Smith Marie, Wilson Richard, O'Brien Sally, Tufarelli Cristina, Anderson Susan I, O'Sullivan Saoirse Elizabeth
Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.
PLoS One. 2015 Sep 28;10(9):e0136546. doi: 10.1371/journal.pone.0136546. eCollection 2015.
The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.
内源性大麻素系统在骨骼中表达，尽管其在调节骨骼生长中的作用存在争议。许多研究已经直接研究了内源性大麻素对破骨细胞功能的影响，但很少有研究探讨它们在人类成骨细胞功能中的作用，而这正是本研究的目的。从接种开始，用浓度递增的花生四烯乙醇胺或2-花生四烯酸甘油酯处理人类成骨细胞1至21天。测量细胞增殖（DNA含量）和分化（碱性磷酸酶（ALP）、胶原蛋白和骨钙素分泌以及钙沉积）。花生四烯乙醇胺和2-花生四烯酸甘油酯在4天后显著降低成骨细胞增殖，并伴有ALP浓度依赖性增加。抑制内源性大麻素降解酶以增加内源性大麻素水平导致ALP产生类似增加。2-花生四烯酸甘油酯也降低了骨钙素分泌。用2-花生四烯酸甘油酯进行长期（21天）处理后，胶原蛋白含量降低，但钙沉积没有变化。花生四烯乙醇胺不影响胶原蛋白或骨钙素，但减少了钙沉积。花生四烯乙醇胺增加了磷酸化CREB、ERK 1/2和JNK的水平，而2-花生四烯酸甘油酯增加了磷酸化CREB和Akt的水平。RT-PCR显示在人类成骨细胞中CB2和TRPV1表达，但CB1不表达。花生四烯乙醇胺诱导的人类成骨细胞分化变化不依赖于CB1和CB2，TRPV1拮抗剂可部分降低这种变化，ERK 1/2和JNK的抑制也可降低这种变化。我们的结果表明，花生四烯乙醇胺和2-花生四烯酸甘油酯明显参与调节人类成骨细胞的活性，花生四烯乙醇胺增加早期细胞分化，2-花生四烯酸甘油酯增加早期但降低晚期成骨细胞特异性分化标志物。
