Therapeutic effect and mechanism of the topical preparation of baicalein on atopic dermatitis
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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.
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Keywords:
- atopic dermatitis /
- baicalein /
- topical preparations /
- inflammatory factors /
- NF-κB p65
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特应性皮炎(atopic dermatitis, AD)发病机制复杂，涉及遗传缺陷与表皮屏障、表观遗传改变、免疫调节紊乱等多种因素[1]。目前，特应性皮炎的治疗方法以抑制过度的炎症反应为主，主要使用的药物包括皮质类固醇、钙调磷酸酶抑制剂、抗组胺药物、Janus激酶 (JAK) 抑制剂和免疫抑制剂等[2]。上述药物临床疗效显著，但各自具有其特殊的不良反应。如，外用皮质类固醇存在反弹发作的风险、长期使用易造成全身不良反应等[3]；JAK抑制剂可增加患者严重心脏相关事件、癌症、血栓和死亡的风险[4]。基于以上原因，需要开发新的药物治疗特应性皮炎。
得益于丰富的中药资源及研究基础，天然药物是新药及新药先导化合物的重要来源[5]。多项研究表明，白鲜碱、姜黄素、白藜芦醇等化合物可通过作用于NF-κB信号通路，缓解特应性皮炎炎症反应[6]。研究显示黄芩对皮肤炎症有良好的作用效果[7]，而以黄芩为配伍的中药方剂如小柴胡汤、清热除湿汤、龙肝泻胆汤等可应用于特应性皮炎的治疗[8−10]。但由于中药汤剂相较于其他剂型（如外用制剂，注射剂等）有着煎液体积较大、味苦，服用、携带不方便及不宜大量制备等缺点。而外用制剂治疗皮肤炎症可直接作用于靶部位发挥药理作用，降低药物的不良反应，患者用药依从性高。因此，本研究通过建立小鼠特应性皮炎模型，研究黄芩素外用制剂对小鼠特应性皮炎的改善作用，并探究相关作用机制，以期为治疗特应性皮炎提供更加安全有益的选择。
1. 材 料
1.1 药品与试剂
黄芩素外用制剂 （南京芩领医药科技有限公司）；左西替利嗪 (levocetirizine, LD) ，卡泊三醇(calcipotriol , MC903, 美国MedChemexpress公司)；2,4-二硝基-1-氟苯(1-fluoro-2,4-dinitrobenzene, DNFB, 德国Sigma公司)；苏木精-伊红(HE)染液（武汉赛维尔生物科技有限公司）；甲苯胺蓝染色液（北京索莱宝科技有限公司）；其他化学试剂均为市售分析纯。
蛋白质预染Marker，实时荧光定量PCR实验(qPCR)试剂（南京诺唯赞生物公司）；100×青链霉素混合液 (以色列Bioind公司)；BCA试剂盒、RIPA裂解液 (美国Thermo公司)；磷酸化STAT1抗体 (美国Cell Signaling Technology公司)；STAT1抗体 (美国Affinity Biosciences公司)；磷酸化RELA/NF-κB p65抗体、RELA/NF-κB p65抗体、β-actin抗体 (美国Santa Cruz Biotechnology公司)；丝聚蛋白抗体 (英国Abcam公司)；FITC anti-M CD4抗体(美国BioLegend公司)；IL-4 抗体(中国Huabio公司)；小鼠屋尘螨特异性IgE (HDM-sIgE) ELISA科研试剂盒（湖南艾方生物科技有限公司）。
1.2 细 胞
人永生化角质形成细胞 (HaCaT细胞) 来源于中国科学院上海生科院细胞库。使用含1%青链霉素混合液 (100×) 和10%FBS的DMEM培养基并置于5% CO2、37 ℃恒温细胞培养箱中培养。
1.3 仪 器
全自动样品快速研磨仪（上海净信实业发展有限公司）；倒置显微镜（日本奥林巴斯公司）；CK-MPA4 Tewameter TM300 (德国CK公司)；全波长酶标仪Multiskan GO (美国Thermo Fisher Scientific公司)；实时荧光定量PCR仪LightCylcler 480Ⅱ (瑞士Roche公司)；高流电源Powerpac HC、小型垂直电泳槽Mini-PROTEAN Tetra、小型Trans-Blot 转印槽 (美国Bio-Rad公司)；医用胶片洗片机OPTIMAX (德国Protec公司)。
1.4 动 物
C57BL/6J 小鼠，SPF级，雌性，体重(20±2) g，6~8 周龄，购于杭州子源实验动物科技有限公司，实验动物生产许可证号：SCXK (浙) 2019-0004。所有动物实验均符合中国药理动物伦理委员会批准的《实验动物管理条例》 (2023-06-30)。
2. 方 法
2.1 特应性皮炎样小鼠模型造模及经皮水分丢失 (transepidermal water loss, TEWL) 检测
2.1.1 卡泊三醇诱导的小鼠特应性皮炎样模型
