二甲氨基乙醇和复方氨基酸延缓大鼠皮肤老化的实验研究
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摘要
研究背景：永葆青春是每个人的梦想和追求。随着全球人口老龄化问题的日益凸显,抗衰老成为人们热衷的话题,尤其是微创面部美容年轻化受到众多求美者的追捧,也是国内、外生命科学领域的研究热点之一。Mesotherapy是目前最常用的微创美容方法之一,在1952年首先由法国医生Michel Pistor提出,1987年获得法国国有医学会认证,2005年通过中国卫生部和检验局认证。其原理就是采用超微渗透技术和／或定位注射技术,定位、定层、定量的把含有多种营养成分、高浓度的皮肤营养物质,直接透过表皮这一生理屏障,输送到真皮和皮下深层组织,从而达到美容、抗衰老的作用,同时完成“护肤、养肤、疗肤”三部曲,其特点是能够突破皮肤这一生理吸收屏障,彻底解决了传统的生活美容及各种美容保养产品不易透过表皮吸收的难题。Mesotherapy不但显效快,效果明显,而且创伤小、恢复期短甚至不需要恢复期,非常适合生活节奏忙碌而又爱美的现代女性和成功人士,是目前盛行的“午休整形”主要内容之一,其引导的“微整形”美容风潮引发了传统医学美容的结构发生重大改变,受到广大爱美人士和整形美容界、皮肤美容界医生热烈的欢迎,迅速风靡全球。尽管Mesotherapy如此盛行,产品亦是花样百出、层出不穷,但有关其基础研究的文献报道却很少,并有部分学者质疑Mesotherapy的作用,认为该方法没有显著的抗衰老的作用,只是一个有着巨大商业利益的美容噱头。本研究即立足与此,以D-半乳糖诱导的化学性衰老大鼠为模型,依据Mesotherapy的治疗理念,通过真皮内注射,把抗衰老药物定位、定量的输送到衰老大鼠的真皮中,观察抗衰老药物延缓皮肤老化的作用并初步探讨其作用机理。
二甲氨基乙醇是(DMAE)是维生素B胆碱的类似物,是乙酰胆碱的前体。在美国最早被用于治疗情绪和运动机能亢进性疾病,现在主要用于增强记忆力、改善儿童的学习和行为障碍。研究显示皮肤即是乙酰胆碱合成、储存、分泌、代谢的部位,又是其效应器。目前DMAE作为较新的皮肤年轻化的产品,因其具有即刻抗衰老、面部提升和抗皱的疗效而受到广泛的关注,是各种抗衰老化妆品中的主要成分之一。已有临床试验证实外用3%DMAE凝胶能显著改善前额皱纹、眶周细纹、唇的厚度和形状,以及整个面部皮肤的外观,对粗糙皱纹、黑眼圈、颈部皮肤松垂亦有一定的改善。而且证实了3%DMAE凝胶连续外用12个月治疗面部中度至中等严重的光老化表现出良好的疗效和安全性。但同时也有学者报道,2.5-1Ommol/L的DMAE即可导致培养的兔成纤维细胞细胞内产生大量空泡,并且具有浓度依赖性；3%(相当于337mmol/L)DMAE外用也能引起兔耳表皮细胞的空泡化,认为较高浓度的DMAE具有一定的细胞毒性。目前临床应用的DMAE均为外用品,其大剂量用药不但增加其相应的副作用和毒性,也增加了病人的医疗负担。因此,本研究依据Mesotherapy的治疗理念,通过真皮内注射小剂量、低浓度的DMAE,直接作用于拟治疗的部位,减低其浓度和用量,同时还将结合复方氨基酸(AA)同步注射以减轻真皮内注射DMAE对靶细胞的细胞毒性,并为其提供多种氨基酸作为营养物质。
衰老是人体机能变缓的必然结果,皮肤和外貌的衰老其最为直观的表现。皮肤老化现象主要表现在以下方面：(1)衰老的皮肤表皮细胞间连接疏松,水合能力下降。新生儿的皮肤含水量最多,随人体机能逐渐老化,皮肤的水合能力也下降,皮肤中含水量也逐渐下降,老年人皮肤含水量仅是正常青年人的75%,因此常表现为皮肤干燥。(2)皮肤的厚度随着年龄的增加而逐渐变薄,到老年期表皮的颗粒层可萎缩至消失,棘细胞生存期缩短。真皮变薄并伴有萎缩：20岁以后真皮纤维细胞数量逐渐减少,胶原总含量每年减少1%,胶原纤维变粗,出现异常交联；同时,密度增大,不易被胶原酶所分解,胶原稳定性增加。衰老皮肤中Ⅰ型胶原合成减少,而Ⅲ型胶原基因表达增加。一般婴儿和年青人皮肤Ⅰ型胶原含量约占80%,Ⅲ型胶原约占10%,Ⅰ型胶原在真皮中聚集成束,交织成网,赋予皮肤机械性与充盈感,维持皮肤张力；Ⅲ型胶原是一种幼稚的胶原纤维,是构成真皮中网状纤维的主要成分。当皮肤衰老时,两者比例逐渐倒置,Ⅰ型胶原蛋白越来越少,Ⅲ型胶原蛋白逐渐增多,真皮中胶原整体由Ⅰ型向Ⅲ型胶原转变。老化的胶原应力传导下降,抗剪切力减弱。真皮乳头层和网状层弹力纤维减少及退化,胶原纤维和弹力纤维排列也渐趋紊乱,引起皱纹、松弛及下垂。(3)生理功能低下,皮脂腺、汗腺功能衰退,汗液与皮脂排除减少,皮肤逐渐失去昔日光泽而变得干燥,血液循环功能减退难以补充皮肤必要的营养。其中,真皮层结构的改变是皮肤老化的主要原因。
根据上述皮肤衰老组织结构和代谢等方面的变化,本研究通过以下六方面来观测皮肤老化的程度：
(1)组织形态学数据测量：测定皮肤表皮、真皮厚度和真皮中胶原纤维密度。
(2)含水量：反映皮肤的水合能力。
(3)胶原蛋白含量：羟脯氨酸是胶原蛋白中一种重要而且含量稳定的氨基酸,约占其总量的13.4%,仅在弹力蛋白中含量很少,并且不存在于在其它蛋白中。因此,测定羟脯氨酸的含量是检测组织中胶原蛋白含量的可靠而准确的方法,其含量的多少可间接反映出组织中胶原蛋白的含量。
(4)胶原合成代谢：检测Ⅰ型胶原蛋白、Ⅲ型胶原蛋白mRNA的表达水平。
(5)胶原分解代谢：基质金属蛋白酶(MMP-1)又称胶原酶-1、成纤维细胞胶原酶或间质胶原酶,可降解Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ型胶原明胶及蛋白多糖。MMP-1在体外可由多种正常细胞(如成纤维细胞、巨噬细胞、内皮细胞和上皮细胞)产生,但在正常休眠组织中MMP-1水平极低,一般难以检测到。在体内,MMP-1主要在生理和病理条件下,如组织生长、重构期间表达,广泛发挥其生物学功能。在正常成人组织中MMP-1表达量极少,但在病理情况下,如创伤愈合、修复或重塑过程中,MMP-1表达量增加。基质金属蛋白酶抑制剂(TIMP)是MMP的天然抑制物,到目前为止共发现其家族有四种,即TIMP-1、TIMP-2、 TIMP-3和TIMP-4。TIMP-1和TIMP-2可直接与活化的MMP形成紧密的1-1复合体,从而抑制其活性。
(6)成纤维细胞增殖情况：增殖细胞核抗原(PCNA)由Miyachi等在1978年在系统性红斑狼疮患者的血清中首次发现并命名,其只存在于正常增殖细胞和肿瘤细胞内,以后的研究发现PCNA与细胞DNA合成关系密切,在细胞增殖的启动上起重要作用,是反映细胞增殖状态的良好指标。
根据以上众多学者、专家的研究基础,本研究依据Mesotherapy的治疗理念,观察0.1%DMAE、0.2%DMAE与复方氨基酸注射液(AA)联合应用对D-半乳糖诱导的亚急性化学性衰老大鼠皮肤组织形态和胶原代谢的影响,以期初步探讨其抗衰老的作用和机理。
方法：80只wistar大鼠随机分入正常对照组、衰老对照组和衰老治疗组,衰老治疗组共分为6组,即衰老生理盐水(NS)组、衰老AA组、衰老0.1%DMAE组、衰老0.2%DMAE组、衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组,每组10只大鼠。在衰老治疗组和衰老对照组大鼠颈背部皮下注射D-半乳糖125mg/kg.d,连续42天,建立亚急性化学性衰老大鼠皮肤模型。从第18天开始,在大鼠两侧臀部事先文身标记的直径3cm圆形皮肤区域内多点(10点)、均匀地真皮内分别注射0.2%DMAE+AA、0.1%DMAE+AA、0.2%DMAE、0.1%DMAE、AA、NS各lml,每周一次,连续4周,即实验42天后,处死动物,切取皮肤标本,通过HE和Masson染色观察各组皮肤组织学变化,免疫组化法检测各组皮肤PCNA的表达情况。生化方法定量检测各组大鼠皮肤中含水量、羟脯氨酸含量,并同时采用RT-PCR法检测各组皮肤组织中Ⅰ、Ⅲ型胶原蛋白、MMP-1、和TIMP-1mRNA的表达水平。应用simple PCI专业图像分析软件测定各组皮肤表皮、真皮厚度、真皮中胶原纤维密度值和PCNA阳性染色灰度值。应用SPSS13.0软件进行实验数据的统计学分析。计量数据用x±s表示,组间差异比较采用单因素方差分析检验,P<0.05表明差异有统计学意义。
