脂联素改善间歇性低氧性肺动脉高压大鼠离体肺动脉舒张功能及其机制

Adiponectin improves diastolic functions of isolated pulmonary artery of intermittent hypoxia pulmonary hypertension rats and its mechanism

目的研究重组人球状脂联素(g Ad)对低氧性肺动脉高压(HPH)大鼠离体肺动脉舒张功能的影响并研究其作用机制。方法将24只雄性SD大鼠随机分为正常对照组、HPH 2周组(HPH2W)、HPH 4周组(HPH4W),每组8只。正常对照组动物在正常环境中饲养,低氧组大鼠以间歇性低压低氧法建立HPH模型。低氧组模型建立后,以右心导管法测定平均肺动脉压(m PAP)、平均右心室压(mRVP),称重测量右心室/左心室+室间隔(RV/LV+S)、右心室/体质量(RV/BW);ELISA检测试剂盒检测血清g Ad及NO浓度。右肺下叶肺组织经HE染色后观察肺小动脉血管显微结构的改变。取大鼠左、右肺动脉干制备血管环,行离体灌流实验,观察不同浓度的乙酰胆碱及硝普钠诱导的血管舒张作用。取HPH4W组动物肺外肺动脉干,分组进行孵育(HPH4W组:Krebs液孵育;HPH4W+g Ad组:g Ad 2μg/ml孵育;HPH4W+g Ad+L-NAME组:g Ad 2μg/ml+L-NAME 0.5 mmol/L孵育),孵育后观察不同浓度的乙酰胆碱及硝普钠诱导的血管舒张作用,然后收集作用于血管环的灌流液,检测NO产物。取HPH4W组大鼠肺动脉血管按上述分组进行孵育,然后行Western blot检测AMPK、Akt、e NOS等信号蛋白分子的表达及磷酸化水平。结果与正常对照组相比,HPH组大鼠的m PAP、mRVP、RV/LV+S、RV/BW均显著增高(P<0.05);血清g Ad、NO水平下降(P<0.05);光镜下肺动脉平滑肌及弹力纤维层增生,管壁增厚,管腔狭窄。与正常对照组比较,HPH组大鼠肺动脉对乙酰胆碱诱导的血管舒张作用明显减弱(P<0.05),正常对照组最大舒张率69.94%,HPH2W组最大舒张率48.79%,HPH4W组最大舒张率仅42.09%。经g Ad体外孵育后,血管环内皮依赖的舒张作用明显增强,HPH4W组最大舒张率可达到46.35%,加入L-NAME组的血管环舒张作用被显著阻断。各组大鼠对硝普钠诱导的血管舒张均有良好反应,无统计学意义。经g Ad孵育后的血管环组织AMPK、Akt、e NOS磷酸化水平、灌流液NO产物均增加(P<0.05),L-NAME可阻断g Ad增强e NOS磷酸化和NO水平的作用。结论 g Ad对间歇性低氧性肺动脉高压大鼠的肺动脉内皮依赖性的血管舒张功能具有确切的直接保护作用。AMPK/Akt/e NOS/NO信号通路可能是g Ad发挥肺动脉保护作用的分子机制。

AIM To investigate the effect of recombinant human globular adiponectin （gAd） on isolated pulmonary artery of intermittent hypoxic pulmonary hypertension （HPH） rats and the underlying mechanisms. METHODS Twenty four male Sprague Dawley （SD） rats were divided randomly into three groups ： control group, 2-week HPH （HPH2W） group and 4-week HPH （HPH4W） group. Rats in control group were fed in normal environments and rats in hypoxia groups became HPH models using intermittent hypobaric hypoxia method. After establishing the hypoxia models, mean pulmonary artery pressure （mPAP） and mean right ventricular pressure （mRVP） were measured using right cardiac catheter. Weight ratio of right ventricle and left ventricle ＋ ventricular septum （RV/LV ＋ S） and the weight ratio of right ventricle and body weight （RV/BW） were also measured and the gad and NO concentrations in blood serum were detected using ELISA detection kit. Microstructure changes of pulmonary small artery were observed after H/E staining on the inferior lobe of the right lung. Left and right pulmonary artery trunks of the rat were taken to make vascular circles. Isolated perfusion experiment was then carried out and the vascular diastolic function induced by acetylcholine and sodium nitroprussiate under varied con- centrations was observed. The pulmonary artery trunks were removed from the lungs in HPH4W group and were incubated by grouping （ HPH4W group ： incubated in Krebs fluid ; HPH4W ＋ gAd group ： incubated in gad 2 μg/ml, HPHgW ＋ gad ＋ L-NAME group： incubated in gAd 2 μg/ml ＋ L-NAME 0. 5 mmol/L）. After incubation, the author observed the vascular diastolic functions induced by acetylcholine and sodium nitroprussiate under varied concentrations. The peffusate was then collected, which acted on the vascular circle and detected NO product. The author took the pulmonary arterial vessels in the HPH4W group, divided them into groups according to the above method, incubated them and then conducted Western blot to detect the expressions and phosphorylation levels of AMPK, Akt and eNOS. RESULTS Compared with those in control group, mPAP, mRVP, RV/LV ＋ S and RV/BW all significantly increased （ P 〈 0. 05 ） and serum levels of gad and NO decreased （ P 〈 0.05 ）. Under light micro- scope, smooth muscle and elastic fiber layer of pulmonary artery proliferated, the wall of the vessels thickened and lumen of the vessels was narrow. Compared with those in control group, vascular diastolic functions of the pulmonary artery in HPH rats induced by acetylcholine decreased significantly （P 〈 0.05）. The maximal diastolic rate in control group was 69.94% , 48.79% in HPH2W group and only 42. 09% in HPH4W group. After gAd incubation in vitro, the diastolic function dependent on vascular circle endothelium increased significantly, the maximal diastolic rate reached 46.35% in HPH4W group and the vascular circle diastolic function in L-NAME group was obviously blocked. The reflection of vasodilation of rats induced by sodium nitroprussiate in all three groups was good with no statistically significant difference. Through gAd incubation, the phosphorylation levels of AMPK, Akt and eNOS of the vascular circle tissue and the NO product of perfusate all increased （ P 〈 0. 05）. L-NAME blocked the gAd effect of increasing eNOS phosphorylation and NO level. CONCLUSION gad has a direct protective effect on the vascular diastolic function dependent on the pulmonary artery endothelium in intermittent hypoxia pulmonary hypertension rats. The signal channels of AMPK/Akt/eNOS/NO may be the molecular mechanism through which gad exerts the protective effect on the pulmonary artery.