选用 35只6~8 周龄雌性 C57BL/6J 小鼠，适应性饲养1周后，随机分成7组，分别为对照组 (Control)、模型组 (MC903)、左西替利嗪阳性药组 (LD, 0.578 mg/kg)、黄芩素外用制剂基质组 (Base material)以及黄芩素外用制剂低、中、高剂量组 (0.45、4.5、45 mg/kg)。对照组耳部涂抹无水乙醇，除对照组外，各组耳部涂抹MC903连续造模12 d。造模期间测量记录耳部经皮水分丢失并拍摄耳部照片。
2.1.2 2,4-二硝基-1-氟苯诱导的小鼠特应性皮炎样模型
选用 25只6~8 周龄雌性 C57BL/6J 小鼠，适应性饲养1周后，随机分成5组，分别为对照组、模型组 (DNFB)、左西替利嗪阳性药组、黄芩素外用制剂基质组、黄芩素外用制剂组 (45 mg/kg)。小鼠造模前1天 (day 0) 背部脱毛，次日背部用移液器均匀滴加0.5% DNFB 50 μL (丙酮-橄榄油，3∶1配制)，第 3日重复第1日操作，从第8日起每隔1日给予0.3% DNFB 40 μL造模。第2日开始给药治疗，阳性药组灌胃给药，黄芩素外用制剂组背部涂抹黄芩素外用制剂。每日称量体重，每5天记录背部皮肤经皮水分丢失 (TEWL) 并拍摄皮肤照片。对皮肤照片按照严重程度[每种症状：0 （无）、1 （轻度）、2 （中度） 和3 （重度）] 对4个维度 (干燥/脱屑、出血/红疹、溃烂/表皮脱落、水肿) 进行评分。
2.2 血清免疫球蛋白E (IgE)含量的测定
小鼠眼球取血后离心取血清，用酶联免疫吸附试验检测IgE表达水平。
2.3 动物皮肤组织炎症因子的测定
皮肤组织研磨后提取RNA，实时荧光定量PCR实验检测炎症因子胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin, TSLP) 的mRNA表达水平。引物序列见表1。
Table 1. Primer sequences for real-time qPCRBiological indicator Forward primer (5'→3') Reverse primer (5'→3') TSLP GCAAATCGAGGACTGTGAGAGC TGAGGGCTTCTCTTGTTCTCCG GAPDH CATCACTGCCACCCAGAAGACTG ATGCCAGTGAGCTTCCCGTTCAG 2.4 HE染色
用4%多聚甲醛溶液对皮肤样本进行过夜固定后进行石蜡包埋切片制作5 μm厚的组织病理切片，经HE染色后二甲苯透明，中性树胶封片后用倒置显微镜观察。
2.5 甲苯胺蓝染色
石蜡切片脱蜡水化，甲苯胺蓝染色液染色，无水乙醇分色后梯度乙醇脱水，二甲苯透明，中性树脂封片，镜下观察并记录肥大细胞形态及数量。
2.6 皮肤组织屏障蛋白及Th2细胞的检测
切片脱蜡和复水后，修复抗原后，用 5% 山羊血清封闭1 h，用丝聚蛋白抗体、involucrin 抗体、FITC anti-M CD4 抗体、IL-4 抗体在4 ℃下孵育过夜，二抗室温孵育1 h，加抗荧光淬灭剂后封片，用荧光显微镜观察。
2.7 黄芩素治疗特应性皮炎的网络药理学分析
以“Baicalein”为关键词，通过Pubchem (https://pubchem.ncbi.nlm.nih.gov/) 结合Swiss Target (http://swisstargetprediction.ch/) 预测Baicalein作用靶点基因；以“Atopic dermatitis”为关键词进行检索，在Genecards (https://www.genecards.org/)、OMIM (https://www.omim.org/) 和DisGeNET (https://disgenet.cn/) 数据库中得到特应性皮炎的潜在治疗靶点，通过黄芩素的作用靶点与特应性皮炎治疗靶点之间的交集绘制疾病基因和Baicalein靶点Venny图。
在既往研究数据的基础上，应用 STRING (https://cn.string-db.org/) 数据库对输入基因的相互关系进行整理和预测，绘制PPI蛋白互作网络图。通过Cytoscape 3.9.1进行可视化处理。
使用 DAVID (https://david.ncifcrf.gov/) 数据库对黄芩素治疗特应性皮炎的潜在基因靶点进行GO富集分析。
2.8 qPCR检测黄芩素对人源永生化角质形成细胞 (HaCaT) 炎症因子表达的影响
取对数生长期HaCaT细胞，分别设置空白组 (Control)、模型组 (Model)、黄芩素给药组 (20 μmol/L)。细胞铺板后，给予IFN-γ (25 ng/mL)+TNF-α (25 ng/mL)或Poly (I:C) (50 μg/mL)+TNF-α (20 ng/mL)进行刺激并加药，24 h后收集细胞并提取RNA，测定HaCaT细胞中细胞因子的mRNA表达水平。引物序列见表2。
Table 2. Primer sequences for real-time qPCRBiological indicator Forward primer (5'→3') Reverse primer (5'→3') CCL17 TTGTAACTGTGCAGGGCAGG TGAACACCAACGGTGGAGGT CCL22 GAAGCCTGTGCCAACTCTCT GGGAATCGCTGATGGGAACA CTSS TGGGCTTTCAGTGCTGTGGG TCAATGATGTACTGGAAAGC IL-4 CCGTAACAGACATCTTTGCTGCC GAGTGTCCTTCTCATGGTGGCT TSLP GAA AGCTCTGGAGCATCAGG AGGGAACATACGTGGACACC IL-6 AGACAGCCACTCACCTCTTCAG TTCTGCCAGTGCCTCTTTGCTG TNF-α CTCTTCTGCCTGCTGCACTTTG ATGGGCTACAGGCTTGTCACTC GAPDH GAAGGCTCATGACCACAGT GGATGCAGGGATGATGTTCT 2.9 蛋白免疫印迹实验
