目的:探究mitoNEET能否抑制铁死亡引起的棕色脂肪细胞线粒体代谢功能紊乱.方法:采用原代C57BL/6J小鼠棕色脂肪细胞构建体外模型,Western blot检测相关蛋白,试剂盒检测细胞中铁离子和ATP含量,荧光显微镜及流式细胞术检测线粒体膜电位和活性氧(ROS)水平.结果:与对照组相比,铁死亡诱导剂erastin处理组棕色脂肪细胞中铁死亡相关蛋白ACSL4表达增加了1.13倍,SLC7A11和GPX4表达分别减少了27.33%和25.33%(P<0.05);mitoNEET蛋白含量下降了42.14%(P<0.05).试剂盒检测结果显示,erastin组细胞铁离子含量较对照组升高了1.80倍,但ATP含量下降了14.95%(P<0.05).荧光显微镜及流式细胞术结果显示,erastin组细胞的线粒体膜电位分别下降了52.18%和61.31%(P<0.05),线粒体ROS含量增加了80.97%(P<0.05),线粒体能量代谢相关蛋白Nrf1、PGC-1α、MFN2和UCP1表达水平分别下降了20.98%、15.17%、15.03%和34.22%(P<0.05).荧光显微镜及流式细胞术结果显示,与对照组相比,LV-mitoNEET组过表达mitoNEET促使erastin诱导的棕色脂肪细胞的线粒体膜电位分别增加17.61%和96.05%,线粒体ROS的生成减少了24.48%(P<0.05).Western blot结果表明,mitoNEET慢病毒转染组棕色脂肪细胞中mitoNEET的含量增加了11.19倍(P<0.05),提示病毒转染成功;在erastin处理下,与对照组相比,LV-mitoNEET组棕色脂肪细胞中铁死亡相关蛋白ACSL4表达下降了37.95%,SLC7A11和GPX4表达分别增加了77.82%和66.30%(P<0.05);线粒体代谢相关蛋白MFN2、PGC-1α、Nrf1和UCP1的表达分别上调了79.06%、72.89%、40.14%和31.68%(P<0.05).试剂盒检测结果显示,与对照组相比,LV-mi-toNEET组细胞铁离子含量降低了43.50%,ATP含量增加了33.50%(P<0.05).结论:mitoNEET过表达抑制铁死亡引起的棕色脂肪细胞线粒体代谢紊乱.
AIM:To investigate the potential impact of mitoNEET[mitochondrial protein containing Asn-Glu-Glu-Thr(NEET)sequence]on mitochondrial metabolism in brown adipocytes,and to elucidate its underlying mecha-nism.METHODS:An in vitro model of primary mouse brown adipocytes was established.Western blot were utilized to detect relevant proteins,and iron ion and ATP content was measured using kits.Mitochondrial membrane potential and re-active oxygen species(ROS)were assessed by fluorescence microscopy and flow cytometry.RESULTS:The expression of the ferroptosis-related protein ACSL4 increased by 1.13 times in ferroptosis inducer erastin treatment group,whereas the expression of SLC7A11 and GPX4 decreased by 27.33%and 25.33%,respectively,compared with control group(P<0.05).The expression of Nrf1,PGC-1α,MFN2 and UCP1 proteins,related to mitochondrial energy metabolism,de-creased by 20.98%,15.17%,15.03%and 34.22%,respectively(P<0.05).Additionally,the mitoNEET protein con-tent was significantly reduced by 42.14%(P<0.05).The iron ion content in erastin group was substantially increased by 1.80 times compared with control group.However,a notable decrease in ATP content of 14.95%was seen(P<0.05).The results obtained from fluorescence microscopy and flow cytometry demonstrated a significant decrease in the mitochon-drial membrane potential of brown adipocytes in erastin group,with reductions of 52.18%and 61.31%(P<0.05),re-spectively.A substantial increase in mitochondrial ROS content of 80.97%was seen(P<0.05).Western blot analysis of overexpressed stable strains revealed a significant elevation in mitoNEET levels in brown adipocytes following lentivirus transfection,exhibiting an increase of 11.19 times(P<0.05),thus confirming successful transfection.The LV-mitoNEET group exhibited a significant decrease of 37.95%in the expression of ferroptosis-related protein ACSL4 in brown adipose cells compared with control group.Additionally,there was a notable increase of 77.82%and 66.3%in the expression of SLC7A11 and GPX4,respectively(P<0.05).Up-regulation was observed in the expression of MFN2(79.06%),PGC-1α(72.89%),Nrf1(40.14%),and UCP1(31.68%)(P<0.05).The test results demonstrated that the LV-mitoNEET group experienced a reduction of 43.5%in iron ion content compared with control group while exhibiting an increase of 33.5%in ATP content(P<0.05).The results obtained from fluorescence microscopy and flow cytometry demonstrated that mitoNEET overexpression led to a significant increase in the mitochondrial membrane potential of erastin-induced brown adipocytes,with increments of 17.61%and 96.05%,respectively.Additionally,mitoNEET overexpression effec-tively reduced the production of mitochondrial ROS by 24.48%(P<0.05).CONCLUSION:Our findings suggest that mitoNEET overexpression can effectively inhibit the disruption of mitochondrial energy metabolism caused by ferroptosis-induced death of brown adipocytes.
郑红玉;田震;王燕霞;周坤;任重;周支香;熊文昊;郑核;姜志胜
南华大学衡阳医学院心血管疾病研究所,动脉硬化学湖南省重点实验室,动脉硬化性疾病湖南省国际科技创新合作基地,湖南 衡阳 421001邵阳中心医院(南华大学附属邵阳医院)肝胆外科,湖南 邵阳 422000
临床医学
mitoNEET蛋白;棕色脂肪细胞;线粒体;铁死亡
mitoNEET protein;brown adipocytes;mitochondria;ferroptosis
《中国病理生理杂志》 2024 (005)
852-861 / 10
国家自然科学基金资助(No.91839103;No.81670429);湖南省重点研发计划(湖南创新型省份建设专项经费资助)(No.2020SK2105)
10.3969/j.issn.1000-4718.2024.05.010
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