miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

摘要 目的：探讨miR-20b-5p对氧糖剥夺（OGD）/Hemin处理的脑微血管内皮细胞（BMVEC）功能的影响及机制。方法：将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine 2000试剂对细胞进行相应的转染处理。BMVEC转染后，将BMVEC再分为Control组、OGD/Hemin组（O/H组）、OGD/Hemin+agomir-NC组（O/H+agomir-NC组）、OGD/Hemin+agomir-miR-20b-5p组（O/H+agomir-miR-20b-5p组）、OGD/Hemin+antagomir-NC组（O/H+antagomir-NC组）和OGD/Hemin+antagomir-miR-20b-5p组（O/H+antagomir-miR-20b-5p组）。Control组BMVEC正常培养，其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖，TUNEL染色检测BMVEC凋亡，Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平。使用Iron Assay试剂盒检测Fe²⁺含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果：与Control组和agomir-NC组比较，agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高（P<0.05）。与Control组和antagomir-NC组比较，antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低（P<0.05）。与Control组比较，O/H组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。与O/H组和O/H+agomir-NC组比较，O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高，细胞活力升高，TUNEL阳性率和Bax蛋白表达水平降低，Bcl-2蛋白表达水平升高，迁移数量升高，SOD和GSH-Px活性升高，MDA含量降低，Fe²⁺含量和PTGS2的蛋白表达水平降低，GPX4的蛋白表达水平升高，MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低（P<0.05）。与O/H组和O/H+antagomir-NC组比较，O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。结论：本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。

miR-20b-5p Targets MAPK1 to Regulate Ferroptosis of Brain Microvascular Endothelial Cells during Intracerebral Hemorrhage

ABSTRACT Objective: To investigate the effects and mechanism of miR-20b-5p on the function of brain microvascular endothelial cells (BMVEC) treated with oxygen glucose deprivation (OGD) /Hemin. Methods: BMVEC were divided into Control group, agomir-NC group, agomir-miR-20b-5p group, antagomir-NC group and antagomir-miR-20b-5p group. The cells were transfected with Lipofectamine 2000 reagent. After BMVEC transfection, BMVEC was divided into Control group, OGD/Hemin group (O/H), OGD/Hemin+agomir-NC group (O/H+agomir-NC), OGD/Hemin+agomir-miR-20b-5p group (O/H+agomir-miR-20b-5p), OGD/Hemin+antagomir-NC group (O/H+antagomir-NC) and OGD/Hemin+antagomir-miR-20b-5p group (O/H+antagomir-miR-20b-5p). BMVEC in Control group were cultured normally, and BMVEC in other groups were treated with OGD/Hemin. BMVEC proliferation was detected by MTT assay. BMVEC apoptosis was detected by TUNEL staining. BMVEC migration was detected by Transwell. The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured using a commercial kit. Fe²⁺ content was determined using Iron Assay kit. The levels of miR-20b-5p and MAPK1 mRNA were detected by qRT-PCR. The protein expression levels of MAPK1, Bax, Bcl-2, glutathione peroxidase 4 (GPX4) and prostaglandin peroxidase synthase 2 (PTGS2) were detected by Western blot. Fluorescence intensity of MAPK1 was detected by immunofluorescence staining. Results: Compared with Control group and agomir-NC group, the level of miR-20b-5p in BMVEC of agomir-miR-20b-5p group increased(P<0.05). Compared with Control group and antagomir-NC group, the level of miR-20b-5p in BMVEC of antagomir-miR-20b-5p group decreased(P<0.05). Compared with Control group, miR-20b-5p level in BMVEC of O/H group decreased, cell viability decreased, TUNEL positive rate and Bax protein expression level increased, Bcl-2 protein expression level decreased, the number of migration decreased, SOD and GSH-Px activity decreased, MDA content increased, Fe²⁺ content and PTGS2 protein expression level increased, GPX4 protein expression level decreased, the mRNA and protein expression level and the relative fluorescence intensity of MAPK1 increas(P<0.05). Compared with O/H group and O/H+agomir-NC group, the miR-20b-5p level in BMVEC of O/H+agomir-miR-20b-5p group increased, the cell viability increased, the positive rate of TUNEL and the expression level of Bax protein decreased, the expression level of Bcl-2 protein increased, the number of migration increased, the activity of SOD and GSH-Px increased, the content of MDA decreased, the content of Fe²⁺ and the protein expression level of PTGS2 decreased, the protein expression level of GPX4 increased, the mRNA and protein expression level and the relative fluorescence intensity of MAPK1 decreased(P<0.05). Compared with O/H group and O/H+antagomir-NC group, miR-20b-5p level in BMVEC of O/H+antagomir-miR-20b-5p group decreased, cell viability decreased, TUNEL positive rate and Bax protein expression level increased, Bcl-2 protein expression level decreased, the number of migration decreased, SOD and GSH-Px activity decreased, MDA content increased, Fe²⁺ content and PTGS2 protein expression level increased, GPX4 protein expression level decreased, the mRNA and protein expression level and the relative fluorescence intensity of MAPK1 increased(P<0.05). Conclusion: This study suggests that upregulation of miR-20b-5p inhibits the ferroptosis pathway by inhibiting the expression of MAPK1 in OGD/Hemin-treated BMVEC.