自噬在葛根素促成骨细胞分化调节中的作用

Autophagy participates in puerarin-induced osteoblast differentiation

目的探讨自噬在葛根素调节h FOB1.19人成骨细胞分化过程中的作用。方法将成骨细胞分为A组(成骨分化诱导组)、B组(A组+10^(-8)mol/L葛根素3 h)和C组[B组+5 mmol/L自噬抑制剂3-甲基嘌呤(3-MA)1 h]。采用碱性磷酸酶(ALP)和茜素红染色检测成骨细胞的分化及钙化;透射电镜检测成骨细胞内自噬小体的改变情况;免疫荧光染色检测自噬微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)在细胞内的分布情况;Western blotting检测成骨细胞内成骨分化和自噬相关蛋白的表达情况。结果ALP和茜素红染色显示,葛根素(10^(-8) mol/L)能够明显促进成骨细胞的分化,加入3-MA后葛根素的促成骨细胞分化作用受到抑制;葛根素促进成骨细胞自噬小体增加,3-MA抑制自噬小体增加;免疫荧光显示葛根素促进LC3-Ⅱ在细胞核表面的聚集,加入3-MA后,这种现象被抑制;葛根素促进成骨分化蛋白矮小相关转录因子2(RUNX 2)、骨保护素(OPG)和自噬相关蛋白LC3-Ⅱ、BECLIN 1的表达,3-MA抑制LC3-Ⅱ和BECLIN 1的表达。结论葛根素通过自噬调节成骨细胞分化。

Objective To explore the role of autophagy in regulating h FOB1.19 human osteoblast differentiation by puerarin.Methods These experiments incorporated three different conditions:A)basic osteogenic differentiation-inducing conditions,B)conditions in A+10^(-8) mol/L puerarin for 3 h,and C)conditions in B+5 mmol/L autophagy inhibitor 3-methyladenine(3-MA)for 1 h.Alkaline phosphatase(ALP)and Alizarin red staining were used to detect osteoblast differentiation and calcification.Transmission electron microscopy was used to detect changes in autophagosomes in osteoblasts.Immunofluorescence was used to detect subcellular distribution of the key autophagy regulatory protein microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ)in osteoblasts.Western blotting was used to detect expression of osteogenic differentiation and autophagy related proteins in osteoblasts.Results ALP and alizarin red staining showed that puerarin(10^(-8) mol/L)promoted osteoblast differentiation,and 3-MA inhibited osteoblast differentiation.Puerarin also promoted an increase in autophagosomes in osteoblasts,whereas 3-MA inhibited the puerarin-induced increase in autophagosomes.Immunofluorescence showed that puerarin promoted LC3-Ⅱaggregation on the nuclear surface,which was inhibited by addition of 3-MA.Puerarin promoted RUNX 2,OPG,LC3-Ⅱand BECLIN 1 expression,while 3-MA inhibited expression of these proteins.Conclusion Puerarin regulates osteoblast differentiation through autophagy.