药学学报  2015, Vol. 50 Issue (9): 1128-1134   PDF    
白杨素对野百合碱诱导的肺动脉高压大鼠右心室NOX4及NF-κB表达的影响
李先伟 , 过波, 沈媛媛, 杨解人    
皖南医学院药理学教研室, 安徽 芜湖 241002
摘要: 观察白杨素(chrysin, 5,7-二羟基黄酮)对野百合碱(monocrotaline, MCT)诱导的肺动脉高压(pulmonaryarterial hypertension, PAH)大鼠右心室重构的影响及其机制。Sprague-Dawley大鼠适应性喂养1周,随机分为正常对照组、MCT组、MCT+chrysin (50 mg·kg-1·d-1)及MCT+chrysin (100 mg·kg-1·d-1)剂量组。MCT (60 mg·kg-1)皮下注射诱导PAH大鼠模型。连续给药4周后,右颈外静脉插管测定大鼠右心室收缩压(RVSP)、平均肺动脉压(mPAP)。分离大鼠右心室(RV)、左心室+室间隔(LV+S)并称重,剥离大鼠胫骨并测量其长度,计算RV/(LV+S)及RV/胫骨长度的比值。HE染色观察右心室病理学变化, Masson染色观察右心室胶原沉积的变化。比色法测定右心室总抗氧化能力(T-AOC)和丙二醛(MDA)含量。qPCR、Western blot和(或)免疫组化检测右心室collagen I、collagen III、NADPH氧化酶4(NADPH oxidase 4, NOX4)、核转录因子κB (nuclear factor-kappa B, NF-κB) mRNA和蛋白表达。结果发现,白杨素连续给药4周后能明显降低MCT诱导的PAH大鼠RVSP及mPAP,减轻RV/(LV+S)及RV/胫骨长度的比值,改善右心室病理变化,降低右心室胶原的沉积及collagen I、collagen III的表达,提高右心室T-AOC水平,降低NOX4的表达及MDA含量,抑制NF-κB的表达。结果表明,白杨素能缓解MCT诱导的PAH大鼠右心室重构,其机制可能与其抑制NOX4介导的氧化应激损伤,进而抑制NF-κB所介导的胶原沉积有关。
关键词: 白杨素     肺动脉高压     右心室重构     NADPH氧化酶4     核转录因子κB    
Effect of chrysin on expression of NOX4 and NF-κB in right ventricle of monocrotaline-induced pulmonary arterial hypertension of rats
LI Xian-wei , GUO Bo, SHEN Yuan-yuan, YANG Jie-ren    
Department of Pharmacology, Wannan Medical College, Wuhu 241002, China
Abstract: The aim of the present study is to investigate the protective effect of chrysin (5,7-dihydroxyflavone) on right ventricular remodeling in a rat model of monocrotaline-induced pulmonary arterial hypertension (PAH). PAH rats were induced by a single injection of monocrotaline (60 mg·kg-1, sc) and were administered with chrysin (50 or 100 mg·kg-1·d-1) for 4 weeks. At the end of experiment, the right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored via the right jugular vein catheterization into the right ventricle. Right ventricle (RV) to left ventricle (LV) + septum (S) and RV to tibial length were calculated. Right ventricular morphological change was observed by HE staining. Masson 's trichrome stain was used to demonstrate collagen deposition. The total antioxidative capacity (T-AOC) and malondialdehyde (MDA) levels in right ventricle were determined according to the manufacturer 's instructions. The expressions of collagen I, collagen III, NADPH oxidase 4 (NOX4) and nuclear factor-kappa B (NF-κB) were analyzed by immunohistochemisty, qPCR and (or) Western blot. The results showed that chrysin treatment for 4 weeks attenuated RVSP, mPAP and right ventricular remodeling index (RV/LV+S and RV/Tibial length) of PAH rats induced by monocrotaline. Furthermore, monocrotaline-induced right ventricular collagen accumulation and collagen I and collagen III expression were both significantly suppressed by chrysin. The expressions of NOX4, NF-κB and MDA contents were obviously decreased, while the T-AOC was significantly increased in right ventricule from PAH rats with chrysin treatment. These results suggest that chrysin ameliorates right ventricular remodeling of PAH induced by monocrotaline in rats through its down-regulating of NOX4 expression and antioxidant activity, and inhibiting NF-κB expression and collagen accumulation.
Key words: chrysin     pulmonary arterial hypertension     right ventricular remodeling     NADPH oxidase 4     nuclear factor-kappa B    

