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黄芩素外用制剂对特应性皮炎的治疗作用及其机制

王登, 孙中英, 郭青龙, 李晰, 白宇杰, 魏立彬, 何远

王登,孙中英,郭青龙,等. 黄芩素外用制剂对特应性皮炎的治疗作用及其机制[J]. 中国药科大学学报,2025,56(1):99 − 109. DOI: 10.11665/j.issn.1000-5048.2024070302
引用本文: 王登,孙中英,郭青龙,等. 黄芩素外用制剂对特应性皮炎的治疗作用及其机制[J]. 中国药科大学学报,2025,56(1):99 − 109. DOI: 10.11665/j.issn.1000-5048.2024070302
WANG Deng, SUN Zhongying, GUO Qinglong, et al. Therapeutic effect and mechanism of the topical preparation of baicalein on atopic dermatitis[J]. J China Pharm Univ, 2025, 56(1): 99 − 109. DOI: 10.11665/j.issn.1000-5048.2024070302
Citation: WANG Deng, SUN Zhongying, GUO Qinglong, et al. Therapeutic effect and mechanism of the topical preparation of baicalein on atopic dermatitis[J]. J China Pharm Univ, 2025, 56(1): 99 − 109. DOI: 10.11665/j.issn.1000-5048.2024070302

黄芩素外用制剂对特应性皮炎的治疗作用及其机制

基金项目: 国家自然科学基金项目(No.82002907);枣庄英才集聚工程;泰山产业领军人才工程;山东省中央引导地方科技发展资金项目(YDZX2023082)
详细信息
    通讯作者:

    何远: Tel:13813902328 E-mail:yuanhe0822@cpu.edu.cn

    #王登与孙中英为共同第一作者

  • 中图分类号: R967

Therapeutic effect and mechanism of the topical preparation of baicalein on atopic dermatitis

Funds: This study was supported by the National Natural Science Foundation of China (No. 82002907); Zaozhuang Talent Gathering Project; Taishan Industry Leading Talent Project; and the Guiding Funds of Central Government for Supporting the Development of the Local Science and Technology of Shandong Province (YDZX2023082)
  • 摘要:

    为评价黄芩素外用制剂对特应性皮炎的治疗效果,构建了卡泊三醇及2,4-二硝基-1-氟苯诱导的特应性皮炎样小鼠模型,利用皮肤组织染色等实验确定了黄芩素外用制剂的疗效。结果显示,其可缓解病变皮肤组织表皮增厚、修复受损的皮肤屏障蛋白,并抑制病变组织辅助性T细胞2 (Th2)浸润及肥大细胞浸润和活化。通过网络药理学分析了黄芩素可通过干预多条特应性皮炎致病相关信号通路从而治疗特应性皮炎。随后,通过细胞机制实验确定黄芩素可降低角质形成细胞的炎症因子的mRNA水平并抑制NF-κB p65及STAT1蛋白的磷酸化,从而抑制炎症进程。研究表明,黄芩素外用制剂可通过下调角质形成细胞内NF-κB的磷酸化水平,从而降低角质形成细胞炎症因子的表达,而缓解小鼠特应性皮炎样症状,为黄芩素外用制剂对于特应性皮炎等皮肤炎症性疾病治疗提供思路和理论基础。

    Abstract:

    To evaluate the therapeutic effect of baicalein topical preparation on atopic dermatitis, we first constructed two atopic dermatitis-like mouse models induced by calcipotriol and 1-fluoro-2,4-dinitrobenzene to assess their therapeutic effect with skin tissue staining and other experiments. It was found that topical preparation of baicalein could alleviate epidermal thickening of diseased skin tissues, repair damaged skin barrier proteins, and inhibit T helper 2 cells (Th2) infiltration and mast cell infiltration and activation in lesional sites. Cyberpharmacology was utilized to analyze whether baicalein could treat atopic dermatitis by interfering with multiple pathogenesis-associated pathways. Results indicated that baicalein reduced the mRNA levels of inflammatory factors and inhibited the phosphorylation of NF-κB p65 and STAT1 proteins in keratinocyte cells. Together, the topical preparation of baicalein may be effective in alleviating atopic dermatitis-like symptoms in mice by down-regulating the phosphorylation level of NF-κB in keratinocytes, thereby decreasing the expression of inflammatory factors in keratinocytes, which provides an idea and a theoretical basis for the topical preparation of baicalein for the treatment of inflammatory skin diseases such as atopic dermatitis.

