AA is a non-scarring alopecia that manifests mainly as localized, diffuse, or generalized alopecia[23]. The main cause of AA may be related to the breakdown of the HF IP, while immune, genetic, psychiatric, and environmental factors may also lead to AA [24].
In this study, 90 active components of CX were screened, and Thymol was identified as the key active ingredient in treating AA. Thymol is a monoterpenoid phenolic compound, which has important functions such as antioxidation, anti-inflammation, analgesia, and local anesthesia[25] It has been proven significant in treating cardiovascular and cerebrovascular diseases, digestive system disorders, immune diseases, cancer, and more[26]. Inflammation and oxidative stress are the inducing factors of many immune diseases including AA[27]. In this study, hDPCs were treated with IFN- γ to simulate the pathophysiological environment of AA. The results of the CCK-8 assay showed that thymol could increase the activity of hDPCs in a concentration-dependent manner and decrease the inhibition rate of IFN-γ on cells. IFN-γ is a crucial inflammatory factor for AA. It can increase the expression of MHC class I chain-related protein A (MICA) and certain chemokines like IL-15 in HF cells, which results in the breakdown of the HF's immune protection mechanism[28]. MICA is the ligand of NKG2D cells (such as CD8+T cells and NK cells), which can be stimulated by oxidative stress in HFs[29]. IL-15 is a key immunostimulatory cytokine and mast cell regulator[30]. In this study, the RT-PCR experiment found that IFN-γ increases the expression of MICA and IL-15 genes in HF cells. In contrast, the expression level decreased significantly after Thymol treatment. This study also confirmed that Thymol inhibited the expression of IL6, IL-1 β, and caspase-1. Both IL-6 and IL-1 β are the key factors in inducing oxidative stress. IL-6 plays an important role in innate immunity, it participates in the activation of the JAK-STAT signal pathway in the inflammatory pathway, and it has been found that both IL-1 β and Th1 play a catalytic role in the pathogenesis of AA[31]. Caspcase-1 is an inflammatory caspase that is mainly involved in inflammatory stimulation in immune cells[32]. Some studies have found that the expression of caspase-1 in HF residues and inflammatory cells of patients with AA is significantly increased[33]. The results indicate that Thymol may treat AA by reducing various inflammatory cytokines and ameliorating oxidative stress.
In addition, this study also experimentally examined the changes in the expression of BDNF in each experimental group. BDNF has immunomodulatory effects on non-neural cells such as mast cells and keratinocytes[34]. The results by RT-PCR showed that its expression was elevated in the blank and Thymol-treated groups, while the levels in the disease group showed lower levels. This result is contrary to the pathological hypothesis; however, it is not an isolated result. The study by Dawoud observed a negative correlation between serum BDNF and the duration of AA, and it is hypothesized that patients with longstanding skin disorders, such as AA, may develop severe negative moods that are manifested in the expression of BDNF levels[35]. Furthermore, a potential link between low levels of BDNF and major depression has been confirmed by clinical studies[36]. Therefore, it can be inferred that Thymol may improve the depressive state of patients with AA by modulating the expression of BDNF and positively affecting the psychological state of patients, thus inhibiting the development of AA.
We screened the core target genes of CX on AA by network pharmacology: IL6, IL1B, IL10, IFNG, CCL2, TNF, INS, IL4, CRP, TGFB1, ALB, TP53, and BDNF. IL6, IL1B, IL10, and IL4 belong to the same family of interleukins, which can participate in the systemic inflammatory response and the process of immune regulation[37]. The cytokine IL-6 is involved in innate immunity and activation of the JAK-STAT signalling pathway and has been found to catalyse the cytokines Th1 and Th17, which are involved in the pathogenesis of AA[38]. The pathogenesis of AA is closely related to the dysregulation of Th1-type cytokines (IFN-γ, TNF), and Th2-type cytokines (IL-4, IL-5, IL-6, IL-9, IL-10)[39], whereas IL-10 is considered to be an anti-inflammatory agent that inhibits the production of various cytokines, such as IL-2, IL-3, and IFN-γ, which are mainly produced by Th1[40]. IL-4 stimulates the transformation of Th cells into Th1 cells[41], suggesting the use of IL-4 as a potential therapeutic target and biomarker of activity in AA.IFNG, or IFN-γ, is involved in developing several autoimmune skin diseases such as AA, vitiligo and systemic lupus erythematosus[42]. IFN-γ induces a runaway of the follicular immune amnesty mechanism, which is considered a CCL2 is secreted by immune cells and is involved in their immune cell infiltration and migration; its expression can be induced by IL-4, TGF-β, IFN-γ, etc. TNF is a major pro-inflammatory cytokine that is responsible for mediating a variety of autoimmune inflammatory disorders[43], and has a significant inhibitory effect on hair growth in humans[44]. CRP is a key marker of the inflammatory response[45]; some clinical studies have found that patients with AA may have significantly elevated CRP levels in their serum[46].TGFB1 plays an important role in the metastasis of tumours and the immune-suppressive response[47]. During the anagen phase, TGFB1 can reduce IFN-γ-induced MHC class I expression in HFs, leading to a restoration of the HF's immune privilege[48].ALB, or albumin, is mainly involved in plasma osmolality regulation, transport of compounds, and antioxidant reduction reactions to prevent oxidative damage[49]. Some studies have shown that oxidative stress may be closely associated with the pathogenesis of AA[50], so dysregulation of the antioxidant reduction function of ALB may be an important factor in the development of AA.BDNF, or brain-derived neurotrophic factor, has an immunomodulatory effect on non-neuronal cells, such as mast cells and keratinocytes[51].BDNF also stimulates follicular keratin leading to apoptosis and regulates macrophage activity, which is an important pathophysiological factor in the pathogenesis of AA.BDNF inhibits the elongation of the hair shaft accelerates the anagen process in the HF and promotes the transition of the HF to the regression phase[52], which may also be its role in the pathogenesis of AA.
This experiment was carried out based on the JAK-STAT pathway as an entry point in the results derived from network pharmacology. It is activated by many cytokines including interferon, interleukins, colony-stimulating factors, etc., which in turn are involved in inflammatory responses, innate and acquired immune responses, and cell growth[53]. Factors on the JAK-STAT signalling pathway, such as JAK1/2, are thought to promote T-cell-mediated inflammatory response, which in turn induces the production of cytokines, such as IFN-γ as well as IL-15[54]. Some of the key genes in the JAK-STAT pathway, including STAT1, STAT3, JAK1, and JAK3, are highly expressed during the catagen and telogen phases of hair growth, and are suppressed in the early anagen phase hair growth[55]. Kwack et al. showed that IL-6 was expressed at higher levels in balding dermis compared to non-balding dermis. Also, injection of recombinant IL-6 into anagen skin induced premature onset of regression, suggesting that IL-6 is an important mediator of hair growth inhibition[56].Western blot results showed that the Thymol intervention group could significantly down-regulate IL6, JAK1, and STAT1 expression. The above results suggest that the pathogenesis of AA-induced by IFN-γ may be closely related to the activation of the JAK-STATsignaling pathway, and Thymol can significantly reduce the cellular inflammatory response of IFNγ-induced AA model, and the mechanism of its therapeutic effect on AA may be related to the modulation of JAK-STAT signalling pathway.