肺动脉高压 (pulmonary arterial hypertension,PAH) 是一种临床常见的以肺血管阻力进行性增加并伴有不可逆的血管构型重建为特征的疾病,肺血管阻力和肺动脉压力的持续升高,最终可导致呼吸衰竭、肺心病、心力衰竭甚至死亡,故防治肺动脉高压和右心室重构具有重要的临床意义[1, 2]。研究表明氧化应激在PAH右心重构中发挥了重要的作用,其中NADPH氧化酶4 (NADPH oxidase 4,NOX4) 诱 导的氧化应激损伤参与了PAH右心室重构,作者前期的研究也发现在野百合碱诱导的PAH大鼠右心室重构中NOX4的表达明显升高[3, 4]。另外,研究还发现核转录因子κB (nuclear factor-kappa B,NF-κB) 的活化参与了PAH心血管重构,而NOX4介导的氧化应激可能通过增强对NF-κB的抑制蛋白IκB (inhibitor kappa B) 的解离,激活NF-κB与DNA的结合活性,进一步激活NF-κB相关的转录因子[5],促进细胞因子、生长因子、趋化因子、炎症介质和黏附分子等相关基因表达,造成细胞外基质合成增多,降解减少,促进细胞间质纤维化,参与了PAH心血管重构[6]

白杨素 (chrysin) 是一种天然的黄酮类化合物,其化学结构为5,7-二羟基黄酮 (5,7-dihydroxyflavone)。近年来动物实验和体外实验发现其具有抗炎、抗氧化、抗凋亡及抑制细胞增殖等作用,主要的原因与其抑制NF-κB的活化有关[7, 8, 9]。而作者前期的研究发现白杨素通过抑制NOX4介导的氧化应激损伤而减轻PAH肺血管重构[10]。但目前关于白杨素对PAH右心室重构的保护作用未见文献报道。基于以上的研究背景,本研究以野百合碱诱导的肺动脉高压右心室重构为模型,选择NOX4和NF-κB为靶点,探讨白杨素对肺动脉高压大鼠右心室重构的保护作用及其机制。

材料与方法 动物

健康雄性Sprague-Dawley (SD) 大鼠,体重180~220 g (上海杰恩捷实验动物有限公司),微生物等级SPF级 (上海实验动物质量监督监测站),许可证号SCXK (沪) 2013-0006。

