Amygdalin ameliorates alopecia areata on C3H/HeJ mice by inhibiting inflammation through JAK2/STAT3 pathway

Evidence has demonstrated that Chinese medicine formula Xuefu Zhuyu decoction can markedly promote the formation of new hair in patients and mice with alopecia areata (AA). Amygdalin is one of the active components of Xuefu Zhuyu decoction, but its therapeutic effects and the underlying mechanisms on AA remains largely unrevealed. Therefore, this study aims to investigate the therapeutic effects and to probe its molecular mechanisms of inflammation and immune regulation on AA model of C3H/HeJ mice. The C3H/HeJ female mice were divided into control, AA, rusolitinib (60 mg/kg), and amygdalin groups (60, 90, and 120 mg/kg, 0.2 ml/10 g, i.g.). The optical microscope was used to observe the feature of the local skin, and the number of lanugo and terminal hair. H&E staining was performed to determine the degree of pathological damage to the skin. ELISA was performed to detect levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in mice serum. Flow cytometry was carried out to analyze the CD4+CD25+FOXP3+, CD4+ and CD8+ of skin tissue. And the levels of CD4+ and CD8+, p-JAK/JAK2, p-STAT3/STAT, and SOCS3 were detected by immunohistochemistry. Western blot and qRT-PCR were employed to examine the expression levels of IL-6, TNF-α, IFN-γ, JAK2, p-JAK, STAT, p-STAT3 and SOCS3 proteins and genes in skin tissues. Compared with AA group, amygdalin immensely increased the number of vellus hairs and decreased the number of terminal hairs determined by skin microscopy and H&E staining. ELISA, western blot and qRT-PCR data showed that the levels of IL-6, TNF-α and IFN-γ in serum and skin tissues of AA mice were significantly increased, while amygdalin administration dramatically restrained the contents of the three pro-inflammatory factors. Flow cytometry and immunohistochemistry hinted that amygdalin observably enhanced the number of CD4+CD25+FOXP3+ and CD4+ cells, while inhibited the number of CD8+ positive cells in mice with AA. Moreover, amygdalin signally reduced JAK2/STAT3 pathway-related protein and gene levels in AA mice. Amygdalin could inhibit inflammatory response and improve immune function in the treatment of AA. The underlying molecular mechanism may be related to inhibition of JAK2/STAT3 pathway.