中药新药与临床药理2026，Vol.37Issue(3)：395-403,9.DOI:10.19378/j.issn.1003-9783.2026.03.002
补肾化痰方调控mTOR/NLRP3通路介导自噬促进小鼠胚胎成骨细胞骨形成
Bushen Huatan Formula Promotes Bone Formation in Mouse Embryonic Osteoblasts via Regulation of the mTOR/NLRP3 Pathway-Mediated Autophagy
摘要
Abstract
Objective To investigate the mechanism by which Bushen Huatan Formula induces autophagy to promote bone formation in mouse embryonic osteoblasts(MC3T3-E1),based on the mammalian target of rapamycin(mTOR)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)pathway.Methods MC3T3-E1 cells were treated with different concentrations(0,0.01,0.05,0.1,0.5,1,2 mg·mL-1)of Bushen Huatan Formula for 12,24,48,and 72 hours.Cell proliferation was assessed using the CCK-8 assay to determine the optimal intervention concentration and time.MC3T3-E1 cells in the logarithmic growth phase were randomly divided into a blank control group,a Bushen Huatan Formulagroup(0.1 mg·mL-1),a metformin group(0.5 mmol·L-1),and a rapamycin group(100 nmmol·L-1),each treated for 48 hours.Osteogenic activity was evaluated using alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining.Western Blot was used to detect the protein expression levels of Runt-related transcription factor 2(Runx2),interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-alpha(TNF-α),mTOR,p-mTOR,and NLRP3.RT-qPCR was used to measure the mRNA expression levels of TNF-α,IL-6,and IL-1β.The number of autophagosomes in cells was observed by transmission electron microscopy.Autophagic flux was assessed by transient transfection of an LC3 plasmid using LipofectamineTM 2000.Results Compared with the blank control group,the Bushen Huatan Formula group,metformin group,and rapamycin group showed significantly increased ALP and ARS staining positive areas(P＜0.01),significantly upregulated Runx2 protein expression(P＜0.01),significantly downregulated protein and mRNA expression of TNF-α,IL-6,and IL-1β(P＜0.01),a significantly increased number of autophagosomes and autolysosomes in cells(P＜0.05,P＜0.01),and significantly decreased protein expression levels of p-mTOR/mTOR and NLRP3(P＜0.01).No statistically significant differences were observed in the aforementioned indicators among the Bushen Huatan Formula group,metformin group,and rapamycin group(P＞0.05).Conclusion Bushen Huatan Formula can enhance autophagic activity,downregulate the expression of inflammatory factors,and promote bone formation in MC3T3-E1 cells by inhibiting the mTOR/NLRP3 pathway.关键词
补肾化痰方/小鼠胚胎成骨细胞/骨形成/自噬/炎症因子/mTOR/NLRP3 通路Key words
Bushen Huatan Formula/mouse embryonic osteoblasts/bone formation/autophagy/inflammatory factors/mTOR/NLRP3 pathway分类
医药卫生引用本文复制引用
熊梦欣,朱伟,陈颖庆,张昊雪,蒋泽宇,向楠..补肾化痰方调控mTOR/NLRP3通路介导自噬促进小鼠胚胎成骨细胞骨形成[J].中药新药与临床药理,2026,37(3):395-403,9.基金项目
国家自然科学基金项目(82305350,82074416) （82305350,82074416）
湖北省自然科学基金项目(2022CFD153,2024AFB343) （2022CFD153,2024AFB343）
湖北省中医药管理局中医药青年人才项目(ZY2023Q031). （ZY2023Q031）
