Elucidation of aging mechanism of hyaluronan knock-down mouse and its application to the evaluation for effect of anti-aging reagents

透明质酸(HA)是一种高相对分子质量的非硫酸化糖胺聚糖，作为细胞外基质的主要成分存在于多种组织中，在器官再生、伤口愈合和抗衰老等方面发挥着重要作用。我们已经报道了4-甲基伞形酮(MU)特异性地抑制HA的合成。在本研究中，我们首先研究了HA相关基因的表达，如HA合成酶(HAS-1、HAS-2、HAS-3)、透明质酸酶(Hyal-1、Hyal-2、Hyal-3、Hyal-4)和HA受体(CD44、rhamm)。我们已经证明，在HA基因敲除的小鼠中，HAS-2基因在表皮和真皮中的转录都降低了，而rhamm在表皮中的转录也降低了。这些数据表明，MU处理特异性地下调HAS-2和RHAMM诱导的HA产生的基因表达，导致HA基因敲除小鼠皮肤衰老，如皱纹形成和皮肤脱水。接下来，我们利用dna微阵列a…检测了61种HA基因敲除小鼠的特异性基因。更多的分析。具体地说，在HA基因敲除小鼠中，Toll样受体3基因强烈下调，这表明炎症诱导导致了以皱纹形成为特征的皮肤衰老。癌基因如酪氨酸磷酸酶、细胞周期蛋白Y和Rho的表达也在HA基因敲除小鼠中下调。我们以前的研究表明，MU通过抑制HA的产生来抑制恶性肿瘤细胞的转移。因此，这些结果表明，下调这些癌基因的表达，至少在一定程度上参与了抑制转移。另一方面，基质分解蛋白3的基因表达强烈上调，这是细胞外基质成分如蛋白多糖和胶原的降解酶。这些数据表明，细胞外基质的降解导致了HA基因敲除小鼠皱纹的形成。最后，我们对HA及其不同相对分子质量的寡糖进行了给药。结果表明，仅皮内注射高分子透明质酸可延缓皮肤衰老，而口服或局部给药对老化皮肤无明显影响。此外，在HA基因敲除小鼠中引入衰老相关基因，如COL5A1、COL7A1、RHAMM、Toll样受体3等，并没有减少皮肤衰老的发生，这表明单独使用每个基因并不有效，多基因组合的鸡尾酒疗法对于治疗HA基因敲除小鼠的皮肤衰老是必要的。

Hyaluronan(HA), a non-sulfated glycosaminoglycan of high molecular mass, presents in various tissues as a major component of the extracellular matrix and plays an important role in regeneration, wound healing, and anti-aging of organs. We have previously reported that 4-methylumbelliferone(MU) specifically inhibits HA synthesis. In this study, we first investigated the expression of HA-related gene, such as HA synthase(HAS-1, HAS-2, HAS-3), hyaluronidase(Hyal-1, Hyal-2, Hyal-3, Hyal-4), and HA receptor(CD44, RHAMM). We have demonstrated that HAS-2 gene transcription in both epidermis and dermis, and RHAMM transcription in epidermis were reduced in HA-knock-down mice. These data suggested that MU treatment specifically downregulates gene expressions of HAS-2 and RHAMM induced the HA production, resulting in skin aging, such as wrinkle formation and skin dehydration in HA knockdown mice. Next, we have detected sixty one kinds of HA knock-down mice specific genes by using DNA microarray a … More nalysis. Specifically, toll-like receptor 3 gene was strongly down-regulated in HA knock-down mice, suggesting induction of inflammation resulted in skin aging characterize by wrinkle formation. Oncogene expressions such as of tyrosine phosphatase, cyclin Y, and Rho were also down-regulated in HA-knock down mice. Our previous studies have demonstrated MU suppressed the metastasis of malignant tumor cells by inhibiting of HA production. Therefore, these results suggested that down-regulation of these oncogene expression, at least in part, were involved in inhibition of metastasis. On the other hand, gene expression of stromelysin 3, depredating enzyme of extracellular matrix components such as proteoglycan and collagen, strongly up-regulated. These data suggested that the degradation of extracellular matrix resulted in wrinkle formation of HA knock-down mice. Finally, we have administered HA and its oligosaccharides with various molecular weight. As the result, only intradermal injection of high molecular HA decreased skin aging, whereas no change was observed in aged skin by orally or topically administration. Also, skin aging was not reduced by introduction of aging-related gene which was down-regulated in HA knock-down mice, such as COL5A1, COL7A1, RHAMM, toll-like receptor 3, suggesting alone administration of each gene was not effective and cocktail therapy, the combination of multiple genes, was necessary of treatment of skin aging in HA knock-down mice Less