细胞样本收集后加RIPA裂解液并用测定总蛋白浓度。随后，将蛋白质样品进行SDS-PAGE电泳，转膜，室温封闭1 h后加入一抗 (磷酸化STAT1抗体；STAT1抗体；磷酸化RELA/NF-κB p65抗体、RELA/NF-κB p65抗体、β-actin抗体) 于４℃过夜，后加入二抗，室温孵育1 h，滴加发光液显色后在暗室中使用X射线胶片进行显影。
2.10 数据处理与分析
使用GraphPad Prism 9.5软件进行统计学分析，采用Student’s t-test对两组之间的差异进行直接比较，并使用方差分析(ANOVA)进行多组比较。P<0.05 表示具有统计学显著差异。
3. 结 果
3.1 黄芩素外用制剂缓解MC903诱导的特应性皮炎样小鼠模型病症
为了评价黄芩素外用制剂对特应性皮炎的治疗作用，采用MC903诱导的特应性皮炎样小鼠模型来进行实验(图1-A)。结果表明，黄芩素外用制剂及阳性药使MC903诱导小鼠的特应性皮炎样病变得到显著缓解(图1-B~图1-D)。耳缘组织的qPCR检测结果表明，造模组的TSLP转录表达水平上升，而给药组显著性地降低造模组的TSLP表达量 (图1-E)。以上结果表明，黄芩素外用制剂可缓解MC903诱导的小鼠特应性皮炎样病变，且呈剂量依赖性。
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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 $, n=5) ###P<0.001 vs control group; *P<0.05，**P<0.01, ****P<0.0001 vs MC903 group; n.s.: no significance3.2 黄芩素外用制剂缓解DNFB诱导的特应性皮炎样小鼠模型病症
为了进一步确定黄芩素外用制剂对于全身性特应性皮炎的治疗效果，构建了DNFB诱导特应性皮炎样小鼠模型进行药效评价(图2-A)。实验结果表明，黄芩素外用制剂组可显著缓解DNFB诱导小鼠的特应性皮炎样皮肤病变(图2-B)，并显著性地抑制淋巴结增大，缓解TEWL，降低皮肤损伤指数及血清IgE含量，提示了黄芩素外用制剂可以缓解DNFB诱导的全身炎症 (图2-C~图2-G)。其次，相较于造模组，LD组对TEWL无明显影响，而给药组可显著缓解TEWL。以上结果表明，在DNFB诱导的小鼠特应性皮炎样病变模型中，黄芩素外用制剂同样可显著地抑制小鼠特应性皮炎样病变。
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Figure 2. Therapeutic effect of the topical preparation of baicalein on AD-like mouse models induced by 1-fluoro-2,4-dinitrobenzene (DNFB)A: Schematic diagram of AD-like mouse models induced by DNFB; B: Photographs of back lesions in mice after modeling and drug administration; C: Photographs of inguinal lymph nodes in mice; D: Quantification of inguinal lymph node length in mice; E: TEWL monitoring of backs of mice; F: Scoring of AD-like lesions on backs of mice for the four dimensions of dryness/deflagration, hemorrhage/rash, ulceration/epidermal detachment, edema scored according to conditions 0 (absent), 1 (mild), 2 (moderate), and 3 (severe) ($ \bar{x}\pm s $, n=5); G: Mouse serum IgE content ##P<0.0001 vs control group; *P<0.05, **P<0.01, ****P<0.0001 vs DNFB group3.3 黄芩素外用制剂缓解特应性皮炎样皮肤组织病理学改变
小鼠病变组织病理切片组织化学染色结果显示对照组小鼠皮肤真表皮结构完整，组织病理无异常改变，而造模组皮损部位表现为毛细血管扩张，角化过度且角化不全，棘层向下延伸呈钉状，表皮层、真皮层炎性细胞浸润增加，给药组皮损部位组织病理学改变得到明显改善 (图3-A, B)。对表皮增厚进行测量，结果发现，黄芩素外用制剂可抑制表皮增厚 (图3-C, D)。