试剂与仪器

白杨素 (chrysin,5,7-二羟基黄酮,分子式: C15H10O4,纯度≥98%,上海纯优生物科技有限公司); 野百合碱 (monocrotaline,MCT,美国Sigma公司)。Masson染色试剂盒 (南京凯基生物科技发展有限公司); 总抗氧化能力 (T-AOC)、丙二醛 (MDA) 及BCA蛋白检测试剂盒 (江苏碧云天生物技术研究所); TRIzol RNA提取试剂盒 (美国Invitrogen公司); PrimeScriptTM RT reagent Kit、Power SYBR Green PCR Master Mix (大连宝生物工程有限公司),引物设计合成 (上海生工生物工程有限公司)。小鼠抗大鼠GAPDH、NOX4及NF-κB单克隆抗体 (美国Santa Cruz公司); 小鼠抗大鼠collagen I及collagen III单 克隆抗体 (美国Abcam公司); LunimataTM Crescendo发光液 (美国Millipore公司)。MedLab多导生理记录仪 (型号: MedLab-TA6008,南京美易科技有限公司); BioPhotometer核酸测定仪 (BioPhotometer D30)、Eppendorf离心机 (5417R) (德国Eppendorf公司); 梯度PCR仪(Veriti™ 96)、实时荧光定量PCR仪 (7300) (美国ABI公司); 全波长酶标仪 (DTX880,美国Beckman Coulter公司); 垂直电泳系统 (Mini-PROTEAN Tetra + PowerPac Basic)、湿转电泳槽 (Mini Trans- Blot®)、半干转印系统转印槽 (Trans-Blot®)、化学发光成像系统 (ChemiDoc XRS+) (美国Bio-Rad公司); 石蜡切片机 (RM-20160)、组织包埋机 (EG1150) (德国Leica公司); Nikon显微镜 (Eclipse 80i,日本Nikon公司)。

肺动脉高压大鼠模型的建立及实验分组

SD大鼠适应性喂养1周,按体重随机分为: 正常对照组 (control)、MCT组、MCT+chrysin (50 mg·kg−1) 剂量 组和MCT+chrysin (100 mg·kg−1) 剂量组,每组8只。MCT (60 mg·kg−1) 单次皮下注射复制肺动脉高压动物模型[11]。Chrysin用羧甲基纤维素钠充分混匀,每日上午8∶00~10∶00灌胃给药,连续给药4周。所有大鼠实验期间自由进食、饮水。室内通风良好,相对湿度40%~70%,温度(20± 3) ℃,光照12/24 h。

血流动力学指标的测定

给药4周后,大鼠用戊巴比妥钠 (60 mg·kg−1) 腹腔注射麻醉。将连有压力换能器的聚乙烯导管 (直径0.5 mm) 经右颈外静脉插入右心室及肺动脉,用MedLab多导生理记录仪分别记录右心室收缩压 (RVSP) 和平均肺动脉压 (mPAP)[11]

组织标本采集与处理

血流动力学数据检测完毕后,颈动脉采血后处死大鼠,立即开胸迅速分离心脏,置于预冷的PBS中洗涤干净,另取大鼠右后肢 胫骨; 准确分离右心室、左心室加室间隔,称重,计算右心室 (RV)/(左心室+室间隔) (LV+S) 质量比; 用游标卡尺测量胫骨长度,计算RV/胫骨长度比值[3, 12]。取右心室心尖部固定于4% 多聚甲醛以备石蜡切片; 准确称取100 mg右心室置于液氮中研磨后加入Trizol (1 mL),−80 ℃冰箱保存,用于相关指标mRNA表达水平检测; 剩余右心室置于液氮中研磨后加入组织裂解液冰上裂解30 min,12 000 r·min−1,4 ℃离心10 min,提取蛋白,用于相关指标蛋白表达水平的检测。

右心室组织病理学及胶原沉积的观察

取大鼠右心室心尖部以4% 多聚甲醛固定48 h,石蜡包埋切片,进行HE及Masson染色,分别观察右心室病理变化及胶原沉积情况。Masson胶原染色按试剂盒说明书进行。

生化指标测定

采用Ferric Reducing Ability of Plasma法和硫代巴比妥酸法分别检测右心室T-AOC及MDA含量,具体步骤详见T-AOC及MDA检测试剂盒说明书。BCA法测定右心室蛋白浓度。