  • 透明质酸(hyaluronic acid, HA)是由N-乙酰氨基葡萄糖(N-acetyl-glucosamine, GlcNAc)和D-葡萄糖醛酸(D-glucuronic acid, GlcUA)通过β-1,4糖苷键交替连接而成的高分子线性糖胺聚糖。HA在生理条件下荷高负电,具高极性、可高度水合等聚电解质特性[1]。当HA的主链基团或分子量增长至一定数值,HA因分子内和分子间形成的大量氢键而呈现线团状。因此,HA具有黏弹性和剪切稀化等流变学特征[2]。除了具有良好的理化性质,HA还具有生物相容和可降解性、分化簇44(cluster of differentiation 44, CD44)靶向性、炎症调节等独特的生物学特性。因此,HA是医药研发领域的重要原材料。

    然而,HA的分子内和分子间氢键易受体液稀释、蛋白、盐等复杂成分的影响。因此,未化学改性的HA经注射后难以形成稳定的凝胶[3]。此外,人类基因组含有6种透明质酸酶(hyaluronidase, HAase)。HAase通过切割GlcNAc和GlcUA之间的β-1,4糖苷键降解HA[4]。大多数组织中的主要HAase为透明质酸氨基葡糖苷酶1(hyaluronoglucosaminidase 1, HYAL-1)和透明质酸氨基葡糖苷酶2(hyaluronoglucosaminidase 2, HYAL-2)。虽然,已有文献表明HAase对HA的特异性降解作用可被用于构建基于HA骨架的、病灶靶向的尺寸可变型或触发释药型药物传递系统[5]。但是,由于血清含有大量HYAL-1,静脉注射的HA可被HYAL-1快速清除。可见HA的医药应用受限于机体的生理复杂性。

    化学改性可调控HA的理化性质、增强其功能性,从而拓展其医药应用范围。但化学改性在改变HA理化性质的同时,亦可能影响其生物学特性。本综述结合HA生物学性质的产生机制、以及其衍生化方法和表征手段总结并讨论化学改性对HA生物学性质的影响,为合理的HA化学改性研究提供参考。

    与在高尔基体内合成的其他蛋白聚糖和糖胺聚糖不同,HA是由真核生物质膜内表面上的透明质酸合成酶(hyaluronan synthase, HAS)催化合成[6]。HAS以尿苷二磷酸-葡糖醛酸和尿苷二磷酸-N-乙酰氨基葡萄糖为原料,在细胞质内膜连续合成HA,并通过跨膜通道将新生HA挤出至胞外基质[7]。HAS1和HAS2具中等活性,负责合成高分子量透明质酸(high molecular weight hyaluronic acid, HMWHA); HAS3蛋白则具有最高活性,负责合成低分子量透明质酸(low molecular weight hyaluronic acid, LMWHA)。HMWHA和LMWHA的分子量分别大于100 kD及介于10~100 kD;而小于25个双糖单元的HA则被称为透明质酸寡糖(hyaluronan oligosaccharides, oHA)[7]。不同分子量的HA表现出截然不同的生物学特性。

    CD44是由单一基因编码、不同亚型表达的受体蛋白。不同亚型CD44的N端均具有两个透明质酸结合结构域(hyaluronic acid binding domain, HABD)[8]:Link结构域和BX7B基序(B代表赖氨酸或精氨酸,X7代表任意非酸性氨基酸)[9]。HA的羧基与HABD的至少两个丙氨酸残基(Ala102,Ala103)相互作用,伯羟基则与HABD的至少一个酪氨酸残基(Tyr109)相互作用[1011]。因此,“HA-CD44”特异性结合力随HA分子量的增大而增强。CD44识别HA所介导的胞吞作用也高度依赖HA的分子量,分子量小的HA更容易被细胞内化[1213]。虽然CD44介导HA内吞的具体机制尚未被完全解析,但是现有研究已证明脂筏参与了该内吞过程:HA结合CD44并激活脂筏相关功能,从而驱动能量依赖性的细胞膜穴样内陷[14]。HA分子量造成的内化效率差异可能与膜内陷的难易程度相关[15]

