Alopecia, also called hair loss, is caused by hair follicle atrophy that can lead to abnormal alopecia (Dhariwala and Ravikumar, 2019; Baek et al., 2022). Approximately 40% of women worldwide suffer from hair loss, while the proportion of hair loss in males is much higher at 85% (Zito and Raggio, 2022). Although hair loss is not life-threatening, excessive shedding can negatively impact mental health and quality of life, leading to depression and other health conditions (Aukerman and Jafferany, 2022; Macbeth et al., 2022). Hair growth is cyclical and includes three consecutive phases: anagen (growth phase), catagen (regression phase), and telogen (resting phase) (Paus and Foitzik, 2004; Lin et al., 2022). Regulation of the hair growth cycle is a common strategy for treating hair loss. In fact, two drugs, minoxidil and finasteride, have been approved by the United States Food and Drug Administration for this purpose. However, due to the associated adverse events, patient compliance for these drugs is poor. For example, finasteride can significantly reduce libido, cause erectile dysfunction, and abnormal sexual function (Arca et al., 2004; Sato and Takeda, 2012; Dou et al., 2022). Moreover, the pathological mechanism of hair loss is complex, making effective treatment with a single compound challenging. Hence, a need exists for the development of more effective and less toxic therapeutic options.

Traditional Chinese Medicine (TCM) has a long history of clinical application for myriad conditions and diseases, representing a unique health resource. In particular, various TCM preparations based on plant monomers have exhibited excellent results in promoting hair growth (Kang et al., 2012; Kang et al., 2019; Her et al., 2022), encouraging a more in-depth investigation of such treatments. This approach has the advantage of providing multi-component, multi-target synergistic effects while with low toxicity. Tianma Guoteng decoction (TGD) is a TCM preparation described in the book “New Meaning of Miscellaneous Diseases Treatment”. TGD contains 11 ingredients, namely: Gastrodia elata Blume [Orchidaceae; Gastrodiae Rhizoma, GR 9 g], Uncaria rhynchophylla (Miq.) Miq. Ex Havil. [Rubiaceae; Uncariae Ramulus cum Uncis, UR 12 g], Haliotis diversicolor Reeve [Haliotidae; Haliotidis Concha, HC 18 g], Gardenia jasminoides J. Ellis [Rubiaceae; Gardeniae Fructus, GF 9 g], Scutellaria baicalensis Georgi [Lamiaceae; Scutellariae Radix, SR 9 g], Eucommia ulmoides Oliv. [Eucommiaceae; Eucommiae Cortex, EC 9 g], Leonurus japonicus Houtt. [Lamiaceae; Leonuri Herba, LH 9 g], Taxillus chinensis (DC.) Danser [Loranthaceae; Taxilli Herba, TH 9 g], Reynoutria multiflora (Thunb.) Moldenke [Polygonaceae; Polygoni Multiflori Caulis, PMC 9 g], Faria cocos (Schw.) Wolf [Polyporaceae; Poria, PA 9 g], and Cyathula officinalis K.C. Kuan [Amaranthaceae; Cyathulae Radix, CR 12 g]. Pharmacological studies have demonstrated that TGD exhibits anti-oxidant, anti-apoptotic, and neuroprotective (Liu et al., 2015; Xian et al., 2016) effects. Moreover, it has proven effective in the treatment of hypertension, Parkinson’s disease, and hair loss (Zhang et al., 2003; Tsai et al., 2014; Liu et al., 2015; Deng et al., 2022). In fact, many components of TGD have exhibited beneficial therapeutic effects on hair loss. For example, baicalin (the active ingredient of HQ) effectively treats hair loss via promotion of hair follicle development through activation of the Wnt/β-catenin pathway (Xing et al., 2018). Meanwhile, quercetin (the active ingredient in DZ) is used to prevent and treat hair loss by promoting the expression of growth factors (Wikramanayake et al., 2012; Kim et al., 2020). Hence, TGD has potential as an effective complementary and alternative medicine option for hair loss treatment, however, a complete list of its bioactive components, and the associated therapeutic mechanisms, remain to be characterized.

TCM formulas are characterized by their multi-component composition that provides multi-target synergistic actions. However, the use of traditional pharmacological methods to study the biological mechanisms of these medicines is limited. In contrast, network pharmacology is a new strategy that combines computer technology and systems biology to explore associated pathways (Zhou et al., 2020; Wei et al., 2022). Compared with traditional approaches, this technique can decipher the complex relationships between biological systems, medicine, and diseases in a multidimensional manner from a network perspective (Zhang et al., 2019). Herein, we describe a dual-dimensional pharmacological network utilizing the active components of TGD as selected from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP) database and detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). We propose potential bioactive compounds involved in the anti-alopecia mechanism using these multiple perspectives, and subsequently validating our data with molecular docking, in vivo experiments, and in vitro analyses (Figure 1).