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Figure 3. Pathologic observations on epidermal lesions in AD-like mouse modelsA: Representative images of HE staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of HE staining of AD-like dorsal lesion tissue induced by DNFB; C: Quantification of HE-stained epidermal thickness of AD-like ear lesions in mice induced by MC903; D: Quantification of HE-stained epidermal thickness of AD-like dorsal lesion in mice induced by DNFB ($ \bar{x} \pm s$, n=5) ####P<0.0001 vs control group; **P<0.01, ****P<0.0001 vs MC903 or DNFB group3.4 黄芩素外用制剂抑制特应性皮炎样小鼠皮肤组织内肥大细胞浸润
IL-4 和 IL-13 可激活肥大细胞，释放过敏介质，促进特应性皮炎发展[11]。因此，本研究也检测了皮肤组织内肥大细胞的浸润程度，并发现相比于 MC903 造模组，给药组可以明显地降低病变皮肤组织中的肥大细胞浸润数量 (图4-A，B)，并显著地降低肥大细胞脱颗粒比率，提示了给药组也抑制了活化的肥大细胞(图4-C)。DNFB 诱导的不同组别的特应性皮炎样小鼠皮肤组织中也观察到相同趋势 (图4-D, E)。综上，黄芩素外用制剂可以抑制特应性皮炎样病变小鼠病变皮肤组织中肥大细胞的浸润，降低肥大细胞活化程度。
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Figure 4. Pathologic observations of mast cell staining at skin lesions in AD-like mouse modelsA: Representative images of toluidine blue staining of mouse AD-like ear lesion tissue induced by MC903; B: Quantification of mast cell infiltration in mouse AD-like ear lesion tissue induced by MC903; C: Quantification of mast cell degranulation ratio in mouse AD-like ear lesion tissue induced by MC903; D: Quantification of mast cell infiltration in mouse AD-like dorsal lesion tissue induced by DNFB; E: Quantification of mast cell degranulation ratio in mouse AD-like dorsal lesion tissue induced by DNFB ($ \bar{x}\pm s $, n=5)####P<0.0001 vs control group; *P<0.05, ****P<0.0001 vs MC903 or DNFB group; PVF: Positive cells in the visual field (500 μm×890 μm)3.5 黄芩素外用制剂修复特应性皮炎诱导的损伤皮肤屏障
皮肤屏障受损会导致皮肤屏障蛋白缺失，尤其是丝聚蛋白和内披蛋白，从而导致特应性皮炎形成及加重[12]。通过对特应性皮炎样小鼠的组织皮肤屏障蛋白免疫荧光染色的定量，造模组小鼠皮肤屏障蛋白含量相比对照组下降，且呈不连续状，提示皮肤屏障受损，而给药组可改善皮肤屏障蛋白不连续状的情况 (图5)。以上结果表明，黄芩素外用制剂可修复小鼠特应性皮炎样病变导致的皮肤屏障蛋白受损。
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Figure 5. Immunofluorescence staining of skin barrier proteins at mouse lesionsA: Representative images of involucrin immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of Filaggrin immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; C: Representative images of involucrin immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB; D: Representative images of filaggrin immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB3.6 黄芩素外用制剂抑制特应性皮炎样病变部位Th2细胞浸润程度
