mitoNEET过表达抑制铁死亡引起的棕色脂肪细胞线粒体代谢功能障碍OA北大核心CSTPCD

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.