HDAC6调控HSC70乙酰化水平对卵巢切除术后骨质疏松骨量丢失的影响  

Effects of HDAC6 regulation of HSC70 acetylation level on bone loss in osteoporosis after oophorectomy

作　　者：李慧 雷洁赟 张丽 李双雪 雷垣生[4] 李峰 Li Hui;Lei Jieyun;Zhang Li;Li Shuangxue;Lei Yuansheng;Li Feng(Department of Gynaecology and Obstetrics,People’s Hospital Affiliated to Shanxi Medical University,Taiyuan 030012,China;The University of Sheffield,Sheffield City England,S11WB;Department of Respiratory and Medicine,People’s Hospital Affiliated to Shanxi Medical University,Taiyuan 030012,China;Department of Neurology,Second Clinical School of Medicine,Shanxi Medical University,Taiyuan 030001,China;Department of General surgery,Taigang General Hospital,Taiyuan 030003,China)

机构地区：[1]山西医科大学附属人民医院妇产科,太原030012 [2]谢菲尔德大学,英国S11WB [3]山西医科大学附属人民医院呼吸内科,太原030012 [4]山西医科大学第二临床医学院神经内科,太原030001 [5]太钢总医院普外科,太原030003

出　　处：《中华内分泌外科杂志》2022年第2期190-195,共6页Chinese Journal of Endocrine Surgery

基　　金：国家卫健委"十三五"规划重点课题(YYWS2404)。

摘　　要：目的探讨组蛋白乙酰化酶6(histone deacetylase 6,HDAC6)通过结合热休克蛋白70(heat-shock protein 70,HSC70)的启动子区域调控其乙酰化对卵巢切除术(oopherectomy,OOX)诱导的骨质疏松(osteoporosis,OP)骨量丢失的影响。方法采用OOX法建立小鼠OP模型。将小鼠分为假手术组、OOX组、OOX+shHDAC6组、OOX+shNC组和OOX+shHDAC6+shHSC70组。采用micro-CT系统和Western blot实验检测小鼠右股骨的骨微观参数及成骨细胞特异性转录因子蛋白表达水平。体外实验中,采用Westwen blot、碱性磷酸酶(ALP)染色和茜素红S(ARS)染色实验测定HDAC6和HSC70对MC3T3-E1细胞成骨分化的影响。采用qRT-PCR检测HDAC6和HSC70在组织或细胞中的表达水平。HDAC6和HSC70间的关系采用ChIP实验进行分析。结果与Sham组比较,OOX组小鼠右股骨骨矿物质密度(bone mineral density,BMD)、骨小梁数(trabecular bone number,Tb.N)、骨小梁厚度(trabecular thickness,Tb.Th)和骨体积分数(bone volume fraction,BV/TV)表达降低,骨小梁分离度(trabecular bone separation,Tb.Sp)表达升高,Osterix(OSX)和Runt相关转录因子2(runt-related transcription factor 2,RUNX2)的蛋白表达水平降低。此外,与Sham组比较(1±0.11),OOX组HDAC6表达(2.33±0.19)升高(t=10.56,P<0.001)。与pcDNA3.1-NC组比较,pcDNA3.1-HDAC6组细胞OSX和RUNX2的蛋白水平、ALP活性和矿化面积降低(均P<0.05)。ChIP分析显示与pcDNA3.1-NC组(5.26±0.47)比较,pcDNA3.1-HDAC6组HSC70启动子区域的乙酰化水平(2.37±0.21)显著降低(t=9.72,P<0.001)。与pcDNA3.1-HDAC6组比较,pcDNA3.1-HDAC6+pcDNA3.1-HSC70组细胞中OSX和RUNX2的表达升高,ALP活性和矿化的增加(均P<0.05)。与OOX+shHDAC6组比较,OOX+shHDAC6+shHSC70组OSX和RUNX2的蛋白水平、BMD、Tb.N、Tb.th和BV/TV表达降低,Tb.Sp表达升高(均P<0.05)。结论HDAC6能调控HSC70的乙酰化水平进而影响OOX诱导的OP骨量丢失,抑制HDAC6能显著改善OP骨量丢失。Objective To investigate the effects of histone deacetylase 6(histone deacetylase 6,HDAC6)on oopherectomy(OOX)induced osteoporosis(OP)bone loss by binding to the promoter region of heat-shock protein 70(HSC70)and regulating it’s acetylation.Methods OP mouse model was established by using OOX methods.Then the mice were divided into sham operation group,OOX group,OOX+shHDAC6 group,OOX+shNC group and OOX+shHDAC6+shHSC70 group.The micro-CT system and Western blot experiment were used to detect the bone microscopic parameters of the mouse right femur and the protein expression levels of osteoblast-specific transcription factors.In vitro experiments,Westwen blot,alkaline phosphatase(ALP)staining and Alizarin Red S(ARS)staining were used to determine the effects of HDAC6 and HSC70 on the osteogenic differentiation of MC3T3-E1 cells.QRT-PCR was used to detect the expression levels of HDAC6 and HSC70 in tissue or cells.The relationship between HDAC6 and HSC70 was analyzed by ChIP experiment.Results Compared with sham group,the expression of bone mineral density(BMD),trabecular bone number(Tb.N),trabecular thickness(Tb.th)and bone volume fraction(BV/TV)in the right femur of OOX group mice were decreased,the expression of TB.Sp was increased,protein expression of OSX and RUNX2 was increased.At the same time,compared with sham group(1±0.11),the expression of HDAC6 was increased in OOX group(2.33±0.19)(t=10.56,P<0.001).Compared with pcDNA3.1-NC group,the protein level of Osterix(OSX)and runt-related transcription factor 2(RUNX2),ALP activity and mineralized area in pcDNA3.1-HDAC6 group were decreased(all P<0.05).ChIP analysis showed that compared with the pcDNA3.1-NC group(5.26±0.47),the acetylation level of the HSC70 promoter region in the pcDNA3.1-HDAC6 group(2.37±0.21)was significantly reduced(t=9.72,P<0.001).Compared with pcDNA3.1-HDAC6 group,the expression of OSX and RUNX2,ALP activity and mineralization were increased in pcDNA3.1-HDAC6+pcDNA3.1-HSC70 group(all P<0.05).Compared with OOX+shHDAC6 group,the expression

关 键 词：HDAC6 HSC70 乙酰化 卵巢切除术 骨质疏松 骨量丢失 

分 类 号：R73[医药卫生—肿瘤]