当归多糖改善大鼠心肌缺血再灌注损伤的体内外观察及其机制探讨

Angelica sinensis polysaccharide ameliorates MIRI in rats in vitro and its mechanism

目的建立大鼠心肌缺血再灌注损伤(MIRI)的体内、体外模型,观察当归多糖(ASP)对大鼠MIRI的改善作用并探讨其机制。方法在体内研究,成年SD大鼠随机分为假手术组、MIRI组、ASP低剂量组、ASP高剂量组,除假手术组外,均采用冠状动脉左前降支结扎再灌注的方法建立MIRI模型,ASP低、高剂量组在建模后分别给予50、100 mg/kg的ASP灌胃,假手术组及MIRI组给予等量生理盐水灌胃,持续4周;采用ELISA法检测各组血清心肌损伤标志物肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)及炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β;Western blotting法检测各组心肌组织凋亡蛋白B淋巴细胞瘤2(Bcl-2)、Bcl-2关联X蛋白(Bax)、裂解的半胱胺酸蛋白酶(cleaved Caspase-3)及toll样受体4(TLR4)/核因子κB(NF-κB)通路相关蛋白TLR4、磷酸化核蛋白(p-p65)、磷酸化NF-κB抑制蛋白α(p-IκBα)表达。在体外研究,将大鼠心肌细胞分为对照组、H/R组、H/R+ASP低剂量组、H/R+ASP高剂量组,对照组正常培养不做处理,其他3组均建立体外心肌细胞缺氧复氧(H/R)模型,建模后H/R+ASP低、高剂量组分别加入浓度为5、10µg/mL的ASP,H/R组不做处理;采用MTT比色法检测各组心肌细胞活性,ELI-SA法检测各组心肌细胞炎症因子TNF-α、IL-6、IL-1β,Western blotting法检测各组心肌细胞凋亡蛋白Bax、Bcl-2、cleaved Caspase-3及TLR4/NF-κB通路蛋白TLR4、p-p65、p-IκBα表达。结果在体内研究,血清心肌损伤标志物LDH、CK-MB水平及血清炎症因子TNF-α、IL-6、IL-1β水平MIRI组>ASP低剂量组>ASP高剂量组>假手术组(P均<0.05);心肌组织Bax、cleaved Caspase-3蛋白表达MIRI组>ASP低剂量组>ASP高剂量组>假手术组,心肌组织Bcl-2蛋白表达假手术组>ASP高剂量组>ASP低剂量组>MIRI组(P均<0.05);心肌组织TLR4/NF-κB通路相关蛋白TLR4、p-p65、p-IκBα表达MIRI组>ASP低剂量组>ASP高剂量组>假手术组(P均<0.05)。在体外研究,心肌细胞活性对照组>H/R+ASP高剂量组>H/R+ASP低剂量组>H/R组(P均<0.05);心肌细胞炎症因子TNF-α、IL-6、IL-1β水平H/R组>H/R+ASP低剂量组>H/R+ASP高剂量组>对照组(P均<0.05);心肌细胞Bax、cleaved Caspase-3蛋白表达H/R组>H/R+ASP低剂量组>H/R+ASP高剂量组>对照组,心肌细胞Bcl-2蛋白表达对照组>H/R+ASP高剂量组>H/R+ASP低剂量组>H/R组(P均<0.05);心肌细胞TLR4/NF-κB通路相关蛋白TLR4、p-p65、p-IκBα表达H/R组>H/R+ASP低剂量组>H/R+ASP高剂量组>对照组(P均<0.05)。结论ASP在体内、体外缺血再灌注模型中均可改善大鼠MIRI,其机制可能与抑制TLR4/NF-κB通路激活、减轻炎症反应、减少细胞凋亡有关。

