The antiinflammatory effects of Xuefu Zhuyu decoction on C3H/HeJ mice with alopecia areata

As a traditional and typical prescription of prominently activating blood circulation to remove blood stasis, Xuefu Zhuyu decoction (XZD) consists of 15 kinds of herbal medicine. Clinical investigations have showed that XZD could significantly promote the new hair generation of alopecia areata (AA) patients characterized by Qi stagnation and blood stasis. The purpose of this study was executed to determine whether the mechanisms by which XZD stimulated newborn hair were related to its anti-inflammatory effects. Clinical AA individuals were recruited to confirm the efficies of XZD. High performance liquid chromatography (HPLC) analysis was performed to qualitatively and quantitatively determine the contents of 15 compounds in XZD. Schrodinger molecular docking and in vivo surface plasmon resonance (SPR) techniques were used to evaluate the potential binding properties of compounds to target proteins. C3H/HeJ mice were randomly assigned to groups control, AA, and the XZD administration (6.5, 13.0 and 26.0 g/kg/d). Except for mice in control group, all the mice in the other groups were treated with a 21-day chronic unpredictable mild stress (CUMS) induced AA. Hematoxylin-eosin (H&E) staining was performed to determine the degree of pathological damage to the skin. Enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) and in serum and skin tissues. Western blot, immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to examine the expression levels of IL-6, IL-1β, TNF-α and osteopontin proteins and genes in skin tissues. XZD could visibly promote hair regeneration of AA patients. The potential active ingredients in XZD prescription included at least amygdalin, hydroxysafflor yellow A, kaempferide, ferulic acid, catalpol, verbascoside, β-ecdysone, platycodin D, paeoniflorin, naringin, neohesperidin, liquiritin, glycyrrhizic acid, saikosaponin A and saikosaponin D. The results of molecular docking and SPR analysis showed that verbascoside, liquiritin, kaempferide and amygdalin showed the best potential binding properties with IL-6, IL-1β, TNF-α and osteopontin, respectively. Pathological evaluation showed that compared with the CUMS group, the administration of XZD significantly promoted hair regeneration, evidenced by increased number of skin hair follicles in C3H/HeJ AA mice. Compared with control group, ELISA data showed that the levels of IL-6, IL-1β and TNF-α in serum and skin tissues of CUMS induced AA mice were significantly increased, while XZD administration dramatically restrained the contents of the three pro-inflammatory factors. Western blot, immunohistochemistry, and qRT-PCR results further demonstrated that XZD administration notably down-regulated the protein and gene expression levels of osteopontin, IL-6, IL-1β and TNF-α in comparation with CUMS group. XZD could dramatically ameliorate CUMS-induced AA damage in the skin of C3H/HeJ mice, possibly by suppressing the levels of IL-6, IL-1β, TNF-α and osteopontin.