基于巨噬细胞极化调控心肌细胞代谢重塑探讨暖心康改善心肌梗死小鼠心肌纤维化的机制

To Explore the Mechanism of Nuanxinkang on Alleviating Myocardial Fibrosis in Mice with Myocardial Infarction Through Regulation of Myocardial Cell Metabolic Remodeling Based on Macrophage Polarization

目的基于巨噬细胞极化调控心肌细胞代谢重塑探讨暖心康(红参、毛冬青)改善心肌梗死小鼠心肌纤维化的机制。方法(1)将C57BL/6J雄性小鼠随机分为3组:假手术组、模型组、暖心康组(1.65 g·kg^(-1)),每组10只。采用结扎冠状动脉术复制心肌梗死小鼠模型。灌胃给药,每日1次,连续4周。采用Masson染色法检测心肌组织纤维化;qPCR法检测心脏组织中LDHA、HK2、CD36、CPT-1A、PPAR-γ、PGC-1α、Ndufa8、SDHd、Cyc1、Cox4i2、CD86、iNOS、TNF-αmRNA表达水平;ELISA法检测血清炎症因子IL-1β的水平。(2)采用脂多糖诱导RAW264.7巨噬细胞向促炎型巨噬细胞极化,制备促炎型巨噬细胞上清液,将其作用于HL-1心肌细胞模型。HL-1心肌细胞分组与RAW264.7巨噬细胞分组相同:空白血清对照组(含5%空白血清+5%胎牛血清的培养基)、暖心康含药血清组(含5%暖心康含药血清+5%胎牛血清的培养基)、脂多糖组(含5%空白血清+5%胎牛血清的培养基+200μg·mL^(-1)脂多糖)和暖心康含药血清+脂多糖组(含5%暖心康含药血清+5%胎牛血清的培养基+200μg·mL^(-1)脂多糖),均干预24 h。采用油红O染色检测心肌细胞中脂质分布情况;DCFH-DA荧光探针检测细胞ROS水平。结果(1)与模型组比较,暖心康组小鼠心脏组织胶原沉积面积显著减少(P<0.01),且梗死区域局部室壁损伤程度减轻;心脏组织中LDHA、HK2、CD36、CPT-1A、PPAR-γ、Ndufa8、SDHd、Cyc1、Cox4i2、CD86、iNOS、TNF-αmRNA表达水平显著降低(P<0.05,P<0.01,P<0.001),PGC-1αmRNA表达水平明显升高(P<0.05);血清IL-1β水平显著降低(P<0.001)。(2)与脂多糖组比较,暖心康含药血清+脂多糖组HL-1心肌细胞胞浆内的脂滴沉积明显减少(P<0.05),ROS水平显著降低(P<0.001)。结论暖心康能够减轻心肌梗死小鼠的心肌纤维化及心室重构,其作用机制可能与抑制促炎型巨噬细胞极化,降低炎症水平,减少ROS生成,降低心脏组织糖酵解水平,改善氧化磷酸化和脂肪酸代谢有关。

Objective To explore the mechanism of Nuanxinkang(NXK,composed of Ginseng Radix et Rhizoma Rubra and Ilex Pubescen)on alleviating myocardial fibrosis in mice with myocardial infarction by orchestrating myocardial cell metabolic remodeling based on macrophage polarization.Methods(1)C57BL/6J male mice were randomly divided into three groups:sham operation group,model group and NXK administration group(1.65 g·kg^(-1)),with 10 rats in each group.The myocardial infarction model of mice was induced by ligating coronary artery.The drug administration group was given NXK once a day for four weeks.Masson staining was used to detect myocardial fibrosis,and quantitative PCR was used to detect the mRNA expression levels of LDHA,HK2,CD36,CPT-1A,PPAR-γ,PGC-1α,Ndufa8,SDHd,Cyc1,Cox4i2,CD86,iNOS and TNF-αin cardiac tissues.The content of inflammatory factor IL-1βin serum was detected by ELISA.(2)Lipopolysaccharide(LPS)was used to induce the polarization of RAW264.7 macrophages into pro-inflammatory subtype.The supernatant fluid of pro-inflammatory macrophages was prepared and subsequently acted on HL-1 cardiomyocytes.RAW264.7 macrophages and HL-1 cardiomyocytes were divided into control serum group(culture medium with 5%blank serum and 5%fetal bovine serum),NXK-contained serum group(culture medium with 5%NXK-contained serum and 5%fetal bovine serum),LPS treatment group(culture medium with 5%blank serum,5%fetal bovine serum and 200μg·mL^(-1)LPS)and NXK-contained serum+LPS treatment group(culture medium with 5%NXK-contained serum,5%fetal bovine serum and 200μg·mL^(-1)LPS).After 24-h intervention in each group,the lipid in the cells was labeled by Oil Red O staining,and the reactive oxygen species(ROS)level of the cells was detected by DCFH-DA fluorescent probe.Results(1)Compared with the model group,collagen deposition area in cardiac tissue of mice was remarkably reduced(P<0.01)and local ventricular wall damage in the infarcted area was alleviated in NXK group.Also,the mRNA expressions of LDHA,HK2,CD36,CPT-1A,PPAR-γ,Ndufa8,SDHd,Cyc1,Cox4i2,CD86,iNOS and TNF-αwere significantly decreased(P<0.05,P<0.01,P<0.001).The mRNA expression of PGC-1αwas significantly increased(P<0.05).Serum IL-1βlevel was obviously decreased(P<0.001).(2)Compared with the LPS group,the deposition of lipid droplets in cytoplasm of HL-1 cardiomyocytes in NXK-contained serum+LPS treatment group was remarkably reduced(P<0.05)and ROS level decreased significantly(P<0.001).Conclusion NXK can alleviate myocardial fibrosis and ventricular remodeling after myocardial infarction in mice,and its mechanism may be related to inhibiting the polarization of pro-inflammatory macrophages,alleviating inflammation level,reducing the production of ROS,reducing the level of glycolysis in cardiac tissue,and improving oxidative phosphorylation and fatty acid metabolism.