结果：
1.动物大体表现：在大鼠颈背部皮下注射D-半乳糖(D-gal)125mg/kg.d,连续42天,7个衰老组大鼠表现出体型瘦小,体重减轻,被毛枯黄,精神萎靡、嗜睡,反应迟钝,活动减少,行动迟缓等一系列老化征象。
2.组织学表现：衰老对照组表皮变薄,细胞层数减少；真皮变薄,皮肤胶原纤维排列疏松,胶原纤维厚度变薄,胶原束细长、断裂、走向平直。其中衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组上述改变较衰老对照组轻。与衰老对照组相比,衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组、衰老0.2%DMAE组表皮厚度增高有显著性意义(P均<0.05)；衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组真皮的厚度增高有显著性意义(P均<0.01)；衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组、衰老0.2%DMAE组和衰老0.1%DMAE组真皮中胶原纤维的密度增加有显著性意义(P均<0.01)。然而,与正常对照组相比,所有衰老组的表皮、真皮厚度和胶原纤维密度的减少均有统计学意义(P均<0.01)。
3.含水量：各组之间皮肤含水量的差异无统计学意义(P均>0.05)。
4.羟脯氨酸含量：与其他衰老组相比,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组皮肤中羟脯氨酸含量增多有显著性意义(P均<0.05)。正常对照组羟脯氨酸含量与衰老0.2%DMAE+AA组的差异无显著性意义(P>0.05),衰老0.2%DMAE+AA组、衰老0.1%DMAE+AA组之间差异无显著性意义(P>0.05)。
5.胶原合成代谢的变化：
(1)Ⅰ型胶原蛋白mRNA表达水平：与衰老对照组相比,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组皮肤中Ⅰ型胶原蛋白mRNA表达水平升高有统计学意义(P均<0.01)。与正常对照组相比,衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组表达水平的差异无显著性意义(P均>0.05),而其他衰老组的降低有显著性意义(P均<0.01)。
(2)Ⅲ型胶原蛋白mRNA表达水平：与衰老对照组比较,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组、衰老0.2%DMAE组和衰老AA组皮肤中Ⅲ型胶原蛋白mRNA表达水平升高有显著性意义(P均<0.01)。而和正常对照组相比,所有衰老组的表达水平降低有统计学意义(P均<0.01)。
6.胶原分解代谢的变化：
(1)MMP-1mRNA表达水平：与衰老对照组相比,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组皮肤中MMP-1mRNA表达水平升高有显著性意义(P均<0.01)。与正常对照组比较,衰老对照组、衰老NS组MMP-1mRNA表达水平降低有显著性差异(P均<0.05),而衰老AA组、衰老0.1%DMAE组、衰老0.2%DMAE组、衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组表达水平的差异无统计学意义(P均>0.05)。和衰老0.2%DMAE+AA组相比,正常对照组和衰老0.1%DMAE+AA组皮肤中MMP-1mRNA表达水平的差异无显著性意义(P均>0.05),而其他衰老各组表达水平的降低有显著性意义(P均<0.01)。与衰老0.1%DMAE+AA组比较,衰老对照组、衰老NS组和衰老AA组表达水平的降低有显著性意义(P均<0.05),而与其他组的差异无显著性意义(P均>0.05)。
(2)TIMP-1的mRNA表达水平：与衰老对照组比较,正常对照组TIMP-1的mRNA表达水平的升高有显著性意义,而其他衰老治疗组表达水平的差异无显著性意义(P均>0.05)。与正常对照组相比,7个衰老组TIMP-1的mRNA表达水平的降低有显著性意义(P均<0.01)。
7.成纤维细胞PCNA表达情况：与衰老对照组比较,正常对照组PCNA表达的升高有显著性意义(P均<0.01),而其他衰老治疗组PCNA表达的差异无显著性意义(P均>0.05)。与正常对照组相比,7个衰老组PCNA表达降低有显著性意义(P均<0.01)。
结论：
1.在大鼠颈背部皮下注射D-半乳糖125mg/Kg/d,连续42天,可使大鼠体型瘦小,体重减轻,被毛枯黄,精神萎靡、嗜睡,反应迟钝,活动减少,行动迟缓。组织学显示其表皮变薄,细胞层数减少；真皮变薄,皮肤胶原纤维排列疏松,密度降低,胶原束细长、断裂、走向平直。皮肤中胶原蛋白含量减少,胶原合成代谢和分解代谢均显著下降,成纤维细胞增殖亦显著降低。大鼠的大体形态、皮肤组织学、胶原代谢和细胞增殖状态的改变,均显示出老化的特征,提示D-半乳糖诱导的亚急性化学性衰老大鼠皮肤模型造模成功。
2.真皮内注射0.1%DMAE+AA、0.2%DMAE+AA两组复合溶液可使衰老大鼠皮肤的表皮和真皮厚度增加,真皮中胶原纤维密度增加,组织学上表现为表皮、真皮的增厚,胶原排列致密,具有延缓衰老大鼠皮肤老化的作用。
3. DMAE和AA对正常大鼠和衰老各组大鼠皮肤的保湿活性没有影响。
4.真皮内注射0.1%DMAE+AA和0.2%DMAE+AA能显著提高衰老大鼠皮肤中的羟脯氨酸含量,其中0.2%DMAE+AA的作用更为明显,可达到与正常大鼠相近的水平,提示该两组复合溶液可显著提高衰老大鼠皮肤中胶原蛋白的含量,具有抗老化的作用。
5.真皮内注射0.2%DMAE+AA、0.1%DMAE+AA显著上调Ⅰ型胶原蛋白mRNA表达水平,且与正常大鼠皮肤中Ⅰ型胶原蛋白mRNA水平无显著性差异,说明该两组复合药物注射于真皮中能增加衰老大鼠皮肤的Ⅰ型胶原蛋白的合成,可提高整体胶原中Ⅰ型胶原蛋白的比例,达到正常健康大鼠的水平,提示这两组复合药物溶液具有延缓皮肤老化的作用。
同时,尽管这两组复合药物溶液能显著提高衰老大鼠皮肤中的Ⅲ型胶原蛋白mRNA表达水平,但正常大鼠Ⅲ型胶原蛋白mRNA表达水平仍显著高于衰老各组的水平,与其他文献报道的衰老皮肤中Ⅲ型胶原蛋白含量和比例增加不一致,可能是因为D-半乳糖所致的亚急性化学性衰老的模型和正常生理性衰老的皮肤胶原代谢的特征不同所致。
6.真皮内注射0.2%DMAE+AA、0.1%DMAE+AA使衰老大鼠皮肤中MMP-1的]mRNA表达水平显著升高,且与正常大鼠的表达水平相近。而MMP-1的抑制剂TIMP-1在衰老各治疗与衰老对照组无显著性差别,均明显低于正常对照。说明该两组复合药物溶液注射于真皮中能促进衰老大鼠皮肤的胶原分解代谢,使老化、变性的胶原降解,以修复和重新构建衰老皮肤,接近或达到正常健康大鼠的组织结构,提示这两组复合药物溶液具有一定的抗衰老作用。
