CN117064896A - Cdk5抑制剂在制备皮肤创伤愈合药物中的应用 - Google Patents
Cdk5抑制剂在制备皮肤创伤愈合药物中的应用 Download PDFInfo
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Abstract
本发明提供了一种CDK5抑制剂在制备皮肤创伤愈合药物中的应用，属于药物技术领域。本发明提供的CDK5抑制剂在制备皮肤创伤愈合药物中的应用，通过使用CDK5抑制剂，促进了肺泡巨噬细胞的杀菌、抗炎等多种潜在生物学作用，使用含有CDK5抑制剂的缓释凝胶外涂小鼠皮肤伤口，愈合进程显著加快，说明抑制CDK5活性可以发挥抗炎和促进创伤皮肤组织修复的作用。
Description
技术领域
本发明涉及药物技术领域，特别涉及CDK5抑制剂在制备皮肤创伤愈合药物中的应用。
背景技术
皮肤创面愈合是临床常见的棘手问题，目前仍然缺乏较好的治疗方法，了解其潜在分子机制并开发有效的治疗靶点和药物十分迫切。皮肤创面愈合的复杂生理特征包括：炎症、细胞增殖和组织重塑三个阶段，其中早期炎症阶段主要是炎性细胞浸润并发挥抗感染和清洁伤口，为组织修复提供清洁环境的作用，其中巨噬细胞在炎症期和增殖期以及肉芽组织的形成中发挥关键作用。巨噬细胞极化往往与皮肤伤口愈合的生理进程相关，主要分为促炎性M1(表达炎症相关因子IL-1β、TNF-α等)和抗炎组织修复型M2(表达IL-10、CD163等)两种表型，当巨噬细胞由M1向M2表型转换时将抑制炎症反应并促进组织修复。新近研究发现，巨噬细胞极化与皮肤伤口愈合密切相关。
丝氨酸/苏氨酸细胞周期素依赖蛋白激酶5(CDK5)是细胞周期蛋白激酶家族中的一个非典型成员，其主要功能是参与中枢神经系统的发育、功能和相关疾病。近年来研究发现，巨噬细胞中CDK5表达介导LPS诱导的NF-κB激活参与炎症反应；与此同时，CDK5在多种肿瘤细胞中也发挥促细胞增殖的作用。目前，在皮肤创伤愈合过程中，CDK5信号是否发生紊乱，介导巨噬细胞的表型转化并影响皮肤创面修复的研究仍未见报道。
发明内容
有鉴于此，本发明目的在于提供CDK5抑制剂在制备皮肤创伤愈合药物中的应用，本发明提供的CDK5抑制剂在制备皮肤创伤愈合药物中的应用，可显著加快皮肤愈合进程，促进创伤皮肤的组织修复。
为了实现上述目的，本发明提供以下技术方案：
CDK5抑制剂在制备皮肤创伤愈合药物中的应用。
优选地，所述CDK5抑制剂为Roscovitine。
优选地，所述皮肤创伤包括但不限于炎症伤口、刀伤、溃烂、糖尿病并发症溃烂、烧伤、烫伤、冻伤。
优选地，所述CDK5抑制剂在药物中的浓度为1.5g/L～2.5g/L。
优选地，所述Roscovitine可制备成缓释凝胶，所述缓释凝胶的制备包括以下步骤：
S1、将PF127粉末溶于缓冲溶液中，4℃搅拌至澄清，得到PF127溶液，4℃保藏；
S2、将Roscovitine溶解于无水乙醇中，得到Roscovitine溶液；
S3、将Roscovitine溶液与PF127溶液混合，4℃搅拌过夜，22℃～37℃下静置，得到Roscovitine缓释凝胶。
优选地，所述S1中的PF127溶液的浓度为0.2g/mL～0.4g/mL，缓冲溶液为PBS缓冲液。
优选地，所述S2中的Roscovitine溶液的浓度为0.4mg/mL～0.6mg/mL。
优选地，所述S3中的Roscovitine溶液与PF127溶液的体积比为(1～2)：(23～25)。
有益技术效果：本发明提供了一种CDK5抑制剂在制备皮肤创伤愈合药物中的应用，本发明通过使用CDK5抑制剂，促进了肺泡巨噬细胞的杀菌、抗炎等多种潜在生物学作用，使用含有CDK5抑制剂的缓释凝胶外涂小鼠皮肤伤口，愈合进程显著加快，说明抑制CDK5活性可以发挥抗炎和促进创伤皮肤组织修复的作用。
附图说明
