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郭瑾,王梓仪,张倩,孟骊冲,胡志希.基于miR-139/Wnt/β-catenin信号通路探讨参附注射液对慢性心力衰竭大鼠心肌纤维化的保护作用[J].湖南中医药大学学报英文版,2024,44(2):197-205.[Click to copy
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| 基于miR-139/Wnt/β-catenin信号通路探讨参附注射液对慢性心力衰竭大鼠心肌纤维化的保护作用 |
| 郭瑾,王梓仪,张倩,孟骊冲,胡志希 |
| (湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 探讨参附注射液对盐酸异丙肾上腺素（isoprenaline hydrochloride，ISO）诱导的慢性心力衰竭（chronic heart failure，CHF）大鼠心肌纤维化的作用与机制。方法 采用随机数字表法将45只SD大鼠随机分为正常组9只，造模组36只，采用ISO背部皮下多点注射14天制备CHF大鼠模型，再正常饲养14 d通过模型验证和评价，期间死亡大鼠5只，未成模大鼠4只。将造模成功的27只大鼠按随机数字表法分为3组：模型组（腹腔注射6 mL·kg-1氯化钠注射液+灌胃10 mL·kg-1蒸馏水）、参附注射液组（腹腔注射6 mL·kg-1参附注射液+灌胃10 mL·kg-1蒸馏水）、卡托普利组（腹腔注射6 mL·kg-1生理盐水+灌胃10 mL·kg-1蒸馏水，蒸馏水含8.8 mg·kg-1卡托普利，相当于临床等效量）。药物干预15 d后，检测各组大鼠心功能及心肌纤维化的相关指标：超声心动图、体质量、心脏质量指数及左心室质量指数；ELISA法检测氨基末端脑钠肽前体（N-terminal pro brain natriuretic peptide，NT-proBNP）；HE染色、Masson染色检测心肌组织病理形态及纤维化状态；RT-qPCR检测心肌组织中miR-139、Wnt家族成员3a（Wnt family member3a，Wnt3a）、β-连环蛋白（β-catenin）、糖原合成酶激酶3β（glycogen synthase kinase 3β，GSK3β）、Ⅰ型胶原蛋白（typeⅠcollagen，Col-Ⅰ）、Ⅲ型胶原蛋白（typeⅢcollagen，Col-Ⅲ）及α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）mRNA的表达；Western blot检测心肌组织中Wnt3a、β-catenin、GSK3β、Col-Ⅰ、Col-Ⅲ、α-SMA、MMP-2、MMP-9蛋白表达。结果 与正常组比较，模型组左室射血分数（left ventricular ejection fraction，LVEF）、左室短轴缩短率（left ventricular fraction shortening，LVFS）、体质量降低（P<0.01），左室舒张末期内径（left ventricular end diastolic diameter，LVEDD）、左室收缩末期内径（left ventricular end systolic diameter，LVESD）、NT-proBNP、心脏质量指数、左心室质量指数升高（P<0.01），miR-139表达量下降（P<0.01），Wnt3a、β-catenin、GSK3β、Col-Ⅰ、Col-Ⅲ、α-SMA、MMP-2、MMP-9蛋白和m RNA表达升高（P<0.01）；细胞排列紊乱，心肌纤维化明显，伴有明显的炎症浸润。与模型组比较，参附注射液组LVEF、LVFS、体质量升高（P<0.01或P<0.05），LVEDD、LVESD、NT-proBNP、心脏质量指数、左心室质量指数降低（P<0.01），miR-139表达量升高（P<0.01），Wnt3a、β-catenin、GSK3β、Col-Ⅰ、Col-Ⅲ、α-SMA、MMP-2、MMP-9蛋白和mRNA表达降低（P<0.01）；细胞排列趋于整齐，纤维结缔组织增生及炎症细胞浸润现象减少，胶原纤维沉积减少，纤维化程度减轻。结论 参附注射液能改善CHF大鼠心功能及心肌纤维化状态，其机制可能与调控miR-139/Wnt/β-catenin信号通路有关。 |
| 关键词: 慢性心力衰竭 参附注射液 miR-139 Wnt/β-catenin 心肌纤维化 |
| DOI：10.3969/j.issn.1674-070X.2024.02.004 |
| Received:July 12, 2023 |
| 基金项目:国家自然科学基金项目（82274412）；广东省重点领域研发项目（2020B1111100001）；湖南省教育厅项目（21A0230，21B0361）。 |
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| Protective effects of Shenfu Injection on myocardial fibrosis in rats with chronic heart failure based on miR-139/Wnt/β-catenin signaling pathway |
| GUO Jin,WANG Ziyi,ZHANG Qian,MENG Lichong,HU Zhixi |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To investigate the mechanism of action of Shenfu Injection (SFI) on myocardial fibrosis in rats with chronic heart failure (CHF) induced by isoprenaline (ISO).Methods Forty-five SD rats were divided into normal group (n=9) and modeling group (n=36) by random number table method.CHF rat model was prepared by 14-day subcutaneous multi-point injection of ISO on the back,followed by 14-day normal feeding to validate and evaluate the model.During this period,five rats died and four rats were not