AIM:To investigate whether melatonin can ameliorate acute myocardial infarction(AMI)by in⁃hibiting ferroptosis.METHODS:H9C2 cells were cultured in AnaeroPack system with low sugar and serum-free medium for 10 h to con...AIM:To investigate whether melatonin can ameliorate acute myocardial infarction(AMI)by in⁃hibiting ferroptosis.METHODS:H9C2 cells were cultured in AnaeroPack system with low sugar and serum-free medium for 10 h to construct a cell model of AMI.Then cells were treated with melatonin and ferroptosis inducer erastin.The cell activity,reactive oxygen species(ROS),lipid peroxidation,mitochondrial membrane potential(MMP),and ferroptosis related protein expression were detected.A rat model of AMI induced by isoprenaline(ISO)injection was established to evaluate the effects of melatonin,in which the myocardial infarction size,cardiac injury,pathological changes,oxidative stress,iron ion and ferroptosis related protein expression were examined.RESULTS:Melatonin decreased the oxidative stress,lipid peroxidation and expression of ferroptosis protein in cardiomyocytes induced by hypoxia,but these effects could be impeded by the ferroptosis inducer erastin.Furthermore,in vivo experiments,we also found that melatonin im⁃proved the myocardial infarction size,cardiac injury,pathological changes,oxidative stress,and alleviated iron ion accu⁃mulation and ferroptosis.CONCLUSION:The cardioprotective effects of melatonin in AMI are associated with the inhi⁃bition of ferroptosis.展开更多
目的构建人T细胞活化核因子C2(NFATc2)基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter,检测其转录活性,探究二甲双胍和脂多糖对其转录活性的影响。方法利用UCSC网站查找人NFATc2基因的启动子序列并设计上下游引物PCR扩增人NFATc2...目的构建人T细胞活化核因子C2(NFATc2)基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter,检测其转录活性,探究二甲双胍和脂多糖对其转录活性的影响。方法利用UCSC网站查找人NFATc2基因的启动子序列并设计上下游引物PCR扩增人NFATc2基因启动子片段;用限制性内切酶KpnⅠ和HindⅢ双酶切质粒pGL3-basic,将人NFATc2基因启动子片段插入到pGL3-basic质粒,重组质粒命名为pGL3-NFATc2-promoter。将pGL3-NFATc2-promoter与内参质粒pRL-TK共转染293F细胞,检测其荧光素酶活性。同时构建人NFATc2基因启动子不同片段长度的报告基因载体并进行荧光素酶活性检测,分别给予不同浓度的二甲双胍和脂多糖处理24 h后检测二甲双胍和脂多糖对NFATc2转录活性的影响。进一步突变NFATC2基因启动子上转录因子RUNX2的结合位点探究二甲双胍和脂多糖对NFATc2的转录调控作用。结果研究成功构建了不同片段长度(2170、2077、1802、1651、1083、323 bp)的人NFATC2基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter,经酶切及测序鉴定完全正确。将不同片段长度的pGL3-NFATc2-promoter转染到293F细胞,发现pGL3-1651 bp具有最高转录活性,荧光素酶活性约为pGL3-2170 bp的3.3倍(1.8433±0.1457 vs 0.5467±0.0850)。中浓度(5 mmol/L)和高浓度(10 mmol/L)的二甲双胍分别上调pGL3-1651 bp的转录活性多达2.5、3倍(1.3467±0.1601 vs 0.8867±0.0321,1.8124±0.2771 vs 0.8867±0.0321)。不同剂量的脂多糖均能上调pGL3-1651 bp的转录活性不低于1.6倍(1.4813±0.0616 vs 0.8867±0.0321)。突变pGL3-1651 bp上的RUNX2结合位点后,二甲双胍和脂多糖上调的pGL3-1651 bp转录活性均受到抑制,转录活性下降(2.1667±0.1527 vs 1.233±0.1155;2.3667±0.2887 vs 1.1333±0.3786)。结论pGL3-NFATc2-promoter在293F细胞中能被转录激活,并证实脂多糖和二甲双胍转录激活pGL3-NFATc2-promoter依赖于转录因RUNX2。展开更多
