罗格列酮对野百合碱诱导肺动脉高压大鼠的作用及机制探讨

【作者】 王晓芳；

【导师】 陆慰萱； 郭子建； 施举红；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 [背景]肺动脉高压(pulmonary hypertension, PH)是一组以肺血管阻力持续增加为特征的具有潜在破坏力的严重疾病,动脉型肺动脉高压(pulmonary arterial hypertension,PAH)是PH的最常见类型之一。血管重构是PH发生发展的中心环节,目前PH的治疗药物虽可改善患者的临床症状,但不能抑制和逆转肺血管重构发生。近年研究发现,PH的发病与过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ, PPARγ)表达降低相关,PPARγ的配体能够阻止或逆转PH的发生发展。罗格列酮是PPARγ的人工合成配体,具有抗炎、抑制细胞增殖、诱导细胞凋亡分化等作用。本研究拟通过野百合碱(monocrotaline, MCT)诱导PAH的大鼠模型,观察罗格列酮对野百合碱诱导PAH大鼠的治疗作用。[目的]通过观察罗格列酮对MCT诱导PAH大鼠一般状况、肺血流动力学及肺组织病理变化的影响,评估罗格列酮对MCT诱导的已形成PAH大鼠的治疗作用。[方法]1.实验动物分组：雄性Sprague-Dawley大鼠40只,随机分为4组,每组10只：①对照组(C组)：颈背部皮下注射生理盐水1次；②PAH模型组(P组)：颈背部皮下注射野百合碱(60mg/kg) 1次,并于注射后第21天至第34天连续给予等体积生理盐水灌胃(约1.5ml/d);③罗格列酮高剂量治疗PAH组(PRH组)：野百合碱(60mg/kg)颈背部皮下注射1次,并于注射后第21天至第34天连续给予罗格列酮(每日5mg/kg)灌胃；④罗格列酮低剂量治疗PAH组(PRL组)：野百合碱(60mg/kg)颈背部皮下注射1次,并于注射后第21天至第34天连续给予罗格列酮(每日2.5mg/kg)灌胃。2.于第35天行右心导管检查测定肺动脉平均压(mPAP)、右心室收缩压(RVSP);分离心脏,沿室间隔分离右心室,分别称量右心室、左心室+室间隔,计算右心室肥厚指数(RVHI)。3.肺组织切片HE染色及Elastin Van Gieson(ET+VG)染色,应用Image Pro Plus 6.0病理图像分析系统测定并计算各组大鼠肺小动脉管壁中膜厚度占管壁外径的百分比(WT%)。4.采用t检验及单因素方差分析比较组间差异,P<0.05为差异具有统计学意义,P<0.01为差异具有非常显著统计学意义。[结果]1.罗格列酮治疗对MCT诱导PAH大鼠一般情况的影响从注射MCT 2周后开始,P组、PRH组及PRL组的部分大鼠开始出现皮毛凌乱无光泽、呼吸急促、活动迟缓的表现,上述情况随时间延长而加重；从注射MCT3周后陆续出现大鼠死亡现象,C组、P组、PRH组及PRL组大鼠的病死率依次为0、50%、50%及30%;注射MCT 3周及5周后,C组大鼠体重分别为(386±14)g及(475±18)g;P组大鼠体重[(335±28)g,(316±28)g]、PRH组大鼠体重[(331±22)g,(320±43)g]及PRL组大鼠体重[(327±30)g,(296±41)g]均较C组大鼠降低,差异具有非常显著统计学意义(P<0.01),而P组、PRH组及PRL组之间大鼠体重差异无统计学意义(P>0.05)。2.罗格列酮治疗对MCT诱导PAH大鼠mPAP、RVSP及RVHI的影响C组大鼠mPAP、RVSP及RVHI依次为(21.66±2.43) mmHg, (43.76±3.64) mmHg及0.28±0.02；P组大鼠mPAP、RVSP及RVHI[(61.57±7.28) mmHg, (108.65±9.97) mmHg,0.70±0.03]:均较C组大鼠显著升高,差异具有非常显著统计学意义(P<0.01)；与P组大鼠比较,PRH组大鼠mPAP、RVSP、RVHI[(58.83±9.47) mmHg, (106.61±11.67) mmHg,0.63±0.07]和PRL组大鼠mPAP、RVSP、RVHI [(56.31±8.44) mmHg, (102.90±11.69) mmHg,0.63±0.07]虽略降低,但差异无统计学意义(P>0.05),且仍均显著高于C组大鼠(P<0.01)；PRH组和PRL组大鼠之间,mPAP、RVSP及RVHI差异均无统计学意义(P>0.05)。3.罗格列酮治疗对MCT诱导PAH大鼠肺小动脉重构的作用C组大鼠肺组织切片HE染色可见各级肺动脉管壁结构清楚,血管周围无炎症细胞浸润,ET+VG染色可见内外弹力板平滑无挤压；P组、PRH组及PRL组大鼠肺组织切片镜下表现相似,HE染色可见3组大鼠肌型肺小动脉管壁增厚,平滑肌增生肥厚,管腔狭窄,血管外膜肿胀,血管周围炎性细胞浸润,腺泡内肺微动脉“肌型化”,ET+VG染色可见内弹力板挤压呈“波浪状”改变。C组大鼠肺小动脉管壁中膜厚度占管壁外径的百分比(WT%)为(8.82±3.23)%,P组、PRH组及PRL组大鼠肺小动脉WT%[(55.22±5.72)%,(50.92±5.18)%,(52.45±6.45)%]均较C组大鼠显著增高,差异具有非常显著统计学意义(P<0.01)；而与P组大鼠相比,PRH组大鼠及PRL组大鼠WT%略降低,但差异无统计学意义(P>0.05)；不同剂量罗格列酮治疗组(PRH组及PRL组)大鼠WT%差异无统计学意义(P>0.05)。[结论]1.罗格列酮治疗不能降低野百合碱诱导大鼠已形成的明显增高的肺动脉平均压和右心室收缩压。2.罗格列酮治疗不能逆转野百合碱诱导大鼠已发生的肺血管重构及右心室肥厚。[背景]肺动脉高压(PH)的发病机制极其复杂,至今仍未完全阐明。近年研究认为炎症和免疫机制在PH发生发展中起重要作用。罗格列酮属于噻唑烷二酮类药物(TZDs),是过氧化物酶体增殖物激活受体γ(PPARy)的人工合成配体,其抗炎及免疫调节作用已在多种炎症疾病动物模型中得到证实。本研究拟通过野百合碱(MCT)诱导动脉型肺动脉高压(PAH)的大鼠模型,观察罗格列酮抗炎作用对早期肺血管病变的保护作用。[目的]1.通过观察大鼠一般状况、肺血流动力学及肺组织病理变化,评估早期应用罗格列酮对MCT诱导PAH大鼠的保护作用。2.通过测定MCT诱导PAH大鼠肺组织白细胞介素6 (interleukin 6, IL-6)、肿瘤坏死因子α(tumor necrosis factor a, TNF-α)及单核细胞趋化蛋白(monocyte chemotactic protein 1, MCP-1)的变化,探讨早期应用罗格列酮对MCT诱导PAH大鼠炎症发病机制的影响。[方法]1.实验动物分组：雄性Sprague-Dawley大鼠32只,随机分为4组,每组8只：①对照组(N组)：颈背部皮下注射生理盐水1次；②PAH模型组(M组)：颈背部皮下注射野百合碱(60mg/kg) 1次,并于注射当天(第0天)开始给予等体积生理盐水(约1.5ml/d)连续灌胃21天；③罗格列酮高剂量干预PAH组(MRH组)：野百合碱(60mg/kg)颈背部皮下注射1次,并于注射当天(第0天)开始给予罗格列酮(每日5mg/kg)连续灌胃21天；④罗格列酮低剂量干预PAH组(MRL组)：野百合碱(60mg/kg)颈背部皮下注射1次,并于注射当天(第0天)开始给予罗格列酮(每日2.5mg/kg)连续灌胃21天。2.于第21天行右心导管检查测定肺动脉平均压(mPAP)、右心室收缩压(RVSP),颈内动脉插管测定体循环平均动脉压(MAP)；分离心脏,沿室间隔分离右心室,分别称量右心室、左心室+室间隔,计算右心室肥厚指数(RVHI)。3.肺组织切片行HE染色,做血管周围炎症评分；行Elastin Van Gieson (ET+VG)染色,测定并计算大鼠肺小动脉管壁中膜厚度占管壁外径百分比(WT%)；行a-SMA免疫组化染色,测定并计算腺泡内肺微动脉肌化百分比及a-SMA平均光密度值,评估非肌性血管肌化程度。4. ELISA方法测定肺组织匀桨IL-6、TNF-α和MCP-1水平。5.采用t检验及单因素方差分析比较组间差异,P<0.05为差异具有统计学意义,P<0.01为差异具有非常显著统计学意义。[结果]1.罗格列酮早期干预对MCT诱导PAH大鼠一般情况的影响注射MCT 2周后开始,M组的部分大鼠开始出现皮毛凌乱无光泽、轻微喘息、活动迟缓表现。