新型Nrf2激活剂CBR-470-1对地塞米松诱导的成骨细胞的作用机制  

Mechanism of the novel Nrf2 activator CBR-470-1 on dexamethasone-induced osteoblasts

作　　者：薛飞 唐洪辉 季峰 岳海涛 梁锦前[2] XUE Fei;TANG Honghui;JI Feng;YUE Haitao;LIANG Jinqian(Huai'an Clinical College,Xuzhou Medical University,Huai'an 223300,Jiangsu,China;Department of Orthopaedics,Peking Union Medical College Hospital,Peking Union Medical College,Chinese Academy of Medical Science,Beijing 100730,China)

机构地区：[1]徐州医科大学淮安临床学院,江苏淮安223300 [2]中国医学科学院北京协和医学院北京协和医院骨科,北京100730

出　　处：《中华骨与关节外科杂志》2025年第1期70-79,共10页Chinese Journal of Bone and Joint Surgery

基　　金：国家自然科学基金委员会面上项目(82072477)。

摘　　要：目的:探讨新型Nrf2激活剂CBR-470-1对DEX诱导的成骨细胞的作用机制。方法:体外培养小鼠MC3T3-E1成骨细胞,分为对照组、DEX组、CBR-470-1组和联合组,实时荧光定量PCR(qRT-PCR)检测Nrf2和Keap1基因表达、蛋白质免疫印迹实验(WB)检测NRF2和KEAP1蛋白水平、半胱天冬酶活性检测细胞凋亡、TUNEL法检测细胞凋亡、CCK-8法检测细胞活性、乳酸脱氢酶(LDH)检测细胞毒性。进一步利用CRISPR/Cas9基因编辑技术建立Nrf2基因敲除小鼠MC3T3-E1成骨细胞系、通过慢病毒构建Nrf2/PGK1基因敲低小鼠MC3T3-E1成骨细胞,分为对照组、DEX组和联合组,检测Nrf2/ARE基因表达、细胞凋亡、细胞活性及细胞毒性。结果:CBR-470-1组的Keap1和Nrf2 mRNA表达与对照组比较,差异均无统计学意义(P均>0.05)。CBR-470-1组NRF2蛋白水平高于对照组,差异有统计学意义(P<0.05)。对照组、CBR-470-1组和联合组Caspase-3/Caspase-9活性均显著低于DEX组;对照组、CBR-470-1组和联合组TUNEL阳性核细胞数均少于DEX组;对照组、CBR-470-1组和联合组的细胞相对活性均显著高于DEX组;对照组、CBR-470-1组和联合组的细胞存活率均显著低于DEX组,差异均有统计学意义(P均<0.05)。敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞中Nrf2/ARE mRNA表达缺失;敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞中ARE mRNA相对表达均低于正常细胞。正常细胞与敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞的DEX组、联合组细胞相对活性均低于对照组;DEX组、联合组中,敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞的细胞相对活性均低于正常细胞;正常细胞与敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞的DEX组、联合组细胞存活率均高于对照组;敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞的细胞存活率均低于正常细胞;正常细胞与敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞的DEX组、联合组的TUNEL阳性核细胞数均高于对照组;敲低/敲除Nrf2的小鼠MC3T3-E1成骨细胞的TUNEObjective:To investigate the mechanism of the novel Nrf2 activator CBR-470-1 on dexamethasone(DEX)-induced osteoblasts.Methods:Mouse MC3T3-E1 osteoblasts were cultured in vitro and divided into four groups:control,DEX,CBR-470-1 and combination groups.Quantitative Real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of Nrf2 and Keap1,and Western blot(WB)was used to detect the protein levels of NRF2 and KEP1.Caspase activity was measured to assess apoptosis,and the terminal deoxynucleotidyl transferase dUTP Nick-End Labeling(TUNEL)assay was used to detect apoptosis.Cell viability was assessed using the Cell Counting Kit-8(CCK-8)assay,and cytotoxicity was measured using the lactate dehydrogenase(LDH)assay.Additionally,CRISPR/Cas9 gene editing was used to establish Nrf2/PGK1 knockout MC3T3-E1 osteoblast cell lines from mice.Lentivirus was used to construct Nrf2/PGK1 knockdown MC3T3-E1 osteoblast cell lines.Both knockout and knockdown cell lines were divided into control,DEX and combination groups.The qRT-PCR was used to detect the expression of ARE and HO-1 genes,and the TUNEL and CCK-8 assay were used to detect apoptosis and cell viability,respectively.LDH was used to measure cytotoxicity.Results:The expression of Keap1 and Nrf2 mRNA in the CBR-470-1 group compared with the control group showed no statistically significant differences(all P>0.05).However,the protein level of NRF2 in the CBR-470-1 group was significantly higher than that in the control group,with a statistically significant difference(P<0.05).The activities of Caspase-3/Caspase-9 in the control group,CBR-470-1 group,and combination group were all significantly lower than those in the DEX group;the number of TUNEL-positive nuclei cells in the control group,CBR-470-1 group,and combination group were fewer than those in the DEX group;the relative cell activity in the control group,CBR-470-1 group,and combination group was significantly higher than that in the DEX group;and the cell survival rates in the control group,CBR-470-1 g

关 键 词：地塞米松 细胞凋亡 Nrf2通路 CBR-470-1 

分 类 号：R681.8[医药卫生—骨科学]