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OA
Bushen Huatan Formula Promotes Bone Formation in Mouse Embryonic Osteoblasts via Regulation of the mTOR/NLRP3 Pathway-Mediated Autophagy
目的 基于哺乳动物雷帕霉素蛋白(mTOR)/核苷酸结合寡聚化结构域样受体蛋白 3(NLRP3)通路探讨补肾化痰方诱导小鼠胚胎成骨细胞(MC3T3-E1)自噬促进骨形成的作用机制.方法 以不同浓度(0、0.01、0.05、0.1、0.5、1、2 mg·mL-1)补肾化痰方分别干预MC3T3-E1 细胞 12、24、48、72 h后,采用CCK-8 法检测细胞增殖情况,筛选补肾化痰方最佳干预浓度及时间.将对数生长期MC3T3-E1 细胞随机分为空白组、补肾化痰方组(0.1 mg·mL-1)、二甲双胍组(0.5 mmol·L-1)、雷帕霉素组(100 nmol·L-1),分别干预 48 h.采用碱性磷酸酶(ALP)染色法及茜素红(ARS)染色法检测MC3T3-E1 细胞成骨活性;Western Blot法检测细胞Runt相关转录因子 2(Runx2)、白细胞介素 6(IL-6)、白细胞介素 1β(IL-1β)、肿瘤坏死因子α(TNF-α)、mTOR、p-mTOR、NLRP3 蛋白表达水平;RT-qPCR法检测细胞TNF-α、IL-6、IL-1β mRNA表达水平;透射电镜观察细胞中自噬小体的数量;LipofectamineTM 2000 瞬时转染LC3 质粒检测细胞自噬流.结果 与空白组比较,补肾化痰方组、二甲双胍组、雷帕霉素组MC3T3-E1 细胞的ALP、ARS染色阳性表达面积均显著增加(P＜0.01),Runx2蛋白表达显著上调(P＜0.01),TNF-α、IL-6、IL-1β蛋白及mRNA表达均显著下调(P＜0.01),细胞中的自噬小体及自噬溶酶体数量均明显增多(P＜0.05,P＜0.01),p-mTOR/mTOR、NLRP3 蛋白表达水平均显著降低(P＜0.01).补肾化痰方组、二甲双胍组、雷帕霉素组之间比较,上述指标的差异无统计学意义(P＞0.05).结论 补肾化痰方能够通过抑制mTOR/NLRP3 通路增强MC3T3-E1 细胞的自噬活性,下调炎症因子表达,促进MC3T3-E1 细胞骨形成.
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.
熊梦欣;朱伟;陈颖庆;张昊雪;蒋泽宇;向楠
湖北省中医院妇产科,湖北 武汉 430061||中医肝肾研究及应用湖北省重点实验室,湖北 武汉 430061||湖北中医药大学附属医院,湖北 武汉 430061武汉体育学院,湖北 武汉 430079湖北中医药大学,湖北 武汉 430065湖北中医药大学,湖北 武汉 430065湖北中医药大学,湖北 武汉 430065湖北中医药大学,湖北 武汉 430065
医药卫生
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
Bushen Huatan Formulamouse embryonic osteoblastsbone formationautophagyinflammatory factorsmTOR/NLRP3 pathway
《中药新药与临床药理》 2026 (3)
395-403,9
国家自然科学基金项目(82305350,82074416)湖北省自然科学基金项目(2022CFD153,2024AFB343)湖北省中医药管理局中医药青年人才项目(ZY2023Q031).
评论