CN117414356A - 6-姜酚在制备促进毛囊乳突细胞衰老产品中的应用 - Google Patents
6-姜酚在制备促进毛囊乳突细胞衰老产品中的应用 Download PDFInfo
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Abstract
本发明涉及一种6‑姜酚的新用途,具体涉及6‑姜酚在制备促进毛囊乳突细胞衰老产品中的应用,属于生物医药领域。本发明通过实验发现6‑姜酚能够通过抑制毛囊乳突细胞线粒体自噬,促进毛囊乳突细胞衰老,进而抑制毛囊乳突细胞增殖,抑制毛发生长。同时发现6‑姜酚处理后毛囊根鞘外泌体能通过传递LncRNA H19,抑制Pink1/Parkin介导的线粒体自噬,进而促进毛囊乳突细胞衰老。本发明可为临床治疗多毛症或毛发过多提供新的潜在分子靶点,具有非常好的临床应用潜力,可为抑制毛发生长提供新的治疗手段。
Description
技术领域
本发明涉及一种6-姜酚的新用途,具体涉及6-姜酚在制备促进毛囊乳突细胞衰老产品中的应用,属于生物医药领域。
背景技术
6-姜酚是生姜中含量最为丰富的一类活性成分目前已经确定其具有抗氧化、抗肿瘤、抗炎、强心、降压、降血脂、降血糖、抗凝等多种生物学活性。其中6-姜酚抗肿瘤的作用最受关注。其药理作用显示其具有散寒发汗、化痰止咳、和胃、止呕等作用,6-姜酚能刺激胃肠粘膜,使胃肠道充血,消化能力增强,能有效的治疗吃寒凉食物过多而引起的腹胀、腹痛、腹泻、呕吐等。还具有抗凝、抗氧化、升压强心、降血脂、抗动脉粥样硬化、保肝利胆、消炎、镇咳、防晕止吐、对中枢神经抑制等多种功效,杀虫、抗病原微生物和杀菌防腐作用。
中国科技文献“6-姜酚对毛发生长影响的实验研究”探讨了生姜提取物6-姜酚对毛发生长的影响,其发现6-姜酚可抑制毛发生长,其机制可能是通过增加DPCs中Bax/Bcl-2蛋白的比值促进DPCs凋亡而实现。
中国发明专利CN115554380B公开了一种通过作用于毛囊组织防治脱发的组合物及其用途,所述组合物由黑桑根提取物、姜提取物和荚膜黄芪根提取物组成。本发明还提供了上述组合物在制备提高毛囊组织活性的产品中的用途,将所述产品直接涂抹或喷洒到头皮上促使休止期的毛囊进入再生周期,促进毛发生长。该发明专利还提供了上述组合物在制备提高体外培养的毛囊组织活性的药物中的用途,将所述组合物制成组织培养液作用于体外培养毛囊组织能够提高培养的毛囊组织的活性,显著提高移植毛囊的存活率。
发明内容
本发明通过实验发现6-姜酚能够通过抑制毛囊乳突细胞线粒体自噬,促进毛囊乳突细胞衰老,进而抑制毛囊乳突细胞增殖,抑制毛发生长,同时发现6-姜酚处理后毛囊根鞘外泌体能通过传递LncRNA H19,抑制Pink1/Parkin介导的线粒体自噬,进而促进毛囊乳突细胞衰老。基于此,提出一种6-姜酚的新用途。
本发明实施例1研究了6-姜酚处理与LncRNA H19表达、线粒体自噬以及毛囊乳突细胞衰老的相关性。实验中对小鼠背部进行脱毛,采用1mg/mL的6-姜酚涂抹处理,10天后检测小鼠背部毛发生长状况,检测毛囊长度和毛囊数量,检测毛囊衰老情况,检测毛囊组织中LncRNA H19表达,检测毛囊组织线粒体自噬水平。通过以上实验探究6-姜酚处理与LncRNAH19表达、线粒体自噬以及毛囊乳突细胞衰老的相关性。6-姜酚处理后毛囊根鞘外泌体通过传递LncRNA H19,抑制Pink1/Parkin介导的线粒体自噬,进而促进毛囊乳突细胞衰老。具体实验结果如下:
(1)采用β-半乳糖苷酶染色试剂盒检测小鼠毛囊衰老情况,发现6-姜酚组的β-半乳糖苷酶检测值显著高于对照组,这表明,6-姜酚组可以显著促进细胞衰老。
(2)通过qPCR检测皮肤组织中LncRNA H19水平,结果发现6-姜酚组的皮肤组织中LncRNA H19表达水平均显著高于对照组;通过Western blot检测线粒体自噬相关蛋白Pink1、Parkin、LC3II/I表达水平以及Co-IP检测Parkin磷酸化水平显示,6-姜酚组的P-parkin水平显著低于对照组。
本发明实施例2中通过体外分离培养毛囊外根鞘细胞,进行6-姜酚处理并分离外泌体。采用外泌体处理毛囊乳突细胞的同时干预毛囊乳突细胞线粒体自噬水平,检测毛囊乳突细胞增殖、凋亡与衰老情况。通过以上实验揭示6-姜酚后毛囊外根鞘外泌体可通过向毛囊乳突细胞传递LncRNA H19抑制毛囊乳突细胞线粒体自噬,进而促进毛囊乳突细胞衰老,抑制毛发生长。
本发明实施例3研究了6-姜酚对毛囊外根鞘细胞外泌体LncRNA H19的调控的体外实验,通过体外分离培养毛囊外根鞘细胞进行6-姜酚处理,检测毛囊外根鞘细胞中LncRNAH19表达水平;分离培养毛囊外根鞘外泌体,检测毛囊外根鞘外泌体中LncRNA H19表达水平;分离培养毛囊乳突细胞,采用毛囊外根鞘外泌体处理,检测毛囊乳突细胞增殖、凋亡、衰老情况;检测毛囊乳突细胞线粒体自噬水平;在毛囊乳突细胞中过表达LncRNA H19,检测毛囊乳突细胞增殖、凋亡与衰老;检测毛囊乳突细胞线粒体自噬水平。实验结果如图1所示,通过以上实验明确6-姜酚可以通过调控毛囊外根鞘细胞外泌体LncRNA H19促进毛囊乳突细胞衰老。
基于上述实验研究成果,本发明具体技术方案如下:
本发明请求保护一种6-姜酚的新应用,具体为6-姜酚在制备促进毛囊乳突细胞衰老产品中的应用。
如上所述的6-姜酚的应用,所述的促进毛囊乳突细胞衰老产品为包含6-姜酚的外用制剂。本领域技术人员在熟知本发明所述的应用下,单独或者与其他组分配伍制备成外用制剂为本领域的常规技术。
如上所述的6-姜酚的应用,6-姜酚能够抑制毛囊乳头状细胞增殖并促进细胞凋亡。
如上所述的6-姜酚的应用,所述的促进毛囊乳突细胞衰老产品用于治疗多毛症或毛发过多。
如上所述的6-姜酚的应用,所述的促进毛囊乳突细胞衰老产品中6-姜酚的作用靶点为毛囊外根鞘细胞外泌体LncRNA H19水平。6-姜酚促进毛囊乳突细胞衰老通过LncRNAH19介导,6-姜酚通过传递LncRNA H19抑制毛囊乳突细胞增殖同时促进毛囊乳突细胞凋亡。
如上所述的6-姜酚的应用,6-姜酚的作用机制为通过促进毛囊根鞘细胞内及毛囊根鞘细胞外泌体中LncRNA H19的表达,升高毛囊外根鞘细胞外泌体LncRNA H19水平,进而抑制Pink1/Parkin介导的线粒体自噬,进而抑制毛囊乳突细胞增殖同时促进毛囊乳突细胞凋亡。
本发明相对于现有技术的创新点主要包括以下三点:
1.本发明通过深入探究天然药物单体6-姜酚抑制毛发生长的具体机制,可为临床治疗多毛症或毛发过多提供新的潜在分子靶点,具有非常好的临床应用潜力。
2.本发明探究了6-姜酚能通过调控毛囊根鞘外泌体内容物含量,进而促进毛囊乳突细胞的衰老,可为抑制毛发生长提供新的治疗手段。本发明实施例1-3证明,6-姜酚通过调控毛囊外根鞘细胞外泌体LncRNA H19促进毛囊乳突细胞衰老。6-姜酚促进毛囊根鞘细胞内及毛囊根鞘细胞外泌体中LncRNA H19的表达。6-姜酚通过促进后毛囊外根鞘外泌体向毛囊乳突细胞传递LncRNA H19抑制毛囊乳突细胞线粒体自噬,进而促进毛囊乳突细胞衰老。