分别于第1周、第2周、第3周时测定大鼠体重,N组大鼠体重依次为(287±12)g,(331±15)g及(375±19)g;M组大鼠体重[(261±17)g,(276±24)g,(319±29)]、MRH组大鼠体重[(268±8)g,(275±15)g,(328±14)g]及MRL组大鼠体重[(261±8)g,(284±11)g,(329±12)g]均较N组大鼠明显降低,差异具有统计学意义(P<0.01或P<0.05),而M组、MRH组和MRL组之间,大鼠体重差异均无统计学意义(P>0.05)。2.罗格列酮早期干预对MCT诱导PAH大鼠mPAP、RVSP、MAP及RVHI的作用MCT皮下注射3周后,N组大鼠mPAP、RVSP及RVHI依次为(17.13±3.30)mmHg, (40.33±5.77) mmHg及0.25±0.02；与N组大鼠比较,M组大鼠mPAP、RVSP及RVHI[(37.00±4.98) mmHg, (67.04±4.87) mmHg,0.40±0.02]均显著增高,差异具有非常显著统计学意义(P<0.01);MRH组大鼠及MRL组大鼠mPAP、RVSP、RVHI分别为[(26.88±3.55)mmHg, (50.09±5.61)mmHg,0.33±0.03]和[(28.29±3.60)mmHg, (55.71±4.85) mmHg,0.33±0.02],均低于M组大鼠,差异具有非常显著统计学意义(P<0.01),但仍高于N组大鼠(P<0.01或P<0.05)；MRH组与MRL组大鼠之间mPAP、RVSP及RVHI无统计学差异(P>0.05)。N组、M组、MRH组及MRL组大鼠之间MAP[(87.81±7.78)mmHg,(88.23±9.29)mmHg,(90.17±6.82) mmHg, (89.88±9.08) mmHg]差异无统计学意义(P>0.05)。3.罗格列酮早期干预对MCT诱导PAH大鼠肺血管重构的作用光镜下观察肺组织HE染色切片可见M组大鼠肌型肺小动脉管壁明显增厚,平滑肌增生肥厚,管腔狭窄,腺泡内肺微动脉“肌型化”,并伴有肺泡间隔增宽,肺泡内巨噬细胞浸润。肺组织ET+VG染色可见M组大鼠肌型肺小动脉内弹力板挤压呈“波浪状”改变。N组大鼠WT%为(8.91±2.30)%,M组大鼠WT%[(45.52±5.48)%]较N组大鼠显著增高,差异具有非常显著统计学意义(P<0.01);而MRH组及MRL组大鼠WT%[(13.11±3.91)%,(16.70±1.68)%]较M组大鼠显著降低(P<0.01),其中MRL组大鼠WT%仍高于N组大鼠(P<0.05);MRH组大鼠WT%较MRL组进一步降低,但差异无统计学意义(P>0.05)。肺组织切片a-SMA免疫组化染色显示：N组大鼠肺腺泡内微动脉肌化百分比及a-SMA平均光密度值分别为(19.91±2.27)%及0.22±0.04；与N组大鼠比较,M组大鼠肺腺泡内微动脉肌化百分比及a-SMA平均光密度值[(67.55±2.95)%,0.49±0.03]均显著升高,差异具有非常显著统计学意义(P<0.01)；MRH组大鼠、MRL组大鼠肺腺泡内微动脉肌化百分比及α-SMA平均光密度值分别为[(51.74±2.67)%,0.31±0.02]、[(54.73±2.39)%,0.38±0.03],均低于M组大鼠,差异具有非常显著统计学意义(P<0.01),但仍高于N组大鼠(P<0.01)；MRH组大鼠肺微动脉肌化百分比较MRL组大鼠进一步降低,但差异无统计学意义(P>0.05),而MRH组大鼠α-SMA平均光密度值低于MRL组大鼠,差异具有统计学意义(P<0.05)。4.罗格列酮早期干预对MCT诱导PAH大鼠肺血管周围炎症的影响MCT注射3周后,M组大鼠肺组织切片HE染色显示肺小动脉周围有明显的炎症细胞浸润,N组大鼠肺小动脉周围炎症评分为0.40±0.16；与N组大鼠比较,M组大鼠肺小动脉周围炎症评分(3.24±0.41)显著升高,差异具有非常显著统计学意义(P<0.01)；与M组大鼠比较,MRH组大鼠及MRL组大鼠肺小动脉周围炎症评分(1.26±0.22,1.22±0.36)显著降低(P<0.01),但仍高于N组大鼠(P<0.01)；不同剂量罗格列酮干预组(MRH组和MRL组)大鼠之间肺小动脉周围炎症评分差异无统计学意义(P>0.05)。5.罗格列酮早期干预对MCT诱导PAH大鼠肺组织匀浆中IL-6、TNF-α和MCP-1水平的影响N组大鼠肺组织匀浆IL-6、TNF-α及MCP-1水平依次为(64.97±17.65) pg/ml, (56.51±14.92) pg/ml及(65.18±23.37) pg/ml; M组大鼠上述指标[(459.40±94.77)pg/ml, (436.46±62.20) pg/ml, (6265.53±2014.83) pg/ml]较N组大鼠均升高,差异具有非常显著统计学意义(P<0.01)；MRH组大鼠和MRL组大鼠肺组织匀浆IL-6、TNF-a及MCP-1水平分别为[(102.94±45.28) pg/ml, (244.50±41.01) pg/ml, (979.28±428.71) pg/ml]和[(156.97±61.01) pg/ml, (249.92±61.23) pg/ml, (1107.56±408.32) pg/ml],均低于M组大鼠,差异具有非常显著统计学意义(P<0.01),其中TNF-a及MCP-1水平仍高于N组大鼠(P<0.01或P<0.05),而IL-6水平与N组大鼠差异无统计学意义(P>0.05)；MRH组大鼠肺组织匀浆IL-6、TNF-α及MCP-1水平较MRL组大鼠进一步降低,但差异无统计学意义(P>0.05)。[结论]1.罗格列酮早期应用可以延缓野百合碱诱导肺动脉高压大鼠的肺动脉平均压和右心室收缩压的升高,其改善肺血流动力学的作用与剂量有关。2.罗格列酮早期应用能够延缓野百合碱诱导肺动脉高压大鼠的肺血管重构,减轻右心室肥厚,此效应与剂量有关。3.罗格列酮早期应用能够减轻野百合碱诱导肺动脉高压大鼠肺小动脉周围炎症,降低大鼠肺组织IL-6、TNF-a和MCP-1水平,可能与其对肺血管的保护作用有关。[背景]细胞外基质(extracellular matrix, ECM)重构是肺动脉高压(PH)主要的病理学特征之一。研究显示,ECM稳态的破坏与血管重构密切相关,而基质金属蛋白酶(matrix metalloproteinases, MMPs)及其抑制物组织金属蛋白酶抑制剂(tissue inhibitor of metalloproteinases, TIMPs)是ECM稳态的主要生理调节因子,已被证实在PH患者及动物模型的肺血管重构中发挥重要作用。近期大量体内外实验研究结果提示,过氧化物酶体增殖物激活受体γ(PPARγ)参与了MMPs和TIMPs的调控过程。本研究拟通过野百合碱(MCT)诱导动脉型肺动脉高压(PAH)大鼠模型,观察罗格列酮早期干预对PPARγ的激动作用及对MMP-2、MMP-9和TIMP-1的影响,初步探讨其改善早期肺血管病变的机制。[目的]1.观察罗格列酮早期干预对MCT诱导PAH大鼠肺组织PPARγ表达的影响。2.观察罗格列酮早期干预对MCT诱导PAH大鼠肺组织MMP-2、MMP-9和TIMP-1的表达及MMP-2、MMP-9活性的影响,初步探讨罗格列酮对PH的作用机制。[方法]1.实验动物分组：同第二部分。2.于第21天处死动物,留取肺组织。肺组织切片行PPARγ、MMP-2和MMP-9免疫组化染色,观察PPARγ、MMP-2和MMP-9在大鼠肺组织的定位表达情况。3.采用荧光定量PCR的方法,检测大鼠肺组织PPARγ、MMP-2、MMP-9及TIMP-1的mRNA表达情况。4.采用明胶酶谱法,检测大鼠肺组织MMP-2及MMP-9的活性。5.采用t检验及单因素方差分析比较组间差异,P<0.05为差异具有统计学意义,P<0.01为差异具有非常显著统计学意义。[结果]1.罗格列酮早期干预对MCT诱导PAH大鼠肺组织PPARγ表达的影响免疫组化染色下,PPARγ阳性细胞主要分布于肺血管内膜,表现为细胞浆及细胞核染成棕黄色。N组大鼠肺小动脉内膜PPARγ部分表达,M组大鼠肺小动脉内膜PPARγ表达量降低,MRH组及MRL组大鼠肺小动脉内膜PPARγ表达量增高。