Qian Huanzhu, Ye Zhen, Hu Yu, Chen Liulin, Li Linzhen, Qin Kaihua, Ye Qiaobo, Zuo Xiaohong
School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Health Preservation and Rehabilitation College, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
J Ethnopharmacol. 2025 Feb 11;341:119347. doi: 10.1016/j.jep.2025.119347. Epub 2025 Jan 10.
Dahuang-Gancao decoction (DGD) is a traditional Chinese medicinal formula that is recorded in the Synopsis of the Golden Chamber, and is widely used to treat damp-heat in the body. Since the pathological factors of androgenetic alopecia (AGA) also reflect damp-heat blockage, DGD has great potential for the treatment of AGA and has been used effectively in clinical practice.
The aim of the study was to investigate whether external application of DGD could promote the activation and proliferation of hair follicle stem cells (HFSCs) and improve AGA through the Wnt/β-catenin pathway.
The main chemical components of DGD-contained serum were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database search. Cell Counting Kit-8 (CCK8) was used to investigate the appropriate concentration. Hair regeneration was assessed by hair growth score and histopathological staining. The proliferation of HFSCs and the activation of Wnt/β-catenin pathway were detected by Western blot, immunofluorescence staining, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). The AGA mouse model was induced by external application of testosterone (T). Immunofluorescence staining was performed to localize HFSCs by CK15, followed by staining with Ki67, β-catenin, and Cyclin D1, respectively.
The results illustrated that the 10% DGD group and the 10% DGD + HLY78 group could significantly promote the expression of Wnt10b and β-catenin and the proliferation of HFSCs in vitro, while the 10% DGD + IWR-1 group could reverse the promotion effect of DGD. Animal experiments showed that compared with the model group (T group), DGD promoted hair follicles to enter the anagen phase, as evidenced by an increase in hair growth score, an increase in the number of hair follicles in hematoxylin and eosin (HE) staining, and a significant increase in the ratio of the number of anagen follicles to the total number of hair follicles (AF/AF + TF). In addition, DGD upregulated the expression of Wnt/β-catenin signaling pathway proteins in the skin tissues of AGA mice. It also promoted the proliferation of HFSCs and the expression of β-catenin and Cyclin D1 cytokines in the region of HFSCs.
Both oral and external application of DGD can promote the proliferation of HFSCs by activating the Wnt/β-catenin signalling pathway. External application of DGD can promote the hair follicles to enter the anagen phase, which can ameliorate the symptoms of alopecia in AGA mice. Therefore, compared to oral DGD, external application of DGD is an effective and safer way of administration for the treatment of AGA.
大黄甘草汤（DGD）是《金匮要略》中记载的一种中药方剂，广泛用于治疗体内湿热。由于雄激素性脱发（AGA）的病理因素也反映了湿热阻滞，大黄甘草汤在治疗AGA方面具有很大潜力，并已在临床实践中得到有效应用。
本研究旨在探讨外用大黄甘草汤是否能通过Wnt/β-连环蛋白信号通路促进毛囊干细胞（HFSCs）的激活和增殖，并改善AGA。
采用液相色谱-串联质谱（LC-MS/MS）和数据库检索鉴定含大黄甘草汤血清的主要化学成分。使用细胞计数试剂盒-8（CCK8）研究合适的浓度。通过毛发生长评分和组织病理学染色评估毛发生长情况。采用蛋白质免疫印迹法、免疫荧光染色、实时逆转录定量聚合酶链反应（RT-qPCR）和酶联免疫吸附测定（ELISA）检测HFSCs的增殖和Wnt/β-连环蛋白信号通路的激活情况。通过外用睾酮（T）建立AGA小鼠模型。采用细胞角蛋白15（CK15）进行免疫荧光染色定位HFSCs，随后分别用Ki67、β-连环蛋白和细胞周期蛋白D1进行染色。
结果表明，10%大黄甘草汤组和10%大黄甘草汤+HLY78组能显著促进体外Wnt10b和β-连环蛋白的表达以及HFSCs的增殖，而10%大黄甘草汤+IWR-1组可逆转大黄甘草汤的促进作用。动物实验表明，与模型组（T组）相比，大黄甘草汤可促进毛囊进入生长期，表现为毛发生长评分增加、苏木精-伊红（HE）染色中毛囊数量增加以及生长期毛囊数与毛囊总数之比（AF/AF+TF）显著增加。此外，大黄甘草汤上调了AGA小鼠皮肤组织中Wnt/β-连环蛋白信号通路蛋白的表达。它还促进了HFSCs的增殖以及HFSCs区域中β-连环蛋白和细胞周期蛋白D1细胞因子的表达。
口服和外用大黄甘草汤均可通过激活Wnt/β-连环蛋白信号通路促进HFSCs的增殖。外用大黄甘草汤可促进毛囊进入生长期，改善AGA小鼠的脱发症状。因此，与口服大黄甘草汤相比，外用大黄甘草汤是治疗AGA的一种有效且更安全的给药方式。