Objective To establish in vivo and in vitro models of myocardial ischemia-reperfusion injury(MIRI)in rats,to observe the improvement effect of Angelica polysaccharide(ASP)on MIRI rats and to explore its mechanism.Methods In vivo study,adult SD rats were randomly divided into the sham group,MIRI group,low-dose ASP group and high-dose ASP group.Except the sham group,the MIRI models were established by ligation and reperfusion of the left an⁃terior descending branch of coronary artery in the other groups.After modeling,the rats in the low-dose and high-dose ASP groups were given 50 and 100 mg/kg ASP by intragastric administration,once a day,for 4 weeks,respectively.Rats in the sham group and MIRI group were given the equal volume of normal saline.Serum markers of myocardial injury such as creatine kinase-MB(CK-MB),lactate dehydrogenase(LDH),tumor necrosis factor-α(TNF-α),interleukin6(IL-6),and IL-1βwere detected by ELISA.The expression levels of apoptotic proteins Bax,Bcl-2,cleaved Caspase-3,and TLR4/NF-κB pathway-related proteins TLR4,p-p65,and p-IκBαwere detected by Western blotting.In vitro study,H/R cardiomycetes(derived from neonatal cell culture)were divided into the control group,hypoxia reoxygenation(H/R)group,low-dose H/R+ASP group and high-dose H/R+ASP group.The control group was cultured normally without treat⁃ment,and in vitro cardiomyocyte H/R models were established in the other three groups.After modeling,the low-dose and high-dose H/R+ASP groups were added with 5 and 10μg/mL ASP,respectively,and the H/R group was not treated.MTT colorimetric assay was used to detect cardiomyocyte activity,and ELISA was used to detect inflammatory cytokines TNF-α,IL-6 and IL-1β.Western blotting was used to detect apoptotic proteins Bax,Bcl-2,cleaved Caspase-3,and TLR4/NFκB pathway proteins TLR4,p-p65,and p-IκBαin each group.Results In vivo,the levels of serum myocardi⁃al injury markers(LDH and CK-MB)and serum inflammatory factors(TNF-α,IL-6 and IL-1β)ranked from high to low:MIRI group,low-dose ASP group,high-dose ASP group,and sham group(all P<0.05).Myocardial Bax and cleaved Cas⁃pase-3 protein expression ranked from high to low:MIRI group,low-dose ASP group,high-dose ASP group,sham group,and myocardial Bcl-2 protein expression ranked from high to low:sham group,high-dose ASP group,low-dose ASP group,MIRI group(all P<0.05).The expression levels of TLR4/NF-κB pathway-related proteins TLR4,P-P65 and P-iκBαin the myocardial tissues were higher in the MIRI group than in the low-dose ASP group,which were higher than those of the high-dose ASP group,followed by the sham operation group(all P<0.05).In vitro study,cardiomyocyte activity ranked from high to low:control group,high-dose H/R+ASP group,low-dose H/R+ASP group,and H/R group(all P<0.05);the levels of cytokines TNF-α,IL-6 and IL-1βin H/R group were higher than those in H/R+ASP low-dose group,which were higher than those in the high-dose H/R+ASP group,followed by the control group(all P<0.05).Bax and cleaved caspase-3 protein expression ranked from high to low:H/R group,low-dose H/R+ASP group,high-dose H/R+ASP group,and control group.Cardiac Bcl-2 protein expression ranked from high to low:control group,high-dose H/R+ASP group,low-dose H/R+ASP group,and H/R group(all P<0.05);the expression levels of TLR4/NF-κB pathway-related proteins(TLR4,P-P65 and P-iκbαin cardiomyoma cells)in the H/R group were higher than those in the low-dose H/R+ASP,which were higher than those of the high-dose H/R+ASP group,and those were higher in the high-dose H/R+ASP group than the control group(all P<0.05).Conclusion ASP can improve MIRI in both in vivo and in vitro ischemia-re⁃perfusion models,and its mechanism may be related to inhibiting the activation of TLR4/NFκB pathway,alleviating in⁃flammation,and reducing apoptosis.