    不同分子量的HA靶向结合CD44后,可以通过不同信号通路对肿瘤起到不同的药理作用。HMWHA和oHA可抑制肿瘤生长和转移,LMWHA则加速肿瘤恶化。具体而言,HMWHA与细胞膜表面的CD44结合并导致CD44聚集,从而激活肿瘤抑制性Hippo通路以调节细胞增殖和凋亡。相反,LMWHA结合CD44并竞争性抑制“HMWHA-CD44”介导的部分缺陷1b蛋白(partitioning-defective 1b, PAR1b)募集,造成HMWHA对Hippo信号通路的激活作用被抑制。oHA则结合CD44并抑制PI3K/Akt细胞存活通路,减少磷酸化Akt对B细胞淋巴瘤-2相关死亡促进因子(B-cell lymphoma-2-associated death promoter, BAD)、叉头转录因子(forkhead transcription factor, FKHR)等的抑制作用,有效诱导肿瘤细胞凋亡[16]

    HA还可主动靶向透明质酸介导的运动受体(receptor for hyaluronan mediated motility, RHAMM)、Toll样受体2(Toll-like receptors 2, TLR2)、TLR4、淋巴管内皮透明质酸受体1(lymphatic vessel endothelial hyaluronan receptor 1, LYVE-1)、透明质酸内吞受体(hyaluronan receptor for endocytosis, HARE)和Layilin蛋白(LAYN)[9],参与多项细胞信号的传导。LYVE-1和HARE通过Link结构域结合HA[1718]。缺乏Link结构域的RHAMM、TLR2和TLR4则通过两个规范BX7B基序与HA结合[1921]。LAYN不仅不含Link结构域,而且含有两个可阻碍其结合HA的非规范BX7B基序。然而,LAYN仍能特异性结合HA,只是具体的结合机制尚不明晰[2223]

    本文仅总结并讨论HA的化学改性对机制相对清晰且应用相对广泛的“HA-CD44”靶向性的影响。

    HMWHA限制炎症细胞的趋化、吞噬作用以及炎症介质和裂解酶的自由运动,具抗炎特性,可促进伤口愈合[24];而LMWHA促进单核细胞转变为巨噬细胞,提高胰岛素样生长因子1(insulin-like growth factor 1, IGF-1)和白细胞介素-1β(interleukin-1β, IL-1β)的分泌水平,起促炎作用。基于HMWHA良好的黏弹性和炎症抑制能力,临床已使用关节腔注射型Hyalgan®(500~730 kD)治疗骨关节炎[2526]。但需指出的是,HA基于分子量发生促炎、抗炎性质转变的机制尚不明确。

    HYAL-1常见于血清、组织基质和溶酶体,可降解各种分子量的HA。HYAL-1的最适pH为3~4.5。因此,HYAL-1在溶酶体内活性最强,在pH大于4.5时其酶活力为最大效力的50%~80%。HYAL-2主要锚定于质膜表面富含CD44的脂筏区域。HYAL-2仅在低pH条件下具有酶活力,其最适pH为4[4]。HMWHA结合脂筏区域的CD44并激活Na+-H+交换蛋白,从而形成胞外局部酸性环境。随后,HYAL-2被激活并降解HMWHA至20 kD左右的分子片段。HA片段被CD44介导内吞,并于溶酶体内被HYAL-1降解为四糖。四糖最终被β-葡萄糖醛酸酶和β-N-乙酰葡萄糖胺酶降解为单糖[13,15]

    HA富含羟基、羧基、酰胺基等官能团,可通过酰胺化、酯化、开环、交联等原理进行化学改性,从而制备功能化HA衍生物以扩大其应用范围[27]。HA不同修饰位点的化学改性结构式见图1

    图  1  透明质酸(hyaluronic acid, HA)结构式和改性位点(红色)