7.采用相同的治疗途径,单独在衰老大鼠真皮内注射0.2%DMAE、0.1%DMAE、AA、NS,在羟脯氨酸含量,以及Ⅰ型胶原蛋白、MMP-1mRNA表达水平与衰老对照组均无显著性差异,说明单用DMAE和AA无效,只有两者结合,才具有促进Ⅰ型胶原蛋白合成、老化和变性胶原分解,重塑皮肤结构,延缓皮肤老化的功效。
8.真皮内注射0.1%DMAE可使衰老真皮中胶原纤维密度增加,真皮内注射0.2%DMAE可使衰老大鼠表皮厚度和真皮中胶原纤维密度增加,提示0.1%DMAE和0.2%DMAE可能对衰老皮肤具有一定的刺激作用,浓度越高刺激性越强,使真皮中胶原纤维增粗,表皮增厚,而对胶原代谢没有影响。
9.真皮内注射0.2%DMAE+AA、0.1%DMAE+AA无诱导成纤维细胞增殖的作用,没有细胞过度增殖导致变异和癌变的隐患,仅通过改善成纤维细胞的胶原合成和分解代谢提高衰老大鼠皮肤中胶原蛋白的含量和增加Ⅰ型胶原蛋白mRNA表达而达到延缓皮肤衰老的作用,提示其抗衰老作用是安全的。
综上所述,本研究通过在D-半乳糖诱导的化学性衰老大鼠真皮内注射0.2%DMAE+AA、0.1%DMAE+AA发现,一方面该两组复合药物具有促进其Ⅰ、Ⅲ型胶原蛋白合成的作用,使羟脯氨酸含量和Ⅰ型胶原蛋白mRNA表达水平接近于正常生长的大鼠水平,增加衰老大鼠皮肤中Ⅰ型胶原含量,恢复年轻状态皮肤的胶原比例；另一方面,促进衰老大鼠皮肤中老化、变性胶原纤维的降解,重新塑造、构建衰老皮肤的组织结构,使之接近正常生长健康大鼠的状态。两方面共同作用使衰老的大鼠表皮、真皮厚度增加,胶原纤维丰富,排列致密,恢复年轻状态皮肤的组织结构特点,具有延缓衰老的作用；同时,这两组复合溶液没有诱导成纤维细胞增殖的作用,推测其抗衰老的作用主要是通过改善衰老皮肤的胶原代谢来实现,其安全性得到初步验证,但其深入的作用机制还有待于进一步的研究。
本研究依据Mesotherapy的治疗理念,在老化的大鼠皮肤真皮层内注射小剂量、低浓度的DMAE和AA复合溶液,探索其改善衰老皮肤胶原代谢和皮肤的组织结构的作用。该方法不需要大量口服全身用药,一方面减少药物的用量和浓度,降低治疗成本,同时能减少其相应的副作用和毒性；另一方面将药物直接运输至靶器官,克服了传统护肤产品不易透过表皮屏障,难于吸收的缺陷,使其抗衰老的疗效更加直接、显著。目前DMAE均为外用产品,而且已经报道临床应用具有稳定、显著、安全的抗衰老疗效,如果结合Mesotherapy的独特优势,将会具有更加直接、有效的延缓皮肤老化的作用。本研究将为DMAE进一步、多途径的抗衰老临床应用奠定一定的实验理论基础。
Keeping youth forever is an eternal topic and dream of human being. In the recent years, mesotherapy has been arousing everyone's interest as an anti-aging strategy. Pioneered by the French physician Dr. Michel Pistor in1952, Mesotherapy is a minimally invasive procedure that is widely used in Europe and elsewhere to treat various injuries and medical conditions. It was recognized by the French National Academy of Medicine in1986as an integral part of traditional medicine, and has been becoming highly popular within the public. In China, Mesotherapy was approved by Ministry of Health and CFDA. Mesotherapy, a safe, simple, less painful procedure which is one of the so-called "lunchtime cosmetic procedures", requires no recovery time and is perfect for professionals and successful people in the fast-paced modern life. Although there are a wide range of products of Mesotherapy, less foundimental reasearchs have been proceeded. Moreover, there is some controversy over its efficacy and safety.
DMAE is an analog of vitamin B choline that has been used to treat a number of conditions affecting the brain and central nervous system. There is some evidence that DMAE may be helpful for attention deficit hyperactivity disorder (ADHD). More widely marketed today as a memory and mood enhancer, DMAE is said to improve intellectual functioning. The basis for such claims probably stems from its purported ability to increase levels of a neurotransmitter called acetylcholine, although this has not been proven. Studies suggest that the skin is an active site of acetylcholine synthesis, storage, secretion, metabolism, and receptivity. The role of DMAE as a modulator of acetylcholine-mediated functions in the skin remains to be elucidated. DMAE is receiving more attention as a new skincare regimen today for its acute effects of anti-aging, anti-wrinkle and skin firmness. From three recent clinical reports,3%DMAE facial gel formula has been shown to be efficacious in the mitigation of forehead lines and periorbital fine wrinkles, and in improving lip fullness and shape and the overall appearance of facial skin. The safety concern of the long-term application of DMAE gel for up to1year has also been further documented.
