摘要
目的观察内质网过度应激中转录激活因子4(ATF4)对骨质疏松的影响,并探讨可能的分子学机制。方法将2对ATF4过表达杂合子小鼠进行扩增繁殖后,通过鼠尾鉴定法筛选出ATF4过表达纯合子小鼠和野生型小鼠。将12周龄的野生型小鼠和ATF4过表达纯合子小鼠分为正常野生小鼠组(对照野生组),内质网应激通路激动剂衣霉素(TM)干预野生小鼠组(TM野生组)、ATF4过表达纯合子组(ATF4^(+/+)组)和衣霉素干预ATF4过表达纯合子组(TM ATF4^(+/+)组),每组10只。干预6周后剥离小鼠股骨,采用小动物显微影像系统(microCT)检测股骨骨密度(BMD)、骨小梁骨体积分数(Tb.BV/TV)、骨小梁数量(Tb.N)和骨小梁间隔(Tb.Sp)的变化;采用苏木精-伊红(HE)染色观察各组小鼠股骨的病理形态;采用实时荧光定量PCR和蛋白质印迹法检测各组小鼠股骨中Runt相关转录因子2(Runx2)、成骨相关转录因子(Osx)、骨钙素(Ocn)、ATF4和C/EBP同源蛋白(CHOP)的mRNA和蛋白表达。结果MicroCT检测结果显示,与对照野生组比较,其他3组小鼠的BMD、Tb.BV/TV和Tb.N均降低,Tb.Sp增加,差异均有统计学意义(P<0.05);与TM野生组比较,TM ATF4^(+/+)组小鼠的BMD、Tb.BV/TV和Tb.N均降低,Tb.Sp增加,差异均有统计学意义(P<0.05)。HE染色结果显示,与对照野生组比较,其他3组小鼠的骨小梁相对面积减小,差异均有统计学意义(P<0.05)。实时荧光定量PCR和蛋白质印迹检测结果显示,与对照野生组比较,其他3组小鼠的成骨相关因子Runx2、Osx和Ocn的表达减少,而ATF4和CHOP的表达增加,差异均有统计学意义(P<0.05);与TM野生组比较,TM ATF4^(+/+)组小鼠的Runx2、Osx和Ocn的表达减少,而ATF4和CHOP的表达增加,差异均有统计学意义(P<0.05)。结论内质网过度应激可通过促进ATF4/CHOP信号通路表达抑制骨形成,从而导致骨质疏松。
Objective To explore the effect of activating transcription factor 4(ATF4)in endoplasmic reticulum overstress on osteoporosis and its molecular mechanism.Methods After 2 pairs of heterozygous mice with ATF4 overexpression were multiplied,homozygous mice with ATF4 overexpression and wild-type mice were screened by the tail identification method.The 12-week-old wild-type mice and ATF4 overexpression homozygous mice were divided into normal wild mice group(control wild group),endoplasmic reticulum stress(ERS)pathway agonist tunicamycin(TM)intervention wild mice group(TM wild group),ATF4 overexpression homozygotes group(ATF4^(+/+) group)and TM intervention homozygous ATF4 overexpression group(TM ATF4+/+group),each with 10 animals.After 6 weeks of intervention,the femur was taken and microCT was used to detect the change of the distal femur bone mineral density(BMD),trabecular bone volume fraction(Tb.BV/TV),number of trabecular bones(Tb.N),and trabecular septum(Tb.Sp).HE staining was used to observe the pathological morphology of mouse femurs in each group.Real-time fluorescence quantitative PCR and Western blotting were used to detect the mRNA and protein expression of osteogenic factors Runt-related transcription factor 2(Runx2),Osterix(Osx),osteocalcin(Ocn),ATF4 and CCAAT/enhancer-binding protein homologous protein(CHOP).Results MicroCT results showed that compared with the control wild group,the BMD,Tb.BV/TV and Tb.N of mice in the other 3 groups were significantly reduced,and Tb.Sp was significantly increased,all with a statistical significance(P<0.05).Compared with the TM wild group,the BMD,Tb.BV/TV and Tb.N of the TM ATF4^(+/+) group were significantly reduced,and Tb.Sp was significantly increased,all with a statistical significance(P<0.05).The results of HE staining showed that compared with the control wild group,the area of trabecular bones in the other 3 groups was significantly reduced,all with a statistical significance(P<0.05).The results of real-time fluorescence quantitative PCR and Western blotting sh
作者
李少华
张铁山
邢克炎
何新庄
李玉伟
Li Shaohua;Zhang Tieshan;Xing Keyan;He Xinzhuang;Li Yuwei(Department of Orthopaedics,General Hospital of Yongcheng Coal Group,Shangqiu 476600,Henan,China;Department of Orthopaedics,Luohe Central Hospital,Luohe 462000,Henan,China)
出处
《脊柱外科杂志》
2021年第4期259-264,共6页
Journal of Spinal Surgery
基金
河南省医学科技攻关计划项目(201504092)。
关键词
骨质疏松
内质网
应激
转录激活因子4
信号传导
Osteoporosis
Endoplasmic reticulum
Stress
Activating transcription factor 4
Signal transduction