TCM contains a variety of biologically active compounds and has been widely used in the treatment of various diseases due to its beneficial therapeutic effects, few side effects, and its multi-component and multi-target synergistic value (Bu et al., 2020; Luan et al., 2020; Wang et al., 2021). However, assessing the efficacy of compound herbal therapies can be challenging. Emerging network pharmacology is an ideal tool for the study of the mechanisms underlying TCM treatment, which is helpful to promote the development of TCM preparations (Sucher, 2013; Nogales et al., 2022). Network pharmacology combines computer science with biological information, constructing networks using algorithms, theoretical data, and experimental data; this allows the purposeful and directional investigation of the therapeutic mechanisms of TCM on a molecular and biological level (Zhao et al., 2019). In our study, we expanded on conventional network pharmacology to construct a dual-dimensional network for analyzing the mechanism of TGD treatment of alopecia, using both theoretical and experimental data. This method seeks to evaluate the authenticity and reliability of the action targets of TGD for alopecia. A total of 39 bioactive components of TGD were detected by UHPLC-MS/MS. Subsequently, a bioactive component-target network was constructed to predict the protein targets and bioactive components of TGD for treating hair loss. Comprehensive GO, KEGG, and molecular docking analyses revealed the binding capacity between the target proteins and bioactive components, as well as the potential signaling pathway underlying the TGD anti-alopecia effects. After analysis of the networks, we used a murine model of alopecia confirmed the efficacy of TGD in vivo in promoting hair growth. Finally, Western blot and immunofluorescence analysis were performed to verify the result of network pharmacologic prediction, elaborate in vitro and in vivo the mechanisms of TGD against alopecia.

A total of 251 active ingredients were detected in the TGD extracts using UHPLC-MS/MS, of which 39 bioactive components satisfied the condition of OB ≥ 30% and DL ≥ 0.18. According to the analysis of bioactive component-target network and references, quercetin, luteolin, fisetin, wogonin, oroxylin A, boldine, tetrahydroalstonine, and galangin were likely the most important bioactive compounds associated with the anti-alopecia activity of TGD, which relative contents in TGD extract were 0.16%, 0.35%, 0.002%, 2.03%, 1.17%, 0.12%, 0.45% and 0.06% respectively (Supplementary Table S6). Among them, Quercetin, luteolin, wogonin, oroxylin A and tetrahydroalstonine have high relative contents in TGD extract. There is a good correlation between the content of compound and its importance. Furthermore, one of the causes of hair loss is oxidative stress due to increased free radicals in the hair follicle (Ablon, 2021). Quercetin has a significant antioxidative role in many diseases (Cebecioglu et al., 2019; Tang et al., 2019; Zou et al., 2021). Quercetin also can stimulate the proliferation of human dermal papilla cells by activating the MAPK/CREB signaling pathway, thereby promoting the production of growth factors, and stimulating the elongation of human hair follicles (Kim et al., 2020). Studies have shown that quercetin can promote hair growth in C3H/HeJ mice and stimulate the proliferation of epithelial cells, which is considered as a potential hair protectant (Towatari et al., 2002; Wikramanayake et al., 2012; Dario et al., 2020). Luteolin has been shown to significantly promote hair regeneration in vitro (Shin et al., 2018; Taguchi et al., 2019). The anti-alopecia activities of fisetin have been validated in vivo and in vitro studies, which might be achieved by enhancing telomerase reverse transcriptase (TERT) levels (Kubo et al., 2020; Ogawa et al., 2021). Moreover, Quercetin, luteolin and fisetin also have significant anti-androgen effect (Khan et al., 2008; Das et al., 2019; Wu et al., 2021). It is noteworthy that quercetin, luteolin, fisetin, and wogonin are flavonoids, which have a significant effect on protecting hair follicles, and constitute a class of natural medicines with great potential to prevent hair loss (Bassino et al., 2020). Wogonin and oroxylin A existed in Scutellaria baicalensis Georgi, and tetrahydroalstonine is unique ingredient in Uncaria rhynchophylla (Miq.) Miq. Ex Havil. Studies have shown that extract of Scutellaria baicalensis Georgi can effectively inhibit the expression of androgen receptor, thus inhibiting hair loss (Kim et al., 2014). Oroxylin A have been shown that could reduce the expression of inflammatory factors in human dermal fibroblasts thereby inhibiting the damage to human dermal fibroblasts (Nakajima et al., 2001). As for wogonin, oroxylin A and tetrahydroalstonine these compounds get more attention on regulating hormone levels activity (Roquebert and Demichel, 1984; Chi and Kim, 2005; Wang et al., 2013). Galangin can promote hair darkening in mice in vitro, but the specific mechanism needs further study (Huo et al., 2014). Collectively, all of which indicate that these ingredients have potent research value in promoting hair growth activity, and TGD is a TCM preparation with positive therapeutic anti-alopecia effects.

