织NLRP3、半胱氨酸天冬氨酸特异性蛋白酶-1(Caspase-1)、和凋亡相关的斑点样蛋白(ASC)蛋白表达水平。酶联免疫吸附法(ELISA)检测肺泡灌洗液(BALF)中白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)表达水平。组间比较使用t检验,多组比较采取单因素方差分析。结果再灌注2 h后,HE染色可见移植组肺组织明显炎性细胞浸润、肺泡结构破坏、肺水肿、肺泡腔内渗出明显伴有一些红细胞;MFG-E8组肺组织炎性细胞浸润明显减少,未见明显肺水肿,肺泡结构基本完整。再灌注2 h后,MFG-E8组动脉PaO_(2)/FiO_(2)显著高于移植组[(314.35±58.13)mmHg比(212.76±42.19)mmHg,t=4.473,P<0.05];MFG-E8组肺组织ROS水平、细胞凋亡指数和W/D值均明显低于移植组[102.37±20.78比145.72±29.34、(20.36±5.37)%比(28.13±4.64)%、6.15±1.23比8.51±1.83,t=3.813、3.462、3.385,P<0.05]。移植组大鼠肺组织NLRP3、Caspase-1和ASC蛋白水平表达明显高于对照组(0.42±0.18比0.20±0.11、0.28±0.12比0.12±0.10、0.65±0.15比0.40±0.13,t=3.298、3.239、3.983,P<0.05);MFG-E8组大鼠肺组织NLRP3、Caspase-1和ASC蛋白水平表达显著低于移植组(0.25±0.12比0.42±0.18、0.15±0.10)比(0.28±0.12、0.44±0.10比0.65±0.15,t=2.485、2.632、3.684,P<0.05)。移植组大鼠肺泡灌洗液中IL-1β和IL-18蛋白水平表达明显高于对照组[(105.74±34.73)pg/ml比(70.8±7.25)pg/ml、(128.56±23.82)pg/ml比(93.48±14.53)pg/ml,t=3.114、3.975,P<0.05];MFG-E8组大鼠肺泡灌洗液中IL-1β和IL-18蛋白水平表达显著低于移植组[(80.16±8.35)pg/ml比(105.74±34.73)pg/ml、(95.50±13.18)pg/ml比(128.56±23.82)pg/ml,t=2.265、3.840,P<0.05]。结论MFG-E8可能通过下调ROS/NLRP3信号通路,抑制NLRP3炎症小体活化,减轻氧化应激和免疫炎性反应,发挥抗移植肺缺血再灌注损伤作用。

Objective To investigate the effects of milk fat globule epidermal growth factor 8(MFG-E8)on reactive oxygen species(ROS)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway in lung grafts ischemic reperfusion injury and mechanisms.Methods A total of 50 male Sprague Dawley(SD)rats were randomly divided into control group,transplant group and MFG-E8 group.Rat left lung transplantation model was established by a modified"cuff-like"technique.Rats in MFG-E8 group were given rhMFG-E820μg/kg via the tail vein 30 min before transplantation,and in the control group and transplant group,rats were treated with the same amount of normal saline.The pathological symptoms of lung grafts were determined by Hematoxylin-eosin(HE)staining.Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)assay was used to detect the apoptosis index of lung grafts.The oxygenation and gas exchange of lung grafts were measured by an automatic blood gas analyzer under occlusion of the right main pulmonary artery and bronchus for 5 min after 2 h reperfusion,as well as measurement of ROS level and Wet/Dry lung weight ratio.Western blotting was used to detect the expressions of NLRP3,cysteinyl aspartate-specific protease-1(Caspase-1)and apoptosis-associated speck-like protein containing a CARD(ASC).The levels of interleukin-1β(IL-1β)and IL-18 in bronchoalveolar lavage fluid(BALF)were determined by enzyme linked immunosorbent assay(ELISA).The t-test was used to compare between two groups,and ANOVA was used to compare between multiple groups.Results HE staining showed that there was extensive alveolar hemorrhage,neutrophil infiltration and interstitial thickening in the transplant group,and no obvious alveolar exudates,edema and neutrophil infiltration observed in the MFG-E8 group.Compared to the transplant group,the PaO2/FiO2 was improved significantly in the MFG-E8 group[(314.35±58.13)vs.(212.76±42.19)mmHg,t=4.473,P<0.05].The content of ROS,AI and Wet/Dry lung weight ratio in the MFG-E8 group were significantly lower than those in the transplant group[102.37±20.78 vs.145.72±29.34,(20.36±5.37)%vs.(28.13±4.64)%,(6.15±1.23)vs.(8.51±1.83),t=3.813,3.462,3.385,P<0.05].Compared to the control group,the expression of NLRP3,Caspase-1 and ASC was increased significantly in the transplant group(0.42±0.18 vs.0.20±0.11,0.28±0.12 vs.0.12±0.10,0.65±0.15 vs.0.40±0.13,t=3.298,3.239,3.983,P<0.05).Compared to the transplant group,the expression of NLRP3,Caspase-1,ASC,IL-1βand IL-18 was decreased significantly in the MFG-E8 group(0.25±0.12 vs.0.42±0.18,0.15±0.10 vs.0.28±0.12,0.44±0.10 vs.0.65±0.15,t=2.485,2.632,3.684,P<0.05).Compared to the control group,the expression of IL-1βand IL-18 was increased significantly in the transplant group[(105.74±34.73)pg/ml vs.(70.8±7.25)pg/ml,(128.56±23.82)pg/ml vs.(93.48±14.53)pg/ml,t=3.114,3.975,P<0.05].Compared to the transplant group,the expression of IL-1βand IL-18 was decreased significantly in the MFG-E8 group[(80.16±8.35)pg/ml vs.(105.74±34.73)pg/ml,(95.50±13.18)pg/ml vs.(128.56±23.82)pg/ml,t=2.265,3.840,P<0.05].Conclusion MFG-E8 can alleviate lung graft ischemic reperfusion injury by inhibiting the activation of NLRP3 inflammasome and oxidative stress inflammatory response via down-regulating the ROS/NLRP3 signaling pathway.