【作者】 杨波；

【导师】 毕志刚；

【作者基本信息】 南京医科大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 紫外线(ultraviolet,UV)辐射引起的皮肤老化称为皮肤光老化,皮肤光老化是长波紫外线(ultraviolet A,UVA)和中波紫外线(ultraviolet B,UVB)共同作用的结果。以往的研究中,更多集中于UVA在皮肤光老化中的作用,而UVB对于皮肤光老化的影响关注较少。近年来,随着人们生活水平的提高和户外活动的增加,皮肤光老化的防护逐渐受到人们重视。植物抗氧化剂等有效成分抗光老化作用的研究较为引人注目。反式玉米素(trans-Zeatin,tZ)是从玉米种子中提取的一种植物激素,具有抗氧化、抗凋亡和抗炎等生物学活性。对自然老化的皮肤成纤维细胞有延缓其衰老的作用。反式玉米素是否对皮肤光老化具有防护作用?目前,国内外尚未有研究报道。本研究分三个部分探讨中波紫外线辐射对人皮肤成纤维细胞(human skin fibroblasts,HSFs)表达基质金属蛋白酶-1 ( matrix metalloproteinase-1 , MMP-1 )、基质金属蛋白酶-3 ( matrix metalloproteinase-3, MMP-3)和Ⅰ型前胶原(type I procollagen)的影响,反式玉米素对此影响的保护作用及分子机制。研究目的:第一部分探讨中波紫外线(UVB)辐射对体外培养的人皮肤成纤维细胞产生MMP-1和MMP-3的影响;反式玉米素对此影响的保护作用。第二部分探讨反式玉米素干预UVB辐射人皮肤成纤维细胞表达MMP-1的信号转导通路机制。第三部分探讨UVB对体外培养的人皮肤成纤维细胞表达、合成Ⅰ型前胶原的影响,反式玉米素的干预作用及机制。研究方法:用MTT方法检测反式玉米素对人皮肤成纤维细胞以及对UVB辐射的人皮肤成纤维细胞活性的影响;用ELISA方法检测细胞培养上清中MMP-1、MMP-3的分泌量,Ⅰ型前胶原蛋白合成的变化;用RT-PCR方法检测人皮肤成纤维细胞Ⅰ型前胶原mRNA表达;用Western blot方法检测MMP-1和MMP-3的蛋白表达水平,细胞内丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)(ERK1/2、JNK、p38)、c-Jun蛋白磷酸化表达水平,以及细胞内TGFβ、Smad3、Smad7蛋白表达量的变化。研究结果:第一部分:在150μM浓度以下,反式玉米素对人皮肤成纤维细胞活性无影响,在600μM浓度,反式玉米素对人皮肤成纤维细胞活性有明显影响,具有细胞毒性作用;反式玉米素(20μM、40μM、80μM)呈剂量依赖性提高UVB辐射后的皮肤成纤维细胞活性;UVB辐射可显著增加皮肤成纤维细胞MMP-1和MMP-3的蛋白表达水平;反式玉米素预处理,呈剂量依赖性,明显抑制UVB辐射的皮肤成纤维细胞MMP-1的蛋白表达量,而对MMP-3的蛋白表达则没有明显影响;未经UVB辐射,反式玉米素预处理后,对皮肤成纤维细胞MMP-1和MMP-3的蛋白表达量均无明显影响。第二部分:UVB辐射后,细胞内ERK1/2、JNK、p38 MAPK和c-Jun蛋白磷酸化活化,与未辐射对照组比较,磷酸化蛋白表达水平显著增加。反式玉米素在80μM浓度时,显著抑制UVB辐射后ERK1/2磷酸化活化;不同浓度反式玉米素(20μM、40μM、80μM)呈剂量依赖性,显著抑制JNK和c-Jun磷酸化活化;反式玉米素在40μM和80μM浓度时,呈剂量依赖性抑制UVB辐射后p38 MAPK磷酸化活化。MAPK信号抑制剂(PD98059,ERK1/2信号抑制剂、SP600125,JNK信号抑制剂、SB203580,p38 MAPK信号抑制剂)分别能显著抑制UVB辐射后ERK1/2、JNK、p38 MAPK磷酸化蛋白表达水平。PD98059(20μM)、SP600125(10μM)、SB203580(10μM)及反式玉米素(80μM)能明显抑制UVB辐射后皮肤成纤维细胞MMP-1蛋白表达。第三部分:与未辐射对照组比较,UVB辐射后,细胞Ⅰ型前胶原mRNA表达和蛋白分泌量,及细胞内TGFβ、Smad3蛋白表达量均显著减少,而Smad7蛋白表达量则明显增加;反式玉米素在40μM、80μM浓度,呈剂量依赖性,明显增加UVB辐射后皮肤成纤维细胞Ⅰ型前胶原mRNA表达和蛋白分泌量;细胞未经紫外线辐射,仅在不同浓度反式玉米素处理条件下,对细胞Ⅰ型前胶原mRNA表达和蛋白分泌量均无明显影响;反式玉米素呈剂量依赖性上调UVB辐射后皮肤成纤维细胞TGFβ、Smad3蛋白表达,下调Smad7蛋白表达。研究结论:UVB辐射,可以上调人皮肤成纤维细胞MMP-1和MMP-3的蛋白表达水平,抑制人皮肤成纤维细胞Ⅰ型前胶原mRNA表达和蛋白合成;UVB辐射,通过MAPK(ERK1/2、JNK、p38)/AP-1(c-Jun)信号传导通路,促进人皮肤成纤维细胞MMP-1蛋白表达;反式玉米素通过MAPK(ERK1/2、JNK、p38)/AP-1(c-Jun)信号传导通路,抑制UVB辐射的人皮肤成纤维细胞表达MMP-1;反式玉米素通过TGFβ/Smad信号传导通路,上调UVB辐射的人皮肤成纤维细胞Ⅰ型前胶原表达和蛋白合成,从而对皮肤光老化具有防护作用。更多还原

