摘要
目的:通过非靶向心肌代谢组学结合实验验证,探讨二至丸减轻去卵巢小鼠心肌损伤的作用靶点.方法:选用去卵巢小鼠模型,将40只C57BL/6雌性小鼠随机分为假手术组、模型组、雌激素组(戊酸雌二醇,0.13 mg·kg-1)、二至丸低、高剂量组(3.12、9.36 g·kg-1),每组8只,各给药组分别给予相应剂量药物灌胃,假手术组及模型组给予等体积蒸馏水灌胃,给药12周.超声心动图检测小鼠心功能、苏木素-伊红(HE)染色观察小鼠心肌形态学改变,酶联免疫吸附测定法(ELISA)检测小鼠雌激素水平,N端前脑钠素(NT-proBNP)、超敏肌钙蛋白T(hs-TnT)水平,总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、白细胞介素(IL)-1β、IL-18及肿瘤坏死因子-α(TNF-α)水平;采用超高效液相色谱-四级杆-静电场轨道阱高分辨质谱法(UPLC-Q-Exactive Orbitrap MS)对小鼠心肌组织进行非靶向代谢组学分析,并筛选差异代谢物,富集代谢通路;实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠心肌组织磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)mRNA的表达水平,蛋白免疫印迹法(Western blot)检测小鼠心肌组织PI3K、Akt、磷酸化(p)-Akt的蛋白表达水平.结果:与假手术组比较,模型组小鼠心功能异常,心肌纤维间隙增大、心肌细胞萎缩、肌浆凝聚、偶见肌纤维溶解或断裂,血清中雌激素水平显著降低、心肌损伤标志物NT-proBNP、hs-TnT水平显著升高,炎症因子IL-1β、IL-18、TNF-α水平显著升高,TG、TC、LDL-C水平显著升高,HDL-C水平显著降低(P＜0.01);与模型组比较,二至丸组小鼠心功能异常和心肌组织病理性损伤明显改善,二至丸组小鼠雌激素水平显著升高,心肌损伤标志物NT-proBNP、hs-TnT和炎症因子IL-1β、IL-18、TNF-α水平显著降低,TG、TC、LDL-C水平显著降低,HDL-C水平显著升高(P＜0.01).非靶向心肌代谢组学结果显示,模型组与假手术组间162个差异代谢物中的31个在二至丸给药后出现回调,主要为甘油磷脂代谢物;通路富集结果显示,二至丸主要影响甘油磷脂代谢、PI3K/Akt通路及环磷酸鸟苷(cGMP)/蛋白激酶G(PKG)等多条代谢通路.与假手术组比较,模型组小鼠心肌组织甘油磷脂代谢物中11种磷脂酰胆碱(PC)和5种磷脂酰乙醇胺(PE)水平升高(P＜0.05,P＜0.01),心肌组织PI3K、p-Akt mRNA和蛋白表达降低(P＜0.05,P＜0.01);与模型组比较,二至丸组小鼠心肌组织11种PC和5种PE水平明显降低(P＜0.05,P＜0.01),心肌组织PI3K、p-Akt mRNA和蛋白表达升高(P＜0.01).结论:二至丸可以减轻去卵巢小鼠心肌组织病理性损伤、改善心功能异常、改善血脂代谢紊乱,降低心肌损伤特异性标志物和炎症因子水平,涉及心脏内多条信号通路和代谢途径,其中甘油磷脂代谢途径和PI3K/Akt通路可能具有关键作用.
Abstract
Objective:To explore the target of Erzhiwan in reducing myocardial injury in ovariectomized mice through non-targeted myocardial metabolomics combined with experimental verification.Methods:Ovariectomized mouse model was selected,40 female C57BL/6 mice were randomly divided into sham operation group,model group,estrogen group(estradiol valerate,1.3× 10-4g·kg-1),Erzhiwan low and high dose groups(3.12,9.36 g·kg-1),with 8 mice in each group.Each administration group was given the corresponding dose of Erzhiwan by gavage,and the sham operation group and model group were given equal volume of distilled water by gavage for 12 weeks.Echocardiography was used to detect cardiac function,hematoxylin-eosin(HE)staining was used to observe myocardial morphological changes,and enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of estrogen,N-terminal pro-brain natriuretic peptide(NT-proBNP),hypersensitive troponin T(hs-TnT),total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),interleukin(IL)-1β,IL-18 and tumor necrosis factor-α(TNF-α).The non-targeted metabolomics of mouse myocardium were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry(UPLC-Q-Exactive Orbitrap MS),and the differential metabolites and corresponding metabolic pathways were obtained.The mRNA expression levels of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt)in mouse myocardial tissues were detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and the protein expression levels of PI3K,Akt,phosphorylated(p)-Akt were detected by Western blot.Results:Compared with the sham operation group,the model group showed abnormal cardiac function,increased myocardial fiber space,cardiomyocyte atrophy,sarcoplasmic aggregation,and occasional dissolution or rupture of muscle fiber,the level of estrogen in the serum was decreased,the levels of NT-proBNP,hs-TnT,IL-1β,IL-18,TNF-α,TG,TC and LDL-C were increased,and the level of HDL-C was decreased(P＜0.01).Compared with the model group,Erzhiwan could increase the level of estrogen,improve the abnormal cardiac function,reduce the pathological injury of myocardial tissue,decrease the levels of myocardial injury markers(NT-proBNP,hs-TnT)and inflammatory factors(IL-1β,IL-18,TNF-α),decrease the levels of TG,TC,LDL-C,and increased the level of HDL-C(P＜0.01).The results of non-targeted myocardial metabolomics showed that 31 of the 162 differential metabolites between the model group and sham operation group were significantly adjusted after administration of Erzhiwan,which were mainly glycerol phospholipid metabolites.Pathway enrichment results showed that Erzhiwan mainly affected glycerophospholipid metabolic pathway,PI3K-Akt pathway,cyclic guanosine monophosphate(cGMP)-protein kinase G(PKG)pathway and other metabolic pathways.Compared with the sham operation group,the levels of phosphatidylcholine(PC,11 types)and phosphatidylethanolamine(PE,5 types)in mouse myocardial tissue of the model group were increased(P＜0.05,P＜0.01),and the mRNA and protein expressions of PI3K and p-Akt were decreased(P＜0.05,P＜0.01).Compared with the model group,the levels of PC(11 types)and PE(5 types)were decreased(P＜0.05,P＜0.01)in myocardial tissue of Erzhiwan group,the mRNA and protein expressions of P13K and p-Akt were elevated(P＜0.01).Conclusion:Erzhiwan can alleviate the pathological injury of myocardium in ovariectomized mice,improve the abnormal cardiac function,improve lipid metabolism disorder,and reduce the levels of myocardial injury markers and inflammatory factors,which involves a number of signaling and metabolic pathways in the heart,among which glycerophospholipid metabolism pathway and PI3K/Akt pathway may have key roles.
国家科技期刊平台
NSTL
维普
万方数据