新型抗炎介质Maresin-1在肺动脉高压中的作用和机制研究

【作者】 刘敏；

【导师】 陈丽红；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2023， 博士

【摘要】 背景:肺动脉高压(pulmonary arterial hypertension,PAH)是一种严重的心血管病理生理综合征,肺血管重构、右心室功能障碍是PAH的主要病理特征。持续性的血管收缩,血管腔狭窄或闭塞,导致肺血管阻力进行性增加,肺动脉压力持续升高,进而继发右心室衰竭甚至死亡。PAH在临床上引起了广泛关注,目前PAH的临床治疗药物主要通过改善血管舒张功能而实现,包括前列环素及其类似物、内皮素受体拮抗剂、磷酸二酯酶抑制剂、鸟苷酸环化酶激动剂、钙离子通道阻滞剂等。这些药物虽然可以在一定程度上减轻患者的临床症状,延缓疾病进展,但尚不能从根本上抑制PAH的发生。因此,深入探究PAH发生发展的病理机制,探求新的具有高效稳定治疗效果的药物分子靶点迫在眉睫。近年来,研究表明,肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)过度增殖和线粒体功能异常可以促进肺血管重构,在PAH中发挥重要作用,是PAH发生发展的重要病理因素之一,抑制PASMCs过度增殖已成为逆转肺血管重构和治疗PAH的新靶点。新型抗炎介质Maresin-1(macrophage mediators in resolving inflammation,MaR1)是巨噬细胞在炎症消退阶段由内源性二十二碳六烯酸(docosahexenoicacid,DHA)通过脂氧酶等氧化途径合成的具有强烈抗炎作用的脂质介质。MaR1可以抑制炎性因子释放、中性粒细胞浸润、氧化应激反应,促进巨噬细胞吞噬、组织再生,减轻组织损伤,在众多心血管疾病中具有保护作用,例如动脉粥样硬化、腹主动脉瘤、高血压、糖尿病以及心室重构和心律不齐等。本课题旨在探究MaR1在PAH发生发展中的作用并明确其作用机制,为PAH的干预和治疗提供新思路。目的:明确MaR1在PAH发生发展过程中的作用并探究其作用机制。方法:体内实验,使用野百合碱(monocrotaline,MCT)诱导大鼠肺动脉高压(pulmonary hypertension,PH)模型或低氧联合SU5416(HySu)诱导小鼠PH模型,分别在造模全程或造模最后一周,给与MaR1(100mg/day)和(或)BOC-2(ALXR的特异性拮抗剂,50μg/kg)腹腔注射,探究MaR1及其受体ALXR在PAH发生发展中的作用及MaR1对PAH的治疗作用。采用血流动力学检测,即直接经右心室插管测量右心室收缩压,评估肺高压程度。取心脏组织剥离右心室及左心室和室间隔称重,计算右心室肥厚指数。形态学评估肺血管重构,主要包括肺组织切片HE染色及α-SMA染色及血管肌化程度评估。肺组织切片行DHE和MitSOX Red染色,检测肺组织内活性氧(reactive oxygen species,ROS)的产生。使用ATP检测试剂盒和SOD酶活性检测试剂盒,检测肺组织匀浆中ATP含量和SOD酶活性。提取肺组织蛋白,Western blot检测增殖相关蛋白PCNA和CyclinD1以及线粒体稳态信号通路相关蛋白的表达。细胞实验,使用原代提取的大鼠PASMCs,3%低氧处理后,给予MaR1和(或)LGR6、ALXR、RORα的特异性拮抗剂或shRNA腺病毒处理,明确MaR1及其三种受体在低氧诱导的PASMCs增殖和线粒体氧化应激损伤中的作用。结合RNA-seq高通量测序寻找可能的分子机制并进行验证。细胞功能检测主要包括:CCK-8实验用于检测细胞活力,Western blot检测增殖相关蛋白线粒体稳态信号通路相关蛋白表达,DHE和Mito SOX Red荧光探针检测ROS产生,JC-1和TMRM探针检测细胞线粒体膜电位的变化,ATP检测试剂盒和SOD酶活性检测试剂盒检测ATP含量和SOD酶活性等。结果:低氧诱导的PASMCs给与MaR1处理发现,MaR1可显著抑制低氧诱导的PASMCs增殖和线粒体氧化应激损伤,提示MaR1可能会通过改善PASMCs的线粒体稳态,进而减轻血管重构,降低肺动脉高压。体内实验结果证实,PH模型动物血清中MaR1的含量显著下降。而且,无论是MCT大鼠PH模型,还是HySu小鼠PH模型,与单纯造模组相比,造模全程给与MaR1处理均可显著降低模型动物的右心室收缩压和右心室肥厚指数,并抑制肺血管重构,减轻氧化应激损伤,改善线粒体稳态。此外,即使仅在PH造模最后一周给与MaR1处理,MaR1也同样可以显著降低MCT大鼠的右心室收缩压和右心室肥厚指数,并减轻肺血管重构,提示MaR1对已形成的PH也具有一定治疗作用。进一步,在HySu小鼠PH模型中给与MaR1受体ALXR的特异性拮抗剂BOC-2预处理,我们发现MaR1对PH的保护作用可被BOC-2明显阻断,表明MaR1是通过ALXR受体在PH中发挥保护作用。为探索MaR1/ALXR参与PH和肺血管重构的具体分子机制,我们对MaR1处理的PASMCs进行RNA-seq高通量测序分析发现,MaR1可显著抑制低氧诱导的与线粒体功能密切相关的热休克蛋白HSP90的表达增高,而BOC-2抑制剂则可以阻断MaR1对HSP90的抑制作用。体内和体外实验进一步证实,使用HSP90线粒体特异性拮抗剂可部分恢复被BOC-2阻断的MaR1对PASMCs增殖和线粒体氧化应激损伤的保护作用,提示线粒体HSP90可能是MaR1/ALXR保护PH和肺血管重构的重要分子靶点。结论:新型抗炎介质MaR1对肺动脉高压的发生发展具有延缓和治疗作用,其具体机制为MaR1可作用于PASMCs上的ALXR受体,抑制低氧诱导的热休克蛋白HSP90的表达,从而改善线粒体稳态,减轻氧化应激损伤,抑制PASMCs的过度增殖,减轻肺血管重构。靶向MaR1/ALXR/HSP90轴有望为肺动脉高压的防治提供新思路。更多还原