【Abstract】 Skin photoaging is induced by ultraviolet (UV) irradiation, which results from the comitant effects of UVA and UVB. The mechanism of skin photoaging had been concentrated in the study of UVA in the past years. While the effects of UVB had been little mentioned. In recent years, with the improvement of living standards and increase of outdoor activities, people have attached more importance to the protection of skin photoaging. Studies of the photoaging preventive effects of botanical antioxidants have been gained considerable attention. Trans-Zeatin is a plant hormone,purified from zea mays, which possesses a variety of biological actions, such as anti-oxidant properties, anti-apoptosis activity and anti-inflammatory effects. Trans-Zeatin also has gerontomodulatory, youth preserving and antiaging effects on serially passaged adult skin fibroblasts undergoing aging in vitro. Although many studies have revealed the functions of trans-Zeatin, its effects on skin photoaging has not yet been previously reported. In this study (including three parts), we have attemped to investigate the effect and action mechanisms of trans-Zeatin on matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3) and type I procollagen expression in UVB- irradiated human skin fibroblasts (HSFs).Objective: In partⅠ, we explored the effects of UVB on MMP-1, MMP-3 expression and the protective effects of trans-Zeatin against UVB-induced MMP-1, MMP-3 expression in human skin fibroblasts (HSFs) in vitro. In partⅡ, we investigated the signaling pathways of UVB-induced MMP-1 expression and trans-Zeatin against UVB-induced MMP-1 expression in HSFs. In partⅢ, we examined the effects of trans-Zeatin on type I procollagen synthesis in UVB-irradiated HSFs and revealed the possible mechanism of its action. Methods: The cell viability of trans-Zeatin on HSFs and UVB-exposed HSFs was detected by MTT assay. Amounts of MMP-1, MMP-3 and type I procollagen in the culture supernatants were quantitated by enzyme-linked immunosorbent assay kits. The expression of type I procollagen mRNA was detected by RT-PCR. The change of ERK1/2, JNK, p38 MAPK and c-Jun phosphorylation, and MMP-1, TGFβ, Smad3 as well as Smad7 expression in cells were determined by Western blot.Results:PartⅠ: We found that trans-Zeatin did not affect cell viability at the concentration lower than 150μM. While the concentration was over 600μM, trans-Zeatin affected the cell viability and had cytotoxic effect on HSFs. Trans-Zeatin (20-80μM) dose-dependently enhanced the cell viability in HSFs irradiated by UVB. The results also showed that UVB irradiation significantly enhanced MMP-1 and MMP-3 expression. Pretreatment with trans-Zeatin markedly inhibited UVB-induced MMP-1 expression in a dose-dependent manner versus the only UVB-irriadiated group. However, pretreatment with trans-Zeatin almost had no effect on UVB-induced MMP-3 expression. Moreover, trans-Zeatin itself did not change the expression of MMP-1 and MMP-3 in HSFs.PartⅡ: The results showed that ERK1/2, JNK, p38 MAPK and c-Jun phosphorylations were significantly enhanced by UVB irradiation, compared with the control group. Pretreatment with trans-Zeatin (80μM) markedly inhibited UVB-induced ERK1/2 phosphorylation. Trans-Zeatin(20-80μM) dose-dependently inhibited UVB-induced JNK and c-Jun phosphorylations. Trans-Zeatin also inhibited UVB-induced p38 MAPK phosphorylation in a dose-dependent manner at the concentration of 40μM and 80μM. As expected, PD98059, a specific ERK1/2 inhibitor , SP600125, a specific JNK inhibitor and SB203580, a specific p38 MAPK inhibitor effectively inhibited UVB-induced phosphorylations of ERK1/2, JNK and p38 MAPK respectively. Moreover, PD98059(20μM), SP600125(10μM), SB203580(10μM)and trans-Zeatin(80μM) significantly suppressed UVB-induced MMP-1 secretion, which is consistent with the above results.PartⅢ: In this study, the results indicated that mRNA expression and protein secretion of type I procollagen as well as synthesis of TGFβand Smad3 were significantly reduced by UVB irradiation, while synthesis of Smad7 was markedly enhanced by UVB irradiation, compared with the control group. Trans-Zeatin(40μM-80μM) dose-dependently increased mRNA expression and protein secretion of type I procollagen in UVB-irradiated HSFs. However, trans-Zeatin itself did not change type I procollagen mRNA expression and protein secretion. Moreover, Trans-Zeatin also increased synthesis of TGFβand Smad3, and reduced synthesis of Smad7 in HSFs irradiated by UVB.Conclusion: Our study demonstrates that UVB irradiation could significantly enhance MMP-1 and MMP-3 expression , and suppress type I procollagen expression and secretion in HSFs. UVB-induced MMP-1 expression is mediated by MAPK(ERK1/2、JNK、p38)/AP-1(c-Jun)signaling pathways. Trans-Zeatin could inhibit UVB-induced MMP-1 expression, which may be mediated by the inhibition of MAPK(ERK1/2、JNK、p38)/AP-1(c-Jun) signaling pathways in HSFs. Trans-Zeatin also could promote type I procollagen expression and synthesis, which may be mediated by stimulation of the TGFβ/Smad signaling in UVB-exposed HSFs. Therefore, we suggest that trans-Zeatin might be considered as a potential agent for the management of skin photoaging.更多还原