研究表明，特应性皮炎患者的皮肤角质形成细胞可表达高水平的TSLP并激活Th2型的炎症反应，有助于特应性皮炎皮肤免疫应答的启动和恶化[13]。本研究在两种小鼠特应性皮炎样病变模型中观察到，造模组小鼠皮肤Th2细胞(CD4+/IL-4+)浸润数量相比对照组明显增多，给药组可显著性地减少Th2细胞在病变皮肤组织中的浸润数 (图6)。结果提示，黄芩素外用制剂可显著抑制特应性皮炎样病变部位Th2细胞浸润程度。
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Figure 6. Immunofluorescence staining of Th2 cells in mouse skin lesionsA: Representative images of CD4+/IL4+ Th2 immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of CD4+/IL4+ Th2 immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB; C: Quantification of Th2 cell infiltration in mouse AD-like ear lesion tissues induced by MC903; D: Quantification of Th2 cell infiltration in mouse AD-like dorsal lesion tissues induced by DNFB ($ \bar{x}\pm s $, n=5) ####P<0.0001 vs control group; **P<0.01, ****P<0.0001 vs MC903 or DNFB group3.7 网络药理学筛选出黄芩素治疗特应性皮炎的潜在治疗靶点
网络药理学分析结果显示，黄芩素治疗特应性皮炎的潜在治疗靶点共50个 (图7-A)。随后，利用STRING 数据库和Cytoscape 软件建立了黄芩素-特应性皮炎调控靶点图 (图7-B)。最后对凝练靶点进行GO富集并分析发现，潜在相关靶点主要富集于对外源性刺激的反应、炎症反应、刺激性C型凝集素受体信号通路、白细胞迁移及固有免疫应答这几条通路 (图7-C)。
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Figure 7. Cyberpharmacologic analysis of baicalein in the treatment of atopic dermatitisA: Intersection of atopic dermatitis disease targets with corresponding targets of baicalein; B: Interaction map of the protein "baicalein-AD target"; C: GO enrichment analysis of biological process (BP) as a potential therapeutic target of baicalein for AD treatment3.8 黄芩素抑制角质形成细胞的炎症相关因子的表达
外用制剂主要通过透皮吸收对特应性皮炎发挥作用，因此角质形成细胞为相关性最高的靶细胞。多重细胞因子，尤其IFN-γ和TNF-α刺激角质形成细胞可促进角质形成细胞活化，并上调多种炎症相关因子加重炎症，尤其是Th2相关趋化因子[14]。细胞实验结果表明，给药组处理可明显下调由IFN-γ+TNF-α上调的角质形成细胞产生的细胞因子，例如CCL17、CCL22、CTSS、IL-6和TNF-α (图8-A~图8-E)。此外，Poly (I:C)+TNF-α 刺激角质形成细胞可诱导Th2相关炎症因子上调[15]。实验结果表明，给药组可显著性地抑制Poly(I:C)+TNF-α所上调细胞因子表达，包括IL-4、TSLP、CCL17和CCL22 (图8-F~图8-I)。以上结果表明，黄芩素可抑制角质形成细胞中促炎细胞因子的表达，从而可能缓解特应性皮炎症状。
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Figure 8. Effect of baicalein on the expression of related chemokines and cytokines in HaCaT cells ($ \bar{x}\pm s $, n=3)A-E: Expression of CCL17, CCL22, CTSS, IL-6 and TNF-α mRNA after IFN-γ+TNF-α treatment of HaCaT cells; F-I: Expression of IL-4, TSLP, CCL17 and CCL22 mRNA after Poly(I:C)+TNF-α treatment of HaCaT cells #P<0.05, ####P<0.0001 vs control group; *P<0.05, ***P<0.001, ****P<0.0001 vs IFN-γ+TNF-α or Poly(I:C)+TNF-α group3.9 黄芩素下调人源角质形成细胞内NF-κB和STAT1磷酸化水平
