【作者】 张学娅； 黄熙； 任平； 许东云； 张颖； 罗珊珊； 王晓雨；

【机构】 昆明医学院第一附属医院； 中南大学湘雅医院中西医结合研究所； 成都市中西医结合医院；

【摘要】 目的:冠心Ⅱ号(GXIII)是治疗冠心病的有效中药复方,其药物组成为丹参、川芎、红花、赤芍、降香按照2:1:1:1:1比例构成。丹川红(DCH)煎剂是GXII子方,其药物组成为丹参、川芎、红花按照2:1:1比例构成。本研究探索:(1)长期小剂量GXII经由Akt信号通路抑制心肌凋亡的心脏保护机理;(2)GXII急性灌胃经由NF-κB诱导抗炎的心脏保护机理;(3)DCH煎剂经由Akt/NF-κB通路诱导的抗炎作用是否与吸收生物活性成分FAT(阿魏酸、羟基红花黄色素A、丹参素)相关。方法:(1)SD大鼠被随机分为四组:假手术组、对照组、口服GXII剂量组(2.5 g/kg/d和0.5 g/kg/d)。大鼠灌胃GXII或等容积0.9%NaCl qd×15d。大鼠急性心肌梗塞(acute myocardialinfarction,AMI)造模前30 min最后一次灌胃GXII或0.9%NaCl,除了假手术组,其余大鼠均结扎冠状动脉。AMI 24-h后采集大鼠心脏,应用组织化学染色法测定24-h心肌梗塞面积(IZ)。大鼠AMI 3-h后采集心脏,应用末端脱氧核苷酸转移酶介导的原位缺口末端标记法(TUNEL)检测大鼠心肌缺血区心肌细胞凋亡指数;免疫组化法检测心肌细胞凋亡调控基因Bcl-2,Bax比值;免疫印迹法测定心肌细胞胞浆总Akt(total Akt)与磷酸化Akt(p-Akt)活性,心肌细胞胞浆和线粒体中的细胞色素C(cytochrome C)释放;比色法测定心肌细胞caspase-3,caspase-9活性。(2)SD大鼠被随机分为四组:假手术组、对照组、口服GXII剂量组(15 g/kg,5 g/kg)。大鼠AMI造模前30 min一次性灌胃GXII或等容积0.9%NaCl。除了假手术组,其余大鼠均结扎冠状动脉。AMI 24-h后采集大鼠心脏,测定IZ。大鼠AMI 3-h后,采集心脏及血清,应用免疫印迹法测定心肌细胞胞浆与胞核的核因子(Nuclearfactor-kappa B,NF-κB)表达,酶联免疫法(ELISA)测定C反应蛋白(CRP)、白介素-6(IL-6)、肿瘤坏死因子(TNF-α)血清水平。(3)SD大鼠被随机分为四组:假手术组、对照组、DCH煎剂(13.33 g/kg)组、FAT组(阿魏酸F 3.14 mg/kg;羟基红花黄色素A 48.76 mg/kg和丹参素T 17.08 mg/kg)。大鼠AMI造模前30 min一次性灌胃DCH煎剂或FAT。除了假手术组,其余大鼠均结扎冠状动脉。AMI 24-h后采集大鼠心脏,测定IZ。大鼠AMI 3-h后,采集心脏及血清,用ELISA法测定CRP血清水平;免疫印迹法测定心肌细胞胞浆与胞核的NF-κB表达,心肌细胞胞浆total Akt与p-Akt活性。结果:(1)与对照组相比,长期小剂量口服GXII(2.5g/kg)降低IZ、降低心肌细胞凋亡指数、增加p-Akt活性、升高Bcl-2/Bax比值、抑制cytochrome C释放、抑制caspase-3,caspase-9活性。GXII(0.5g/kg)在上述指标则无治疗效果。(2)与对照组相比,GXII(15g/kg)能降低IZ、降低NF-κB表达、降低血清TNF-α,IL-6,CRP水平。GXII(5g/kg)在上述指标则无治疗效果。(3)与对照组相比,DCH煎剂及FAT都能降低IZ、增加p-Akt活性、降低NF-κB表达、降低血清CRP水平。结论:(1)长期小剂量预先灌胃GXII降低IZ,通过激活心肌细胞Akt酶活性,进一步经由调节Bcl-2家族蛋白表达,cytochrome C介导的Caspases通路,从而起到抗心肌细胞凋亡作用。(2)一次性灌胃GXII降低IZ,经由抑制NF-κB蛋白表达,降低前炎性细胞因子IL-6,TNF-α水平,进一步降低炎症指标血清CRP水平,从而起到抗炎样心脏保护作用。(3)DCH煎剂及其成分FAT都可降低IZ,经由Akt/NF-κB通路降低血清CRP水平,达到抗炎性心脏保护作用。DCH煎剂抗炎作用的效应成分是FAT。上述研究表明:GXII对心肌梗塞大鼠的心脏保护作用可能经由Akt信号通路,通过抑制凋亡与炎症机制来发挥;Akt信号通路是GXII心脏保护作用的上游靶点;FAT是GXII抗凋亡、抗炎作用的吸收生物活性成分。更多还原