    酰胺化和酯化是最常见的HA羧基化学改性方式。例如,将疏水性抗肿瘤药紫杉醇(paclitaxel, PTX)的羟基与HA羧基偶联,形成两亲性高分子以提高PTX的水溶性和靶向性[28];酰肼类等双端氨基化合物可用作交联剂,基于酰胺反应促使HA形成分子内或分子间交联。Seprafilm®可吸收防粘连薄膜(隔膜)是由HA羧基和羧甲基纤维素发生酯化交联而成;Monovisc®则是由HA羧基和6-位伯羟基发生分子内酯化交联而形成的水凝胶,用于关节腔注射以缓解关节炎疼痛。此外,HA还能以四丁基铵盐的形式与烷基卤化物、甲苯磺酸酯等物质通过烷基化反应生成酯键[29]

    HA的6-位羟基通常与羧基类、烷基琥珀酸酐或甲基丙烯酸酐等酸酐类、酰氯等物质发生酯化反应。同时,HA的分子内交联反应同样可在6-位羟基上进行。例如,交联剂二乙烯基砜(divinyl sulfone, DVS)在室温下可与HA的6-位羟基快速反应以形成HA凝胶,且交联度可由HA的分子量和浓度、反应pH、HA/DVS质量比来控制[27]。Juvéderm® ULTRA是以1,4-丁二醇二缩水甘油醚(1,4-butanediol diglycidyl ether, BDDE)高度交联HA而形成的均质凝胶,适用于面部真皮组织中层至深层注射,以纠正中度鼻唇沟皱纹;Juvéderm® VOLIFT和Juvéderm® VOLUMA则利用BDDE交联长链和短链HA,适用于面部深层注射,因其平衡弹性和内聚力的范围更大而塑形效果更佳。

    乙酰氨基经脱乙酰化可被还原为游离氨基,从而实现HA的相关衍生化。但基于硫酸肼的HA脱乙酰化反应通常需在55 ℃下进行,可使分子链严重断裂。因此,该法不属于HA的常见化学改性方式[29]

    HA主链糖环和末端糖环亦可经开环反应产生醛基。醛基常与伯胺类化合物生成亚胺键,亚胺键可被进一步还原为胺[30]。该法常用于构建交联凝胶。然而,开环反应可显著改变HA的骨架结构,使其主链刚性减弱而更易降解。

    红外和紫外吸收光谱法、波谱法是初步确证HA是否成功改性的主要手段。例如,HA的羧基被酰胺化后,红外光谱1560 cm−1处可见更强的酰胺Ⅱ键特征峰[31]1H NMR则最常用于表征HA的特征氢峰(羧基氢或6-位伯羟基氢)以确认修饰位点的结构变化。此外,高分辨率13C NMR可用于区分HA的碳原子级数(伯、仲、叔和季),从而明确解析相应碳位的原子取代情况和结构的精细变化。但是,蛋白质、多糖等大分子的结构解析常受限于严重的谱峰重叠。二维核磁共振谱可将重叠的一维谱峰展开呈现于二维平面,从而显著提高峰归属辨析的准确率[32]

    分子量检测法亦在高分子结构确证中起重要的辅助作用。研究人员通常利用体积排阻色谱法分离不同分子量的HA衍生物,再以多角度激光散射仪为检测器准确测定其绝对重均分子量及分布。散射仪的散射光强度和角度变化均与HA衍生物的分子量相关。

    HA分子量的多分散性和改性位点的不可控性促使HA衍生物的精确结构表征和构效关系研究难度剧增[9,33]。同时,研究人员对相关领域关注不足。因此,HA衍生物构效关系的科学研究体系尚未形成。本文从受体靶向性、生物可降解性、促炎或抗炎特性、免疫原性四方面总结并讨论现有研究中的化学改性对HA生物学性质的影响和相应的研究手段。

    HA的羧基及伯羟基在CD44和HABD的靶向结合过程中起关键作用。同时,已有研究指出HA至少需6个连续双糖单元才能与HABD结合[34]。因此,羧基及伯羟基位点的过度修饰可显著削弱CD44对HA的特异性识别能力[910]