网络药理学分析结果表明炎症相关通路中NF-κB为关键靶点，也有其他研究表明STAT1与Th2相关趋化因子表达密切相关[16]。因此，本研究细胞模型上检测了相关信号通路。结果表明，IFN-γ+TNF-α处理HaCaT细胞后使得NF-κB p65和STAT1水平上升且NF-κB p65和STAT1的磷酸化水平也上升。黄芩素处理后对IFN-γ+TNF-α处理HaCaT细胞的NF-κB p65和STAT1水平无明显影响，但可以降低磷酸化NF-κB p65和STAT1的水平 (图9)。综上，黄芩素可以下调NF-κB p65 和STAT1的磷酸化水平。
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Figure 9. Effect of baicalein on NF-κB and STAT1 mediated signalings in HaCaT cells ($ \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 group4. 讨 论
在特应性皮炎急性期，皮肤病变组织中Th2细胞浸润增多，而在特应性皮炎的慢性期，皮损中还可见Th1、Th17和Th22的混合炎症浸润[2]。MC903能激活Th2型的炎症反应。 DNFB诱导的小鼠模型会引起Th1和Th17主导的炎症[17]。本研究通过构建两种动物实验模型可更全面地评价黄芩素外用制剂对特应性皮炎急慢性期的治疗作用，且结果表明，黄芩素外用制剂在两种特应性皮炎药物常用评价动物模型中均取得显著疗效，证明黄芩素外用制剂对特应性皮炎的治疗有巨大的潜在应用价值。
此外，特应性皮炎患者的角质形成细胞可释放TSLP诱导机体Th2反应发生并驱动 IgE 合成，从而导致特应性皮炎过敏症状[11]。因此，阻断角质形成细胞及TSLP的释放，可以有效地打断该发病循环。本研究发现黄芩素外用制剂可抑制特应性皮炎样小鼠模型中TSLP的转录水平表达，抑制病变部位的Th2细胞浸润，并减少肥大细胞的浸润和活化。这可能是由于外用制剂通过透皮作用，直接作用于病变部位的特性，影响皮肤角质形成细胞炎症免疫应答。
此外，为了探索黄芩素外用制剂治疗特应性皮炎的作用机制。本研究主要关注胸腺活化调节趋化因子(CCL17) 与巨噬细胞衍生趋化因子 (CCL22)。已有相关报道表明，其在特应性皮炎患者的皮肤组织和血浆中升高，可有效趋化募集表达 CCR4 的 Th2 淋巴细胞到炎症部位，参与特应性皮炎的发生发展[18−19]。而组织蛋白酶 S (CTSS) 可通过激活蛋白酶激活受体2 (PAR2) 在特应性皮炎等炎症性皮肤病中发挥瘙痒作用[20]。本研究结果显示，黄芩素可显著降低以上细胞因子的mRNA水平，提示了黄芩素可能通过下调这些趋化因子从而降低特应性皮炎的炎性细胞的募集。
此外，黄芩素可影响多种炎症相关信号通路治疗炎症性疾病，包括cAMP-PKA-NF-κB/CREB通路[21]，NF-κB和STAT信号通路等[22−23]。网络药理学分析提示黄芩素可影响NF-κB信号通路治疗特应性皮炎，实验结果显示黄芩素可抑制NF-κB p65及STAT1的磷酸化，提示黄芩素可通过NF-κB p65及STAT1信号通路抑制HaCaT细胞炎症因子的表达。
综上所述，黄芩素外用制剂可通过作用于小鼠病变皮肤，缓解小鼠皮肤TEWL，保护小鼠病变皮肤的皮肤屏障，降低角质形成细胞的TSLP表达，减少病变组织中Th2细胞浸润、肥大细胞浸润和激活以及B细胞IgE释放。其机制可能为抑制角质形成细胞中NF-κB p65的磷酸化，抑制炎症因子的表达来缓解特应性皮炎症状。黄芩素外用制剂对特应性皮炎具有良好的治疗作用。
药学院郝海平教授团队熊晶课题组发表调控代谢
重塑促进肝再生的新靶标研究成果近日，权威期刊EMBO JOURNAL发表了药学院郝海平教授团队熊晶课题组的最新研究成果——SLC13A2 promotes hepatocyte metabolic remodeling and liver regeneration by enhancing de novo cholesterol biosynthesis，报道了调控代谢重塑促进肝再生的新靶标。药学院2021级硕士生石丽、2024级博士生陈浩、2023级硕士生张雨欣为本文共同第一作者，药学院熊晶研究员和郝海平教授为本文共同通信作者，中国药科大学为本文唯一通信单位。 该研究工作受到国家自然科学基金（No. 82273982，82070883）、江苏省自然科学基金（No. BK20221525）以及中国药科大学高层次引进人才科研项目的支持。 全文链接：https://www.embopress.org/doi/full/10.1038/s44318-025-00362-y。 -
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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 $, n=5)####P<
0.0001 vs control group; *P<0.05, ****P<0.0001 vs MC903 or DNFB group; PVF: Positive cells in the visual field (500 μm×890 μm)![]()
Figure 5. Immunofluorescence staining of skin barrier proteins at mouse lesions