【Abstract】 BackgroundPulmonary arterial hypertension(PAH)is a progressive and life-threatening disease characterized by severe pulmonary arterial remodeling,leading to increased pulmonary vascular resistance and pulmonary artery pressure,and ultimately,to right ventricular failure.Increasing evidence of PAH pathophysiology has contributed to several effective therapies including drugs targeting prostacyclin,endothelin and nitric oxide pathways with selective pulmonary vasodilator properties.Although the current therapeutic drugs significantly improved symptoms,they are limited in reversing pulmonary vascular remodeling and decreasing mortality.Thus identifying novel treatments targeted pulmonary vascular remodeling in PAH is still critically needed.Growing evidence has illustrated that pulmonary artery smooth muscle cells(PASMCs)overproliferation and mitochondrial dysfunction are important elements contributing to pulmonary vascular remodeling and the pathophysiology of PAH.Inhibition of excessive proliferation of PASMCs,improve mitochondrial homeostasis may be new therapeutic targets for reversing pulmonary vascular remodeling and PAH.Macrophage mediators in resolving inflammation(Maresins)are a family of specialized pro-resolving lipid mediators(SPMs)derived from the ω-3 fatty acid docosahexaenoic acid(DHA).MaR1 is the first member of Maresins family and plays critical role in protecting against many cardiovascular diseases,such as atherosclerosis,abdominal aortic aneurysm,hypertension,diabetes,myocardail infarction and arrhythmia etc.However,the effect of MaR1 on the PAH development are not clear.And the relationship of MaR1 and mitochondrial homeostasis in the pathogenesis of PAH has not been investigated as well.Here we aimed to explore the role and mechanism of MaR1 in the development of PAH and provide a new therapeutic target for the treatment of PAH.ObjectiveThe aim of this study was to clarify the role of MaR1 in the progression of PAH and investigate the underlying mechanisms.MethodsFor in vivo experiments,monocrotaline(MCT)-induced rat and hypoxia+SU5416(HySu)-induced mouse models of pulmonary hypertension(PH)were used.MaR1(100mg/day)and/or BOC-2(a specific antagonist of ALXR,50μg/kg)were used to investigate the role of MaR1 and its receptor ALXR in the development and therapeutic effect of PH.The right ventricular systolic pressure(RVSP)was measured directly by right ventricular intubation.The heart was collected and the ventriculars were carefully dissected and weighed.RV hypertrophy was assessed by Fulton index measurements(weight of RV/weight of left ventricle plus septum [RV/LV+S]).HE and α-SMA staining of lung tissue sections were used to detect the vascular remodeling index and the degree of vascular muscularization.DHE and Mit SOX Red staining were performed on lung tissue sections to detect reactive oxygen species(ROS)production.The ATP assay and total SOD assay kits were used to measure ATP levels and SOD activity in lung tissues.Lung tissue proteins were extracted,and the expressions of proliferation-related proteins PCNA,CyclinD1 and mitochondrial homeostasis related proteins were detected by Western blot.For in vitro cell culture experiment,primary extracted rat pulmonary