图1为CDK5表达和磷酸化修饰参与小鼠皮肤创伤愈合过程；其中图1A为不同时间点WT小鼠皮肤伤口愈合变化(第0d、3d、5d、7d)；图1B为第0d、3d、5d、7d皮肤伤口组织中p-CDK5和CDK5蛋白的表达，每组n＝5只，*P<0.05、**P<0.01与0d比较，Scale bar＝0.2cm；
图2为CDK5介导炎症过程、血管增生和组织重塑调节皮肤创伤愈合；其中，图2A左图为不同组小鼠在不同时间点(第0d、3d、5d、7d)皮肤伤口愈合变化，Scale bar＝0.2cm；图2A右图为不同时间点下定量统计结果，每组n＝5，*P<0.05、**P<0.01与WT比较；图2B左图代表性免疫组化染色不同组小鼠第5d伤口皮肤组织中α-SMA(成纤维细胞)、MAC-3(巨噬细胞)和CD31(内皮细胞)表达，图2B右图为定量分析两组小鼠第5d皮肤伤口愈合组织中α-SMA(成纤维细胞)、MAC-3(巨噬细胞)和CD31(内皮细胞)阳性表达细胞数；每组n＝5只，**P<0.01与WT组比较，Scale bar＝2μm；HPF：高倍视野，即：目镜十倍，物镜四十倍的情况下所观察到范围；
图3为CDK5表达缺失诱导M2型巨噬细胞极化并促进伤口愈合；其中，图3A左图为两组小鼠皮肤损伤后0d、3d、5d、7d的愈合情况，Scale bar＝0.2cm；图3A右图为两组小鼠不同时间点下定量统计结果，每组n＝5只，*P<0.05与WT比较；图3B为Westernblot检测两组小鼠第5d皮肤伤口愈合组织中IL-1β和CD163的蛋白表达；图3C左图为免疫双荧光染色观察两组小鼠第5d伤口处IL-1β(红色)和CD163(绿色)的原位表达；图3C右图为2组IL-1β和CD163阳性细胞百分比定量统计结果；每组n＝5只，*P<0.05、**P<0.01与WT组比较，Scalebar＝1μm；
图4为CDK5活性抑制促进小鼠皮肤伤口愈合；其中，图4A为Roscovitine干预小鼠背部全层皮肤切割伤模型示意图；图4B为比较两组小鼠不同时间点下皮肤伤口愈合情况；每组n＝5只，*P<0.05与Saline组相比，Scale bar＝0.2cm。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。本发明实施例及试验例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得；本发明实施例及试验例中所用的方法，如无特殊说明，均为常规方法。
实施例1
利用Pluronic F127制备Roscovitine皮肤外涂药物缓释凝胶。称固体PF127粉30g，溶于PBS中，4℃搅拌至澄清，定容至100mL。将Roscovitine50mg先溶解于1mL无水乙醇中，混合于24mLPluronie F127凝胶中，4℃搅拌过夜，37℃静置，制成终浓度为2g/L的Roscovitine缓释凝胶备用。
实施例2
利用Pluronic F127制备Roscovitine皮肤外涂药物缓释凝胶。称固体PF127粉20g，溶于PBS中，4℃搅拌至澄清，定容至100mL。将Roscovitine60mg先溶解于1mL无水乙醇中，混合于23mLPluronie F127凝胶中，4℃搅拌过夜，22℃静置，制成终浓度为2.5g/L的Roscovitine缓释凝胶备用。
实施例3
利用Pluronic F127制备Roscovitine皮肤外涂药物缓释凝胶。称固体PF127粉40g，溶于PBS中，4℃搅拌至澄清，定容至100mL。将Roscovitine40mg先溶解于1mL无水乙醇中，混合于25mLPluronie F127凝胶中，4℃搅拌过夜，30℃下静置，制成终浓度为1.5g/L的Roscovitine缓释凝胶备用。
试验例1
1材料与方法
1.1实验材料
1.2实验动物及分组
