【摘要】 目的 探讨紫堇醇灵碱是否通过调节巨噬细胞极化改善骨髓间充质干细胞（bone marrow mesenchyml stem cell,BMSC）迁移、成骨分化。方法 CCK-8选取紫堇醇灵碱最佳实验浓度，采用脂多糖（lipopolysaccharide,LPS）诱导RAW264.7向M1型极化，设置对照组（0μmol/L）和低、中、高浓度实验组（1、4、8μmol/L），加入相应紫堇醇灵碱，通过流式、免疫荧光、实时荧光定量PCR及Western blot测定炎症相关指标的表达，活性氧（reactive oxygen,ROS）实验检测ROS含量变化。设置划痕-对照组、划痕-Cor组、划痕-LPS组、划痕-LPS+Cor组进行细胞划痕实验，观察各组细胞迁移情况；设置成骨-对照组、成骨-Cor组、成骨-LPS组、成骨-LPS+Cor组进行成骨分化检测，观察碱性磷酸酶（alkaline phosphatase,ALP）含量。结果 流式细胞检测和免疫荧光检测显示，对照组、低浓度组、中浓度组、高浓度组CD86表达逐渐降低，CD206表达逐渐升高。qRT-PCR检测和Western blot检测显示，对照组、低浓度组、中浓度组、高浓度组M1相关基因肿瘤坏死因子α、白细胞介素1表达逐渐下调，M2相关基因精氨酸酶1、白细胞介素10表达逐渐上调；且与对照组比较，高浓度组M1下调和M2上调变化最显著（P<0.01）。ROS实验显示，低浓度组、中浓度组、高浓度组细胞内DCFH-DA荧光强度逐渐下降，高浓度组下降最显著（P<0.001）。细胞划痕实验显示，划痕-LPS+Cor组、划痕-LPS组、划痕-Cor组、划痕-对照组细胞迁移率依次降低。ALP染色及定量分析显示，成骨培养14 d时，成骨-LPS+Cor组ALP活性明显高于成骨-LPS组、成骨-Cor组、成骨-对照组（P<0.001）。结论 高浓度（8μmol/L）的紫堇醇灵碱能有效抑制RAW264.7向M1型转化，促进RAW264.7向M2型转化，并且其在介导RAW264.7极化促进BMSC迁移、成骨分化效果显著，为进一步研究紫堇醇灵碱的抗炎机制及治疗骨损伤相关疾病提供了一个新的思路。更多还原

【Abstract】 Objective To investigate whether corynoline enhances the migration and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) through the regulation of macrophage polarization.Methods The CCK-8 assay was employed to determine the optimal experimental concentration of corynoline.RAW264.7 cells were induced with lipopolysaccharide(LPS) to polarize into the M1 phenotype.The study included a control group(0 μmol/L) and three experimental groups treated with low,medium,and high concentrations of corydaline(1,4,and 8 μmol/L,respectively).The expression of inflammation-related indicators was determined by flow cytometry,immunofluorescence,qRT-PCR and Western blot.The reactive oxygen species(ROS) experiment was used to detect the changes in ROS content.Cell scratch experiments were conducted in the scratch-control group,scratch-Cor group,scratch-LPS group,and scratch-LPS+Cor group to observe the cell migration in each group.Osteogenic differentiation was detected in the osteogenesis-control group,osteogenesis-Cor group,osteogenesis-LPS group and osteogenesis-LPS+Cor group to observe the content of alkaline phosphatase(ALP).Results Flow cytometry and immunofluorescence detection showed that the expression of CD86 gradually decreased in the control group,low-concentration group,medium-concentration group and high-concentration group,while the expression of CD206 gradually increased.qRT-PCR detection and Western blot detection showed that the expressions of MI-related genes tumor necrosis factor-a and interleukin-1 were gradually down-regulated in the control group,low-concentration group,medium-concentration group and high-concentration group,while the expressions of M2-related genes arginase 1 and interleukin-10 were gradually up-regulated.Moreover,compared with the control group,the changes of M1 down-regulation and M2 up-regulation in the high-concentration group were the most significant(P<0.01).The ROS experiment showed that the fluorescence intensity of DCFH-DA in cells gradually decreased in the low-concentration group,medium-concentration group and high-concentration group,with the most significant decrease in the high-concentration group(P<0.001).The cell scratch assay showed that the cell migration rates of the scratch-LPS+Cor group,the scratch-LPS group,the scratch-Cor group,and the scratch-control group decreased successively.ALP staining and quantitative analysis showed that at 14 days of osteogenic culture,the ALP activity in the osteogenesis-LPS+Cor group was significantly higher than that in the osteogenesis-LPS group,the osteogenesis-Cor group,and the osteogenesis-control group(P<0.001).Conclusion A high concentration(8 μmol/L) of corynoline effectively inhibits the polarization of RAW264.7 cells toward the M1 phenotype,promotes their differentiation into the M2 phenotype,and significantly enhances the mediation of RAW264.7 cell polarization to facilitate BMSC migration and osteogenic differentiation.These findings provide a novel insight into the anti-inflammatory mechanism of corynoline and its potential therapeutic applic ation in bone inj ury-related diseases.更多还原