二仙汤含药血清外泌体经FN1促进成骨细胞骨形成

Erxian Decoction(二仙汤)-Containing Serum Exosomes Promoting Bone Formation of Osteoblasts via FN1

目的:探讨二仙汤含药血清外泌体蛋白的变化及其对成骨细胞骨形成的作用。方法:对二仙汤含药血清外泌体进行提取、鉴定。体外培养MC3T3-E1成骨细胞,分为正常对照组、空白血清外泌体2.5、5μg/mL组、二仙汤含药血清外泌体2.5、5、10μg/mL组。CCK8法检测成骨细胞增殖情况;碱性磷酸酶(ALP)染色法观察成骨细胞ALP;茜素红染色观察成骨细胞矿化结节;Western blot法检测细胞中骨形态发生蛋白2(BMP-2)蛋白表达。运用蛋白质组学技术对血清外泌体进行差异性蛋白分析。采用纤连蛋白(FN1)联合空白血清外泌体(3 nmol/L纤连蛋白+5μg/mL、12 nmol/L纤连蛋白+5μg/mL)处理成骨细胞,通过试剂盒检测成骨细胞ALP活力;Western blot法检测FN1对成骨细胞中BMP-2蛋白表达的影响。结果:与空白血清外泌体2.5μg/mL组相比,二仙汤含药血清外泌体组成骨细胞的增殖、ALP染色程度和茜素红染色程度明显升高(P<0.05或P<0.01),BMP-2的蛋白表达明显上调(P<0.05)。从血清外泌体中共鉴定到256个蛋白,其中96个蛋白属于ExoCarta外泌体蛋白质数据库。差异统计分析显示有29个蛋白表达有明显变化(P<0.05)。针对显著差异蛋白进行生物信息学分析,与差异蛋白质相关的分子功能、生物过程和信号通路主要涉及氧化还原、细胞外泌体、PI3K/AKT信号通路、补体和凝血级联反应等过程。显著差异性蛋白主要包括FN1、白蛋白(ALB)、血清对氧磷酶/芳基酯酶1(PON1)和簇蛋白(CLU)等。与空白血清外泌体5μg/mL组相比,纤连蛋白联合空白血清外泌体组(3 nmol/L纤连蛋白+5μg/mL、12 nmol/L纤连蛋白+5μg/mL)成骨细胞ALP活力显著升高(P<0.01),BMP-2的蛋白表达明显上调(P<0.05)。结论:二仙汤含药血清外泌体促进成骨细胞骨形成与血清外泌体中的蛋白变化有关,FN1在其中发挥重要作用。

Objective:To investigate the changes of protein in Erxian Decoction(二仙汤)-containing serum exosomes and the effect on the bone formation of osteoblasts.Methods:Erxian Decoction-containing serum exosomes were extracted and identified.MC3T3-El osteoblasts were cultured in vitro and then divided into control group,normal serum exosome groups(2.5 and 5μg/mL),and Erxian Decoction-containing serum exosome groups(2.5,5,and 10μg/mL).CCK8 method was used to detect osteoblast proliferation.Alkaline phosphatase(ALP)staining was used to observe the ALP of osteoblasts.Alizarin red staining was used to observe mineralized nodules of osteoblasts.Western blot was utilized to detect the expression of bone morphogenetic protein 2(BMP-2)in cells.Differential protein analysis of serum exosomes was performed by using proteomics techniques.Osteoblasts were treated with fibronectin(FN1)combined with normal serum exosomes(3 nmol/L FN1+5μg/mL and 12 nmol/L FN1+5μg/mL),and the ALP activity of osteoblasts was measured by the kit.The effect of FN1 on BMP-2 expression in osteoblasts was detected by Western blot.Results:Compared with those in the normal serum exosome group of 2.5μg/mL,the proliferation,degree of ALP staining,and degree of alizarin red staining were significantly increased in the Erxian Decoctioncontaining serum exosome group(P<0.05 or P<0.01),while the expression of BMP-2 was significantly upregulated(P<0.05).A total of 256 proteins were identified from serum exosomes,96 of which belonged to the ExoCarta exosome protein database.Statistical analysis of differences showed that there were 29 proteins with significant changes in expression(P<0.05).Bioinformatics analysis was performed on significantly differential proteins.The molecular functions,biological processes,and signaling pathways associated with the differential proteins mainly involved the oxidation-reduction process,cellular exosomes,PI3K-Akt signaling pathway,and complement and coagulation cascade reactions.The significantly different proteins included mainly FN1,PON1,albumin(ALB),and clusterin(CLU).Compared with the normal serum exosome group of 5μg/mL,the ALP activity of osteoblasts was significantly upregulated in fibronectin combined with the normal serum exosome groups(3 nmol/L FN1+5μg/mL and 12 nmol/L FN1+5μg/mL)(P<0.01),and the expression of BMP-2 was increased(P<0.05).Conclusion:Erxian Decoction-containing serum exosomes promote the bone formation of osteoblasts,which is associated with protein changes in serum exosomes,in which FN1 plays an important role.