基于皮肤TRP通道探讨三伏贴穴位经皮给药渗透机制的研究

赖蓉蓉; 翟苑好; 林媛媛; 曾瑶; 伍艳; 徐月红

doi:10.14148/j.issn.1672-0482.2022.1035

基于皮肤TRP通道探讨三伏贴穴位经皮给药渗透机制的研究

doi: 10.14148/j.issn.1672-0482.2022.1035

中山大学药学院, 广东广州 510006

基金项目:

国家自然科学基金面上项目 81973491

国家自然科学基金面上项目 81473358

广东省基础与应用基础研究基金 2019A1515011161

广东省中医药局科研项目 20211080

详细信息

作者简介:
赖蓉蓉, 女, 硕士研究生, E-mail: lairr@mail2.sysu.edu.cn

徐月红，教授，博士生导师，中山大学药学院教学实验中心主任。主要从事经皮肤/黏膜给药系统设计与渗透机理的研究。研究方向包括: 基于皮肤渗透和免疫探究三伏贴经皮给药的穴位增强效应机制; 皮肤外用制剂的处方设计和评价; 皮肤外用制剂国内外法规比较等。担任世界中医药联合会经皮给药专业委员会常务理事、广东省药品注册审评专家、广东省药学会药物制剂委员会候任主任委员，广东省中药学会经皮给药专委会副主委等。主持国家自然科学基金和省自然科学基金约10项，主持企业委托课题约30项，协助企业获生产和临床批件7项; 发表论文约50篇。授权专利5项

通讯作者:
徐月红, 女, 教授, 主要从事皮肤/黏膜给药系统设计与渗透机理的研究, E-mail: lssxyh@mail.sysu.edu.cn

中图分类号: R285.5
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出版历程
- 收稿日期: 2022-04-25
- 网络出版日期: 2022-11-19
- 发布日期: 2022-11-10

Study on Enhanced Permeation Mechanism of Sanfu Patch at Acupoints Based on Skin TRP Channels

School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China

摘要

摘要: 目的研究三伏贴中有效成分芥子碱硫氰酸盐(Sinapine thiocyanate, SPT)和延胡索乙素(Tetrahydropalmatine, THP)对瞬时感受器电位离子通道(Transient receptor potential ion channels, TRP)的影响, 以及激活TRP通道后对三伏贴透皮行为的影响, 以探究三伏贴经穴位渗透的机制。方法通过背根神经节细胞Ca²⁺内流实验考察三伏贴有效成分对TRP通道的激活作用。通过Western blot和免疫荧光法比较肺俞穴与非穴位皮肤中TRPV1和TRPA1表达的差异以及给药后TRPA1表达的变化, 并且通过在体滞留和激光共聚焦3D扫描, 研究激活TRP通道对三伏贴穴位透皮行为的影响。结果三伏贴中有效成分SPT可以促进TRP通道介导的Ca²⁺内流。大鼠肺俞穴皮肤TRPV1、TRPA1的表达量显著高于非穴位皮肤(P < 0.05), 三伏贴持续作用后肺俞穴和非穴位皮肤TRPA1的表达量下降。皮肤TRPV1和TRPA1被激活后, 经肺俞穴皮肤渗透的SPT和THP的皮肤滞留量均显著提高(P < 0.05), 但对非穴位皮肤的滞留没有明显影响。结论三伏贴中有效成分SPT对TRP通道具有激活作用, 且肺俞穴皮肤TRPV1、TRPA1表达量显著高于非穴位。穴位高表达TRPV1、TRPA1及三伏贴对TRP通道的激活作用为三伏贴穴位敷贴促透的机理之一。
- 三伏贴 /
- 芥子碱硫氰酸盐 /
- 延胡索乙素 /
- TRPV1 /
- TRPA1
Abstract: OBJECTIVE To clarify the mechanism of per-acupoint penetration of Sanfu patch, study the effects of the active ingredients of Sanfu patch including sinapine thiocyanate (SPT) and tetrahydropalmatine (THP) on TRP channels, and further evaluate the influence of activating TRP channels on the transdermal behavior of Sanfu patch. METHODS The activation of TRP channels in dorsal root neurons (DRG) by the active ingredients of Sanfu patch was investigated by calcium-imaging experiments. The expressions of TRPV1 and TRPA1 in the skin of Feishu (FS) acupoints and non-Feishu (NFS) acupoints were detected by Western blot and immunofluorescence, and the changes of TRPA1 expression after administration with Sanfu patch were also measured by immunofluorescence. The effect of activating TRP channels by Sanfu patch on acupoint transdermal behavior was studied using in vivo transdermal and confocal laser 3D scanning. RESULTS The active ingredient SPT in Sanfu patch could promote the influx of Ca²⁺ mediated by TRP channels. The expression levels of TRPV1 and TRPA1 channels in the skin of FS acupoints were significantly higher than those of NFS acupoints skin (P < 0.05), and TRPA1 channels decreased after sustained treatment with Sanfu patch. After the activation of TRPV1 and TRPA1 channels in the skin, the retention of SPT and THP in the skin of FS acupoints significantly increased (P < 0.05), but there was no significant effect on NFS acupoints skin. CONCLUSION The active ingredient SPT in Sanfu patch has an agonistic effect on TRP channels, and the expression of TRPV1 and TRPA1 channels in FS acupoint skin is higher than those in NFS acupoint. The higher expressions of TRPV1 and TRPA1 channels and their activation by Sanfu patch are one of the mechanisms of its penetration-promoting through acupoint administration.
- Sanfu patch /
- sinapine thiocyanate /
- tetrahydropalmatine /
- TRPV1 /
- TRPA1

