miR-140-5p/BCL2L1在HFOB1.19凋亡、自噬中的作用及补肾健脾活血方的影响  

Role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 and effect of Bushen Jianpi Huoxue Decoction

作　　者：陈桐莹 付赛 李小韵 刘树华 杨贻富 杨东升 曾韵杰 李扬博 罗丹 黄宏兴 万雷 CHEN Tong-ying;FU Sai;LI Xiao-yun;LIU Shu-hua;YANG Yi-fu;YANG Dong-sheng;ZENG Yun-jie;LI Yang-bo;LUO Dan;HUANG Hong-xing;WAN Lei(Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Laboratory of Orthopaedics and Traumatology,Lingnan Medical Research Centre,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;the Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378,China;the Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]广州中医药大学,广东广州510006 [2]广州中医药大学岭南医学研究中心中医骨伤科学实验室,广东广州510006 [3]广州中医药大学第三附属医院,广东广州510378 [4]广州中医药大学第二附属医院,广东广州510006

出　　处：《中国中药杂志》2025年第3期583-589,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82274551,82174395);广州中医药大学“双一流”与高水平大学学科协同创新团队重点项目(2021XK21);广州中医药大学第三附属医院“瞪羚计划”普通项目(KY6032022006);广东省中医药局科研项目(20221178)。

摘　　要：骨质疏松症(osteoporosis,OP)是一种骨重建与骨形成失衡的老年性骨病,补肾健脾活血方针对肾虚脾虚血瘀的病机,以补肾健脾活血为法,对治疗OP有显著效果,微核糖核酸(microRNA,miRNA)、B细胞淋巴瘤-2样蛋白1(B-cell lymphoma-2-like protein 1,BCL2L1)与骨细胞代谢密切相关,故该研究采用双荧光素酶法、聚合酶链反应(polymerase chain reaction,PCR)法检测miR-140-5p与BCL2L1的结合情况;沉默或过表达miR-140-5p后,流式细胞术检测、蛋白免疫印迹(Western blot)观察人胎成骨细胞(human fetal osteoblast cell line 1.19,HFOB1.19)凋亡与自噬的发生与成骨功能;大鼠灌胃补肾健脾活血方后制备含药血清,采用不同浓度的补肾健脾活血方含药血清分别干预含或不含miR-140-5p micmi的HFOB1.19,观察各组细胞的成骨蛋白表达,研究miR-140-5p/BCL2L1在HFOB1.19凋亡、自噬中的作用,并观察补肾健脾活血方对其的影响。双荧光素酶检测显示miR-140-5p与BCL2L1结合;流式细胞术检测、Western blot检测结果显示miR-140-5p促进HFOB1.19细胞凋亡且miR-140-5p抑制HFOB1.19细胞自噬;高、中、低剂量补肾健脾活血方含药血清干预HFOB1.19后,与miR-140-5p NC组相比,miR-140-5p micmic组的成骨蛋白骨钙蛋白(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、转化生长因子β1(transforming growth factor beta 1,TGF-β1)表达下降,而高剂量干预下的micmic组骨形成蛋白2(bone morphogenetic protein 2,BMP2)表达无明显差别。故miR-140-5p/BCL2L1可促进HFOB1.19凋亡与抑制自噬的发生,补肾健脾活血方可通过BMP2影响miR-140-5p的抑成骨作用。Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jia

关 键 词：骨质疏松症 补肾健脾活血方 miR-140-5p BCL2L1 凋亡 自噬 

分 类 号：R285.5[医药卫生—中药学]