骨松益骨方调控Beclin 1/Bcl-2对去卵巢大鼠骨细胞凋亡和自噬的影响  

The osteoprotective effect of GSYG on apoptosis and autophagy of osteocytes by regulating Beclin 1/Bcl-2 in ovariectomized rats

作　　者：有曼 魏立伟 柴爽 郑旭霞 孟璐 张虹 何广宏 秦娜 YOU Man;WEI Liwei;CHAI Shuang;ZHENG Xuxia;MENG Lu;ZHANG Hong;HE Guanghong;QIN Na(Luoyang Orthopedic-Traumatological Hospital of Henan Province(Henan Provincial Orthopedic Hospital),Luoyang 471000,China)

机构地区：[1]河南省洛阳正骨医院(河南省骨科医院),河南洛阳471000

出　　处：《中国骨质疏松杂志》2024年第5期655-661,共7页Chinese Journal of Osteoporosis

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：目的探讨骨松益骨方(GSYG)对去卵巢(OVX)大鼠骨质疏松的作用以及对Beclin 1/Bcl-2介导的骨细胞凋亡与自噬的影响。方法将大鼠随机分成假手术组(Sham)、模型组(Model)、GSYG低、中、高剂量组[1.25、2.5、5 g/(kg·d)]。通过ELISA检测大鼠血清中P1NP和β-CTX的水平;Micro-CT测定股骨骨密度以及微结构的变化;HE染色检查骨组织的形态学变化;TUNEL染色观察骨细胞凋亡;免疫组化观察骨组织中LC3的变化;Western blot法检测大鼠胫骨近端Bcl2、Bax、cleaved caspase-3、cleaved PARP、LC3、Beclin1的表达变化;免疫共沉淀法检测Beclin1和Bcl-2的相互作用。结果与模型组相比,GSYG中、高剂量组的P1NP和β-CTX水平显著降低(P<0.01);GSYG各剂量组骨的微结构明显改善(P<0.01);骨小梁明显增多;骨细胞凋亡明显减少(P<0.01);Bcl-2的表达显著升高(P<0.01),Bax表达显著降低(P<0.05或P<0.01),GSYG高剂量组中的cleaved caspase-3、cleaved PARP的蛋白表达显著降低(P<0.01),GSYG中、高剂量组的LC3-Ⅱ/Ⅰ的比值与Beclin1蛋白表达显著升高(P<0.01);免疫组化染色显示,与模型组相比GSYG各剂量组LC3蛋白显著增加(P<0.05或P<0.01);免疫共沉淀的结果提示,模型组Beclin1和Bcl-2相互作用较强,而GSYG高剂量组中两者结合均减少。结论GSYG对OVX大鼠的骨质疏松发展有抑制作用,其机制与其调控Beclin 1/Bcl-2介导的凋亡与自噬有关。Objective To investigate the effect of GSYG on osteoporosis and the apoptosis and autophagy of the bone cells in ovariectomized rats mediated by Beclin 1/Bcl-2.Methods Rats were divided into five groups randomly,including the sham group,the model group,the GSYG low-dose,medium-dose,and high-dose groups[1.25,2.5,and 5 g/(kg·d)].The levels of P1NP andβ-CTX in serum were determined with ELISA.The changes of bone mineral density and microstructure of the femur were determined with micro-CT.The morphological changes of the rat femurs were observed with HE staining.The apoptosis of osteocytes was observed with TUNEL staining.The changes of LC3 in the bone were observed with immunohistochemical staining.The levels of Bcl2,Bax,cleaved caspase-3,cleaved PARP,LC3,and Beclin1 in the proximal tibia of rats were detected with Western blotting.Finally,the action between Beclin1 acted with Bcl-2 was detected with co-immunoprecipitation.Results Compared to those in the model group,the levels of P1NP andβ-CTX in GSYG medium and high dose groups decreased significantly(P<0.01),and the microstructure of bone the GSYG-treated groups improved significantly(P<0.01).In addition,the number of bone trabeculae increased significantly.Furthermore,osteocyte apoptosis decreased markedly(P<0.01).The expression of Bcl-2 in GSYG groups were markedly up-regulated(P<0.01),while Bax expression was significantly down-regulated(P<0.05 or P<0.01).Besides,the cleaved caspase-3 and cleaved PARP expression were markedly down-regulated in the GSYG high-dose group(P<0.01),while LC3-Ⅱ/Ⅰand Beclin1 expression in medium-dose and high-dose GSYG groups increased significantly(P<0.01).Immunohistochemical staining result showed that LC3 protein increased significantly in response to the GSYG treatment(P<0.05 or P<0.01).The result of co-immunoprecipitation showed that Beclin1 and Bcl-2 had stronger interaction in the model group than that in sham group,while the combination of Beclin1 and Bcl-2 decreased in the GSYG high-dose group.Conclusion GSYG inhibits

关 键 词：骨松益骨方 Beclin 1 BCL-2 凋亡 自噬 

分 类 号：R285.5[医药卫生—中药学]