LIPUS调控FOXO1磷酸化促进氧化损伤的人牙周膜干细胞成骨分化的作用研究  

LIPUS Regulates FOXO1 Phosphorylation to Promote Osteogenic Differentiation of Oxidatively Damaged Human Periodontal Stem Cells

作　　者：唐丽[1] 庞华[2] 杨珂[3] 王冬 姚欢 TANG Li;PANG Hua;YANG Ke;WANG Dong;YAO Huan(Department of Ultrasound,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Nuclear Medicine,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Pediatric Research Institute,Children’s Hospital of Chongqing Medical University,National Clinical Research Center for Child Health and Disorders,Ministry of Education Key Laboratory of Child Development and Disorders,China International Science and Technology Cooperation Base of Child Development and Critical Disorders,Chongqing Engineering Research Center of Stem Cell Therapy,Chongqing 400014,China;Department of Ultrasound,Sichuan Provincial People’s Hospital(Affiliated Hospital of University of Electronic Science and Technology),Sichuan Medical College,Chengdu 610072,China)

机构地区：[1]重庆医科大学第附属第一医院,超声科,重庆400016 [2]重庆医科大学附属第一医院,核医学科,重庆400016 [3]重庆医科大学附属儿童医院儿科研究所,国家儿童健康与疾病临床医学研究中心,儿童发育与疾病教育部重点实验室,中国儿童发育与危重症国际科技合作基地,重庆市干细胞治疗工程技术研究中心,重庆400014 [4]四川省医学院,四川省人民医院(电子科技大学附属医院),超声科,成都610072

出　　处：《中国细胞生物学学报》2024年第3期417-425,共9页Chinese Journal of Cell Biology

基　　金：重庆市自然科学基金(批准号:CSTB2022NSCQ-MSX0812)资助的课题。

摘　　要：该研究探讨低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)对氧化损伤的人牙周膜干细胞(human periodontal ligament stem cells,h PDLSCs)成骨分化的保护作用及机制。采用浓度为300μmol/L的H_(2)O_(2)构建细胞氧化损伤模型,将其分为对照组、H_(2)O_(2)组、LIPUS组和LIPUS+H_(2)O_(2)组。DCFH-DA探针和TBA比色法分别检测各组细胞活性氧(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)水平;碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色分别评估各组细胞早期及晚期成骨分化能力;WB(Western blot)评价抗氧化酶[过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase 2,SOD-2)]、成骨相关蛋白[矮小相关转录因子2(runt-related transcription factor-2,RUNX2)、ALP和骨桥蛋白(osteopontin,OPN)]、叉头框蛋白O1(forkhead box protein O1,FOXO1)和(phospho-FOXO1,p-FOXO1)表达水平。随后采用FOXO1抑制剂AS1842856(100 nmol/L)处理细胞,实验分为对照组、H_(2)O_(2)+二甲基亚砜(dimethyl sulfoxide,DMSO)组、LIPUS+H_(2)O_(2)+DMSO组和LIPUS+H_(2)O_(2)+AS1842856组。CCK-8(cell counting kit-8)法检测细胞活力;DCFH-DA探针检测各组细胞ROS水平,WB检测各组RUNX2、ALP和OPN的蛋白表达情况。结果发现与空白对照组相比,H_(2)O_(2)组ROS与MDA水平均升高,抗氧化酶蛋白表达下调(P<0.0001);ALP和茜素红阳性染色以及成骨相关蛋白表达水平均显著降低(P<0.0001);p-FOXO1/FOXO1蛋白值上升(P<0.0001)。与H_(2)O_(2)组比较,LIPUS+H_(2)O_(2)组的ROS与MDA水平均下降,抗氧化酶蛋白表达上调(P<0.0001);成骨染色及相关蛋白表达量显著增加(P<0.0001);p-FOXO1/FOXO1蛋白值降低(P<0.0001)。相较于LIPUS+H_(2)O_(2)+DMSO组,LIPUS+H_(2)O_(2)+AS1842856组细胞活力下降,p-FOXO1/FOXO1蛋白表达上调,ROS水平升高且成骨分化相关蛋白表达量显著降低(P<0.0001)。研究结果显示LIPUS通过调节FOXO1的磷酸化水平,发挥抗氧化防御作用并提高氧化损伤的h PDLSCs成骨分This study was to investigate the protective effect and mechanism of LIPUS(low-intensity pulsed ul-trasound)on osteogenic differentiation of oxidatively damaged hPDLSCs(human periodontal ligament stem cells).The cellular oxidative damage model was constructed by using H_(2)O_(2) at an action concentration of 300μmol/L,which was divided into the control,H_(2)O_(2),LIPUS and LIPUS+H_(2)O_(2) groups.Cellular ROS(reactive oxygen species)and MDA(malondialdehyde)levels were detected by DCFH-DA probe and TBA colorimetric assay in each group,re-spectively.ALP(alkaline phosphatase)and alizarin red staining were used to assess the early and late osteogenic differentiation capacities of each group,respectively.WB(Western blot)evaluation of antioxidant enzymes[CAT(catalase)and SOD-2(superoxide dismutase 2)],osteogenesis-related protein[Runx2(runt-related transcription factor-2),ALP and OPN(osteopontin)],FOXO1(forkhead box protein O1)and p-FOXO1(phospho-FOXO1).Subsequently,the cells were treated with FOXO1 inhibitor AS1842856(100 nmol/L),and the experiments were divided into control,H_(2)O_(2)+DMSO,LIPUS+H_(2)O_(2)+DMSO,and LIPUS+H_(2)O_(2)+AS1842856 groups.CCK-8(cell counting kit-8)assay detected cell viability,DCFH-DA probe detected the level of cellular ROS in each group,and WB detected the protein expression of RUNX2,ALP,and OPN in each group.Compared with the control group,both ROS and MDA levels were elevated and antioxidant enzyme protein expression was down-regulated in the H_(2)O_(2) group(P<0.0001);ALP and alizarin red positive staining as well as expression of osteogenesis-related protein were significantly reduced(P<0.0001);and the p-FOXO1/FOXO1 protein ratio rose(P<0.0001).Compared with the H_(2)O_(2) group,the LIPUS+H_(2)O_(2) group showed decreased ROS and MDA levels and upregulated expression of antioxidant enzyme proteins(P<0.0001);ALP and alizarin red positive staining as well as protein expression of osteogenesis-related were significantly increased(P<0.0001);and p-FOXO1/FOXO1 protein ratio decreased(P<0.0

关 键 词：低强度脉冲超声 人牙周膜干细胞 氧化损伤 叉头框蛋白O1 成骨分化 

分 类 号：R781.42[医药卫生—口腔医学]