采用6w～8w、雄性C57BL/6J背景的全身基因CDK5敲除杂合子KO-CDK5+/-小鼠和巨噬细胞特异性敲除myeCDK5-/-小鼠以及同笼阴性小鼠(wild type，WT)作为对照，构建小鼠背部全层皮肤切割伤模型。实验小鼠来自赛业模式生物研究中心有限公司[SYXK(苏)2018-0003]，小鼠实验符合河北省中医学院伦理审查(DWLL202203068)。实验分为5组：WT、myeCDK5-/-、Roscovitine和Saline组，每组5只。4％水合氯醛腹腔注射麻醉后脱毛，行全层约1cm×1cm大小的方形皮肤切除，建立皮肤创伤模型。在第0d、3d、5d、7d对伤口进行拍照，使用ImageJ软件测量伤口面积。损伤后第5d取皮肤组织进行总蛋白提取或石蜡包埋。
1.3Western blot分析
取小鼠皮肤伤口愈合组织20mg，加入蛋白裂解液200μL和蛋白酶抑制剂2μL后，4℃研磨皮肤组织并提取总蛋白，BCA法测定蛋白质浓度。40μg总蛋白上样量10％胶浓度SDS-PAGE电泳，半干转至PVDF膜，5％脱脂牛奶封闭，一抗p-CDK5，CDK5，IL-1β，CD163和β-actin孵育过夜(稀释度均为1:1000)。洗膜，二抗室温孵育1h，洗膜，ECL化学发光，Image J软件进行光密度扫描分析蛋白条带。
1.4免疫组织化学染色
皮肤愈合组织在4℃下用4％多聚甲醛固定过夜，梯度脱水并石蜡包埋。5μm石蜡切片，烤片，脱蜡水化，抗原修复后3％H2O2室温孵育3min，10％山羊血清封闭25min，一抗(抗α-SMA、抗MAC-3、抗CD31，稀释比例1:150)4℃孵育过夜。PBS洗涤三次，二抗室温孵育1h，DAB显色后苏木精复染1min，乙醇梯度脱水，二甲苯透明后封片。Leica显微镜高倍镜下(×400)采集图像，ImageJ软件分析α-SMA、MAC-3和CD31阳性细胞百分比。
1.5组织免疫双荧光染色
取皮肤愈合组织浸泡4％多聚甲醛4℃固定30min，转移到30％蔗糖溶液中过夜，OCT胶冷冻包埋，5μm冰冻切片后恢复至常温，PBS洗涤后10％山羊血清室温封闭1h，一抗(鼠源IL-1β抗体，兔源CD163抗体，稀释比例1:100)4℃下共同孵育过夜，PBS洗涤5遍，二抗室温孵育1h，PBS洗片，用含DAPI封片剂封片。Leica荧光显微镜高倍镜下(×400)拍照，ImageJ软件分析IL-1β和CD163阳性细胞百分比。
1.6Roscovitine缓释凝胶
取实施例1～3所得的Roscovitine缓释凝胶，用于涂抹小鼠皮肤伤口。
1.7统计学分析
应用GraphpadPrism 7.0统计软件，采用Gaussian分布法对数据进行正态性检验，符合正态分布的计量资料以x±s表示，多组间比较采用单因素方差分析(ANOVA)，进一步两两比较采用Bonferroni检验，双侧检验P<0.05为差异有统计学意义。
2结果
2.1CDK5通过磷酸化参与皮肤创伤愈合过程
为了证实CDK5信号紊乱是否参与皮肤创伤愈合过程，本发明首先构建了小鼠背部全层皮肤切割伤模型，并观察皮肤创伤后的愈合情况。小鼠创伤后愈合情况如图1A所示。由图1A可以看出，WT小鼠经皮肤创伤手术后，第7d伤口自然愈合面积达80％左右。收集创伤后不同时间点(第0d、3d、5d、7d)的皮肤愈合组织，westernblot检测其中CDK5的蛋白及其磷酸化修饰水平，结果如图1B所示。由图1B可以看出，在皮肤创伤愈合过程中CDK5表达及其磷酸化水平均显著升高(P<0.01)，第5d达到高峰，之后稍有降低，这些结果表明CDK5信号紊乱可能参与小鼠皮肤创伤愈合。
2.2CDK5介导炎症过程、血管增生和组织重塑调节皮肤创伤愈合