artery smooth muscle cells(PASMCs)were used.After 3% hypoxia treatment,cells were given MaR1and/or LGR6,ALXR,RORα specific antagonists or shRNA adenovirus treatment to clarify the role of MaR1 and the three receptors in hypoxy-induced PASMCs proliferation and mitochondrial oxidative stress damage.RNA-seq high-throughput sequencing was performed to explore the possible molecular mechanism.CCK-8 kit was used to detecte cell viability,Western blot was used to detect the expression of CyclinD1 and mitochondrial homeostasis related proteins.DHE and Mito SOX Red fluorescent probes were used to to detect ROS production in PASMCs,mitochondrial membrane potential probes JC-1 and TMRM were used to detect mitochondrial membrane potential.The ATP assay and total SOD assay kits were used to measure the ATP levels and SOD activity in PASMCs.ResultsIn the present study,we demonstrated that MaR1 content was decreased in serum from both MCT-induced PH rats and HySu-induced PH mice,MaR1 treatment delayed the development of experimental PH and attenuated pulmonary vascular remodeling in the two models.Mechanistically,MaR1 treatment significantly decreased mitochondrial injury,inhibited ROS production,increased ATP and SOD levels,and rescued the impaired signaling of mitochondrial dynamics and mitophagy.In primary cultured hypoxia-exposed PASMCs,MaR1 also profoundly suppressed cell proliferation,decreased oxidative stress,increased mitochondrial membrane potential,restored disturbed mitochondrial dynamics,and decreased mitochondrial autophagy.Our data strongly revealed that MaR1 may contribute to PAH prevention by protecting mitochondria.Moreover,we found that abrogating the function of MaR1 receptor ALXR,but not LGR6 or RORα,with BOC-2,significantly abolished the protective effect of MaR1 against PAH development and reduced its therapeutic potential,indicating that ALXR functions as the receptor of MaR1 action on PH.To further decipher the underlying mechanisms of MaR1/ALXR axis on PASMCs proliferation and mitochondrial homeostasis,we compared RNA-seq data of hypoxia-challenged PASMCs treated or not with MaR1 and proved that the MaR1/ALXR axis suppressed hypoxia-induced PASMCs proliferation and pulmonary vascular remodeling through improving HSP90-mediated mitochondrial homeostasis.These observations broaden our in-depth understanding of the role of MaR1 in PAH.Targeting MaR1/ALXR/HSP90 signaling may be used therapeutically in PAH treatment.ConclusionsOur present study provides strong evidence that MaR1 exerts a protective role in both the development and the progression of PAH.Mechanistically,MaR1 inhibits PASMCs proliferation and pulmonary vascular remodeling through improving mitochondrial homeostasis via the MaR1/ALXR/HSP90 axis.Targeting the MaR1/ALXR/HSP90 axis is expected to provide new therapeutic strategy for the prevention and treatment of PAH.更多还原