积雪草苷对脂多糖诱导的成骨细胞增殖和分化的影响

Eficacy of Asiaticoside on Proliferation and Differentiation of Osteoblasts Induced by Lipopolysaccharide

目的:探讨积雪草苷(AS)对脂多糖(LPS)诱导的MC3T3-E1成骨细胞增殖、分化的影响。方法:先用2 mg/L脂多糖处理MC3T3-E1成骨细胞,再用5,10,20,40,80μmoL/L积雪草苷处理脂多糖诱导的MC3T3-E1成骨细胞,检测细胞活性,筛选最佳药物浓度;将MC3T3-E1成骨细胞分为对照组(Control组)、脂多糖组(LPS组)、积雪草苷低、中、高浓度组(AS-L、AS-M、AS-H组)、积雪草苷高浓度+CXCR4抑制剂AMD3100组(AS-H+AMD3100组);Hoechst 33258染色观察细胞核形态;碱性磷酸酶(ALP)染色及碱性磷酸酶活性检测细胞成骨能力;茜素红染色法检测钙结节形成情况;免疫印迹检测分化相关标志物Ⅰ型胶原(COL-Ⅰ)、骨桥蛋白(OPN)、骨钙素(OCN)及SDF-1、CXCR4表达情况。结果:筛选出10,20,40μmoL/L积雪草苷为AS-L、AS-M、AS-H组药物浓度进行后续实验;与对照组比较,脂多糖组MC3T3-E1成骨细胞细胞核固缩深染,呈明显细胞凋亡形态,碱性磷酸酶活性、钙结节形成、COL-Ⅰ、OPN、OCN、SDF-1、CXCR4蛋白表达显著下降(P<0.05);与脂多糖组比较,AS-L、AS-M、AS-H组MC3T3-E1成骨细胞细胞核固缩深染逐渐减轻,凋亡形态细胞逐渐减少,碱性磷酸酶活性、钙结节形成、COL-Ⅰ、OPN、OCN、SDF-1、CXCR4蛋白表达依次显著上升(P<0.05);与AS-H组比较,AS-H+AMD3100组MC3T3-E1成骨细胞细胞核固缩深染加重,凋亡形态细胞明显增多,碱性磷酸酶活性、钙结节形成、COL-Ⅰ、OPN、OCN、SDF-1、CXCR4蛋白表达显著下降(P<0.05)。结论:积雪草苷可通过激活SDF-1/CXCR4信号通路促进MC3T3-E1成骨细胞的增殖与分化。

Objective:To investigate the efficacy of asiaticoside(AS)on proliferation and differentiation of MC3T3-El osteoblasts induced by lipopolysaccharide(LPS).Methods:MC3T3-El osteoblasts were first treated with 2 mg/L LPS,and then LPS induced MC3T3-E1 osteoblasts were treated with 5,10,20,40,and 80μmol/L asiaticoside.Cell activity was measured and the optimal drug concentration was selected.MC3T3-El osteoblasts were separated into control group,li-popolysaccharide group(LPS group),low,medium,and high concentration asiaticoside groups(AS-L,AS-M,AS-H group),and high concentration asiaticoside+CXCR4 inhibitor AMD3100 group(AS-H+AMD3100 group).Hoechst 33258 staining was applied to observe the morphology of the nucleus;alkaline phosphatase(ALP)staining and ALP activ-ity were applied to detect the osteogenic ability of cells;alizarin red staining method was applied to detect the formation of calcium nodules;immunoblotting was applied to detect the expression of differentiation related biomarkers such as collagen type I(COL-I),osteopontin(OPN),osteocalcin(OCN),and SDF-1 and CXCR4.Results:10,20,and 40μmol/L AS were selected as drug concentrations for AS-L,AS-M,and AS-H groups in subsequent experiments;compared with the control group,the nucleus of MC3T3-El osteoblasts in the LPS group showed pyknosis and deep staining,showing a obvi-ous apoptotic morphology,the ALP activity,calcium nodule formation,the expression of COL-I,OPN,OCN,SDF-1,CX-CR4 proteins were greatly decreased(P<0.05).The nuclear pyknosis and deep staining of MC3T3-El osteoblasts in AS-L,AS-M,and AS-H groups gradually decreased compared with the LPS group,the number of apoptotic cells gradually decreased,the ALP activity,calcium nodule formation,the expression of COL-I,OPN,OCN,SDF-1,CXCR4 proteins were obviously increased in sequence(P<0.05).The nuclei of MC3T3-El osteoblasts in the AS-H+AMD3100 group showed increased pyknosis and deep staining compared with the AS-H group,the apoptotic morphological cells obviously increased,the ALP activity,calcium nodule formation,the expression of COL-I,OPN,OCN,SDF-1,and CXCR4 proteins were greatly reduced(P<0.05).Conclusion:Asiaticoside can promote the proliferation and differentiation of MC3T3-E1 osteoblasts by activating SDF-1/CXCR4 signaling pathway.