补阳还五汤通过调控IL-6_STAT3信号通路减轻AngⅡ诱导的H9c2细胞损伤.pdf

投稿网址:2023 年 第23 卷 第24 期2023,23(24):10236-09科 学 技 术 与 工 程Science Technology and EngineeringISSN 16711815CN 114688/T收稿日期:2022-12-20修订日期:2023-06-06基金项目:辽宁中医药大学自然科学课题(2100222080);国家级大学生创新创业训练项目(202210162006);辽宁省教育厅育苗项目(L202025)第一作者:薛亚楠(1991),女,汉族,辽宁葫芦岛人,博士研究生,实验师。研究方向:高血压病因病机及中药防治机理。E-mail:。通信作者:张立德(1959),男,汉族,辽宁朝阳人,教授,博士研究生导师。研究方向:高血压病因病机及中医药防治机制。E-mail:。引用格式:薛亚楠,曲怡,王建波,等.补阳还五汤通过调控 IL-6/STAT3 信号通路减轻 Ang诱导的 H9c2 细胞损伤J.科学技术与工程,2023,23(24):10236-10244.Xue Yanan,Qu Yi,Wang Jianbo,et al.Buyang Huanwu Decoction alleviates Ang induced H9c2 cell injury by regulating IL-6/STAT3signal pathwayJ.Science Technology and Engineering,2023,23(24):10236-10244.医药、卫生补阳还五汤通过调控 IL-6/STAT3 信号通路减轻Ang诱导的 H9c2 细胞损伤薛亚楠1,2,曲怡1,2,王建波1,2,高佳馨1,2,张云雨1,2,张立德1,2(1.辽宁中医药大学中医药创新工程技术中心,沈阳 110000;2.中医脏象理论及应用教育部重点实验室,沈阳 110000)摘 要 探讨补阳还五汤对血管紧张素(angiotensin,Ang)环境下大鼠 H9c2 心肌细胞铁死亡的调控作用及相关机制。采用 1 mol 血管紧张素(Ang)对 H9c2 细胞进行干预,建立高血压性心肌损伤体外模型。将细胞分为正常组、模型组(1 mol Ang)、补阳还五汤低剂量组(1 mol Ang+0.5%补阳还五汤)、补阳还五汤中剂量组(1 mol Ang+1%补阳还五汤)、补阳还五汤高剂量组(1 mol Ang+2%补阳还五汤)和阳性对照组(1 mol Ang+10 mol Fer-1)。细胞计数试剂盒 8(cell counting Kit-8,CCK-8)法检测各组 H9c2 细胞活力;罗丹明标记的鬼笔环肽细胞染色法检测心肌细胞面积;免疫荧光染色法检测各组细胞转录因子 NF-E2 相关因子(transcription factor NF-E2 related factors,Nrf2)蛋白表达;q-PCR 法检测各组细胞白介素-6(interleukin-6,IL-6)、细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM)、单核细胞趋化蛋白-1(monocytechemoattractant protein-1,MCP),和信号传导及转录激活因子 3(signal transducers and activators of transcription 3,STAT3)的基因表达;免疫荧光染色法及 Western blot 法检测 STAT3、磷酸化信号传导及转录激活因子 3(phosphorylation signal transducers andactivators of transcription 3,P-STAT3)的蛋白表达变化。结果表明:补阳还五汤高剂量组明显提高 Ang诱导的 H9c2 细胞活力下降(P 0.01),抑制 Ang诱导的 H9c2 心肌细胞肥大的作用(P 0.01);与模型组相比,补阳还五汤高剂量组能显著逆转Ang诱导的 H9c2 细胞 Nrf2 表达水平的下降,IL-6、STAT3、ICAM、MCP mRNA 表达水平的升高及 STAT3 与 P-STAT3 蛋白水平的升高,与阳性对照组结果一致(P 0.01,P 0.05)。补阳还五汤可能通过上调 Nrf2 的表达而负反馈调节 IL-6/STAT3 信号通路,对 AngII 诱导的 H9c2 大鼠心肌细胞的铁死亡和细胞重塑发挥保护作用。关键词 补阳还五汤;心肌细胞;铁死亡;高血压中图法分类号 R284.2;文献标志码 ABuyang Huanwu Decoction Alleviates Ang Induced H9c2 Cell Injury byRegulating IL-6/STAT3 Signal PathwayXUE Ya-nan1,2,QU Yi1,2,WANG Jian-bo1,2,GAO Jia-xin1,2,ZHANG Yun-yu1,2,ZHANG Li-de1,2(1.Innovative Engineering Technology Center of Traditional Chinese Medicine,Liaoning University ofTraditional Chinese Medicine,Shenyang 110000,China;2.Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications,Shenyang 110000,China)Abstract To investigate the regulatory effect of Buyang Huanwu Decoction on ferroptosis of H9c2 cells in the environment of angio-tensin(Ang)and its related mechanism.1 mol angiotensin(Ang)intervened H9c2 cells to establish an in vitro model ofhypertensive myocardial injury.Cells were divided into normal group and model group(1 mol Ang),low dose Buyang HuanwuDecoction group(1 mol Ang+0.5%Buyang Huanwu Decoction),middle dose Buyang Huanwu Decoction group(1 molAng+1%Buyang Huanwu Decoction),high dose Buyang Huanwu Decoction group(1 mol Ang+2%Buyang Huanwu Decoc-tion)and positive control group(1 mol Ang+10 mol Fer-1).The activity of H9c2 cells in each group was detected by cell投稿网址:counting Kit-8(CCK-8)method.Rhodamine labeled ghost pen cyclic peptide cell staining was used to detect myocardial cell area.Theexpression of NF-E2 related factor(Nrf2)protein was detected by immunofluorescence staining.The gene expression of interleukin-6(IL-6),intercellular adhesion molecule-1(ICAM),monocyte chemoattractant protein-1(MCP),and transcription activating factor 3(STAT3)were detected by q-PCR.The protein expression of STAT3 and P-STAT3 was detected by immunofluorescence staining andWestern blot.The results show that the high dose of Buyang Huanwu Decoction significantly increases the decrease of H9c2 cell activityinduced by Ang(P 0.01),and inhibites the hypertrophy of H9c2 cardiomyocytes induced by Ang(P 0.01).Compared withthe model group,the high dose of Buyang Huanwu Decoction could significantly reverse the decrease of Nrf2 expression level,the in-crease of IL-6,STAT3,ICAM,MCP mRNA expression level and the increase of STAT3 and P-STAT3 protein levels in H9c2 cells in-duced by Ang,which are consistent with the results of the positive control group(P 0.01,P NM_012747.2上游:5-GATTCTTCGCAGGTTGTG-3下游:5-GCAGTATAGCCGATTCCT-361IL-6 NM_012589.2上游:5-CAACTCTTTTCTCATTTCCA-3下游:5-CACTGCCTTCCCTACTTC-3142ICAM NM_012967.1上游:5-CTGTTCAGAAGCACCACC-3下游:5-CTCAGAAGGGGACCAAGT-369MCP NM_031530.1上游:5-TCTTCCTCCACCACTATGC-3下游:5-GCATAGTGGTGGAGGAAG-319-actin NM_031144.3上游:5-CCTCTATGCCAACACAGT-3下游:5-AGCCACCAATCCACACAG-315583201科 学 技 术 与 工 程Science Technology and Engineering2023,23(24)投稿网址:解混合液提取蛋白,二喹啉甲酸(bicinchoninic acid,BCA)法测定蛋白定量。60 g 蛋白上样后用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfatepolyacrylamide gel electrophoresis,SDS-PAGE)电泳,湿转至聚偏氟乙烯(polyvinylidene fluoride,PVDF)膜上,5%脱脂奶粉室温摇床封闭2 h 后孵育一抗4 过夜,TBST 洗涤3 次,二抗室温摇床孵育2 h,三乙醇胺缓冲盐水溶液(tris buffered saline,TBST)洗涤 3 次,增强型化学发光试剂(electrochemiluminescence,ECL)发光液进行显影并拍照,Image J 软件分析灰度值。2 统计学方法采用统计学软件 SPSS 19.0、Image J 和 GraphadPrism 5 对数据进行分析,数据采用均值 标准差(x s)表示,方差齐性检验后,组间比较采用单因素方差分析,P 0.05、P 0.05),结果如图 1(a)所示。使用