荧光定量PCR法检测肺组织PPARγmRNA表达,N组大鼠肺组织PPARγmRNA表达为1.06±0.22,M组大鼠肺组织PPARγmRNA表达(0.57±0.07)较N组大鼠降低(P<0.05)；与M组大鼠比较,MRH组及MRL组大鼠肺组织PPARγmRNA表达[(1.98±0.39),(1.73±0.31)]显著升高(P<0.01),且均高于N组大鼠(P<0.01)；MRH组大鼠肺组织PPARy mRNA表达高于MRL组大鼠,但二者差别无统计学意义(P>0.05)。2.罗格列酮早期干预对MCT诱导PAH大鼠肺组织MMP-2、MMP-9及TIMP-1表达的影响免疫组化染色下,MMP-2、MMP-9阳性细胞主要分布于肺血管壁的内膜、外膜(中膜少量),表现为细胞浆呈棕黄色。M组大鼠肺小动脉MMP-2、MMP-9表达量较N组大鼠升高,MRH组及MRL组大鼠肺小动脉MMP-2、MMP-9表达量较M组降低。荧光定量PCR法检测各组大鼠肺组织MMP-2、MMP-9及TIMP-1 mRNA表达,N组大鼠上述指标依次为0.96±0.08,0.96±0.06,1.02±0.14;与N组大鼠比较,M组大鼠肺组织MMP-2、MMP-9及TIMP-1 mRNA表达(3.30±0.39,7.07±0.54,3.52±0.44)均显著升高(P<0.01)；MRH组大鼠及MRL组大鼠肺组织MMP-2、MMP-9、TIMP-1 mRNA表达分别为(2.00±0.20,3.89±0.65,2.20±0.34)及(2.37±0.17,5.00±0.71,2.58±0.36),均低于M组大鼠(P<0.01),但仍高于N组大鼠(P<0.01)；MRH组大鼠肺组织MMP-2、MMP-9及TIMP-1 mRNA表达均低于MRL组大鼠,其中两组大鼠MMP-9表达的差异具有统计学意义(P<0.05),而MMP-2和TIMP-1表达的差异无统计学意义(P>0.05)。3.罗格列酮早期干预对MCT诱导PAH大鼠肺组织MMP-2、MMP-9活性的影响N组大鼠肺组织MMP-2、MMP-9活性为40669.68±±4978.39,9511.13±3429.93。M组、MRH组和MRL组大鼠肺组织中MMP-2及MMP-9的活性分别为(54893.89±8480.12,23864.69±5121.83)、(52806.28±4759.47,14774.15±2392.96)和(53884.35±4688.02,16199.92±2933.85),均高于N组大鼠(P<0.01或P<0.05)；与M组比较,MRH组及MRL组大鼠肺组织MMP-9活性降低(P<0.01或P<0.05),而MMP-2活性虽较M组略降低,但差异无统计学意义(P>0.05)；MRH组大鼠肺组织MMP-2、MMP-9活性均低于MRL组大鼠,但差异无统计学意义(P>0.05)。[结论]1.野百合碱诱导肺动脉高压大鼠肺组织PPARy mRNA表达下降,罗格列酮可激活PPARy而发挥肺血管保护作用,此效应与剂量有关。2.罗格列酮早期干预可降低野百合碱诱导肺动脉高压大鼠肺组织MMP-2、MMP-9及TIMP-1的mRNA表达水平,此效应与剂量有关。3.罗格列酮早期干预可降低野百合碱诱导肺动脉高压大鼠肺组织MMP-9的活性(而非MMP-2),此效应与剂量有关。这提示罗格列酮可能主要通过调节MMP-9的活性,改善细胞外基质重构而发挥肺血管保护作用。更多还原

【Abstract】 [Backgroud]Pulmonary hypertension (PH) is a group of severe diseases characterized by a progressive increase in pulmonary vascular resistance, and pulmonary arterial hypertension is one of the most common type of PH. The remodeling of pulmonary vessels is a key point of PH. Now the therapeutic drugs can only improve the clinical symptoms of PH patients without inhibition or reversibility of pulmonary vascular remodeling. It has been proved recently that the decreased expression of peroxisome proliferator activated receptor y(PPARy) could play an important role in the pathogenesis of PH, and the ligands of PPARy can inhibit or reverse the development of PH. Rosiglitazone is a synthetic ligand of PPARy, which can produce effects on anti-inflammation, cell proliferation, apoptosis and differentiation. To determine the possible therapeutic effects of rosiglitazone on pulmonary hypertension, we investigated the effects of rosiglitazone on monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) in rats.[Objective]To study the therapeutic effects of rosiglitazone on established severe PAH through observation on changes of general condition, pulmonary hemodynamics and vascular morphology in rats.[Methods]1.40 male Sprague-Dawley rats were randomly divided into four groups:①Group C (n=10):the rats were injected subcutaneously with sodium chloride.②Group P (n=10):the rats were injected subcutaneously with MCT (60mg/kg) and received sodium chloride(1.5ml/d) by daily gavage from day 21st to day 34th after injection of MCT.③Group PRH (n=10):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (5mg/kg-d) by daily gavage from day 21st to day 34th after injection of MCT.④Group PRL (n=10):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (2.5mg/kg-d) by daily gavage from day 21st to day 34th after injection of MCT.2. Right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) were detected by right heart catheter on the day 35th. The right ventricle (RV) was separated from the left ventricle (LV) and septum (S), and weighed. Right ventriclular hypertrophy index (RVHI) was caculated.3. Histopathological changes and the tunica media thickness of small pulmonary arteries were evaluated by hematoxylin and eosin staining and Elastin Van Gieson staining (ET+VG)4. Data were analyzed with the analysis of variance and the t test. Statistical significance was assigned for P<0.05, and extremely significant difference was assigned for P<0.01.