    目前最常用于表征HA衍生物CD44靶向性的策略是荧光标记技术。例如,使用FITC标记的HA衍生物和CD44蛋白共孵育,荧光定量“HA-CD44”结合物以量化HA衍生物与受体的靶向结合力[10];或将荧光探针标记的HA衍生物与过表达CD44受体的细胞共孵育,以荧光显微镜、流式细胞仪等定性或定量胞内荧光强度,从而间接评价其对CD44的靶向性。此外,分子对接研究和结合能计算法可用于HA衍生物与天然HA的CD44靶向性质差异的深入分析。有研究者采用SwissDock和AutoDock对接软件模拟CD44对HA的识别作用[35],通过比较对接能量值分析HA化学改性对结合能的影响。

    Oh等[36]将己二酸二酰肼(adipic dihydrazide, ADH)与HA羧基进行偶联,合成修饰率分别为14%、22%、45%、70%的HA-ADH偶联物。结果表明,当羧基的取代度超过25%,HA衍生物的CD44主动靶向作用显著减弱。Kwon等[11]则将降冰片烯(norbornenes, Nor)或甲基丙烯酸酯(methacrylates, Me)以酰胺键或酯键形式修饰于HA的羧基或6-位伯羟基,相关位点的取代度约为10%、20%、40%。结果表明,无论是何种修饰方式和位点,CD44与HA的结合效率都随疏水基团增多而下降;且在羧基位点修饰Nor的衍生物组的结合效率下降最为显著。此外,相比于同取代度的6-位伯羟基Nor衍生化HA,6-位伯羟基Me衍生化HA因Me的疏水性更强而具有更为紧密的疏水核心,表面暴露更多羧基,从而具有更强的CD44靶向结合能力。

    研究者常使用HAase生物降解实验定量分析HA衍生物的酶解抗性:即在生理条件下将HA衍生物与HAase共孵育,随后通过表征HA衍生物基本理化性质或特性参数的方式,对比评价HA衍生物的酶解抗性。目前最常用的表征手段为凝胶渗透色谱法和重量/黏度测量法,其通过测定HA衍生物在酶解前后的重均分子量大小和分布、重量、黏度等的变化来判断酶解程度[37]

    以HA为骨架的抗肿瘤药物递送系统可被肿瘤组织高表达的HYAL-1和HYAL-2降解,存在药物在胞外过早泄漏的风险。同时,胞内HYAL-1和胞膜HYAL-2对于HA的降解作用具CD44依赖性。因此,HA靶向CD44的关键结合位点(羧酸、6-位伯羟基)亦与HAase的降解活性紧密相关[10]。此外,HA化学改性所引入的基团可造成空间位阻,从而限制HAase结合HA。综上,HA羧基或6-位伯羟基的化学改性可能使HA衍生物获得HAase酶解抗性。Schanté等[38]将HA与不同大小的氨基酸通过酰胺化反应结合,获得各种HA-氨基酸衍生物(HA-amino acid derivatives, HA-aa)。与天然HA相比,HA-aa的HAase抗性显著增强;且小分子氨基酸(丙氨酸、精氨酸)改性的HA-aa相较于大分子氨基酸(苯丙氨酸、酪氨酸)改性的HA-aa酶解抗性低,证明改性基团位阻是影响HA衍生物抗酶解性的重要因素。然而,HA的过度改性,如对羧基进行全化学修饰,可导致HA衍生物不能被HAase降解。因此,HA衍生物的设计与制备需平衡其功能性和基于生物代谢的安全性。

    酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)常用于HA衍生物的促炎或抗炎特性研究。该法首先收集巨噬细胞与HA衍生物共孵育后的培养基,而后利用ELISA测定培养基内促炎细胞因子(TNF-α、IL-6、IL-1β)和抗炎细胞因子(IL-10、IL-4)的浓度。体内抗炎实验是通过对小动物注射完全弗氏佐剂建立慢性炎症模型,而后对模型给以HA衍生物,最后以ELISA检测炎症相关因子的同时观测炎症组织的病理变化,综合评价HA衍生物的促炎或抗炎特性[39]

    现阶段HA及其衍生物促炎或抗炎特性的研究尚未深入到分子机制层面,仍仅是聚焦于HA及其衍生物的炎症调节功效:如联用HA与其他抗炎或促炎物质,实现炎症调节作用的增效;或利用HMWHA修饰具有机体炎症刺激特性的载体或物质,以减弱其炎症刺激性。目前亦缺乏相关研究深入探讨和揭示HA改性对其促炎/抗炎特性的影响和相关分子机制。

