目的 观察益气活血方调控METTL3/HAND2/TGF-β1信号轴对冠心病气虚血瘀证大鼠的影响。方法 将48只雄性SD大鼠随机分为假手术组、模型组、益气活血方组和西药(单硝酸异山梨酯)组，每组12只。除假手术组外，其余各组采用左冠状动脉前降支结扎结合游泳力竭法制备冠心病气虚血瘀证大鼠模型。造模结束后，益气活血方组及西药组灌胃干预28 d。观察大鼠一般状况，采集心电图，超声心动图检测大鼠心功能，血液流变学检测血瘀程度，HE染色观察心肌组织形态，免疫组化法检测心肌组织甲基化转移酶3(METTL3)、转化生长因子(TGF)-β1、Smad2、Smad3 表达，RT-PCR 和 Western blot 检测心肌组织METTL3、心脏神经嵴衍生物表达蛋白2(HAND2)、TGF-β1、Smad2、Smad3 mRNA和蛋白表达。结果 与假手术组比较，模型组大鼠出现精神萎靡、行动迟缓、嗜睡、体质量降低等气虚表现，心电图ST段显著抬高，左室射血分数(LVEF)、左室短轴缩短率(LVFS)下降(P＜0。01)，左室舒张末期容积(LVEDV)、左室收缩末期容积(LVESV)、左室舒张末期内径(LVEDd)、左室收缩末期内径(LVEDs)升高(P＜0。01)，红细胞聚集指数、卡松黏度及全血黏度升高(P＜0。01);心肌细胞肿大、坏死，心肌纤维排列紊乱，心肌组织有炎性细胞浸润，心肌组织METTL3、TGF-β1、Smad2、Smad3 mRNA和蛋白表达均升高(P＜0。05，P＜0。01)，HAND2 mRNA和蛋白表达均降低(P＜0。01)。与模型组比较，益气活血方组和西药组大鼠精神状态明显改善，心电图 ST段明显回落，LVEF、LVFS升高(P＜0。01)，LVEDV、LVESV、LVEDd、LVEDs下降(P＜0。05，P＜0。01)，红细胞聚集指数、卡松黏度及全血黏度下降(P＜0。01);心肌纤维排列整齐，心肌细胞结构及形态改善，炎性细胞浸润减少，心肌组织METTL3、TGF-β1、Smad2、Smad3 mRNA和蛋白表达降低(P＜0。05，P＜0。01)，HAND2mRNA和蛋白表达升高(P＜0。05，P＜0。01)。结论 益气活血方能减轻冠心病气虚血瘀证大鼠心肌组织病理损伤，提高心功能，抑制炎症反应，其作用机制可能与调控METTL3/HAND2/TGF-β1信号轴有关。
Effects of Yiqi Huoxue Prescription in Regulating METTL3/HAND2/TGF-β1 Signaling Axis on Rats with Qi Deficiency and Blood Stasis Syndrome of Coronary Heart Disease
Objective To observe the effects of Yiqi Huoxue Prescription in regulating METTL3/HAND2/TGF-β1 signaling axis on rats with qi deficiency and blood stasis syndrome of coronary heart disease.Methods Totally 48 male SD rats were randomly divided into sham-operation group,model group,Yiqi Huoxue Prescription group and Western medicine(isosorbide mononitrate)group,with 12 rats in each group.Except for the sham-operation group,the animal models of coronary heart disease with qi deficiency and blood stasis syndrome were prepared by the method of ligation of the left anterior descending branch of coronary artery combined with swimming exhaustion.After modeling,Yiqi Huoxue Prescription group and the Western medicine group were gavaged for 28 d.The general conditions of the rats were observed,and electrocardiograms and echocardiograms were collected to detect the cardiac function of the rats,blood rheology was used to detect the degree of blood stasis,HE staining was used to observe the morphology of myocardial tissue,immunohistochemistry was used to detect the expression of METTL3,TGF-β1,Smad2 and Smad3 in myocardial tissue,RT-PCR and Western blot were used to detect the expression of METTL3,HAND2,TGF-β1,Smad2 and Smad3 mRNA and protein in myocardial tissue.Results Compared with the sham-operation group,the model group rats showed symptoms of qi deficiency such as mental lethargy,slow movement,drowsiness,and decreased body mass,significant elevation of ST segment on electrocardiogram,the left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)decreased(P＜0.01),the left ventricular end diastolic volume(LVEDV),left ventricular end systolic volume(LVESV),left ventricular end diastolic diameter(LVEDd)and left ventricular end systolic diameter(LVEDs)increased(P＜0.01),the red blood cell aggregation index,Carcasson viscosity and whole blood viscosity increased(P＜0.01);with enlargement and necrosis of myocardial cells,disordered arrangement of myocardial fibers,and infiltration of inflammatory cells into myocardial tissue,the mRNA and protein expressions of METTL3,TGF-β1,Smad2 and Smad3 in myocardial tissue increased(P＜0.05,P＜0.01),while the mRNA and protein expression of HAND2 decreased(P＜0.01).Compared with the model group,the Yiqi Huoxue Prescription group and the Western medicine group showed significant improvement in the mental state of rats,significant ST segment regression on electrocardiogram,LVEF and LVFS increased(P＜0.01),LVEDV,LVESV,LVEDd and LVEDs decreased(P＜0.05,P＜0.01),with red blood cell aggregation index,Carcasson viscosity and whole blood viscosity decreased(P＜0.01);the arrangement of myocardial fibers was neat,the structure and morphology of myocardial cells were improved,the infiltration of inflammatory cells was reduced,the mRNA and protein expressions of METTL3,TGF-β1,Smad2 and Smad3 in myocardial tissue decreased(P＜0.05,P＜0.01),and the mRNA and protein expression of HAND2 increased(P＜0.05,P＜0.01).Conclusion Yiqi Huoxue Prescription can alleviate the pathological damage of myocardial tissue in rats with coronary heart disease of qi deficiency and blood stasis syndrome,improve heart function and inhibit inflammatory response.Its mechanism may be related to the regulation of the METTL3/HAND2/TGF-β1 signaling axis.
Yiqi Huoxue Prescriptioncoronary heart diseaseqi deficiency and blood stasis syndromeMETTL3HAND2TGF-β1/Smads pathwaycombination of disease and syndromerats
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