3.LncRNA在毛发生长中的作用尚未明确,本发明探究了LncRNA H19能通过与靶蛋白互作,发现其可以影响毛囊乳突细胞线粒体自噬,可为后续LncRNA在毛发生长中的相关研究提供新的思路,具有非常好的创新价值。
附图说明
图1为6-姜酚对毛囊乳突细胞自噬及衰老的影响。其中(A-B)ELISA检测毛囊乳突细胞中β-Gal表达;(B)RT-PCR检测毛囊乳突细胞中H19表达水平;(C-D)WB检测毛囊乳突细胞中Parkin,LC3II,LC3I及p-Parkin的蛋白水平。
图2为6-姜酚对毛囊根鞘细胞及毛囊根鞘细胞外泌体中H19表达影响。其中(A)RT-PCR检测毛囊根鞘细胞中H19表达;(B)WB检测外泌体标记物HSP70,CD63及Tag101;(C)RT-PCR检测毛囊根鞘细胞外泌体中H19表达。
图3为H19介导6-姜酚抑制毛囊乳突细胞自噬。其中(A)RT-PCR检测毛囊根鞘细胞外泌体H19表达;(B-C)WB检测毛囊乳突细胞中Parkin,LC3II,LC3I及p-Parkin的蛋白水平。
图4H19介导6-姜酚抑制毛囊乳突细胞增殖和凋亡,其中(A)ELISA检测毛囊乳突细胞中β-GAL表达;(B)CCK8检测毛囊乳突细胞增殖;(C)流式细胞术检测毛囊乳突细胞凋亡水平。
具体实施方式
以下通过具体实施例进一步描述本发明,但所述领域的技术人员应能知晓,所述实施例并不以任何方式限定本发明专利保护的范围,本发明专利保护的范围以权利要求书限定的范围为准。
实施例1 6-姜酚处理与LncRNA H19表达、线粒体自噬以及毛囊乳突细胞衰老的相关性研究
1)研究对象
对12只8周龄C57BL/6小鼠背部毛发进行脱毛,随机将脱毛小鼠分为对照组和6-姜酚组。脱毛24h后在背部脱毛区域分别涂抹1mg/ml的6-姜酚和溶剂,每次涂抹0.2ml,每天2次,连续10d,观察小鼠背部皮肤及毛发变化情况。停药10d后分别从每只小鼠背部皮肤中随机拔除5根毛干测量其长度。
2)处理与分组
小鼠随机分成两组:
①对照组:涂抹0.2ml溶剂(乙醇),每天2次,连续10天。
②6-姜酚组:涂抹0.2ml 1mg/ml的6-姜酚,每天2次,连续10天。
3)检测指标
①统计小鼠背部新生毛发长度;
定期拍摄小鼠脱毛部位照片,然后根据以下公式通过图像分析评估毛发再生长:%毛发再生长=多毛(黑色)面积/脱毛面积。
②取小鼠背部皮肤,HE染色观察毛囊形态,统计毛囊数量与长度;
③采用β-半乳糖苷酶染色试剂盒检测小鼠毛囊衰老情况;
④qPCR检测皮肤组织中LncRNA H19水平;
⑤Western blot检测线粒体自噬相关蛋白Pink 1、Parkin、LC3II/I表达水平;
⑥Co-IP检测Parkin磷酸化水平。
处理方法:收集组织,加入含有蛋白酶抑制剂的IP裂解液,冰上研磨裂解30min后12000rpm/min 4℃离心20min,取上清。留取少量上清用于后续Western blot分析,其余上清中加入1μg的Parkin蛋白抗体以及50μl protein A/G beads,4℃摇晃孵育过夜。次日,于4℃离心机中3000g离心5min,小心弃去上清,用1ml裂解缓冲液冲洗protein A/G beads 4次,最后加入15μl的2×SDS加样缓冲液,100℃金属浴10min,使蛋白质变性。Western blot检测磷酸化修饰水平。
实验结果如图1所示,其中(A-B)ELISA检测毛囊乳突细胞中β-Gal表达。(B)RT-PCR检测毛囊乳突细胞中H19表达水平。(C-D)WB检测毛囊乳突细胞中Parkin,LC3II,LC3I及p-Parkin的蛋白水平。实验结果显示:
(1)采用β-半乳糖苷酶染色试剂盒检测小鼠毛囊衰老情况,发现6-姜酚组的β-半乳糖苷酶检测值显著高于对照组,这表明,6-姜酚组可以显著促进细胞衰老。
(2)通过qPCR检测皮肤组织中LncRNA H19水平,结果发现6-姜酚组的皮肤组织中LncRNA H19表达水平均显著高于对照组;通过Western blot检测线粒体自噬相关蛋白Pink1、Parkin、LC3II/I表达水平以及Co-IP检测Parkin磷酸化水平显示,6-姜酚组的P-parkin水平显著低于对照组。
根据生物信息学网站(http://pridb.gdcb.iastate.edu/RPISeq/results.php)显示:LncRNA H19和Pink1互作的可能性>0.5。因此推测6-姜酚处理后毛囊根鞘外泌体通过传递LncRNA H19,抑制Pink1/Parkin介导的线粒体自噬,进而促进毛囊乳突细胞衰老。
实施例2 6-姜酚通过调控毛囊外根鞘细胞外泌体LncRNA H19促进毛囊乳突细胞衰老
1)研究对象
体外分离培养毛囊外根鞘细胞和毛囊乳突细胞,具体的分离培养方法为:
①毛囊外根鞘细胞分离培养:将去除真皮鞘的毛囊置于培养皿中,4h~6h后加入少量K-SFM培养液。
②毛囊乳突细胞分离培养:在40倍放大的体视镜下进行毛囊乳头的显微解剖分离,用30G针头将所分的毛囊乳头转移至35mm培养皿中,用针尖将毛囊乳头周围的包膜划破,每皿放置5个,并添加2ml DMEM+20%FBS,待DPCs迁出后每3d换液1次。细胞融合后采用含有0.25%胰酶消化并1:3传代。传代后的DPC均采用DMEM+10%FBS培养,选取第4代DPCs进行后续实验。
2)处理与分组
A、毛囊外根鞘细胞分为以下两组:
①对照组:对照组采用溶剂DMSO处理,72h后,收集细胞和外泌体。
②6-姜酚组:采用10μg/ml 6-姜酚处理72h后,收集细胞和外泌体。
B、毛囊乳突细胞分为以下两组:
①空白组:空白组不进行任何处理;
②对照组:采用DMSO处理的毛囊外根鞘细胞外泌体处理;
②外泌体组:采用6-姜酚处理的毛囊外根鞘细胞外泌体处理。
C、外泌体提取方法如下:
毛囊外根鞘外泌体用无外泌体胎牛血清培养到80%密度后饥饿24h收集细胞上清然后按如下超速离心法收集外泌体。将上清吸出至15ml离心管,500g,5min离心;将离心后上清移至新的离心管内(直接倒出),2000g,20min离心;将离心后上清移至超速离心管(直接倒出),配平,<0.1g,超速离心10000g,30min;将离心后上清移至新的超速离心管(直接倒出),配平,超速离心100000g,70min;弃净上清,只留ev沉淀(肉眼无法看出)。冰filter PBS60-80μl resuspend EV palet,使用tip来回转圈吹打60次(外泌体很难溶解),涡旋混匀后继续吹打10次后收样(收样管需要提前以filter PBS清洗,干燥)。
3)检测指标
①qPCR检测毛囊外根鞘细胞和毛囊乳突细胞中LncRNA H19水平;
②检测毛囊外根鞘细胞外泌体中LncRNA H19水平;
③CCK8检测毛囊乳突细胞增殖水平;
④Annexin/PI染色检测毛囊乳突细胞凋亡水平;
⑤β-半乳糖苷酶试剂盒检测毛囊乳突细胞衰老情况;
⑥Western blot检测毛囊乳突细胞中线粒体自噬相关蛋白Pink1、Parkin、LC3II/I表达水平;
⑦Co-IP检测Parkin磷酸化水平。