免疫组化检测右心室NOX4蛋白表达

右心室石蜡切片,厚度约每片4 μm,常规脱蜡至水,抗原修复液修复20 min,封闭液室温封闭1 h,抗NOX4一抗(1∶200) 4 ℃过夜,二抗(1∶400) 室温孵育2 h,SABC室温孵育1 h,DAB显色,苏木素复染,中性树胶封片观察。结果判断: NOX4主要定位于细胞浆。阳性细胞表达呈黄至棕黄色颗粒。

qPCR检测右心室collagen Icollagen IIINOX4NF-κB mRNA的表达

用Trizol等试剂提取右心室总RNA后,按逆转录试剂盒操作步骤进行RT反应。所得cDNA在实时荧光定量PCR仪上进行反应。按照说明,使用Power SYBR Green PCR Master Mix试剂盒,以2 µL cDNA为模板,以β-actin为内参照,PCR扩增基因片段。引物如表 1。PCR反应参数设 置参照前期的研究方法[13]。数据分析用7300 System SDS Software软件,统计ΔΔCt值,计算RQ值以比较各组mRNA的表达。引物设计见表 1

Table 1 Primer sequences used for determination of NOX4,NF-κB,and collagen I and collagen III gene expression
Western blot检测右心室collagen Icollagen III、NOX4NF-κB蛋白表达

低温提取右心室蛋白,BCA法测定蛋白浓度。每孔上样蛋白50 µg,12% SDS-PAGE分离样品,转膜,封闭,分别滴加β-actin (1∶2 00 0)、NOX4 (1∶500)、NF-κB (1∶200)、collagen I (1∶1 000) 和collagen III (1∶1 000) 一抗4 ℃过夜。洗膜,相应二抗 (1∶2 000) 室温孵育1 h。洗膜后将高灵敏的LunimataTM Crescendo发光剂加到膜的正面,采用Bio-Rad ChemiDoc XRS+成像系统进行拍照,用Image J 1.43 (National Institutes of Health) 软件进行灰度值分析并比较各组蛋白表达差异。

统计学分析

实验数据均以平均值 ± 标准差 (x± s) 表示,统计分析采用SPSS l6.0软件。多组均数比较采用ANOVA及Newman-Keuls-Student多重比较t检验。P < 0.05为差异有统计学意义。

结果 1 白杨素对肺动脉高压大鼠血流动力学指标的影响

与对照组相比,MCT处理组大鼠RVSP、mPAP均显著升高 (P < 0.01)。给予不同剂量白杨素4周后,肺动脉高压大鼠RVSP和mPAP均显著降低,提示白杨素具有改善肺动脉高压血流动力学的作用 (表 2)。

Table 2 Effect of chrysin on RVSP and mPAP in monocrotaline- induced pulmonary hypertension of rats. x± s. **P < 0.01 vs control group; #P < 0.05,##P < 0.01 vs monocrotaline group. RVSP: Right ventricle systolic pressure; mPAP: Mean pulmonary arterial pressure
2 白杨素对肺动脉高压大鼠RV/LV+SRV/胫骨长度比值的影响

因RV/LV+S及RV/胫骨长度比值不受体重及左心室重量影响,是评价右心室肥厚程度的主要指标[3, 12]。与对照组相比,MCT处理组RV/LV+S及RV/胫骨长度的比值均显著升高 (P < 0.01),说明野百合碱皮下注射诱导的肺动脉高压大鼠右心室明显增生肥厚。而给予不同剂量白杨素4周后,上述比值均显著降低,提示白杨素具有抑制肺动脉高压右心室重构的作用 (表 3)。

Table 3 Effect of chrysin on RV/LV+S and RV/tibial length in monocrotaline-induced pulmonary hypertension of rats. x± s. **P < 0.01 vs control group; #P < 0.05,##P < 0.01 vs monocrotaline group. RV: Right ventricle; LV+S: Left ventricle + septum
3 白杨素对肺动脉高压大鼠右心室病理变化的影响

右心室HE染色后于400倍镜下观察,与对照组相比,MCT处理组右心室心肌细胞增生肥大且排列紊乱、稀疏和胞浆肿胀,心肌细胞间隙增宽 (图 1,图中箭头所示),而给予不同剂量白杨素4周后上述病理变化均有不同程度的改善。