为了探讨CDK5表达在小鼠皮肤伤口愈合中的作用，本发明采用KO-CDK5+/-小鼠构建背部全层皮肤切割伤模型，观察CDK5表达对皮肤创伤愈合的影响。结果显示，与WT比较发现，KO-CDK5+/-组小鼠皮肤创伤愈合进程明显减缓，且在第5d差异最为显著(WT：43±5％vs.KO-CDK5：59±4％，P<0.01，图2A)。免疫组化染色小鼠皮肤创伤愈合第5d组织中α-SMA、MAC-3、CD31阳性细胞的表达，结果显示KO-CDK5+/-组小鼠皮肤愈合组织中MAC-3阳性细胞数多于WT组(WT：11±2vs.KO-CDK5+/-：15±2per HPF)，而α-SMA(WT：30±2vs.KO-CDK5+/-：15±1per HPF，P<0.01)和CD31(WT：22±3vs.KO-CDK5+/-：7±1per HPF，P<0.01)阳性细胞数显著低于WT组(图2B)。这些结果表明CDK5表达在小鼠皮肤伤口愈合过程中发挥重要作用。
2.3CDK5缺失诱导M1型巨噬细胞向M2型巨噬细胞表型转换并促进伤口愈合
采用myeCDK5-/-小鼠构建背部全层皮肤切割伤模型并观察小鼠皮肤伤口愈合情况。结果显示，与WT组比较，myeCDK5-/-组小鼠皮肤伤口愈合进程加快(WT：23％±3％vs.myeCDK5-/-：8％±3％，P<0.01，图3A)。IL-1β作为早期促炎因子参与巨噬细胞、内皮细胞和成纤维细胞的增殖及基质合成，随后活化的成纤维细胞迁移到伤口处作为支架，内皮细胞的血管新生供给营养并最终形成肉芽组织，而CD163是M2型巨噬细胞的特征标记蛋白。因此，Western blot检测二者表达结果显示，与WT组比较，myeCDK5-/-组小鼠皮肤伤口愈合组织中IL-1β表达减少(WT：2.5±0.2vs.myeCDK5-/-：1.4±0.2，P<0.01)，而CD163蛋白的表达显著升高(WT：1.0vs.myeCDK5-/-：2.7±0.2，P<0.01)(图3B)；与此同时，免疫荧光共染色IL-1β(红色)和CD163(绿色)阳性细胞数统计结果与Western blot结果一致，IL-1β：WT(25％±1％)vs.myeCDK5-/-(14％±2％)；CD163：WT(10％±3％)vs.myeCDK5-/-(27％±2％)，均P<0.01，如图3C所示。这些结果表明巨噬细胞CDK5表达在伤口愈合过程中发挥促炎作用，而其表达抑制促进巨噬细胞从M1向M2的功能表型转换。
2.4CDK5抑制剂Roscovitine能够加快创伤愈合进程
为了验证CDK5信号调节在小鼠皮肤伤口愈合中的作用，本发明采用Roscovitine缓释凝胶0.5mL(终浓度为2g/L)均匀涂抹小鼠皮肤创面进行干预(图4A)，观察CDK5激酶活性在皮肤伤口愈合过程中的作用。实验结果显示，与Saline组比较，Roscovitine组小鼠皮肤伤口愈合明显加快(Saline：38％±4％vs.Roscovitine：24％±3％，P<0.05，图4B)。
3.讨论
往往过激的炎症反应会导致皮肤伤口愈合减缓甚至损伤加重，而M2抗炎修复型巨噬细胞对皮肤损伤后的快速修复以及内稳态的维持至关重要。本研究发现在小鼠皮肤伤口愈合过程中CDK5信号改变并发挥重要作用，主要结果包括：1)皮肤伤口愈合组织中CDK5蛋白表达及其磷酸化修饰水平均升高；2)巨噬细胞中CDK5表达抑制促进小鼠皮肤伤口愈合与M2型转换相关；3)CDK5活性抑制同样促进小鼠皮肤伤口愈合。据此推测，CDK5信号紊乱影响巨噬细胞M1向M2转换和皮肤伤口的愈合。
CDK5是细胞增殖和凋亡平衡调节的关键子，不仅在中枢神经系统中起作用，而且在炎症、新生血管形成、上皮细胞和癌细胞的迁移、肌生成、糖代谢和胰岛素分泌等生理病理过程中发挥重要作用。本研究发现CDK5蛋白及其磷酸化修饰水平在皮肤创伤愈合过程中升高，第5d达到峰值，提示其信号变化与伤口愈合进程相关。由于CDK5激酶在多种组织细胞类型中均发挥重要作用，包括：内皮细胞和小鼠胚胎成纤维细胞，因此，本发明也观察到了CDK5表达剂量不足型KO-CDK5+/-小鼠皮肤伤口愈合进展缓慢的现象，并且采用免疫组化染色三种关键细胞(巨噬细胞、内皮细胞和成纤维细胞)的结果显示，血管新生和肌纤维细胞分化均显著减少(CD31：7±1vs.22±3；α-SMA：15±1vs.30±2，P<0.01)；值得注意的是，MAC3阳性染色结果并未发现巨噬细胞迁移数量的明显改变，提示在皮肤伤口愈合进程中，CDK5表达可能并不影响巨噬细胞的增殖或迁移。
