Figure 1 Efficacy of ZXAS in the AD-like mice A: representative images of dorsal skin in each group on day 28. A1: NC; A2: MC; A3: CET; A4: LDZ; A5: MDZ; A6: HDZ; B: dermatitis scores were assigned on days 1, 4, 14, and 28; C: scratching frequency was recorded on days 1, 4, 14, and 28 for 30 min; D: skin sections from each group was stained with HE for morphological changes (× 400). D1: NC; D2: MC; D3: CET; D4: LDZ; D5: MDZ; D6: HDZ; E: skin thickness in each group. E1: epidermal thickness was measured in HE-stained sections using the microscope (μm, n = 9). E2: dermis thickness was measured in HE-stained sections using the microscope (μm, n = 9). Scale bar = 20 μm. Measurements were taken from day 0 until day 28. ZXAS: Zhenxin Anshen formula; AD: atopic dermatitis; NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. Data are expressed as mean ± standard deviation (n = 9), aP < 0.01 vs the NC group; bP < 0.01 vs the MC group.

Figure 2 Effect of ZXAS on type 2 inflammatory cytokines and IgE A-E: ELISA assay was performed to analyse the serum levels of (A) IL-4, (B) IL-5, (C) IL-13, (D) TSLP, and (E) IgE. NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. ZXAS: Zhenxin Anshen formula; ELISA: enzyme-linked immunosorbent assay; IL: interleukin; TSLP: thymic stromal lymphopoietin; IgE: Immunoglobulin E. Data are expressed as mean ± standard deviation (n = 9). aP < 0.01 vs the NC group; bP < 0.01, cP < 0.05 vs the MC group.

Figure 3 Inhibitory effects of ZXAS on GRPR, TRPV1, and TRPA1 expression in the spinal cord A-C: Western blot and qRT-PCR were used to analyze the protein and mRNA expression levels of (A) GRPR, (B) TRPV1, and (C) TRPA1 in the spinal cord (n = 6). D-F: immunohistochemistry and optical density analyses was used to detect (D) GRPR-, (E) TRPV1-, and (F) TRPA1-positive area and positive cells (×100 magnification). High magnification was shown as an insert within the black frame (×400) (n = 3). D1, E1, F1: NC; D2, E2, F2: MC; D3, E3, F3: CET; D4, E4, F4: LDZ; D5, E5, F5: MDZ; D6, E6, F6: HDZ. ZXAS: Zhenxin Anshen formula; NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. GRPR: gastrin-releasing peptide receptor; TRPV1: transient receptor potential vanilloid 1; TRPA1: transient receptor potential ankyrin 1; qRT-PCR: quantitative real-time polymerase chain reaction. Data are expressed as mean ± standard deviation. aP < 0.01 vs the NC group; bP < 0.05, cP < 0.01 vs the MC group.

Figure 4 Primary culture of DRG neuron cells and inhibitory effects of ZXAS-medicated serum on TRPV1, TRPA1, PLC expression and concentration of intracytoplasmic Ca2+ in DRG neurons A: the morphology and fluorescence immunocytochemistry of DRG neuron cells. A1: DRG neuron cells were stained with fluorescent markers (×40 magnification). High magnification was shown as an insert within the black frame (×100). A2: β-tubulin was expressed in DRG neuron cell bodies and axon fibers (×400). A3: Nuclei of neurons were stained with DAPI (×400). A4: merged picture from A2 and A3 (×400). B: proliferative capacity of DRG neuron cells in each group was measured using a 450 nm wavelength microplate reader with CCK-8. The relative expression levels of (C) TRPV1 and (D) TRPA1 in DRG neuron cells induced by capsaicin. Their protein levels were examined by Western blotting (E). Detection of (F) PLC and (G) Ca2+ using ELISA and (H) fluo-3AM fluorescence probe. Normal group: DRG neuron cells were pre-treated with 20% blank serum. Capsaicin group: DRG neuron cells were pre-treated with 20% blank serum and stimulated with 1 μM capsaicin. ZXAS group: DRG neuron cells were pre-treated with 10% blank serum and co-treated with 10% ZXAS-medicated serum and 1 μM capsaicin. Capsazepine group: DRG neuron cells were pre-treated with 20% blank serum and co-treated with 1 μM capsazepine and 1 μM capsaicin. ZXAS: Zhenxin Anshen formula; DRG: dorsal root ganglia; DAPI: 4,6-diamino-2-phenyl indole; CCK-8: cell counting kit-8; TRPV1: transient receptor potential vanilloid 1; TRPA1: transient receptor potential ankyrin 1; PLC: phospholipase C; ELISA: enzyme-linked immunosorbent assay. Data are expressed as mean ± standard deviation. aP < 0.05, cP < 0.01 vs the normal group; bP < 0.05, dP < 0.01 vs the capsaicin group.