6-姜酚对毛囊根鞘细胞及毛囊根鞘细胞外泌体中H19的表达水平的实验结果如图2所示,6-姜酚对毛囊根鞘细胞及毛囊根鞘细胞外泌体中H19表达影响。(A)RT-PCR检测毛囊根鞘细胞中H19表达。(B)WB检测外泌体标记物HSP70,CD63及Tag101。(C)RT-PCR检测毛囊根鞘细胞外泌体中H19表达。实验结果显示,6-姜酚促进毛囊根鞘细胞及毛囊根鞘细胞外泌体中H19的表达。
图3所示H19介导6-姜酚抑制毛囊乳突细胞自噬。(A)RT-PCR检测毛囊根鞘细胞外泌体H19表达。(B-C)WB检测毛囊乳突细胞中Parkin,LC3II,LC3I及p-Parkin的蛋白水平,实验结果显示,6-姜酚处理后毛囊外根鞘外泌体通过传递LncRNA H19抑制毛囊乳突细胞自噬。
实施例36-姜酚处理后毛囊外根鞘外泌体通过传递LncRNA H19促进毛囊乳突细胞衰老的具体机制
1)研究对象
体外分离培养毛囊外根鞘细胞和毛囊乳突细胞。
2)处理与分组
毛囊乳突细胞分为以下三组:
①过表达对照组(OE-NC)
②过表达LncRNA H19组(OE-LncRNA H19)
③OE-LncRNA H19+OE-Pink1组
采用Lenti病毒进行质粒转染,48h后收集细胞。
3)检测指标
①CCK8检测毛囊乳突细胞增殖水平;
②Annexin/PI染色检测毛囊乳突细胞凋亡水平;
③β-半乳糖苷酶试剂盒检测毛囊乳突细胞衰老情况;
④Western blot检测毛囊乳突细胞中线粒体自噬相关蛋白Pink1、Parkin、LC3II/I表达水平;
⑤Co-IP检测Parkin磷酸化水平;
⑥提取线粒体蛋白,Western blot检测线粒体蛋白泛素化水平。
⑦RNA-IP检测LncRNA H19与Pink1互作情况;
3.2揭示毛囊外根鞘细胞外泌体促进毛囊乳突细胞衰老的具体机制。
1)研究对象
体外分离培养毛囊外根鞘细胞,进行6-姜酚处理并分离外泌体。
2)处理与分组
毛囊乳突细胞分为以下两组:
①外泌体处理组:采用6-姜酚处理过的毛囊外根鞘细胞外泌体处理细胞;
②外泌体处理+OE-Pink1组:外泌体处理的同时采用Lenti病毒感染进行Pink1过表达。
3)检测指标
①CCK8检测毛囊乳突细胞增殖水平;
②Annexin/PI染色检测毛囊乳突细胞凋亡水平;
③β-半乳糖苷酶试剂盒检测毛囊乳突细胞衰老情况。
图4为H19介导6-姜酚抑制毛囊乳突细胞增殖和凋亡。(A)ELISA检测毛囊乳突细胞中β-GAL表达。(B)CCK8检测毛囊乳突细胞增殖。(C)流式细胞术检测毛囊乳突细胞凋亡水平。
Claims (6)
1.6-姜酚在制备促进毛囊乳突细胞衰老产品中的应用。
2.根据权利要求1所述的6-姜酚的应用,所述的促进毛囊乳突细胞衰老产品为包含6-姜酚的外用制剂。
3.根据权利要求1所述的6-姜酚的应用,其特征在于,所述的促进毛囊乳突细胞衰老产品用于治疗多毛症或毛发过多。
4.根据权利要求1所述的6-姜酚的应用,其特征在于,所述的促进毛囊乳突细胞衰老产品中6-姜酚的作用靶点为毛囊外根鞘细胞外泌体LncRNAH19水平。
5.根据权利要求1所述的6-姜酚的应用,其特征在于,6-姜酚能够抑制毛囊乳头状细胞增殖并促进细胞凋亡。
6.根据权利要求1所述的6-姜酚的应用,其特征在于,6-姜酚的作用机制为通过促进毛囊根鞘细胞内及毛囊根鞘细胞外泌体中LncRNAH19的表达,升高毛囊外根鞘细胞外泌体LncRNAH19水平,进而抑制Pink1/Parkin介导的线粒体自噬,进而抑制毛囊乳突细胞增殖同时促进毛囊乳突细胞凋亡。
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