    化学改性是否会改变HA的免疫原性亦是研究者应当关注的关键问题。免疫原性评价试验通常涉及细胞免疫和体液免疫的激活检测。淋巴细胞的增殖与分化是机体激活细胞免疫的重要过程。在HA衍生物被重复注入小鼠体内之后,取小鼠脾细胞进行体外培养,再采用Alamar Blue法测定T细胞和B细胞的增殖活力、及流式细胞术检测T、B、NK细胞亚群及比例,以综合表征HA衍生物激活机体细胞免疫的效力。体液免疫激活效力的评价通常采用ELISA测定血清免疫球蛋白IgG、IgM、IgA的浓度[40]

    化学改性造成的HA自然构象变化可能影响其免疫原性,刺激机体产生免疫应答,诱发强烈炎症反应从而造成机体损伤;激活的机体免疫亦可加速人体对HA递送系统的清除,从而限制其药效发挥[41]。但已报道的相关研究一般仅利用生物相容性HA对具免疫原性的生物活性物质进行理化修饰,以提高其体内应用的安全性;尚缺乏相关研究探讨和揭示HA改性对其免疫原性的影响和相关机制。

    综上所述,化学改性虽可调控HA的理化性质、增强HA的功能性,但亦可影响其生物学特性。HA功能化的相关研究目前仍较少关注HA衍生物生物学性质的科学表征、及衍生化影响HA生物学性质的具体规律和机制,而相关研究结果将对HA衍生物在医药领域应用的安全性和有效性起关键指导作用。

  • Figure  1.   Therapeutic effect of the topical preparation of baicalein on atopic dermatitis (AD)-like mouse models induced by calcipotriol (MC903)

    A: Schematic diagram of AD-like mouse models induced by MC903; B: Photographs of mouse ear lesions; C: Transepidermal water loss (TEWL) changes in mouse ears during MC903 modeling; D: Quantification of ear margin thickness in mice; E: Tissue milling of thymic stromal lymphopoietin (TSLP) mRNA expression in mouse ears ($ \bar{x}\pm s $, n=5) ###P<0.001 vs control group; *P<0.05,**P<0.01, ****P<0.0001 vs MC903 group; n.s.: no significance

    Figure  2.   Therapeutic effect of the topical preparation of baicalein on AD-like mouse models induced by 1-fluoro-2,4-dinitrobenzene (DNFB)

    A: Schematic diagram of AD-like mouse models induced by DNFB; B: Photographs of back lesions in mice after modeling and drug administration; C: Photographs of inguinal lymph nodes in mice; D: Quantification of inguinal lymph node length in mice; E: TEWL monitoring of backs of mice; F: Scoring of AD-like lesions on backs of mice for the four dimensions of dryness/deflagration, hemorrhage/rash, ulceration/epidermal detachment, edema scored according to conditions 0 (absent), 1 (mild), 2 (moderate), and 3 (severe) ($ \bar{x}\pm s $, n=5); G: Mouse serum IgE content ##P<0.0001 vs control group; *P<0.05, **P<0.01, ****P<0.0001 vs DNFB group

    Figure  3.   Pathologic observations on epidermal lesions in AD-like mouse models

    A: Representative images of HE staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of HE staining of AD-like dorsal lesion tissue induced by DNFB; C: Quantification of HE-stained epidermal thickness of AD-like ear lesions in mice induced by MC903; D: Quantification of HE-stained epidermal thickness of AD-like dorsal lesion in mice induced by DNFB ($ \bar{x} \pm s$, n=5) ####P<0.0001 vs control group; **P<0.01, ****P<0.0001 vs MC903 or DNFB group

    Figure  4.   Pathologic observations of mast cell staining at skin lesions in AD-like mouse models

    A: Representative images of toluidine blue staining of mouse AD-like ear lesion tissue induced by MC903; B: Quantification of mast cell infiltration in mouse AD-like ear lesion tissue induced by MC903; C: Quantification of mast cell degranulation ratio in mouse AD-like ear lesion tissue induced by MC903; D: Quantification of mast cell infiltration in mouse AD-like dorsal lesion tissue induced by DNFB; E: Quantification of mast cell degranulation ratio in mouse AD-like dorsal lesion tissue induced by DNFB ($ \bar{x}\pm s $, n=5)####P<0.0001 vs control group; *P<0.05, ****P<0.0001 vs MC903 or DNFB group; PVF: Positive cells in the visual field (500 μm×890 μm)