【Abstract】 Objective:GuanxinⅡ(GXⅡ) is an effective traditional Chinese formula to treat coronary heart disease.It contains Salvia miltiorrhiza Bge.,Carthamus tinctorius L.,Paeonia lactiflora Pall., Ligusticum chuanxiong Hort.and Dalbergia odorifera T.Chen in a ratio of 2:1:1:1:1.Dan-Chuan-Hong (DCH) decoction comes from GXⅡand contains three herbs:Salvia miltiorrhiza Bge.,Ligusticum chuanxiong Hort.,and Carthamus tinctorius L in a ratio of 2:1:1,respectively.The objective of the study were elucidated:(1) chronic low dose GXⅡinduced the anti-apoptotic mechanism of cardioprotection via the Akt signaling pathway by it;(2) the anti-inflammatory mechanism associated with blockade of nuclear factor-kappa B(NF-κB) by single administration of GXⅡ;(3) whether or not DCH induced anti-inflammatory effect via Akt/NF-κB pathway,and identify absorbed bioactive compounds(ABCs) FAT(ferulic acid,hydroxyl safflor yellow A and tanshinol) was correlated with it.Methods:(1) SD rats were randomly assigned to four groups:GXⅡadministered at 2.5 or 0.5 g raw materials/kg/d,the vehicle control and sham-operated oral 0.9%NaCl.They were pretreated once a day for 15 consecutive days by gavage.Thirty minutes after the last administration,the left anterior descending coronary artery was occluded to induce acute myocardial infarction(AMI) except for the sham-operated rats.Hearts were harvested at the end of a 24-h ischemic period for measurement of infarct size(IZ) by histochemical stain. Hearts were harvested at the end of a 3-h ischemic period for measurement of the number of apoptotic cardiomyocytes by terminal deoxy-nucleotidyl transferase mediated dUTP nick end-labeling(TUNEL) assay;expression of Bcl-2 and Bax protein by immunohistochemical staining;total Akt,p-Akt activation and release of cytochrome c by western blots;and activity of caspase-3 and caspase-9 by colorimetric method.(2) SD rats were randomly assigned to four groups:GXⅡ(15 g/kg,5 g/kg) groups as well as sham-operated and vehicle control.Thirty minutes after giving GXⅡor 0.9%NaCl(p.o.) once,coronary arteries were occluded except for the sham-operated rats.We measured 24-h IZ,3-h expression of NF-κB protein by western blots,and serum levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and C-reactive protein(CRP) by Euzymelinked immunosorbent assay(ELISA).(3) SD rats were randomly assigned to four groups:DCH(13.33 g/kg),FAT(ferulic acid 3.14 mg/kg;hydroxyl safflor yellow A 48.76 mg/kg and tanshinol 17.08 mg/kg) groups as well as sham-operated and vehicle control.Thirty minutes after giving DCH,FAT or 0.9%NaCl(p.o.) once,coronary arteries were occluded except for the sham-operated rats.We measured 24-h IZ,3-h expression of NF-κB protein,total Akt and p-Akt activation by western blots,as well as serum levels of CRP by ELISA.Results:(1) Compared with rats receiving vehicle,chronic low dose GXⅡ(2.5g/kg) significantly reduced IZ and myocardial apoptotic index,increased the ratio of Bcl-2/Bax,inhibited release of cytochrome C,as well as activation of caspase-3 and caspase-9.GXⅡ(0.5 g/kg) did not have a significant effect on the indices described above. (2) Compared with rats receiving vehicle,GXⅡ(15g/kg) reduced IZ,the expression of NF-κB,serum level of TNF-α,IL-6 and CRP.GXII(5 g/kg) did not have a significant effect on the indices described above.(3).Compared with rats receiving vehicle,both of DCH and FAT reduced IZ,increased activation of p-Akt,reduced the expression of NF-κB and serum level of CRP.Conclusion:(1).Pretreatment with chronic low dose GXⅡ(2.5g/kg) significantly reduced IZ via activated Akt kinase,further regulated the balance of Bcl-2 family members and cytochrome C—mediated caspases pathway,thus educed anti-apoptotic effect.(2).GXⅡadministration once against 24-h IZ through inhibited expression of NF-κB protein,reduced proinflammatory cytokines IL-6 and TNF-αlevel,further reduced inflammation marker CRP level,thus educed an anti-inflammatory effect.(3).Both DCH and FAT reduced IZ and down-regulated inflammation marker CRP level via the Akt/NF-κB pathway,thus educed an anti-inflammatory effect.FAT was ABCs of DCH that implicated in the anti-inflammatory effect.All the results indicated that GXⅡ-induced anti-apoptotic and anti-inflammatory effects maybe via activation of Akt.Akt signaling pathway was the upstream target of cardioprotection by GXⅡ.FAT was ABCs of GXⅡthat implicated in anti-apoptotic and anti-inflammatory effects.更多还原