The PPI results identified GSK3β as potential targets for TGD treatment of alopecia. GSK3β is defined as a serine-threonine kinase involved in cell motility and development as well as the regulation of glucose homeostasis. Moreover, GSK3β protein has a key role in the Wnt/β-catenin signaling pathway. GSK3β regulates this pathway by governing the stability and function of β-catenin (Gao and Chen, 2010), which is a substrate of GSK3β and a core protein of the pathway. Furthermore, we found that AR and ESR1 proteins related to hair growth are also included. GSK3β can inhibit the activity of AR (Salas et al., 2004), which is a member of the nuclear receptor superfamily that enters the nucleus to regulate the expression of downstream genes after binding to testosterone or dihydrotestosterone (Li et al., 2019). Dysregulation of intracellular androgen levels can cause hair loss, and ARs mediate the circulation of androgen steroid hormones in cells (Lai et al., 2012). More specifically, ARs phosphorylate β-catenin by activating GSK3β in DPCs; phosphorylated β-catenin is degraded by proteasomes, thereby inhibiting activation of the Wnt/β-catenin pathway (Leirós et al., 2012). Meanwhile, ESR1 is an estradiol receptor that induces β-catenin stabilization by promoting the phosphorylation of GSK3β at serine 9 (Varea et al., 2009). In addition, ESR1 can regulate β-catenin/TCF-mediated transcription, possibly regulating transcription of the Wnt protein, thereby triggering activation of the Wnt/β-catenin pathway (Varea et al., 2010). Finally, we validated binding affinity between eight key bioactive components and core targets (GSK3β, β-catenin) utilizing molecular docking to provide further support for the role of TGD in treating alopecia. Molecular docking outcomes manifested that the eight components had strong intermolecular interactive forces with GSK3β and β-catenin protein.

GO and KEGG enrichment analysis confirmed that the role of TGD in hair follicle regeneration was related with Wnt pathway, hormone response, and regulation of cell migration and movement. Wnt/β-catenin signaling pathway plays a crucial role in embryonic hair follicle morphogenesis and is also one of the important pathways affecting hair follicle development and regeneration during the hair cycle. (Rishikaysh et al., 2014; Kim and Garza, 2017). This signaling pathway promotes hair follicle regeneration by regulating hair follicle cycle. Research shows that Dickkopf-1, as an inhibitor of Wnt/β-catenin signaling pathway, has the effect of promoting hair follicle transition from anagen to telogen phase (Kwack et al., 2012). In addition, inactivation of β-catenin in the dermal papilla also leads to enter telogen phase in advance of hair follicles (Enshell-Seijffers et al., 2010). β-catenin is activated and transferred to the nucleus for expression, which is a key signal mediating transcription and activating Wnt/β-catenin signaling pathway (Logan and Nusse, 2004; Shimomura and Christiano, 2010). GSK3β forms a complex with the scaffolding protein Axin (Axin), casein kinase 1α (CK1), and adenomatous polyposis coli (APC) without Wnt ligand, thereby disrupting the signaling pathway by phosphorylating the Thr41/Ser37/Ser33 site of β-catenin, leading to its proteasomal degradation (Nusse and Clevers, 2017). The transcriptional function of β-catenin is mainly regulated by Axin/GSK-3β/APC complex (Jin et al., 2021). When the Wnt/β-catenin pathway is activated, the complex is disassembled, resulting in translocation of β-catenin from the cytoplasm to the nucleus (Angers and Moon, 2009).

We also evaluated the efficacy of TGD in hair follicle regeneration in C57BL/6J mice. Results showed that the mice entered the telogen after depilation, however, following administration of TGD, they entered anagen at an accelerated rate. Hence, TGD effectively promoted hair growth in C57BL/6J mice. Additionally, treatment of rat vibrissa follicle and DPCs with TGD caused accumulation of β-catenin and LEF1 in the nuclei. Furthermore, TGD treatment significantly stimulated the expression of β-catenin and p-GSK3β, while decreasing that of GSK3β in DPCs and C57BL/6J mice. Hence, TGD may promote the phosphorylation of GSK3β in the cytoplasm, which in turn inhibits β-catenin phosphorylation by GSK3β, ultimately leading to accumulation of β-catenin in the cell nuclei and activation of the Wnt/β-catenin pathway (Figure 11). These results confirm the prediction of network pharmacology and molecular docking analyses, providing experimental reference for the mechanism employed by TGD against alopecia.

Collectively, this study provides several new directions for the development of subsequent therapeutics. For example, ESR1 and AR were identified as targets of TGD for the treatment of hair loss; we postulated that TGD may alleviate hair loss by promoting the expression of estrogens, thus counteracting the increase in androgen levels; however, this conjecture requires further confirmation. Moreover, we have shown that dual-dimensional network pharmacology is an effective strategy that builds on basic network pharmacology for a more accurate investigation into the possible therapeutic mechanisms and compounds of drugs. This will be increasingly important as multi-target therapies become more common as a strategy for the treatment of hair loss.

In conclusion, we show that TGD has positive therapeutic effects on hair growth and demonstrate the reliability of network pharmacology in the study of drug mechanisms. Hence, this study lays the foundation for the development of new therapeutics based on TCM.