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尹煜辉, 李琰, 齐保玉, 刘平, 朱立国, 刘宁, 魏戌.基于有氧糖酵解探究补骨生髓方含药血清通过介导HIF-1α促进MC3T3-E1细胞成骨分化的作用[J].湖南中医药大学学报英文版,2025,45(5):818-824.[Click to copy
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| 基于有氧糖酵解探究补骨生髓方含药血清通过介导HIF-1α促进MC3T3-E1细胞成骨分化的作用 |
| 尹煜辉,李琰,齐保玉,刘平,朱立国,刘宁,魏戌 |
| (中国中医科学院望京医院, 北京 100102;北京中医药大学, 北京 100105) |
| 摘要: |
| 目的 探讨补骨生髓方（BGSSD）含药血清通过缺氧诱导因子-1α（HIF-1α）介导MC3T3-E1细胞有氧糖酵解过程对成骨分化的影响。方法 选取18只SD大鼠随机分为含药血清组和空白血清组，各9只。含药血清组按生药量5.87 g/（kg·d）给予BGSSD药液灌胃，空白血清组给予20 mL/（kg·d）蒸馏水灌胃，连续灌胃9次后收集腹主动脉血液，获取BGSSD含药血清和空白血清。将MC3T3-E1细胞分为空白血清组、含药血清组和有氧糖酵解抑制剂组，并将HIF-1α慢病毒转染稳定的MC3T3-E1细胞作为SiRNA HIF-1α组。空白血清组加入含20％空白血清的α-MEM培养基，含药血清组和SiRNA HIF-1α组均加入含20％ BGSSD含药血清的α-MEM培养基，有氧糖酵解抑制剂组加入含20％ BGSSD含药血清和8 mmol/L抑制剂2-脱氧葡萄糖的α-MEM培养基。采用碱性磷酸酶（ALP）染色法观察4组培养7 d后的黑色沉淀物情况；ELISA检测培养48 h后细胞上清液中ALP和丙酮酸（PA）、乳酸（LA）的含量；qRT-PCR和Western blot检测培养48 h后细胞HIF-1α、Runt相关转录因子2（RUNX2）、丙酮酸脱氢酶激酶1（PDK1） mRNA相对表达水平和蛋白表达量。结果 与空白血清组比较，含药血清组的黑色沉淀物明显增加、颜色加深，ALP、LA、PA含量均升高（P＜0.05），PDK1、RUNX2、HIF-1α mRNA相对表达水平和蛋白表达量均升高（P＜0.05）。与含药血清组比较，SiRNA HIF-1α组和有氧糖酵解抑制剂组的黑色沉淀物减少、颜色变浅，ALP、LA及PA含量均降低（P＜0.05），PDK1、RUNX2的mRNA相对表达水平和蛋白表达量均降低（P＜0.05）；SiRNA HIF-1α组HIF-1α的mRNA相对表达水平和蛋白表达量降低（P＜0.05）。结论 BGSSD可能通过HIF-1α介导有氧糖酵解促进MC3T3-E1细胞成骨分化，其作用机制可能与调控HIF-1α/PDK1通路相关。 |
| 关键词: 骨质疏松症 补骨生髓方 有氧糖酵解 MC3T3-E1细胞 骨形成 |
| DOI：10.3969/j.issn.1674-070X.2025.05.006 |
| Received:December 02, 2024 |
| 基金项目:国家自然科学基金面上项目（82174416）；中国中医科学院基本科研业务费新入职青年科研人员培养专项课题（ZZ17-XRZ-059）；中国中医科学院望京医院自主选题专项课题（WJYY-ZZXT-2023-16）。 |
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| Promotion of osteogenic differentiation of MC3T3-E1 cells by serum containing Bugu Shengsui Decoction through mediating HIF-1α based on aerobic glycolysis |
| YIN Yuhui, LI Yan, QI Baoyu, LIU Ping, ZHU Liguo, LIU Ning, WEI Xu |
| (Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China;Beijing University of Chinese Medicine, Beijing 100105, China) |
| Abstract: |
| Objective To investigate the effects of the Bugu Shengsui Deocoction (BGSSF) containing serum on osteogenic differentiation of MC3T3-E1 cells by mediating aerobic glycolysis through hypoxia-inducible factor-1α (HIF-1α). Methods A total of 18 SD rats were randomly divided into a drug-containing serum group and a blank serum group, with nine rats in each group. The drug-containing serum group was administered BGSSD solution by gavage at a raw drug dose of 5.87 g/(kg·d), while the blank serum group was given distilled water by gavage at a volume of 20 mL/(kg·d). After nine consecutive gavages, blood was collected from the abdominal aorta to obtain serum containing BGSSD and blank serum. MC3T3-E1 cells were divided into a blank serum group, a drug-containing serum group, and an aerobic glycolysis inhibitor group. Additionally, MC3T3-E1 cells stably transfected with HIF-1α lentivirus were designated as the SiRNA HIF-1α group. The blank serum group was cultured in α-MEM medium containing 20% blank serum, while both the drug-containing serum group and the SiRNA HIF-1α group were cultured in α-MEM medium containing 20% serum containing BGSSD. The aerobic glycolysis inhibitor group was cultured in α-MEM medium containing 20% BGSSF containing serum and 8 mmol/L of the inhibitor 2-deoxyglucose. Alkaline phosphatase (ALP) staining was used to observe the black precipitates in the four groups after seven days of culture. ELISA was employed to determine the content of ALP, pyruvic acid (PA), and lactic acid (LA) in the cell supernatant after 48 hours of culture. qRT-PCR and Western blot were utilized to measure the relative mRNA expression levels and protein expression amounts of HIF-1α, Runt-related transcription factor 2 (RUNX2), and pyruvate dehydrogenase kinase 1 (PDK1) in the cells after 48 hours of culture. Results Compared with the blank serum group, the drug-containing serum group exhibited a notable increase in black precipitates with deeper coloration, along with elevated levels of ALP, LA, and PA (P<0.05). Additionally, the relative mRNA expression levels and protein expression amounts of PDK1, RUNX2, and HIF-1α all increased in the drug-containing serum group (P<0.05). Compared with the drug-containing serum group, the SiRNA HIF-1α group and the aerobic glycolysis inhibitor group showed reduced black precipitates with lighter coloration, as well as decreased levels of ALP, LA, and PA (P<0.05). The relative mRNA expression levels and protein expression amounts of PDK1 and RUNX2 also decreased in these two groups (P<0.05). The relative mRNA expression levels and protein expression amounts of HIF-1α reduced in the SiRNA HIF-1α group (P<0.05). Conclusion BGSSD may promote osteogenic differentiation of MC3T3-E1 cells by mediating aerobic glycolysis through HIF-1α, and its mechanism of action might be related to the regulation of the HIF-1α/PDK1 pathway. |
| Key words: osteoporosis Bugu Shengsui Decoction aerobic glycolysis MC3T3-E1 cells bone formation |
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