[Results]1. Effects of rosiglitazone on general condition of rats injected with MCT. From the 14th day after MCT injection, some rats in Group P, Group PRH and Group PRL became weak, short of breath and presented a dull coat, and the condition got worse day by day. Until the day of sacrifice, the case fatality rate of Group C, Group P, Group PRH and Group PRL was 0,50%,50% and 30%, separately. Three weeks and five weeks after MCT injection, the mean body weight of rats in Group C were (386±14) g and (475±18) g, separately. Compared with Group C, the mean body weight of rats in Group P[(335v28)g, (316±28) g], Group PRH [(331±22)g, (320±43)g] and Group PRL [(327±30) g, (296±41) g] decreased extremely significantly (P<0.01). However, the mean body weight of rats among Group P, Group PRH and Group PRL showed no significant difference (P>0.05)2. Effects of rosiglitazone on hemodynamics and right ventricular hypertrophy of rats injected with MCT.The mPAP, RVSP and RVHI of rats in Group C were (21.66±2.43) mmHg, (43.76±3.64) mmHg and 0.28±0.02. Compared with Group C, the mPAP, RVSP and RVHI of rats in Group P[(61.57±7.28) mmHg, (108.65±9.97) mmHg,0.70±0.03] increased extremely significantly (P<0.01). Compared with Group P, the mPAP, RVSP and RVHI of rats in Group PRH[(58.83±9.47)mmHg,(106.61±11.67)mmHg,0.63±0.07] and Group PRL[(56.31±8.44) mmHg, (102.90±11.69) mmHg,0.63±0.07] decreased slightly without significant difference (P>0.05), and the mPAP, RVSP and RVHI of rats in Group PRH and Group PRL were still extremely significantly higher than those of Group C (P<0.01). There was no significant difference between Group PRH and Group PRL on mPAP, RVSP and RVHI (P>0.05)3. Effects of rosiglitazone on pulmonary arterial remodeling of rats injected with MCT.HE staining showed prominent tunica media hypertrophy, smooth muscle proliferation, stenosis in muscular pulmonary arteries and evident muscularization of pulmonary arterioles of rats in Group P, Group PRH and Group PRL. ET+VG staining showed a "wave-like" change on internal elastic lamina of rats in Group P, Group PRH and Group PRL. The tunica media thickness percentage of small pulmonary arteries (WT%)of rats in Group C was (8.82±3.23)%. Compared with Group C, WT% of rats in Group P [(55.22±5.72)%], Group PRH [(50.92±5.18)%] and Group PRL [(52.45±6.45)%] increased extremely significantly (P<0.01). Compared with Group P, WT% of rats in Group PRH and Group PRL decreased slightly without significant difference (P>0.05). No significant difference between Group PRH and Group PRL on WT% was found (P>0.05)[Conclusions]1. Rosiglitazone treatment can not lower established severely elevated mPAP and RVSP in pulmonary arterial hypertension rats induced by MCT.2. Rosiglitazone treatment can not reverse established pulmonary vascular remodeling and right ventricular hypertrophy in pulmonary hypertension rats induced by MCT. [Backgroud]The pathogenesis of pulmonary hypertension (PH) is extremely complicated and it has not been explored clearly till now. It has been proved recently that inflammatory mechanisms might play an important role in the pathogenesis and progression of PH. Rosiglitazone, a kind of thiazolidinedione (TZDs), is the syhthetic ligand of peroxisome proliferator activated receptor y (PPARy). It has been shown that rosiglitazone could exert immunomodulatory and anti-inflammatory effects on various animal diseases models. To determine the possible preventive effects of rosiglitazone on pulmonary hypertension, we investigated the protective effects of rosiglitazone intervention on monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) with focus on anti-inflammation and vascular remodeling.