因此，本发明采用myeCDK5-/-小鼠构建创伤模型并观察皮肤伤口愈合情况，结果显示与WT组比较，myeCDK5-/-小鼠皮肤伤口愈合加速。通过检测皮肤愈合组织中IL-1β和CD163表达以及原位荧光染色证实，M1型巨噬细胞特异蛋白IL-1β较对照组显著减少，而M2型特异蛋白CD163表达相对增多，即浸润的巨噬细胞发生了M1向M2的功能表型转换(从促炎表型向抗炎表型的转变)，增强了创伤组织修复的功能，提示巨噬细胞CDK5表达缺失促进皮肤创伤愈合。这些结果表明，CDK5可能是治疗皮肤创伤愈合的潜在靶点。临床研究显示，CDK5活性化学抑制剂Roscovitine具有促进肺泡巨噬细胞的杀菌、抗炎等多种潜在生物学作用，提示特异性小分子Roscovitine干预可能具有潜在治疗小鼠皮肤伤口愈合的作用。正如所期望的，本发明结果同样发现，Roscovitine伤口外涂组小鼠皮肤伤口愈合进程显著加快，提示抑制CDK5活性同样发挥抗炎和促进创伤皮肤组织修复的作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.CDK5抑制剂在制备皮肤创伤愈合药物中的应用。
2.根据权利要求1所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述CDK5抑制剂为Roscovitine。
3.根据权利要求1所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述皮肤创伤包括但不限于炎症伤口、刀伤、溃烂、糖尿病并发症溃烂、烧伤、烫伤、冻伤。
4.根据权利要求1所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述CDK5抑制剂在药物中的浓度为1.5g/L～2.5g/L。
5.根据权利要求1所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述Roscovitine可制备成缓释凝胶，所述缓释凝胶的制备包括以下步骤：
S1、将PF127粉末溶于缓冲溶液中，4℃搅拌至澄清，得到PF127溶液，4℃保藏；
S2、将Roscovitine溶解于无水乙醇中，得到Roscovitine溶液；
S3、将Roscovitine溶液与PF127溶液混合，4℃搅拌过夜，22℃～37℃下静置，得到Roscovitine缓释凝胶。
6.根据权利要求5所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述S1中的PF127溶液的浓度为0.2g/mL～0.4g/mL，缓冲溶液为PBS缓冲液。
7.根据权利要求5所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述S2中的Roscovitine溶液的浓度为0.4mg/mL～0.6mg/mL。
8.根据权利要求5所述CDK5抑制剂在制备皮肤创伤愈合药物中的应用，其特征在于，所述S3中的Roscovitine溶液与PF127溶液的体积比为(1～2)：(23～25)。
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| WO2013169740A2 (en) * | 2012-05-08 | 2013-11-14 | Trustees Of Dartmouth College | Synthetic triterpenoids and methods for modulating stem/progenitor cell gene expression |
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