摘要
背景:研究表明芍药苷对肝、肾等器官纤维化具有改善作用,尤其是在肝纤维化中表现出突出优势,但芍药苷对于血管紧张素Ⅱ诱导的心肌纤维化的保护作用尚不明确。目的:探讨芍药苷对血管紧张素Ⅱ诱导的心肌成纤维细胞的保护作用及分子机制。方法:在分离培养的SD大鼠乳鼠心肌成纤维细胞中加入血管紧张素Ⅱ(1μmol/L)干预48 h作为模型组;芍药苷低、高剂量组给予不同剂量的芍药苷(50,100μmol/L)预处理2 h,再用血管紧张素Ⅱ处理48 h;SIRT1抑制剂组先用10μmol/L SIRT1抑制剂EX527处理2 h,再用100μmol/L芍药苷处理2 h,最后用血管紧张素Ⅱ处理48 h。采用CCK-8法检测细胞活力,Transwell检测细胞迁移能力,用DHA荧光探针检测细胞内活性氧水平,用试剂盒检测氧化应激标志物水平,Western blot检测纤维化相关基因的蛋白表达,qRT-PCR检测细胞外基质和纤维化相关基因的mRNA表达。结果与结论:①与对照组相比,血管紧张素Ⅱ干预后心肌成纤维细胞的增殖、迁移能力明显提高,细胞内活性氧和丙二醛水平升高,超氧化物歧化酶和过氧化氢酶活性降低,α-平滑肌肌动蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、纤维连接蛋白、结缔组织生长因子、基质金属蛋白酶9的mRNA表达增加;与模型组相比,芍药苷剂量依赖性抑制上述效应改变(P<0.01);②与模型组相比,芍药苷剂量依赖性上调SIRT1的蛋白表达(P<0.001);③与芍药苷高剂量组相比,SIRT1抑制剂组细胞迁移数量、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白表达水平显著增加(P<0.01)。结果表明,芍药苷可能通过上调SIRT1的表达,有效减轻了血管紧张素Ⅱ诱导的心肌成纤维细胞纤维化改变,剂量依赖性地抑制了心肌成纤维细胞氧化应激和细胞外基质沉积,对于心肌成纤维细胞纤维化具有保护作用。
BACKGROUND:Previous studies have shown that paeoniflorin has an ameliorative effect on fibrosis in liver and kidney organs,especially in hepatic fibrosis.However,the protective effect of paeoniflorin on angiotensin Ⅱ-induced fibrosis in cardiac fibroblasts remains unclear.OBJECTIVE:To investigate the protective effects of paeoniflorin on angiotensin Ⅱ-induced extracellular matrix deposition in cardiac fibroblasts and molecular mechanisms.METHODS:Angiotensin Ⅱ(1μmol/L)was added to primary isolated cultured SD rat mammary rat cardiac fibroblasts for 48 hours as the model group.The paeoniflorin low and high dose groups were pretreated with different doses of paeoniflorin(50 and 100μmol/L)for 2 hours,and then treated with angiotensin Ⅱ for 48 hours.The SIRT1 inhibitor group was treated with SIRT1 inhibitor EX52710μmol/L for 2 hours,followed by paeoniflorin(100μmol/L)for 2 hours,and then co-incubation with angiotensin Ⅱ for 48 hours.The cell viability was detected using the cell counting kit-8 method(CCK-8)assay.The cell migration ability was detected by Transwell.The level of intracellular reactive oxygen species was detected by DHA fluorescent probe.The level of oxidative stress markers was detected by relevant kits.Protein expression of fibrosis-related genes was detected by western blot assay.The mRNA expression levels of extracellular matrix and fibrosis-related genes were detected by qRT-PCR.RESULTS AND CONCLUSION:(1)Compared with the control group,the proliferation and migration of cardiac fibroblasts were significantly increased after angiotensin Ⅱ intervention;the intracellular content of reactive oxygen species and malondialdehyde content were elevated;the activities of superoxide dismutase and catalase were decreased,and the mRNA expression levels ofα-smooth muscle actin,type Ⅰ collagen,type Ⅲ collagen,fibronectin,connective tissue growth factor,and matrix metalloproteinase 9 were increased.Compared with the model group,paeoniflorin could dose-dependently inhibit the above effect changes(P<0.01).(2)Compared with the model group,paeoniflorin up-regulated the protein expression of SIRT1 in dose-dependent manner(P<0.001).(3)Compared with the high-dose paeoniflorin group,the number of cell migration and the expression level ofα-smooth muscle actin,type Ⅰ collagen,and type Ⅲ collagen were significantly increased in the SIRT1-inhibitor group(P<0.01).All the experimental results show that paeoniflorin effectively attenuates the angiotensin Ⅱ-induced changes in cardiac fibroblast fibrosis possibly through up-regulating the expression of SIRT1,dose-dependently inhibits cardiac fibroblast oxidative stress and extracellular matrix deposition,and has a protective effect on cardiac fibroblast fibrosis.
作者
纪雅琼
宁忠平
Ji Yaqiong;Ning Zhongping(Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Department of Cardiology,Zhoupu Hospital Affiliated to Shanghai University of Medicine&Health Science,Shanghai 201318,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第25期5382-5389,共8页
Chinese Journal of Tissue Engineering Research
基金
上海市浦东新区卫生健康委员会重点学科群建设项目(PWZxq2022-11),项目负责人:宁忠平
上海市浦东新区心房颤动流行病学调查及分级诊疗模式下房颤全程管理前瞻性队列研究项目(PKJ2021-Y33),项目负责人:宁忠平
上海市浦东新区卫健委临床高峰学科建设项目(PWYgf2021-04),项目负责人:宁忠平。
作者简介
第一作者:纪雅琼,女,1997年生,山东省邹平市人,汉族,上海中医药大学在读硕士,主要从事中西医结合治疗心血管疾病方面的研究。https://orcid.org/0009-0002-4256-3074;通讯作者:宁忠平,硕士,主任医师,上海中医药大学,上海市201203,上海健康医学院附属周浦医院心血管内科,上海市201318。