[Objective]1. To study the protective effects of rosiglitazone intervention on pulmonary arterial hypertension through observation on changes of general condition, pulmonary hemodynamics and vascular morphology in rats injected with MCT.2. To study the changes of pulmonary interleukin 6 (IL-6), tumor necrosis a (TNF-a) and monocyte chemotactic protein 1 (MCP-1) in rats injected with MCT, and explore possible mechanisms of the protective effects of rosiglitazone intervention.[Methods]1.32 male Sprague-Dawley rats were randomly divided into four groups:①Group N (n=8):the rats were injected subcutaneously with sodium chloride.②Group M (n=8):the rats were injected subcutaneously with MCT (60mg/kg) and received sodium chloride(1.5ml/d) by daily gavage from day 0 to day 20th after injection of MCT.③Group MRH (n=8):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (5mg/kg-d) by daily gavage from day 0 to day 20th after injection of MCT.④Group MRL (n=8):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (2.5mg/kg·d) by daily gavage from day 0 to day 20th after injection of MCT.2. On the day 21st, right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP) and mean arterial pressure (MAP) were detected by right heart catheter and internal carotid artery catheter, separately. The right ventricle (RV) was separated from the left ventricle (LV) and septum (S), and weighed. Right ventriclular hypertrophy index (RVHI) was caculated.3. Histopathological changes were evaluated by hematoxylin and eosin staining, and the tunica media thickness of small pulmonary arteries were evaluated by Elastin Van Gieson staining (ET+VG). The muscularization degree of non-muscularized pulmonary arterioles were evaluated by muscularization percentage and mean optical density through a-SMA immunohistochemical staining.4. Interleukin 6, tumor necrosis factor a and monocyte chemotactic protein 1 of lung tissue were detected by ELISA.5. Data were analyzed with the analysis of variance and the t test. Statistical significance was assigned for P<0.05, and extremely significant difference was assigned for P<0.01.[Results]1. Effects of rosiglitazone intervention on general condition of rats injected with MCT.From the 14th day after MCT injection, some rats in Group M became weak, short of breath and presented a dull coat. The mean body weight of Group N on day 7th,14th,21st were (287±12) g, (331±15) g and (375±19) g. Compared with Group N, the mean body weight of Group M[(261±17) g, (276±24) g, (319±29) g], Group MRH [(268±8) g, (275±15)g, (328±14)g] and Group MRL[(261±8) g, (284±11)g, (329±12)g]on day 7th,14th,21st decreased significantly (P<0.01 or P<0.05). However, the mean body weight of rats in Group M, Group MRH and Group MRL showed no significant difference (P>0.05)2. Effects of rosiglitazone intervention on hemodynamic parameters and right ventricular hypertrophy of rats injected with MCT.Three weeks after injection of MCT, the mPAP, RVSP and RVHI of rats in Group N were (17.13±3.30) mmHg, (40.33±5.77) mmHg and 0.25±0.02, separately. Compared with Group N, the mPAP, RVSP and RVHI of rats in Group M [(37.00±4.98) mmHg, (67.04±4.87)mmHg,0.40±0.02]increased extremely significantly (P<0.01).Compared with Group M, the mPAP, RVSP and RVHI of rats in Group MRH [(26.88±3.55) mmHg, (50.09±5.61)mmHg,0.33±0.03] and Group MRL[(28.29±3.60)mmHg, (55.71±4.85) mmHg,0.33±0.02]decreased extremely significantly (P<0.01),but were still higher than those of Group N (P<0.01 or P<0.05). There was no significant difference between Group MRH and Group MRL on mPAP, RVSP and RVHI. No significant difference of MAP among Group N [(87.81±7.78) mmHg], Group M [(88.23±9.29) mmHg], Group MRH [(90.17±6.82) mmHg] and Group MRL [(89.88±9.08) mmHg] was found (P >0.05)3. Effects of rosiglitazone intervention on pulmonary arterial remodeling of rats injected with MCT.HE staining showed obvious tunica media hypertrophy, smooth muscle proliferation, stenosis in muscular pulmonary arteries and evident muscularization of pulmonary arterioles of rats in Group M, ET+VG staining showed a "wave-like" change on internal elastic lamina of rats in Group M. The the tunica media thickness percentage of small pulmonary arteries (WT%) of rats in Group N was (8.91±2.30)%. Compared with Group N, the WT% of rats in Group M [(45.52±5.48)%]increased extremely significantly (P<0.01). Compared with Group M, the WT% of Group MRH [(13.11±3.91)%] and Group MRL [(16.70±1.68)%] decreased extremely significantly (P<0.01), but the WT% of Group MRL was still higher than that of Group N (P< 0.05). Compared with Group MRL, the WT% of Group MRH decreased slightly without significant difference (P>0.05)a-SMA immunohistochemical staining showed that, the muscularization percentage of pulmonary arterioles and a-SMA mean optical density of rats in Group N were [(19.91±2.27)%,0.22±0.04]. Compared with Group N, the muscularization percentage of pulmonary arterioles and a-SMA mean optical density of rats in Group M [(67.55±2.95)%,0.49±0.03] increased extremely significantly (P<0.01).Compared with Group M, the muscularization percentage of pulmonary arterioles and a-SMA mean optical density of rats in Group MRH [(51.74±2.67)%,0.31±0.02] and Group MRL [(54.73±2.39)%,0.38±0.03] decreased significantly (P<0.01), but were still higher than those of Group N (P<0.01). Compared with Group MRL, the muscularization percentage of pulmonary arterioles of rats in Group MRH decreased slightly without significant difference (P> 0.05), and a-SMA mean optical density of rats in Group MRH decreased significantly (P<0.05)4. Effects of rosiglitazone intervention on perivascular inflammation of rats injected with MCT.Three weeks after injection of MCT, HE staining showed obvious inflammatory cells infiltration around the small pulmonary arteries. The perivascular inflammation scale of Group N was 0.40±0.16. Compared with Group N, the perivascular inflammation scale of Group M(3.24±0.41) increased extremely significantly(P<0.01). Compared with Group M, the perivascular inflammation scale of Group MRH (1.26±0.22)and Group MRL(1.22±0.36) decreased extremely significantly(P<0.01). There was no significant difference of perivascular inflammation scale between Group MRH and Group MRL (P>0.05)5. Effects of rosiglitazone intervention on pulmoanry IL-6, TNF-a and MCP-1 level of rats injected with MCT.The pulmonary IL-6, TNF-a and MCP-1 level of rats in Group N were (64.97±17.65) pg/ml, (56.51±14.92) pg/ml and (65.18±23.37) pg/ml, separately. Compared with Group N, the pulmonary IL-6, TNF-a and MCP-1 level of rats in Group M [(459.40±94.77)pg/ml, (436.46±62.20)pg/ml, (6265.53±2014.83)pg/ml] increased extremely significantly(P<0.01). Compared