    Figure  5.   Immunofluorescence staining of skin barrier proteins at mouse lesions

    A: Representative images of involucrin immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of Filaggrin immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; C: Representative images of involucrin immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB; D: Representative images of filaggrin immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB

    Figure  6.   Immunofluorescence staining of Th2 cells in mouse skin lesions

    A: Representative images of CD4+/IL4+ Th2 immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of CD4+/IL4+ Th2 immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB; C: Quantification of Th2 cell infiltration in mouse AD-like ear lesion tissues induced by MC903; D: Quantification of Th2 cell infiltration in mouse AD-like dorsal lesion tissues induced by DNFB ($ \bar{x}\pm s $, n=5) ####P<0.0001 vs control group; **P<0.01, ****P<0.0001 vs MC903 or DNFB group

    Figure  7.   Cyberpharmacologic analysis of baicalein in the treatment of atopic dermatitis

    A: Intersection of atopic dermatitis disease targets with corresponding targets of baicalein; B: Interaction map of the protein "baicalein-AD target"; C: GO enrichment analysis of biological process (BP) as a potential therapeutic target of baicalein for AD treatment

    Figure  8.   Effect of baicalein on the expression of related chemokines and cytokines in HaCaT cells ($ \bar{x}\pm s $, n=3)

    A-E: Expression of CCL17, CCL22, CTSS, IL-6 and TNF-α mRNA after IFN-γ+TNF-α treatment of HaCaT cells; F-I: Expression of IL-4, TSLP, CCL17 and CCL22 mRNA after Poly(I:C)+TNF-α treatment of HaCaT cells #P<0.05, ####P<0.0001 vs control group; *P<0.05, ***P<0.001, ****P<0.0001 vs IFN-γ+TNF-α or Poly(I:C)+TNF-α group

    Figure  9.   Effect of baicalein on NF-κB and STAT1 mediated signalings in HaCaT cells ($ \bar{x}\pm s $, n=3)

    A: Effect of baicalein on phosphorylational levels of NF-κB p65 and STAT1 in HaCaT cells; B: Relative gray-scale quantification of p-STAT1; C: Relative gray-scale quantification of p-NF-κB p65 #P<0.05, ##P<0.01 vs control group; *P<0.05, **P<0.01 vs IFN-γ+TNF-α group

    Table  1   Primer sequences for real-time qPCR

    Biological indicatorForward primer (5'→3')Reverse primer (5'→3')
    TSLPGCAAATCGAGGACTGTGAGAGCTGAGGGCTTCTCTTGTTCTCCG
    GAPDHCATCACTGCCACCCAGAAGACTGATGCCAGTGAGCTTCCCGTTCAG
    下载: 导出CSV

    Table  2   Primer sequences for real-time qPCR

    Biological indicatorForward primer (5'→3')Reverse primer (5'→3')
    CCL17TTGTAACTGTGCAGGGCAGGTGAACACCAACGGTGGAGGT
    CCL22GAAGCCTGTGCCAACTCTCTGGGAATCGCTGATGGGAACA
    CTSSTGGGCTTTCAGTGCTGTGGGTCAATGATGTACTGGAAAGC
    IL-4CCGTAACAGACATCTTTGCTGCCGAGTGTCCTTCTCATGGTGGCT
    TSLPGAA AGCTCTGGAGCATCAGGAGGGAACATACGTGGACACC
    IL-6AGACAGCCACTCACCTCTTCAGTTCTGCCAGTGCCTCTTTGCTG
    TNF-αCTCTTCTGCCTGCTGCACTTTGATGGGCTACAGGCTTGTCACTC
    GAPDHGAAGGCTCATGACCACAGTGGATGCAGGGATGATGTTCT
    下载: 导出CSV
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出版历程
  • 收稿日期:  2024-07-02
  • 修回日期:  2024-08-29
  • 录用日期:  2024-09-04
  • 刊出日期:  2025-02-24

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