Age-related skin changes are inevitable and include thinning, sagging, wrinkling, loss of elasticity and areas of dryness. Histological changes include dermal thinning, collagen thickening with degeneration and reduced amount of fibers, and elastin breaking down. In infant and young adult skin, type Ⅰ collagen is the most abundant protein in the dermis accounting for approximately80%, while type Ⅲ collagen is much less with about10%of dermal mass. When skin becomes aged, the collagen synthesis is reduced for type Ⅰ but up-regulated for type Ⅲ collagen, resulting in an inversed turnover of type Ⅰ/Ⅲ ratio in the skin. Though it is rarely seen in normal adult skin, increased expression of matrix Metalloproteinase-1(MMP-1) is a prominent feature of some pathological situations, such as wound healing, tissue repairing and remodeling. MMP activity is a key driver in wound healing to facilitate the motility of inflammatory cells and to enhance the availability of inflammatory mediators to set the stage for healing. However the tissue inhibitor of metalloproteinase (TIMP), a natural inhibitor of MMP, can restrain the activation of MMP and its activity in the wound healing process by forming a ternary complex with MMP.
Proliferating cell nuclear antigen (PCNA) was first found in serum of SLE patients by Miyachi et al, which only express in nomal proliferating cell or tumor cell. Subsequent studies showed that PCNA is close to DNA synthesis and play an important role in trigger of cell proliferation. It is a ideal indicator of state of cell proliferation.
In order to evaluate potential anti-aging effects of DMAE and compound Amino Acid in Mesotherapy, tissue structure and collagen metabolism of D-galactose (gal) induced aging skin were measured in this study. Their considered mechanism of action in the skin was also described.
Methods:Aging was artificially induced by subcutaneous injection with D-gal at the dose of125mg/kg.d for6weeks (42days).80Wistar rats were randomly divided into aging groups receiving D-gal treatment, including aging control group (D-gal for6weeks); NS (D-gal for6weeks+normal saline for4weeks); AA (D-gal for6weeks+compound amino acid for4weeks);0.1%DMAE (D-gal for6weeks+0.1%DMAE for4weeks);0.2%DMAE (D-gal for6weeks+0.2%DMAE for4weeks);0.1%DMAE+AA (D-gal for6weeks+0.1%DMAE/AA for4weeks);0.2%DMAE+AA (D-gal for6weeks+0.2%DMAE/AA for4weeks) and sham control group which was not given D-gal. At the end of each treatment, histologic changes of aging skin were observed; tissue water content and hydroxyproline were determined according to assay kits; PCNA stains of each group were observed by immunochemical methods; RT-PCR was used to detect mRNA expression of type I/III procollagen, MMP-1and TIMP-1. Simple PCI image analysis software were quantified the thickness of epidermis, dermis, collagen fibre density and PCNA positive stains. Data were expressed as means±standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS13.0software. The results were taken to be statistically significant at a probability level of p<0.05.
Results:
1.Animal:There were some aging phenomenon, such as lean and small, low weight, yellow and no luster fair, thinned skin, decreased activities, neurological impairment in aging group rats after rats were subcutaneous injected with D-gal at the dose of125mg/kg.d for6weeks.
2. Histological changes:When compared to sham control group, aging skin showed decreased thickness for both epidermis and dermis (P<0.05). With aging, the epidermis reduced its numbers of cell layers, and the dermal collagen fibers appeared sparse, slender or broken. Although the histologic changes of0.1%DMAE+AA group and0.2%DMAE+AA group were much better improved than that of aging control group, their indicators were all significantly lower than that of sham control group (P<0.01). In addition, when compared to aging control,0.2%DMAE alone also significantly increased epidermal thickness and density of collagen fiber (P<0.01), and0.1%DMAE increased epidermal thickness (P<0.01).
3. Water contents in aging skin:There was no statistically significant difference in water contents among all groups (P>0.05).
4. Hydroxyproline contents in aging skin:Hydroxyproline contents were apparently increased in aging skin treated with0.1%DMAE+AA or0.2%DMAE+AA complex (P<0.01), which showed a significant difference when compared to all other aging groups. Interestingly, hydroxyproline contents in0.2%DMAE+AA group reached an equivalent level to that of sham control group (P>0.05) even though both groups had much higher hydroxyproline contents than any of other6aging groups (P<0.05). There was no significant difference between0.1%DMAE+AA and0.2%DMAE+AA groups (P>0.05).
5.Changes of collagen synthesis:
(1)Collagen type I expression was greatly increased in response to the treatment of both0.1%DMAE+AA and0.2%DMAE+AA complex (P<0.01). Although there was no statistically significant difference between these two groups and sham control (P>0.05), all other aging groups showed much less expression of collagen type I (P <0.01).
(2) When treated with0.1%DMAE+AA,0.2%DMAE+AA or0.2%DMAE alone, the mRNA expression for collagen type III was also considerably elevated over the aging control group (P<0.01), but still significantly less than sham control (P<0.01).
6.Chagens of collagen catabolism:
(1) MMP-1, a potential key regulator in aging skin, was also observed in this study, and results showed that its mRNA expression significantly increased in rats skin tissue treated with either0.1%DMAE+AA or0.2%DMAE+AA. These up-regulations of MMP-1activity showed a statistically significant difference to that of aging control group (P<0.01), but not to sham control(P>0.05).
(2) In contrast, the transcripts for TIMP-1of all aging groups was down regulated and reached significantly lower levels to that of sham control (P<0.01).
7. Expression of PCNA:PCNA expression of sham control is higher than all aging groups (P<0.01), and there were no difference among every aging group(P>0.05).
Conclusion:
1. Mesotherapy with combined application of DMAE and compound AA could improve the thickness of aging skin, and play an anti-aging role.
2. Both DMAE and compound AA may have limited effects on skin moisturizing which suggests that DMAE and compound AA had limited effects on skin moisturizing. This might be due to a lower level of concentration and shorter action period of local available DMAE in epidermal layer when direct subcutaneous injection was used for DMAE delivery, which differs from tropical application of DMAE.
3. Mesotherapy with combined application of DMAE and compound AA could increase collagen contents of aging skin, especially, effect of0.2%DMAE+AA could be better which increased hydroxyproline contents is equal to sham control.
4. Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting collagen type I synthesis.
5. Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting catabolism to remodel skin texture.
6. Intradermal injection of DMAE or AA alone showed no effects on hydroxyprolin contents or messages for collagen type I and MMP-1in the aging skin. These data indicate that combined application of DMAE and AA is the only way to play their anti-aging action in this D-gal induced aging skin model by modulating collagen type I metabolism and remolding the structure of aging skin.
7. Intradermal injection of0.1%DMAE and0.2%DMAE did increase the dermal collagen fiber density over the aging control group. This result suggest that0.1%DMAE and0.2%DMAE treatment may potentially trigger aging skin to thicken the collagen fibre, but not apparently affect dermal collagen metabolism. Previous studies have shown that cultured rabbit dermal fibroblasts responded to DMAE by massive vacuolization in a concentration-dependent manner. The epidermis of rabbit external ear was also significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to tropical application of3%DMAE. It was suggested that vacuolar cytopathology may not be dissociable from the improvement of skin appearance that is rapidly produced by topically administered DMAE, and could be the cellular basis of the anti-wrinkle effect of DMAE. From our results, the vacuolization of the dermal fibroblast might occur in both0.1%and0.2%DMAE treated aging skin, resulting in cellular swelling and increased collagen fiber density. Epithelial cells of aging skin could also respond to the higher concentration of0.2%DMAE by vacuolization, which presented as increased epidermal thickness. However, these may need further studies to clarify this matter.