with Group M, the pulmonary IL-6, TNF-a and MCP-1 level of rats in Group MRH [(102.94±45.28) pg/ml, (244.50±41.01) pg/ml, (979.28±428.71) pg/ml] and Group MRL [(156.97±61.01) pg/ml, (249.92±61.23) pg/ml, (1107.56±408.32) pg/ml] decreased significantly (P<0.01), but the TNF-a and MCP-1 level were still higher than those of Group N (P<0.05 or P<0.01).Compared with Group MRL, the pulmonary IL-6, TNF-a and MCP-1 level of rats in Group MRL decreased slightly without significant difference (P>0.05)[Conclusions]1. Rosiglitazone intervention may delay the increase of mean pulmonary artery pressure and right ventricular systolic pressure in pulmonary arterial hypertension rats induced by MCT, and the improvement of hemodynamic parameters by rosiglitazone is related to drug dosage.2. Rosiglitazone intervention may delay the remodeling of pulmonary vessels and inhibit right ventricular hypertrophy in pulmonary arterial hypertension rats induced by MCT, and this effect is related to drug dosage.3. Rosiglitazone intervention may exert protective effect on MCT-induced pulmonary arterial hypertension partly by inhibiting the pervascular inflammation and the increase of IL-6, TNF-a and MCP-1 in lung tissue. [Backgroud]Remodeling of extracellular matrix (ECM) is a main pathologic feature of PH. Researches showed that the homeostasis and destruction of ECM were closely related to vascular remodeling. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are chief regulatory factors of ECM homeostasis. It has been proved that MMPs and TIMPs played an important role in pulmonary vascular remodeling in patients or animal models of PH. It is recognized recently that peroxisome proliferator activated receptor y (PPARy) is involved in the regulation of MMPs and TIMPs. To explore the underlying mechanisms of rosiglitazone intervention on PH, we investigated the changes of PPARy, MMP-2, MMP-9 and TIMP-1 expression in rats with monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) after rosiglitazone intervention.[Objective]1. To observe the influence of rosiglitazone intervention on PPARy expression of lung tissue in rats with MCT induced PAH.2. To explore the underlying mechanisms of rosiglitazone intervention on PH through observing the influence of rosiglitazone intervention on pulmonary MMP-2、MMP-9 and TIMP-1 expression and MMP-2、MMP-9 activity in rats with MCT induced PAH.[Methods]1. Grouping of experimental animals:see part 2 of this article.2. All of the animals were sacrificed on day 21st, PPARγ, MMP-2 and MMP-9 expression location on pulmonary histological sections of rats were observed through immunohistochemical staining.3. Expression of PPARy, MMP-2, MMP-9 and TIMP-1 mRNA in lung tissue of rats were detected through fluorescent quantitative PCR.4. The activity of MMP-2 and MMP-9 in lung tissue of rats were detected through gelatin zymography.5. Data were analyzed with the analysis of variance and the t test. Statistical significance was assigned for P<0.05, and extremely significant difference was assigned for P<0.01.