Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo
Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo
复制标题
DOI：
10.1016/j.joca.2009.07.001
复制
发表时间：
2009-12-01
影响因子：
7
通讯作者：
Wada, M.
中科院分区：
文献类型：
--
作者：
Nakatani, S.;Mano, H.;Wada, M.
目的：研究体内和体外条件下Pro-Hyp对软骨细胞的直接作用，以确定Pro-Hyp是胶原水解物中的生物活性多肽。方法：通过对C57BL/6J小鼠关节软骨切片的光镜观察，研究Pro-Hyp对关节软骨的影响。在这项研究中，饲喂了含有过量磷的饲料的小鼠被用作体内模型。该小鼠系表现为软骨细胞丢失，关节软骨厚度减少，软骨下骨异常。采用四甲基偶氮唑盐比色法、碱性磷酸酶染色、艾氏蓝和茜素红染色等方法，观察CH、Pro-Hyp、氨基酸等多肽对软骨细胞增殖、分化、糖胺多糖含量和矿化的影响。用半定量逆转录聚合酶链式反应(RT-PCR)检测ATDC5细胞中软骨形成相关基因的表达。结果：在体内，CH和Pro-Hyp可抑制磷诱导的关节软骨细胞丢失和软骨层变薄。在体外实验中，CH和Pro-Hyp不影响软骨细胞的增殖，但抑制其向矿化软骨细胞的分化。复合氨基酸如脯氨酸、羟脯氨酸和羟脯氨酸并不影响软骨细胞的增殖或分化。此外，CH和Pro-Hyp使ATDC5细胞胞外基质中糖胺多糖的染色面积分别增加2倍和3倍。RT-PCR结果显示，Pro-Hyp可使软骨细胞aggrecan基因表达水平增加近一倍，使RUNX1和骨钙素基因表达水平分别降低三分之二和十分之一。结论：Pro-Hyp是第一个在病理条件下影响软骨细胞分化的生物活性多肽。(C)2009年由爱思唯尔有限公司代表国际骨性关节炎研究会出版。
Objective: To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in collagen hydrolysate (CH).Methods: The in vivo effects of CH and Pro-Hyp intake on articular cartilage were studied by microscopic examination of sections of dissected articular cartilage from treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp, amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and mineralization of chondrocytes were determined by MTT activity and staining with alkaline phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5 cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR).Results: In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by two-thirds and one-tenth, respectively.Conclusion: Pro-Hyp is the first bioactive edible peptide derived from CH to be shown to affect chondrocyte differentiation under pathological conditions. (C) 2009 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.