Table 1.
HPLC parameters for the determination of the two main components in PAMs.
Table 2.
Primer design of chemokines/cytokines in HaCaT cells.
Fig 1.
Diagram depicting the design of the imiquimod-induced skin-inflammation mouse model.
BALB/c mice were divided into five groups (n = 6 per group): (1) Control group, (2) Model group, (3) PAMs group, (4) positive drug [Dexamethasone Acetate Cream (DXM)] group, and (5) 50% ethanol group.
Table 3.
Primer sequences of mouse genes by quantitative real-time PCR.
Fig 2.
Representative HPLC chromatogram of two main active components of PAMs.
a: Standard solution (A); b: PAMs sample (B). The detection wavelengths for hydroxysafflor Yellow A (1), and allantoin (2) in PAMs were at 403 and 224 nm respectively.
Fig 3.
Effects of PAMs on the production of inflammatory cytokines in TNF-α and IFN-γ-induced HaCaT cells.
A: Cells were treated with 3.0% PAMs, and TNF-α and IFN-γ (each 10 ng/mL) for 24 h. RT-qPCR was performed to determine the mRNA expression levels of IL-8, IL-6, MDC and ICAM-1. Values are expressed as mean± S.D of three independent experiments. B: Production of IL-8, IL-6 and MDC was measured in the culture supernatant of cells treated with 3.0% PAMs, and TNF-α and IFN-γ (each 10 ng/mL) for 24 h. ##p < 0.01 vs control cells; **p<0.01 vs TNF-α /IFN-γ-treated cells.
Fig 4.
Inhibitory effects of PAMs on NF-κB activation and nuclear translocation in HaCaT cells.
A: Cellular localization of p65 was analyzed by immunofluorescence staining. HaCaT cells were pretreated with or without 3.0% (v/v) PAMs or 1.5% (v/v) ethanol for 6 h and incubated with TNF-α and IFN-γ (each 10.0 ng/mL) for 30 min, later, the cells were incubated with anti-p65 and Cy3-conjugated secondary antibodies subsequently. Arrows indicate that NF-κB does not translocate to the nucleus. Images are representative of three independent experiments. B: The translocation rate was counted. ##p < 0.01 vs control group; **p < 0.01 vs model group. Bar = 50μm.
Fig 5.
Effects of PAMs on erythema, scaling and thickening in imiquimod-induced mouse skin.
A: Phenotypical presentation of mouse back skin after 6 days’ treatment. B: The scores of scales, thickness, erythema and the cumulative scores are shown for each group. The data are shown as mean± S.D (n = 6). Significant differences compared to model group: *p < 0.05.
Fig 6.
PAMs ameliorates the psoriasis-like symptoms and skin inflammation in imiquimod-induced mice.
A: Histopathological investigation in each group (×100). Black arrows represent the presence of horny layer and granular layer. B: Immunohistochemistry analysis was performed for Ki67 and ICAM-1 protein of the back skin of psoriatic mice, PAMs treatment could effectively decrease the expression of Ki67 and ICAM-1 protein (×100). Red arrows represent the overexpression of Ki67 and ICAM-1 protein. C: Epidermal thickness was measured. Data are the mean values ± SD (n = 5). D: PAMs treatment led to the significant decrease of TNF-α levels in the serum of each group. #p < 0.05 vs control group; *p < 0.05 vs model group. Bar = 100 μm.
Fig 7.
RT-qPCR of cytokines expression in mouse skin samples.
RT-qPCR was performed to determine the mRNA expression levels of IL-8(A), TNF-α(B), ICAM-1(C) and IL-23(D). Values are expressed as mean ± S.D of three independent experiments. #p < 0.05 vs control group; *p < 0.05 vs model group.
Fig 8.
Effects of PAMs on NF-κB inactivation and nuclear translocation in imiquimod-induced mouse skin.
A: Localization of NF-κB P65 protein was visualized in each group under a fluorescence microscope after staining with P65 antibody and FITC-labeled secondary antibody (green), the cell nuclei were stained with DAPI (×100). Images are representative of three independent experiments. B: The translocation rate was counted. #p < 0.05 vs control group; *p < 0.05 vs model group. Bar = 50 μm.
Fig 9.
The proposed mechanisms of the inhibition of imiquimod-induced psoriatic inflammation in mouse by PAMs.