[Results]1. Influence of rosiglitazone intervention on PPARy expression of lung tissue in rats injected with MCT.Immunohistochemical staining showed that the PPARy positive cells mostly located at the intima of the vascular wall, and presented a brown staining of nucleus and cytoplasm. PPARy partly expressed at the intima of small pulmonary arteries in rats of Group N, the PPARy expression at the intima of small pulmonary arteries in rats of Group M decreased significantly. The PPARy expression at intima of small pulmonary arteries in rats of Group MRH and Group MRL increased.Fluorescent quantitative PCR:PPARy mRNA expression of lung tissue in rats of Group N was 1.06±0.22. Compared with Group N, PPARy mRNA expression of lung tissue in rats of Group M (0.57±0.07) decreased significantly (P<0.05). Compared with Group M, PPARy mRNA expression of lung tissue in rats of Group MRH (1.98±0.39) and Group MRL (1.73±0.31) increased extremely significantly (P<0.01), and were higher than that of Group N (P<0.01). Compared with Group MRL, PPARy mRNA expression of lung tissue in rats of Group MRH increased slightly without significant difference (P>0.05)2. Influence of rosiglitazone intervention on MMP-2, MMP-9 and TIMP-1 expression of lung tissue in rats injected with MCT.Immunohistochemical staining showed that the MMP-2, MMP-9 positive cells mostly located in the intima and adventitia of the vascular wall, presented a brown staining of cytoplasm. MMP-2 and MMP-9 expression of small pulmonary arteries in rats of Group M increased significantly when compared with Group N. Compared with Group M, MMP-2 and MMP-9 expression of small pulmonary arteries in rats of Group MRH and Group MRL decreased.Fluorescent quantitative PCR:MMP-2, MMP-9 and TIMP-1 mRNA expression of lung tissue in rats of Group N were 0.96±0.08,0.96±0.06 and 1.02±0.14, separately. Compared with Group N, MMP-2, MMP-9 and TIMP-1 mRNA expression of lung tissue in rats of Group M (3.30±0.39,7.07±0.54,3.52±0.44) increased extremely significantly (P<0.01). Compared with Group M, MMP-2, MMP-9 and TIMP-1 mRNA expression of lung tissue in rats of Group MRH (2.00±0.20,3.89±0.65,2.20±0.34) and Group MRL (2.37±0.17,5.00±0.71,2.58±0.36) decreased extremely significantly (P<0.01), but were still higher than those of Group N(P<0.01). Compared with Group MRL, MMP-9 mRNA expression of lung tissue in rats of Group MRH decreased significantly (P< 0.05), but MMP-2 and TIMP-1 expression only decreased slightly (P>0.05)3. Influence of rosiglitazone intervention on MMP-2 and MMP-9 activity of lung tissue in rats injected with MCT.Gelatin zymography:the MMP-2 and MMP-9 activity of lung tissue in rats of Group N were 40669.68±4978.39 and 9511.13±3429.93. Compared with Group N, the MMP-2 and MMP-9 activity of lung tissue in rats of Group M (54893.89±8480.12, 23864.69±5121.83), Group MRH (52806.28±4759.47,14774.15±2392.96) and Group MRL (53884.35±4688.02,16199.92±2933.85) increased significantly (P<0.01 or P< 0.05). Compared with Group M, the MMP-9 activity of lung tissue in rats of Group MRH and Group MRL decreased significantly (P<0.01 or P<0.05), the MMP-2 activity of lung tissue in rats of Group MRH and Group MRL decreased slightly without significant difference (P>0.05). Compared with Group MRL, the MMP-2 and MMP-9 activity of lung tissue in rats of Group MRH decreased slightly without significant difference (P>0.05)[Conclusions]1. PPARy mRNA expression of lung tissue in rats injected with MCT decreased. Rosiglitazone can activate PPARy to exert protective effects to pulmonary vessels, and this effect is related to drug dosage.2. Rosiglitazone intervention can derease the MMP-2, MMP-9 and TIMP-1 expression of lung tissue in PAH rats injected with MCT, and this effect is related to drug dosage.3. Rosiglitazone intervention can derease the MMP-9 activity of lung tissue in PAH rats injected with MCT (but not MMP-2). It suggests that rosiglitazone may improve the remodeling of extracellular matrix to exert protective effects to pulmonary vessels through regulation of MMP-9 activity.更多还原