8. Mesotherapy with0.2%DMAE+AA,0.1%DMAE+AA,0.2%DMAE,0.1%DMAE, AA, NS could not effect expression of PCNA.
Mesotherapy caused minimum invasion and discomfort, without any posttreatment pain. All patients can return to their everyday activities immediately after treatment. In present study mesotherapy with DMAE+AA compound solution were administered directly and accurately into the region to be treated, and effect of medicines are more immediate and effective at minimum dose with the advantage of avoiding side-effects accompany with systemic administration at high dose.
In conclusion, Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting collagen synthesis and catabolism to remodel skin texture, and improve the thickness of aging skin. At the same time, complex of DAMA and AA could not induce fibroblast proliferation. This new strategy shows a promising potential in slowing skin aging in D-gal induced aging model, while further investigations should be conducted to demonstrate the biological bases of its anti-aging effects.
二甲氨基乙醇是(DMAE)是维生素B胆碱的类似物,是乙酰胆碱的前体。在美国最早被用于治疗情绪和运动机能亢进性疾病,现在主要用于增强记忆力、改善儿童的学习和行为障碍。研究显示皮肤即是乙酰胆碱合成、储存、分泌、代谢的部位,又是其效应器。目前DMAE作为较新的皮肤年轻化的产品,因其具有即刻抗衰老、面部提升和抗皱的疗效而受到广泛的关注,是各种抗衰老化妆品中的主要成分之一。已有临床试验证实外用3%DMAE凝胶能显著改善前额皱纹、眶周细纹、唇的厚度和形状,以及整个面部皮肤的外观,对粗糙皱纹、黑眼圈、颈部皮肤松垂亦有一定的改善。而且证实了3%DMAE凝胶连续外用12个月治疗面部中度至中等严重的光老化表现出良好的疗效和安全性。但同时也有学者报道,2.5-1Ommol/L的DMAE即可导致培养的兔成纤维细胞细胞内产生大量空泡,并且具有浓度依赖性；3%(相当于337mmol/L)DMAE外用也能引起兔耳表皮细胞的空泡化,认为较高浓度的DMAE具有一定的细胞毒性。目前临床应用的DMAE均为外用品,其大剂量用药不但增加其相应的副作用和毒性,也增加了病人的医疗负担。因此,本研究依据Mesotherapy的治疗理念,通过真皮内注射小剂量、低浓度的DMAE,直接作用于拟治疗的部位,减低其浓度和用量,同时还将结合复方氨基酸(AA)同步注射以减轻真皮内注射DMAE对靶细胞的细胞毒性,并为其提供多种氨基酸作为营养物质。
衰老是人体机能变缓的必然结果,皮肤和外貌的衰老其最为直观的表现。皮肤老化现象主要表现在以下方面：(1)衰老的皮肤表皮细胞间连接疏松,水合能力下降。新生儿的皮肤含水量最多,随人体机能逐渐老化,皮肤的水合能力也下降,皮肤中含水量也逐渐下降,老年人皮肤含水量仅是正常青年人的75%,因此常表现为皮肤干燥。(2)皮肤的厚度随着年龄的增加而逐渐变薄,到老年期表皮的颗粒层可萎缩至消失,棘细胞生存期缩短。真皮变薄并伴有萎缩：20岁以后真皮纤维细胞数量逐渐减少,胶原总含量每年减少1%,胶原纤维变粗,出现异常交联；同时,密度增大,不易被胶原酶所分解,胶原稳定性增加。衰老皮肤中Ⅰ型胶原合成减少,而Ⅲ型胶原基因表达增加。一般婴儿和年青人皮肤Ⅰ型胶原含量约占80%,Ⅲ型胶原约占10%,Ⅰ型胶原在真皮中聚集成束,交织成网,赋予皮肤机械性与充盈感,维持皮肤张力；Ⅲ型胶原是一种幼稚的胶原纤维,是构成真皮中网状纤维的主要成分。当皮肤衰老时,两者比例逐渐倒置,Ⅰ型胶原蛋白越来越少,Ⅲ型胶原蛋白逐渐增多,真皮中胶原整体由Ⅰ型向Ⅲ型胶原转变。老化的胶原应力传导下降,抗剪切力减弱。真皮乳头层和网状层弹力纤维减少及退化,胶原纤维和弹力纤维排列也渐趋紊乱,引起皱纹、松弛及下垂。(3)生理功能低下,皮脂腺、汗腺功能衰退,汗液与皮脂排除减少,皮肤逐渐失去昔日光泽而变得干燥,血液循环功能减退难以补充皮肤必要的营养。其中,真皮层结构的改变是皮肤老化的主要原因。
根据上述皮肤衰老组织结构和代谢等方面的变化,本研究通过以下六方面来观测皮肤老化的程度：
(1)组织形态学数据测量：测定皮肤表皮、真皮厚度和真皮中胶原纤维密度。
(2)含水量：反映皮肤的水合能力。
(3)胶原蛋白含量：羟脯氨酸是胶原蛋白中一种重要而且含量稳定的氨基酸,约占其总量的13.4%,仅在弹力蛋白中含量很少,并且不存在于在其它蛋白中。因此,测定羟脯氨酸的含量是检测组织中胶原蛋白含量的可靠而准确的方法,其含量的多少可间接反映出组织中胶原蛋白的含量。
(4)胶原合成代谢：检测Ⅰ型胶原蛋白、Ⅲ型胶原蛋白mRNA的表达水平。
(5)胶原分解代谢：基质金属蛋白酶(MMP-1)又称胶原酶-1、成纤维细胞胶原酶或间质胶原酶,可降解Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ型胶原明胶及蛋白多糖。MMP-1在体外可由多种正常细胞(如成纤维细胞、巨噬细胞、内皮细胞和上皮细胞)产生,但在正常休眠组织中MMP-1水平极低,一般难以检测到。在体内,MMP-1主要在生理和病理条件下,如组织生长、重构期间表达,广泛发挥其生物学功能。在正常成人组织中MMP-1表达量极少,但在病理情况下,如创伤愈合、修复或重塑过程中,MMP-1表达量增加。基质金属蛋白酶抑制剂(TIMP)是MMP的天然抑制物,到目前为止共发现其家族有四种,即TIMP-1、TIMP-2、 TIMP-3和TIMP-4。TIMP-1和TIMP-2可直接与活化的MMP形成紧密的1-1复合体,从而抑制其活性。
(6)成纤维细胞增殖情况：增殖细胞核抗原(PCNA)由Miyachi等在1978年在系统性红斑狼疮患者的血清中首次发现并命名,其只存在于正常增殖细胞和肿瘤细胞内,以后的研究发现PCNA与细胞DNA合成关系密切,在细胞增殖的启动上起重要作用,是反映细胞增殖状态的良好指标。
根据以上众多学者、专家的研究基础,本研究依据Mesotherapy的治疗理念,观察0.1%DMAE、0.2%DMAE与复方氨基酸注射液(AA)联合应用对D-半乳糖诱导的亚急性化学性衰老大鼠皮肤组织形态和胶原代谢的影响,以期初步探讨其抗衰老的作用和机理。
方法：80只wistar大鼠随机分入正常对照组、衰老对照组和衰老治疗组,衰老治疗组共分为6组,即衰老生理盐水(NS)组、衰老AA组、衰老0.1%DMAE组、衰老0.2%DMAE组、衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组,每组10只大鼠。在衰老治疗组和衰老对照组大鼠颈背部皮下注射D-半乳糖125mg/kg.d,连续42天,建立亚急性化学性衰老大鼠皮肤模型。从第18天开始,在大鼠两侧臀部事先文身标记的直径3cm圆形皮肤区域内多点(10点)、均匀地真皮内分别注射0.2%DMAE+AA、0.1%DMAE+AA、0.2%DMAE、0.1%DMAE、AA、NS各lml,每周一次,连续4周,即实验42天后,处死动物,切取皮肤标本,通过HE和Masson染色观察各组皮肤组织学变化,免疫组化法检测各组皮肤PCNA的表达情况。生化方法定量检测各组大鼠皮肤中含水量、羟脯氨酸含量,并同时采用RT-PCR法检测各组皮肤组织中Ⅰ、Ⅲ型胶原蛋白、MMP-1、和TIMP-1mRNA的表达水平。应用simple PCI专业图像分析软件测定各组皮肤表皮、真皮厚度、真皮中胶原纤维密度值和PCNA阳性染色灰度值。应用SPSS13.0软件进行实验数据的统计学分析。计量数据用x±s表示,组间差异比较采用单因素方差分析检验,P<0.05表明差异有统计学意义。
