N-羧甲基離胺酸對於造骨細胞與脂肪細胞分化之作用

Abstract

羧甲基離胺酸(CML)為最終糖化產物(AGEs)的主要成分，在糖尿病以及老化之 病人的血管以及組織中，常常發現有累積的現象而導致其他併發症，如:骨質疏 鬆症。先前研究發現AGEs 可調控骨髓間質幹細胞的分化作用，包括脂肪細胞 分化以及造骨細胞分化。但是至今其分子機制尚未了解。 因此本篇主要探討羧甲基離胺酸對於間質幹細胞分化成造骨細胞以及脂肪細 胞的影響。首先我們先利用MTT 試驗來選定不造成細胞死亡的使用劑量: 25μ g/ml，發現在此劑量下能夠有效抑制造骨細胞分化主要是透過促進RAGE、 iNOS、,NO 產生及表現或是抑制PP38、ALP、BMP-2 及OCN 來抑制骨生成。胺 基胍(AG)以及RAGE 接受器之抗體不但能有效回復抑制骨生成也能回復PP38 等 分化訊號表現。而SNP 加入後則可以抑制PP38 以及ALP 活性，證明NO 在此扮 演重要的角色。接下來我們發現羧甲基離胺酸在此無法增加NO 但是卻能夠造成 細胞內ROS 大量產生，並且能夠有效抑制脂肪細胞分化主要是透過抑制ERK 磷 酸化以及PPAR-γ表現量的訊息路徑。加入NAC 以及RAGE 接受器抗體後則可 以明顯回復抑制情形。表示羧甲基離胺酸的作用是透過ROS 的產生並且扮演著 重要的角色，進而影響了細胞分化的相關蛋白質訊號表現。 在動物實驗方面，我們發現STZ 誘導糖尿病之小鼠組別的血液樣本中AGEs 有明顯增高，而頭部以及脛骨之骨密度以及骨質量皆有下降之趨勢。而經由餵食 AG 飲水(20-25mg/day)的組別不但骨密度及骨質量皆有輕微緩解之外，血液中 AGEs 含量以及小鼠體重皆有些微回復。 綜合以上結果，羧甲基離胺酸對於小鼠間質幹細胞之脂肪細胞分化以及造骨細 胞分化作用上扮演著重要調節之角色。未來的研究，對於間質幹細胞的分化調控 的詳細機制，更值得進一步釐清。

N-carboxymethyllysine (CML), the major component of advanced glycation end products (AGEs), accumulates in vessels and tissues of aging and diabetic patients. In the recent studies, AGEs can regulate mesenchymal stem cell differentiation, But the molecular mechanisms remain unknown. The present studies, CML were used to investigate the effect on osteogenesis and adipogenesis of bone marrow mesenchymal stem cells. The testing doses used in this study were without cytotoxicity. We found that CML( 25μg/ml ) inhibited bone formation via increasing the expression of RAGE(AGEs receptor) and iNOS, as well as NO production, but decreasing the expression of pP38, ALP, BMP-2, and OCN. Aminoguanidine (AG) and RAGE antibody can reverse not only the inhibited effect of CML in osteogenesis but also the signaling pathway. SNP (NO donor) treatment also can inhibit the P38 and ALP activity, suggesting that Nitrite oxide (NO) plays an important role in osteogenesis. Moreover, we also found that 25μg/ml CML can inhibit adipogenesis by downregulating the ERK phosphorylation and PPAR-γexpression. The reactive oxygen species (ROS) was generated after CML treatment. Then, NAC and RAGE antibody can reverse the expression of pERK and PPAR-γand adipogenesis, suggesting that ROS plays an important role in adipogenesis. In vivo study, we examined the elevating of AGEs in serum of STZ-induced diabetic mice. The BMD and BMC of head and tibia are decreased, but AG could partially regulate BMD of STZ-induced diabetic mice. The AGEs amount and the body weight also could be regulated by Aminoguanidine(20-25mg/day). In conclusion, we thought that CML plays an important role in osteogenesis and adipogenesis of MSCs. In the future, the detail mechanism of the CML effect on MSCs differentiation and the relation with osteoporosis are still valuably investigated.