基金Supported by Guangdong Medical Research Foundation(No.A2024382)Guangdong Provincial Bureau of Traditional Chinese Medicine research project(No.20231321)Scientific Research Start Plan of Shunde Hospital,Southern Medical University(No.SRSP2022012,No.SRSP2022016)。
文摘AIM:To investigate whether melatonin can ameliorate acute myocardial infarction(AMI)by in⁃hibiting ferroptosis.METHODS:H9C2 cells were cultured in AnaeroPack system with low sugar and serum-free medium for 10 h to construct a cell model of AMI.Then cells were treated with melatonin and ferroptosis inducer erastin.The cell activity,reactive oxygen species(ROS),lipid peroxidation,mitochondrial membrane potential(MMP),and ferroptosis related protein expression were detected.A rat model of AMI induced by isoprenaline(ISO)injection was established to evaluate the effects of melatonin,in which the myocardial infarction size,cardiac injury,pathological changes,oxidative stress,iron ion and ferroptosis related protein expression were examined.RESULTS:Melatonin decreased the oxidative stress,lipid peroxidation and expression of ferroptosis protein in cardiomyocytes induced by hypoxia,but these effects could be impeded by the ferroptosis inducer erastin.Furthermore,in vivo experiments,we also found that melatonin im⁃proved the myocardial infarction size,cardiac injury,pathological changes,oxidative stress,and alleviated iron ion accu⁃mulation and ferroptosis.CONCLUSION:The cardioprotective effects of melatonin in AMI are associated with the inhi⁃bition of ferroptosis.
文摘目的构建人T细胞活化核因子C2(NFATc2)基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter,检测其转录活性,探究二甲双胍和脂多糖对其转录活性的影响。方法利用UCSC网站查找人NFATc2基因的启动子序列并设计上下游引物PCR扩增人NFATc2基因启动子片段;用限制性内切酶KpnⅠ和HindⅢ双酶切质粒pGL3-basic,将人NFATc2基因启动子片段插入到pGL3-basic质粒,重组质粒命名为pGL3-NFATc2-promoter。将pGL3-NFATc2-promoter与内参质粒pRL-TK共转染293F细胞,检测其荧光素酶活性。同时构建人NFATc2基因启动子不同片段长度的报告基因载体并进行荧光素酶活性检测,分别给予不同浓度的二甲双胍和脂多糖处理24 h后检测二甲双胍和脂多糖对NFATc2转录活性的影响。进一步突变NFATC2基因启动子上转录因子RUNX2的结合位点探究二甲双胍和脂多糖对NFATc2的转录调控作用。结果研究成功构建了不同片段长度(2170、2077、1802、1651、1083、323 bp)的人NFATC2基因启动子荧光素酶报告基因载体pGL3-NFATc2-promoter,经酶切及测序鉴定完全正确。将不同片段长度的pGL3-NFATc2-promoter转染到293F细胞,发现pGL3-1651 bp具有最高转录活性,荧光素酶活性约为pGL3-2170 bp的3.3倍(1.8433±0.1457 vs 0.5467±0.0850)。中浓度(5 mmol/L)和高浓度(10 mmol/L)的二甲双胍分别上调pGL3-1651 bp的转录活性多达2.5、3倍(1.3467±0.1601 vs 0.8867±0.0321,1.8124±0.2771 vs 0.8867±0.0321)。不同剂量的脂多糖均能上调pGL3-1651 bp的转录活性不低于1.6倍(1.4813±0.0616 vs 0.8867±0.0321)。突变pGL3-1651 bp上的RUNX2结合位点后,二甲双胍和脂多糖上调的pGL3-1651 bp转录活性均受到抑制,转录活性下降(2.1667±0.1527 vs 1.233±0.1155;2.3667±0.2887 vs 1.1333±0.3786)。结论pGL3-NFATc2-promoter在293F细胞中能被转录激活,并证实脂多糖和二甲双胍转录激活pGL3-NFATc2-promoter依赖于转录因RUNX2。