结果：
1.动物大体表现：在大鼠颈背部皮下注射D-半乳糖(D-gal)125mg/kg.d,连续42天,7个衰老组大鼠表现出体型瘦小,体重减轻,被毛枯黄,精神萎靡、嗜睡,反应迟钝,活动减少,行动迟缓等一系列老化征象。
2.组织学表现：衰老对照组表皮变薄,细胞层数减少；真皮变薄,皮肤胶原纤维排列疏松,胶原纤维厚度变薄,胶原束细长、断裂、走向平直。其中衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组上述改变较衰老对照组轻。与衰老对照组相比,衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组、衰老0.2%DMAE组表皮厚度增高有显著性意义(P均<0.05)；衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组真皮的厚度增高有显著性意义(P均<0.01)；衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组、衰老0.2%DMAE组和衰老0.1%DMAE组真皮中胶原纤维的密度增加有显著性意义(P均<0.01)。然而,与正常对照组相比,所有衰老组的表皮、真皮厚度和胶原纤维密度的减少均有统计学意义(P均<0.01)。
3.含水量：各组之间皮肤含水量的差异无统计学意义(P均>0.05)。
4.羟脯氨酸含量：与其他衰老组相比,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组皮肤中羟脯氨酸含量增多有显著性意义(P均<0.05)。正常对照组羟脯氨酸含量与衰老0.2%DMAE+AA组的差异无显著性意义(P>0.05),衰老0.2%DMAE+AA组、衰老0.1%DMAE+AA组之间差异无显著性意义(P>0.05)。
5.胶原合成代谢的变化：
(1)Ⅰ型胶原蛋白mRNA表达水平：与衰老对照组相比,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组皮肤中Ⅰ型胶原蛋白mRNA表达水平升高有统计学意义(P均<0.01)。与正常对照组相比,衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组表达水平的差异无显著性意义(P均>0.05),而其他衰老组的降低有显著性意义(P均<0.01)。
(2)Ⅲ型胶原蛋白mRNA表达水平：与衰老对照组比较,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组、衰老0.2%DMAE组和衰老AA组皮肤中Ⅲ型胶原蛋白mRNA表达水平升高有显著性意义(P均<0.01)。而和正常对照组相比,所有衰老组的表达水平降低有统计学意义(P均<0.01)。
6.胶原分解代谢的变化：
(1)MMP-1mRNA表达水平：与衰老对照组相比,正常对照组、衰老0.1%DMAE+AA组、衰老0.2%DMAE+AA组皮肤中MMP-1mRNA表达水平升高有显著性意义(P均<0.01)。与正常对照组比较,衰老对照组、衰老NS组MMP-1mRNA表达水平降低有显著性差异(P均<0.05),而衰老AA组、衰老0.1%DMAE组、衰老0.2%DMAE组、衰老0.1%DMAE+AA组和衰老0.2%DMAE+AA组表达水平的差异无统计学意义(P均>0.05)。和衰老0.2%DMAE+AA组相比,正常对照组和衰老0.1%DMAE+AA组皮肤中MMP-1mRNA表达水平的差异无显著性意义(P均>0.05),而其他衰老各组表达水平的降低有显著性意义(P均<0.01)。与衰老0.1%DMAE+AA组比较,衰老对照组、衰老NS组和衰老AA组表达水平的降低有显著性意义(P均<0.05),而与其他组的差异无显著性意义(P均>0.05)。
(2)TIMP-1的mRNA表达水平：与衰老对照组比较,正常对照组TIMP-1的mRNA表达水平的升高有显著性意义,而其他衰老治疗组表达水平的差异无显著性意义(P均>0.05)。与正常对照组相比,7个衰老组TIMP-1的mRNA表达水平的降低有显著性意义(P均<0.01)。
7.成纤维细胞PCNA表达情况：与衰老对照组比较,正常对照组PCNA表达的升高有显著性意义(P均<0.01),而其他衰老治疗组PCNA表达的差异无显著性意义(P均>0.05)。与正常对照组相比,7个衰老组PCNA表达降低有显著性意义(P均<0.01)。
结论：
1.在大鼠颈背部皮下注射D-半乳糖125mg/Kg/d,连续42天,可使大鼠体型瘦小,体重减轻,被毛枯黄,精神萎靡、嗜睡,反应迟钝,活动减少,行动迟缓。组织学显示其表皮变薄,细胞层数减少；真皮变薄,皮肤胶原纤维排列疏松,密度降低,胶原束细长、断裂、走向平直。皮肤中胶原蛋白含量减少,胶原合成代谢和分解代谢均显著下降,成纤维细胞增殖亦显著降低。大鼠的大体形态、皮肤组织学、胶原代谢和细胞增殖状态的改变,均显示出老化的特征,提示D-半乳糖诱导的亚急性化学性衰老大鼠皮肤模型造模成功。
2.真皮内注射0.1%DMAE+AA、0.2%DMAE+AA两组复合溶液可使衰老大鼠皮肤的表皮和真皮厚度增加,真皮中胶原纤维密度增加,组织学上表现为表皮、真皮的增厚,胶原排列致密,具有延缓衰老大鼠皮肤老化的作用。
3. DMAE和AA对正常大鼠和衰老各组大鼠皮肤的保湿活性没有影响。
4.真皮内注射0.1%DMAE+AA和0.2%DMAE+AA能显著提高衰老大鼠皮肤中的羟脯氨酸含量,其中0.2%DMAE+AA的作用更为明显,可达到与正常大鼠相近的水平,提示该两组复合溶液可显著提高衰老大鼠皮肤中胶原蛋白的含量,具有抗老化的作用。
5.真皮内注射0.2%DMAE+AA、0.1%DMAE+AA显著上调Ⅰ型胶原蛋白mRNA表达水平,且与正常大鼠皮肤中Ⅰ型胶原蛋白mRNA水平无显著性差异,说明该两组复合药物注射于真皮中能增加衰老大鼠皮肤的Ⅰ型胶原蛋白的合成,可提高整体胶原中Ⅰ型胶原蛋白的比例,达到正常健康大鼠的水平,提示这两组复合药物溶液具有延缓皮肤老化的作用。
同时,尽管这两组复合药物溶液能显著提高衰老大鼠皮肤中的Ⅲ型胶原蛋白mRNA表达水平,但正常大鼠Ⅲ型胶原蛋白mRNA表达水平仍显著高于衰老各组的水平,与其他文献报道的衰老皮肤中Ⅲ型胶原蛋白含量和比例增加不一致,可能是因为D-半乳糖所致的亚急性化学性衰老的模型和正常生理性衰老的皮肤胶原代谢的特征不同所致。
6.真皮内注射0.2%DMAE+AA、0.1%DMAE+AA使衰老大鼠皮肤中MMP-1的]mRNA表达水平显著升高,且与正常大鼠的表达水平相近。而MMP-1的抑制剂TIMP-1在衰老各治疗与衰老对照组无显著性差别,均明显低于正常对照。说明该两组复合药物溶液注射于真皮中能促进衰老大鼠皮肤的胶原分解代谢,使老化、变性的胶原降解,以修复和重新构建衰老皮肤,接近或达到正常健康大鼠的组织结构,提示这两组复合药物溶液具有一定的抗衰老作用。
7.采用相同的治疗途径,单独在衰老大鼠真皮内注射0.2%DMAE、0.1%DMAE、AA、NS,在羟脯氨酸含量,以及Ⅰ型胶原蛋白、MMP-1mRNA表达水平与衰老对照组均无显著性差异,说明单用DMAE和AA无效,只有两者结合,才具有促进Ⅰ型胶原蛋白合成、老化和变性胶原分解,重塑皮肤结构,延缓皮肤老化的功效。
8.真皮内注射0.1%DMAE可使衰老真皮中胶原纤维密度增加,真皮内注射0.2%DMAE可使衰老大鼠表皮厚度和真皮中胶原纤维密度增加,提示0.1%DMAE和0.2%DMAE可能对衰老皮肤具有一定的刺激作用,浓度越高刺激性越强,使真皮中胶原纤维增粗,表皮增厚,而对胶原代谢没有影响。
9.真皮内注射0.2%DMAE+AA、0.1%DMAE+AA无诱导成纤维细胞增殖的作用,没有细胞过度增殖导致变异和癌变的隐患,仅通过改善成纤维细胞的胶原合成和分解代谢提高衰老大鼠皮肤中胶原蛋白的含量和增加Ⅰ型胶原蛋白mRNA表达而达到延缓皮肤衰老的作用,提示其抗衰老作用是安全的。
综上所述,本研究通过在D-半乳糖诱导的化学性衰老大鼠真皮内注射0.2%DMAE+AA、0.1%DMAE+AA发现,一方面该两组复合药物具有促进其Ⅰ、Ⅲ型胶原蛋白合成的作用,使羟脯氨酸含量和Ⅰ型胶原蛋白mRNA表达水平接近于正常生长的大鼠水平,增加衰老大鼠皮肤中Ⅰ型胶原含量,恢复年轻状态皮肤的胶原比例；另一方面,促进衰老大鼠皮肤中老化、变性胶原纤维的降解,重新塑造、构建衰老皮肤的组织结构,使之接近正常生长健康大鼠的状态。两方面共同作用使衰老的大鼠表皮、真皮厚度增加,胶原纤维丰富,排列致密,恢复年轻状态皮肤的组织结构特点,具有延缓衰老的作用；同时,这两组复合溶液没有诱导成纤维细胞增殖的作用,推测其抗衰老的作用主要是通过改善衰老皮肤的胶原代谢来实现,其安全性得到初步验证,但其深入的作用机制还有待于进一步的研究。