A: Representative images of involucrin immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of Filaggrin immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; C: Representative images of involucrin immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB; D: Representative images of filaggrin immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB
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Figure 6. Immunofluorescence staining of Th2 cells in mouse skin lesions
A: Representative images of CD4+/IL4+ Th2 immunofluorescence staining of mouse AD-like ear lesion tissue induced by MC903; B: Representative images of CD4+/IL4+ Th2 immunofluorescence staining of mouse AD-like dorsal lesion tissue induced by DNFB; C: Quantification of Th2 cell infiltration in mouse AD-like ear lesion tissues induced by MC903; D: Quantification of Th2 cell infiltration in mouse AD-like dorsal lesion tissues induced by DNFB ($ \bar{x}\pm s $, n=5) ####P<
0.0001 vs control group; **P<0.01, ****P<0.0001 vs MC903 or DNFB group![]()
Figure 7. Cyberpharmacologic analysis of baicalein in the treatment of atopic dermatitis
A: Intersection of atopic dermatitis disease targets with corresponding targets of baicalein; B: Interaction map of the protein "baicalein-AD target"; C: GO enrichment analysis of biological process (BP) as a potential therapeutic target of baicalein for AD treatment
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Figure 8. Effect of baicalein on the expression of related chemokines and cytokines in HaCaT cells ($ \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 indicator Forward primer (5'→3') Reverse primer (5'→3') TSLP GCAAATCGAGGACTGTGAGAGC TGAGGGCTTCTCTTGTTCTCCG GAPDH CATCACTGCCACCCAGAAGACTG ATGCCAGTGAGCTTCCCGTTCAG 
下载: 导出CSV
Table 2 Primer sequences for real-time qPCR
Biological indicator Forward primer (5'→3') Reverse primer (5'→3') CCL17 TTGTAACTGTGCAGGGCAGG TGAACACCAACGGTGGAGGT CCL22 GAAGCCTGTGCCAACTCTCT GGGAATCGCTGATGGGAACA CTSS TGGGCTTTCAGTGCTGTGGG TCAATGATGTACTGGAAAGC IL-4 CCGTAACAGACATCTTTGCTGCC GAGTGTCCTTCTCATGGTGGCT TSLP GAA AGCTCTGGAGCATCAGG AGGGAACATACGTGGACACC IL-6 AGACAGCCACTCACCTCTTCAG TTCTGCCAGTGCCTCTTTGCTG TNF-α CTCTTCTGCCTGCTGCACTTTG ATGGGCTACAGGCTTGTCACTC GAPDH GAAGGCTCATGACCACAGT GGATGCAGGGATGATGTTCT
下载: 导出CSV
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