基于内质网应激诱导的骨细胞凋亡探究骨松强骨方减轻去卵巢小鼠骨丢失的作用与机制  

Effect and mechanism of Gusong Qianggu Decoction on reducing bone loss in ovariectomized mice by targeting endoplasmic reticulum stress-induced apoptosis of osteocytes

作　　者：常宇博 潘博文 魏立伟 柴爽 郑旭霞 张虹 丁强 秦娜 CHANG Yu-bo;PAN Bo-wen;WEI Li-wei;CHAI Shuang;ZHENG Xu-xia;ZHANG Hong;DING Qiang;QIN Na(Department of Degree and Postgraduate Education Luoyang,Henan University of Chinese Medicine,Zhengzhou 450018,China;Luoyang Orthopedic-Traumatological Hospital of Henan Province(Henan Provincial Orthopedic Hospital),Zhengzhou 450016,China)

机构地区：[1]河南中医药大学学位与研究生教育洛阳工作部,河南郑州450018 [2]河南省洛阳正骨医院(河南省骨科医院),河南郑州450016

出　　处：《中国中药杂志》2024年第11期2981-2990,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82174413);河南省中医药科学研究专项重点项目(2021ZY1063);洛阳市医疗卫生领域指导性科技计划项目(2040001A)。

摘　　要：基于内质网应激(ERS)诱导的细胞凋亡探究骨松强骨方(GSQG)减轻去卵巢小鼠骨丢失的作用与机制。小鼠卵巢摘除(OVX)骨质疏松造模完成后,根据随机数字法,将60只小鼠随机分为6组:假手术组,模型组,骨松强骨方低(GSQG-L)、中(GSQG-M)、高剂量(GSQG-H)组,雌二醇(E2)组,每组10只;切除双侧卵巢构建模型。术后1个月开始灌胃给药,连续给药3个月。酶联免疫吸附测定法(ELISA)测定血清中骨形成指标骨钙素(OCN)、Ⅰ型胶原氨基端前肽(PINP)和骨吸收指标Ⅰ型胶原交联羧基端肽(CTX)、抗酒石酸酸性磷酸酶5b(TRAcP-5b)的水平,使用Micro-CT观察股骨远端骨微结构的改变,HE染色观察骨组织形态,RT-qPCR检测胫骨干成骨相关基因Ⅰ型胶原(Col-Ⅰ)、碱性磷酸酶(ALP)、Runt相关转录因子2(Runx2)、骨唾液酸糖蛋白(BSP)、OCN以及破骨相关基因抗酒石酸酸性磷酸酶(TRAP)、活化T细胞核因子(NFATc1)和组织蛋白酶K(CATK)mRNA的表达,TUNEL染色、免疫组化检测骨细胞凋亡情况,蛋白免疫印迹法检测胫骨近端骨组织ERS相关蛋白葡萄糖调节蛋白78(Grp78)、蛋白激酶RNA样内质网激酶(PERK)、磷酸化PERK(p-PERK)、真核翻译起始因子2α(eIF2α)、磷酸化e IF2α(p-eIF2α)、肌醇需求酶1α(IRE1α)、磷酸化IRE1α(p-IRE1α)、激活转录因子6(ATF6)的表达。结果显示,GSQG对去卵巢小鼠血清中OCN、PINP、TRAcP-5b、CTX骨代谢生化指标有明显改善,Micro-CT显示GSQG-H组股骨远端骨微结构明显优于其他组。与模型组比较,HE染色结果显示,GSQG-L组、GSQG-M组、GSQG-H组骨小梁明显变宽,数目增多,网状结构得到了一定程度的恢复,排列仍然整齐,部分间隙轻微增大;TUNEL阳性细胞显著减少(P<0.05,P<0.01);凋亡细胞相关蛋白半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、凋亡因子Bcl-2关联X蛋白(Bax)蛋白表达水平显著降低(P<0.05,P<0.01),Bcl-2蛋白表达显著升高(P<0.05,P<0.01);成骨相关基因Col-Ⅰ、ALP、RunThis study aims to investigate the role and mechanism of Gusong Qianggu Decoction(GSQG)in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress(ERS)-induced apoptosis of osteocytes.After the modeling of osteoporosis in mice with bilateral ovary removal(OVX),60 mice were randomized by the random number method into six groups:sham,model,low-,medium-,and high-dose GSQG(GSQG-L,GSQG-M,and GSQG-H,respectively),and estradiol(E2),with 10 mice in each group.The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the administration lasted for 3 months.Enzyme-linked immunosorbent assay(ELISA)was employed to determine the serum levels of osteocalcin(OCN),procollagen typeⅠN-terminal propeptide(PINP),carboxy-terminal cross-linked telopeptide of typeⅠcollagen(CTX),and anti-tartarte acid phosphatase 5b(TRAcP-5b).Micro-CT was employed to observe the changes in bone microstructure of the distal femur.Hematoxylin-eosin(HE)staining was employed to observe the morphology of the bone tissue.RT-qPCR was conducted to determine the mRNA levels of tibial stem osteogenesis-associated genes[typeⅠcollagen(Col-Ⅰ),alkaline phosphatase(ALP),Runtrelated transcription factor-2(Runx2),bone sialoprotein(BSP),and OCN]and bone-breaking related genes[tartrate-resistant acid phosphatase(TRAP),nuclear factor-activated T cell 1(NFATc1),and cathepsin K(CATK)].TUNEL staining and immunohistochemistry were employed to detect the apoptosis of osteoblasts.Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78(Grp78),protein kinase RNA-like endoplasmic reticulum kinase(PERK),phosphorylated PERK(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2α),phosphorylated eIF2α(p-eIF2α),inositol-requiring enzyme 1 alpha(IRE1α),phosphorylated IRE1α(p-IRE1α),and activating transcription factor 6(ATF6)in the proximal tibial bone tissue.The results showed that GSQG significantly recovered the levels of OCN,PINP,TRAcP-

关 键 词：内质网应激 细胞凋亡 骨松强骨方 骨质疏松 骨细胞 

分 类 号：R285.5[医药卫生—中药学]