modeled.Then the 27 successfully modeled rats were subdivided into three groups by the random number table method:model group (intraperitoneal injection of 6 mL·kg-1sodium chloride injection+gavage of 10 mL·kg-1distilled water),SFI group (intraperitoneal injection of 6 mL·kg-1SFI+gavage of 10 mL·kg-1distilled water),and captopril group (intraperitoneal injection of 6.0 mL·kg-1normal saline+gavage of 10 mL·kg-1distilled water containing 8.8 mg·kg-1captopril which was clinically equivalent).After 15 d of drug intervention,the related indicators of cardiac function and myocardial fibrosis in each group were examined,including echocardiogram,body mass,heart mass index,and left ventricular mass index.ELISA was used to determine N-terminal pro brain natriuretic peptide (NT-proBNP) content;HE staining and Masson staining were performed to observe the histopathological morphology and fibrosis of the myocardial tissue;RT-qPCR was conducted to examine the mRNA expressions of miR-139,Wnt family member 3a (Wnt3a),β-catenin,glycogen synthase kinase 3β (GSK3β),type I collagen (Col-I),typeⅢcollagen (Col-Ⅲ),α-smooth muscle actin (α-SMA),matrix metalloproteinase-2 (MMP-2),and matrix metalloproteinase-9 (MMP-9) in the myocardial tissue;Western blot was used to examine the protein expressions of Wnt3a,β-catenin,GSK3β,Col-I,Col-Ⅲ,α-SMA,MMP-2,and MMP-9 in it.Results Compared with normal group,the left ventricular ejection fraction (LVEF),left ventricular fraction shortening (LVFS)and body mass of rats in model group decreased (P<0.01),while the left ventricular end diastolic diameter (LVEDD),left ventricular end systolic diameter (LVESD),NT-proBNP content,heart mass index,and left ventricular mass index increased (P<0.01);the miR-139 expression decreased (P<0.01),and the protein and mRNA expressions of Wnt3a,β-catenin,GSK3β,Col-I,Col-III,α-SMA MMP-2,and MMP-9 increased (P<0.01);the myocardial tissue showed disordered cell arrangement and significant fibrosis,accompanied by obvious inflammatory infiltration.Compared with model group,the LVEF,LVFS,and body mass of rats in SFI group were higher (P<0.01 or P<0.05),while the LVEDD,LVESD,NT-proBNP content,heart mass index,and left ventricular mass index were lower (P<0.01);the miR-139 expression increased (P<0.01),and the protein and mRNA expressions of Wnt3a,β-catenin GSK3β,Col-I,Col-Ⅲ,α-SMA,MMP-2,and MMP-9 decreased (P<0.01);cell arrangement tended to be neat in the myocardial tissue,with reduced fibrous connective tissue hyperplasia,inflammatory cell infiltration,collagen fiber deposition,and fibrosis degree.Conclusion SFI can improve cardiac function and alleviate myocardial fibrosis in rats with CHF,and its mechanism may be related to the regulation of miR-139/Wnt/β-catenin signaling pathway. |
| Key words: chronic heart failure Shenfu Injection miR-139 Wnt/β-catenin myocardial fibrosis |
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