Figure 1 Hematoxylin-eosin staining of right ventricle in monocrotaline-induced pulmonary hypertension of rats (magnification,×400)
4 白杨素对肺动脉高压大鼠右心室胶原表达的影响

Masson染色显示胶原纤维为蓝色,正常对照组右心室心肌细胞间隙清晰可见,血管旁胶原分布稀疏,着色较淡。与对照组相比,MCT处理组大鼠右心室心肌细胞间质和血管旁有较多明显蓝色深染; 与MCT相比,白杨素各剂量组右心室心肌细胞间质和血管旁的蓝色深染逐渐减少,胶原沉积现象逐渐减轻,说明白杨素对肺动脉高压大鼠右心室心肌胶原沉积有一定的改善作用 (图 2)。同时,经qPCR和Western blot检测发现,MCT处理组大鼠右心室collagen I和collagen III mRNA及蛋白表达水平显著升高 (P < 0.01),而给予不同剂量白杨素4周后collagen I和collagen III的表达明显降低 (图 3)。

Figure 2 Effect of chrysin on collagen accumulation of right ventricle in monocrotaline-induced pulmonary arterial hypertension of rats (Masson’s trichrome stain,×400)

Figure 3 Effect of chrysin on the collagen I and collagen III mRNA and proteins expression of right ventricle in monocrotaline-induced pulmonary hypertension of rats. A and C: The expression of collagen I and collagen IIImRNA was determined by qPCR. B and D: The expression of collagen I and collagen III protein was determined by Western blot. n = 7 722;8,x± s. **P < 0.01 vs control group; #P < 0.05,##P < 0.01 vs MCT group. MCT: Monocrotaline
5 白杨素对肺动脉高压大鼠右心室NOX4表达的影响

经qPCR、Western blot及免疫组化检测发现,MCT处理组右心室NOX4 mRNA及蛋白表达水平明显升高 (P < 0.01)。与MCT组相比,给予不同剂量白杨素4周后均能不同程度下调大鼠右心室NOX4 mRNA及蛋白的表达 (图 4)。

Figure 4 Effect of chrysin on the NOX4 expression of right ventricle in monocrotaline-induced pulmonary hypertension of rats. A: The expression of NOX4 in right ventricle was determined by qPCR. B: The expression of NOX4 in right ventricle was determined by Western blot. C: The expression of NOX4 in right ventricle was determined with immunohistochemisty staining (arrows indicate NOX4 positive staining). n = 7−8,x± s. **P < 0.01 vs control group; #P < 0.05,##P < 0.01 vs MCT group. MCT: Monocrotaline; NOX4: NADPH oxidase 4
6 白杨素对肺动脉高压大鼠右心室MDAT-AOC的影响

与对照组相比,MCT处理组右心室MDA含量显著升高,T-AOC活性明显降低 (P < 0.01); 与MCT组相比,白杨素各剂量组右心室T-AOC活性明显增高,MDA含量显著下降,提示白杨素具有一定的抗氧化作用 (表 4)。

Table 4 Effect of chrysin on MDA and T-AOC level in right ventricle of monocrotaline-induced pulmonary hypertension of rats. x± s. **P < 0.01 vs control group; #P < 0.05,##P < 0.01 vs monocrotaline group. T-AOC: Total antioxidative capacity; MDA: Malondialdehyde
7 白杨素对肺动脉高压大鼠右心室NF-κB表达的影响

经qPCR及Western blot检测发现,MCT处理组右心室NF-κB mRNA及蛋白表达水平明显升高 (P < 0.01)。与MCT组相比,给予不同剂量白杨素4周后均能不同程度下调大鼠右心室NF-κB mRNA及蛋白的表达 (图 5)。