HTML全文

图 1 20 μmol·L^-1 CAP、100 μmol·L^-1 SPT和100 μmol·L^-1 THP对DRG细胞内Ca²⁺浓度的影响

注：A.分别给予20 μmol·L^-1 CAP、100 μmol·L^-1 SPT和100 μmol·L^-1 THP后DRG细胞内Ca²⁺浓度变化(ΔF/F₀); B.分别给予20 μmol·L^-1 CAP和100 μmol·L^-1 SPT前后DRG细胞的CLSM图像, 标尺=20 μm

Figure 1. Effects of 20 μmol·L^-1 CAP, 100 μmol·L^-1 SPT and 100 μmol·L^-1 THP on Ca²⁺ uptaked into DRG cells

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图 2 肺俞穴和非穴位皮肤TRPV1和TRPA1表达的差异

注：A.CLSM观察肺俞穴和非穴位皮肤TRPV1和TRPA1的表达, 标尺=100 μm; B.皮肤中TRPA1、TRPV1蛋白表达量的Western blot结果图和灰度分析；FS.肺俞穴组；NFS.非穴位组；与非穴位组比较，^*P < 0.05。x±s, n=3。

Figure 2. Differences of TRPV1 and TRPA1 expression between Feishu acupoint and non-acupoint skin

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图 3 皮肤TRPA1免疫荧光成像图(标尺=100 μm)

Figure 3. Immuno-fluorescence imaging of TRPA1 in skin (scale bar=100 μm)

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图 4 激活TRPV1对三伏贴敷贴透皮行为的影响

注：A.CAP与三伏贴同时作用于大鼠皮肤6 h后SPT在体皮肤滞留量; B.CAP与三伏贴同时作用于肺俞穴和非穴位皮肤后皮肤不同深度的CLSM图像; C.大鼠不同皮肤深度的荧光半定量图; D.荧光三维重构图, 标尺=200 μm；FS+CAP.CAP和三伏贴联合给予大鼠肺俞穴皮肤组；FS.仅给予三伏贴于大鼠肺俞穴皮肤组；NFS+CAP.CAP和三伏贴联合给予大鼠非穴位皮肤组；NFS.仅给予三伏贴于大鼠非穴位皮肤组；与FS+CAP组比较，^*P < 0.05;与FS组比较，^#P < 0.05。x±s, n=5。

Figure 4. Effects of activating TRPV1 on the transdermal behavior of Sanfu patch

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图 5 激活TRPA1对三伏贴敷贴透皮行为的影响

注：A.AITC与三伏贴同时作用于大鼠皮肤6 h后SPT在体皮肤滞留量; B.AITC与三伏贴同时作用于肺俞穴和非穴位皮肤后皮肤不同深度的CLSM图像; C.大鼠不同皮肤深度的荧光半定量图; D.荧光三维重构图, 标尺=200 μm；FS+AITC.AITC和三伏贴联合给予大鼠肺俞穴皮肤组；FS.仅给予三伏贴于大鼠肺俞穴皮肤组；NFS+AITC.AITC和三伏贴联合给予大鼠非穴位皮肤组；NFS.仅给予三伏贴于大鼠非穴位皮肤组；与FS+AITC组比较，^*P < 0.05;与FS组比较，^#P < 0.05。x±s, n=5。

Figure 5. Effects of activating TRPA1 on the transdermal behavior of Sanfu patch

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