本研究依据Mesotherapy的治疗理念,在老化的大鼠皮肤真皮层内注射小剂量、低浓度的DMAE和AA复合溶液,探索其改善衰老皮肤胶原代谢和皮肤的组织结构的作用。该方法不需要大量口服全身用药,一方面减少药物的用量和浓度,降低治疗成本,同时能减少其相应的副作用和毒性；另一方面将药物直接运输至靶器官,克服了传统护肤产品不易透过表皮屏障,难于吸收的缺陷,使其抗衰老的疗效更加直接、显著。目前DMAE均为外用产品,而且已经报道临床应用具有稳定、显著、安全的抗衰老疗效,如果结合Mesotherapy的独特优势,将会具有更加直接、有效的延缓皮肤老化的作用。本研究将为DMAE进一步、多途径的抗衰老临床应用奠定一定的实验理论基础。
Keeping youth forever is an eternal topic and dream of human being. In the recent years, mesotherapy has been arousing everyone's interest as an anti-aging strategy. Pioneered by the French physician Dr. Michel Pistor in1952, Mesotherapy is a minimally invasive procedure that is widely used in Europe and elsewhere to treat various injuries and medical conditions. It was recognized by the French National Academy of Medicine in1986as an integral part of traditional medicine, and has been becoming highly popular within the public. In China, Mesotherapy was approved by Ministry of Health and CFDA. Mesotherapy, a safe, simple, less painful procedure which is one of the so-called "lunchtime cosmetic procedures", requires no recovery time and is perfect for professionals and successful people in the fast-paced modern life. Although there are a wide range of products of Mesotherapy, less foundimental reasearchs have been proceeded. Moreover, there is some controversy over its efficacy and safety.
DMAE is an analog of vitamin B choline that has been used to treat a number of conditions affecting the brain and central nervous system. There is some evidence that DMAE may be helpful for attention deficit hyperactivity disorder (ADHD). More widely marketed today as a memory and mood enhancer, DMAE is said to improve intellectual functioning. The basis for such claims probably stems from its purported ability to increase levels of a neurotransmitter called acetylcholine, although this has not been proven. Studies suggest that the skin is an active site of acetylcholine synthesis, storage, secretion, metabolism, and receptivity. The role of DMAE as a modulator of acetylcholine-mediated functions in the skin remains to be elucidated. DMAE is receiving more attention as a new skincare regimen today for its acute effects of anti-aging, anti-wrinkle and skin firmness. From three recent clinical reports,3%DMAE facial gel formula has been shown to be efficacious in the mitigation of forehead lines and periorbital fine wrinkles, and in improving lip fullness and shape and the overall appearance of facial skin. The safety concern of the long-term application of DMAE gel for up to1year has also been further documented.
Age-related skin changes are inevitable and include thinning, sagging, wrinkling, loss of elasticity and areas of dryness. Histological changes include dermal thinning, collagen thickening with degeneration and reduced amount of fibers, and elastin breaking down. In infant and young adult skin, type Ⅰ collagen is the most abundant protein in the dermis accounting for approximately80%, while type Ⅲ collagen is much less with about10%of dermal mass. When skin becomes aged, the collagen synthesis is reduced for type Ⅰ but up-regulated for type Ⅲ collagen, resulting in an inversed turnover of type Ⅰ/Ⅲ ratio in the skin. Though it is rarely seen in normal adult skin, increased expression of matrix Metalloproteinase-1(MMP-1) is a prominent feature of some pathological situations, such as wound healing, tissue repairing and remodeling. MMP activity is a key driver in wound healing to facilitate the motility of inflammatory cells and to enhance the availability of inflammatory mediators to set the stage for healing. However the tissue inhibitor of metalloproteinase (TIMP), a natural inhibitor of MMP, can restrain the activation of MMP and its activity in the wound healing process by forming a ternary complex with MMP.