Figure 5 Effect of chrysin on the NF-κB expression of right ventricle in monocrotaline-induced pulmonary hypertension of rats. A: The expression ofNF-κB in right ventricle was determined by qPCR. B: The expression ofNF-κB in right ventricle was determined by Western blot. n = 7−8,x± s. **P < 0.01 vs control group; #P < 0.05,##P < 0.01 vs MCT group. MCT: Monocrotaline; NF-κB: Nuclear factor-kappa B
讨论

最近的研究发现白杨素能抑制血小板源性生长因子诱导的血管平滑肌细胞增殖[14],而作者前期的研究发现白杨素能抑制低氧性PAH肺血管重构[10]。本实验研究结果显示,白杨素连续给药4周后,PAH大鼠的RVSP、mPAP、RV/(LV+S)、RV/胫骨长度和右心室胶原沉积、collagen I及collagen III的表达均显著降低,右心室病理损伤明显减轻,提示白杨素对PAH大鼠具有降低肺动脉压力、减轻右心室重构和纤维化的作用。

肺动脉高压右心室重构的发生机制尚未完全阐明,大量研究表明NOX诱导的氧化应激在肺血管重构及右心重构中起着重要作用[15]。最新研究发现,白藜芦醇及其衍生物通过抑制NOX2及NOX4的表达而抑制低氧诱导的肺动脉高压右心室重构[3]。而前期研究发现在野百合碱诱导的PAH大鼠右心室NOX4的表达显著增高[4],表明NOX4介导的氧化应激损伤参与了肺动脉高压右心室重构。而给予白杨素4周后,PAH大鼠右心室NOX4表达显著降低,MDA含量明显减少,T-AOC含量显著升高。这些结果进一步表明白杨素具有抗氧化作用,可能通过其抗氧化作用,下调NOX4的表达,进而对PAH大鼠右心室重构具有一定的保护作用。

NF-κB是一种核转录因子,普遍存在于真核生物体内,参与生物体内很多重要的生理反应过程。其中氧化应激损伤诱导的NF-κB活化在肺动脉高压的形成进程中发挥重要作用[16, 17]。正常状态下,组织细胞中的NF-κB与其抑制物IκB结合,以无活性形式存在于胞浆中。当组织细胞受到氧化应激损伤,细胞内IκB激酶被激活,后者可使IκB磷酸化而解离,NF-κB的核定位信号暴露出来并迅速发生核移位,启动基因转录造成细胞外基质合成增多,降解减少,促进细胞间质纤维化,参与了PAH心血管重构[5, 6]。而大量的研究表明白杨素的抗炎、抗氧化、抗凋亡及抑制细胞增殖等作用主要是通过抑制了NF-κB活化而实现的[18, 19]。本研究发现,野百合碱诱导的PAH大鼠右心室NF-κB的表达显著增高,而给予不同剂量白杨素4周后均能不同程度下调大鼠右心室NF-κB mRNA及蛋白的表达,表明白杨素可能也是通过影响NF-κB活化而抑制PAH右心室重构。

综上所述,白杨素能够缓解野百合碱诱导的PAH大鼠右心室重构,其机制可能与抑制NOX4的表达,进而降低氧化应激损伤诱导的NF-κB活化有关,但也有文献报道,心肌NOX2的表达升高也参与了肺动脉高压右心室重构的发生与发展[3],而白杨素是否通过抑制NOX2介导的氧化应激损伤进而产生心脏保护作用有待进一步研究。