Proliferating cell nuclear antigen (PCNA) was first found in serum of SLE patients by Miyachi et al, which only express in nomal proliferating cell or tumor cell. Subsequent studies showed that PCNA is close to DNA synthesis and play an important role in trigger of cell proliferation. It is a ideal indicator of state of cell proliferation.
In order to evaluate potential anti-aging effects of DMAE and compound Amino Acid in Mesotherapy, tissue structure and collagen metabolism of D-galactose (gal) induced aging skin were measured in this study. Their considered mechanism of action in the skin was also described.
Methods:Aging was artificially induced by subcutaneous injection with D-gal at the dose of125mg/kg.d for6weeks (42days).80Wistar rats were randomly divided into aging groups receiving D-gal treatment, including aging control group (D-gal for6weeks); NS (D-gal for6weeks+normal saline for4weeks); AA (D-gal for6weeks+compound amino acid for4weeks);0.1%DMAE (D-gal for6weeks+0.1%DMAE for4weeks);0.2%DMAE (D-gal for6weeks+0.2%DMAE for4weeks);0.1%DMAE+AA (D-gal for6weeks+0.1%DMAE/AA for4weeks);0.2%DMAE+AA (D-gal for6weeks+0.2%DMAE/AA for4weeks) and sham control group which was not given D-gal. At the end of each treatment, histologic changes of aging skin were observed; tissue water content and hydroxyproline were determined according to assay kits; PCNA stains of each group were observed by immunochemical methods; RT-PCR was used to detect mRNA expression of type I/III procollagen, MMP-1and TIMP-1. Simple PCI image analysis software were quantified the thickness of epidermis, dermis, collagen fibre density and PCNA positive stains. Data were expressed as means±standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS13.0software. The results were taken to be statistically significant at a probability level of p<0.05.
Results:
1.Animal:There were some aging phenomenon, such as lean and small, low weight, yellow and no luster fair, thinned skin, decreased activities, neurological impairment in aging group rats after rats were subcutaneous injected with D-gal at the dose of125mg/kg.d for6weeks.
2. Histological changes:When compared to sham control group, aging skin showed decreased thickness for both epidermis and dermis (P<0.05). With aging, the epidermis reduced its numbers of cell layers, and the dermal collagen fibers appeared sparse, slender or broken. Although the histologic changes of0.1%DMAE+AA group and0.2%DMAE+AA group were much better improved than that of aging control group, their indicators were all significantly lower than that of sham control group (P<0.01). In addition, when compared to aging control,0.2%DMAE alone also significantly increased epidermal thickness and density of collagen fiber (P<0.01), and0.1%DMAE increased epidermal thickness (P<0.01).
3. Water contents in aging skin:There was no statistically significant difference in water contents among all groups (P>0.05).
4. Hydroxyproline contents in aging skin:Hydroxyproline contents were apparently increased in aging skin treated with0.1%DMAE+AA or0.2%DMAE+AA complex (P<0.01), which showed a significant difference when compared to all other aging groups. Interestingly, hydroxyproline contents in0.2%DMAE+AA group reached an equivalent level to that of sham control group (P>0.05) even though both groups had much higher hydroxyproline contents than any of other6aging groups (P<0.05). There was no significant difference between0.1%DMAE+AA and0.2%DMAE+AA groups (P>0.05).
5.Changes of collagen synthesis:
(1)Collagen type I expression was greatly increased in response to the treatment of both0.1%DMAE+AA and0.2%DMAE+AA complex (P<0.01). Although there was no statistically significant difference between these two groups and sham control (P>0.05), all other aging groups showed much less expression of collagen type I (P <0.01).
(2) When treated with0.1%DMAE+AA,0.2%DMAE+AA or0.2%DMAE alone, the mRNA expression for collagen type III was also considerably elevated over the aging control group (P<0.01), but still significantly less than sham control (P<0.01).
6.Chagens of collagen catabolism:
(1) MMP-1, a potential key regulator in aging skin, was also observed in this study, and results showed that its mRNA expression significantly increased in rats skin tissue treated with either0.1%DMAE+AA or0.2%DMAE+AA. These up-regulations of MMP-1activity showed a statistically significant difference to that of aging control group (P<0.01), but not to sham control(P>0.05).
(2) In contrast, the transcripts for TIMP-1of all aging groups was down regulated and reached significantly lower levels to that of sham control (P<0.01).
7. Expression of PCNA:PCNA expression of sham control is higher than all aging groups (P<0.01), and there were no difference among every aging group(P>0.05).
Conclusion:
1. Mesotherapy with combined application of DMAE and compound AA could improve the thickness of aging skin, and play an anti-aging role.
2. Both DMAE and compound AA may have limited effects on skin moisturizing which suggests that DMAE and compound AA had limited effects on skin moisturizing. This might be due to a lower level of concentration and shorter action period of local available DMAE in epidermal layer when direct subcutaneous injection was used for DMAE delivery, which differs from tropical application of DMAE.
3. Mesotherapy with combined application of DMAE and compound AA could increase collagen contents of aging skin, especially, effect of0.2%DMAE+AA could be better which increased hydroxyproline contents is equal to sham control.
4. Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting collagen type I synthesis.
5. Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting catabolism to remodel skin texture.
6. Intradermal injection of DMAE or AA alone showed no effects on hydroxyprolin contents or messages for collagen type I and MMP-1in the aging skin. These data indicate that combined application of DMAE and AA is the only way to play their anti-aging action in this D-gal induced aging skin model by modulating collagen type I metabolism and remolding the structure of aging skin.
7. Intradermal injection of0.1%DMAE and0.2%DMAE did increase the dermal collagen fiber density over the aging control group. This result suggest that0.1%DMAE and0.2%DMAE treatment may potentially trigger aging skin to thicken the collagen fibre, but not apparently affect dermal collagen metabolism. Previous studies have shown that cultured rabbit dermal fibroblasts responded to DMAE by massive vacuolization in a concentration-dependent manner. The epidermis of rabbit external ear was also significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to tropical application of3%DMAE. It was suggested that vacuolar cytopathology may not be dissociable from the improvement of skin appearance that is rapidly produced by topically administered DMAE, and could be the cellular basis of the anti-wrinkle effect of DMAE. From our results, the vacuolization of the dermal fibroblast might occur in both0.1%and0.2%DMAE treated aging skin, resulting in cellular swelling and increased collagen fiber density. Epithelial cells of aging skin could also respond to the higher concentration of0.2%DMAE by vacuolization, which presented as increased epidermal thickness. However, these may need further studies to clarify this matter.
8. Mesotherapy with0.2%DMAE+AA,0.1%DMAE+AA,0.2%DMAE,0.1%DMAE, AA, NS could not effect expression of PCNA.
Mesotherapy caused minimum invasion and discomfort, without any posttreatment pain. All patients can return to their everyday activities immediately after treatment. In present study mesotherapy with DMAE+AA compound solution were administered directly and accurately into the region to be treated, and effect of medicines are more immediate and effective at minimum dose with the advantage of avoiding side-effects accompany with systemic administration at high dose.
In conclusion, Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting collagen synthesis and catabolism to remodel skin texture, and improve the thickness of aging skin. At the same time, complex of DAMA and AA could not induce fibroblast proliferation. This new strategy shows a promising potential in slowing skin aging in D-gal induced aging model, while further investigations should be conducted to demonstrate the biological bases of its anti-aging effects.
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