参考文献
[1] Chin KM, Rubin LJ. Pulmonary arterial hypertension [J]. J Am Coll Cardiol, 2008, 51: 1527-1538.
[2] Voelkel NF, Quaife RA, Leinwand LA, et al. Right ventricular function and failure: report of a National Heart, Lung, and Blood institute working group on cellular and molecular mechanisms of right heart failure [J]. Circula-tion, 2006, 114: 1883-1891.
[3] Liu B, Luo XJ, Yang ZB, et al. Inhibition of NOX/VPO1 pathway and inflammatory reaction by trimethoxystilbene in prevention of cardiovascular remodeling in hypoxia-induced pulmonary hypertensive rats [J]. J Cardiovasc Pharmacol, 2014, 63: 567-576.
[4] Li XW, Wang XM, Li S, et al. Effects of rutaecarpine on right ventriclar remodeling in rats with monocrotaline-induced pulmonary hypertension [J]. Chin J Appl Physiol (中国应用生理学杂志), 2014, 30: 405-410.
[5] Sawada H, Mitani Y, Maruyama J, et al. A nuclear fac-tor-κB inhibitor pyrrolidine dithiocarbamate ameliorates pulmonary hypertension in rats [J]. Chest, 2007, 132: 1265-1274.
[6] Huang J, Kaminski PM, Edwards JQ et al. Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced pulmonary artery hypertension [J]. Am J Physiol Lung Cell Mol Physiol, 2008, 294: L1250-L1259.
[7] Chaudhuri S, Banerjee A, Basu K, et al. Interaction of flavonoids with red blood cell membrane lipids and proteins: antioxidant and antihemolytic effects [J]. Int J Biol Macromol, 2007, 41: 42-48.
[8] Pushpavalli G, Kalaiarasi P, Veeramani C, et al. Effect of chrysin on hepatoprotective and antioxidant status in D- galactosamine-induced hepatitis in rats [J]. Eur J Pharmacol, 2010, 631: 36-41.
[9] Villar IC, Jiménez R, Galisteo M, et al. Effects of chronic chrysin treatment in spontaneously hypertensive rats [J]. Planta Med, 2002, 68: 847-850.
[10] Li XW, Wang XM, Li S, et al. Effects of chrysin (5, 7- dihydroxyflavone) on vascular remodeling in hypoxia-induced pulmonary hypertension in rats [J]. Chin Med, 2015, 10: 4.
[11] Wei W, Wu MX, Li YJ. Experimental Methodology of Pharmacology (药理实验方法学) [M]. 4th ed. Beijing: People’s Medical Publishing House, 2010: 866−868.
[12] Xiao J, Xu T, Li J, et al. Exercise-induced physiological hypertrophy initiates activation of cardiac progenitor cells [J]. Int J Clin Exp Pathol, 2014, 7: 663-669.
[13] Li XW, Hao W, Liu Y, et al. Effect of sequoyitol on expression of NOX4 and eNOS in aortas of type 2 diabetic rats [J]. Acta Pharm Sin (药学学报), 2014, 49: 329-336.
[14] Lo HM, Wu MW, Pan SL, et al. Chrysin restores PDGF- induced inhibition on protein tyrosine phosphatase and reduces PDGF signaling in cultured VSMCs [J]. J Nutr Biochem, 2012, 23: 667-678.
[15] Jankov RP, Kantores C, Pan J, et al. Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats [J]. Am J Physiol Lung Cell Mol Physiol, 2008, 294: L233-L245.
[16] Zhang Y, Dai L, Wu S, et al. Atorvastatin attenuates involvement of RhoA/Rho-kinase pathway and NF-κB activation in hypoxic pulmonary hypertensive rats [J]. Chin Med J, 2014, 127: 869-872.
[17] Price LC, Caramori G, Perros F, et al. Nuclear factor κ-B is activated in the pulmonary vessels of patients with end-stage idiopathic pulmonary arterial hypertension [J]. PLoS One, 2013, 8: e75415.
[18] Ha SK, Moon E, Kim SY. Chrysin suppresses LPS-stimu-lated proinflammatory responses by blocking NF-κB and JNK activations in microglia cells [J]. Neurosci Lett, 2010, 485: 143-147.
[19] Shao JJ, Zhang AP, Qin W, et al. AMP-activated protein kinase (AMPK) activation is involved in chrysin-induced growth inhibition and apoptosis in cultured A549 lung cancer cells [J]. Biochem Biophys Res Commun, 2012, 423: 448-453.