Imiquimod cream (containing 5% imiquimod) was obtained from Meheco Keyi Pharma Co. Ltd (Hubei, China, batch number: 190301). Depilatory cream was purchased from Reckitt Benckiser (London, United kingdom). Methotrexate and chemical references (salvianolic acid B, tanshinone IIA, berberine hydrochloride, prim-O-glucosylcimifugin, sophocarpidine, and ammonium glycyrrhetate) were purchased from National Institutes for Food and Drug Control of China (Beijing, China). ELISA kits for IL-17A, IL-23, IL-22, IL-6, TNF-α, IL-1β were purchased from Invitrogen (Carlsbad, CA, United States). Primary antibodies used in western blotting analysis were obtained from Cell Signaling Technology (Danvers, MA, United States).

All DB ingredients were commercially available TCM decoction pieces, bought from Tianji pharmaceutical company (Hubei, China). The ingredients were authenticated by Professor Hezhen Wu from Hubei University of Chinese Medicine through morphological identification according to Chinese Pharmacopoeia. The specimens of the 15 ingredients were deposited in the herbarium of Wuhan Hospital of Traditional Chinese and Western Medicine for future reference (voucher number from 20190301 to 20190315 for all ingredients). Detailed information on medicinal parts and processing methods of the materials was listed in Table 1. According to traditional use of TCM prescriptions, the ingredients were mixed together and soaked with 10x weight of distilled water, followed by boiled for 1 h. The decoction was filtrated with gauze, and the medicines were boiled with another 10x weight of distilled water for 1 h. The decoctions from the two cycles were mixed and then concentrated to a solution containing 0.228 g/ml of crude drugs with a rotary evaporator (IKA, Staufen, Germany). Concentrated solution containing 0.228 g/ml of crude drugs was used to treat mice of the high-dosage DB group. The solutions of low-dosage (0.114 g/ml) and middle-dosage (0.057 g/ml) DB were diluted from the high-dose solution with distilled water. The solutions were stored at −20°C before use.

The dosages of DB used in our experiment were calculated from human dosage based on body surface area as described by Xu et al. (2002). Body surface area ratio of mouse (20 g) to human (70 kg) is 0.0026. Recommend dose of DB for an adult was 22 g per day. For a 20 g mouse, the equivalent dose of DB was 22 g × 0.0026 ÷ 0.02 kg = 2.86 g/kg, which was set to be the middle dosage in our experiment. The volume of drug administrated to mice was 0.5 ml, thus the concentration of solution for middle dosage was 2.86 g/kg × 0.02 kg ÷ 0.5 ml = 0.114 g/ml of crude drugs. The low dosage and high dosage were set at half or 2 times of middle dosage. Thus, the concentration of the solution for low, middle and high dosage was 0.057, 0.114, 0.228 g/ml of crude drugs, respectively.

HPLC-MS/MS method was used to analyze the chemical constituents of DB. According to Chinese Pharmacopoeia, salvianolic acid B and tanshinone IIA are used as reference compounds for quality control of Salvia miltiorrhizae radix et rhizome. Berberine hydrochloride, prim-O-glucosylcimifugin, sophocarpidine and ammonium glycyrrhetate are used as reference compounds for Phellodendri amurensis cortex, Saposhnikoviae radix, Sophorae flavescentis radix, Glycyrrhizae radix et rhizoma, respectively (Chinese-Pharmacopoeia-Commission, 2020). And these compounds were reported to be active compounds of these ingredients, respectively (Su et al., 2015; Kreiner et al., 2017; Pastorino and Cornara, 2018; Sun and Lenon, 2019; Zhu and Hou, 2021). Therefore, salvianolic acid B, tanshinone IIA, berberine hydrochloride, prim-O-glucosylcimifugin, sophocarpidine, and ammonium glycyrrhetate were used as standard references. An appropriate amount of each standard compounds were dissolved in a known volume of methanol individually to make the standard stock solutions. The standard working solutions were prepared by diluting the stock solutions with methanol. Then, an appropriate amount of each standard working solution were mixed and diluted with a known volume of methanol to make the mixed standard working solution.

The DB extract was prepared as described above and was filtered through a 0.45 μm filter for the HPLC-MS/MS analysis. The analysis was performed on a Shimadzu LC-20AD liquid chromatography system coupled with an API 4000 mass spectrometer (AB SCIEX, Foster City, United States). A Sunfire C18 column (2.1*150 mm, 3.5 µm particle size, Waters, Milford, United States) was used for the separation of the components. Water and acetonitrile [both containing 0.1% (v/v) formic acid] were used as mobile phase A and B. HPLC analysis was carried out at a flow rate of 0.2 ml/min with a gradient elution (10–30% B from 0 to 20 min; 30–100% B from 20 to 35 min; 100% B from 35 to 40 min).

The quantifications of the chemical constituents of DB were performed with the multiple reaction monitoring (MRM) mode. The turbo spray ion source was operated in the positive ion mode. The source parameters (ion-spray voltage, temperature, gas1, gas2, curtain gas, collision gas) used in the analysis were 5000 V, 500°C, 55, 55, 35, 6 psi. Ion pairs used for multiple reaction monitoring were 741.3/561.3 (salvianolic acid B), 295.3/277.2 (tanshinone IIA), 336.3/321.2 (berberine hydrochloride), 469.1/307.4 (prim-O-glucosylcimifugin), 249.6/148.3 (sophocarpidine) and 840.6/823.6 (ammonium glycyrrhetate). Compounds in the DB were quantified using the standard calibration curves.

Male C57BL/6 mice of 20–25 g weight and 8-weeks age were obtained from Laboratory Animal Center of Huazhong University of Science and Technology (Hubei, China). A total number of 60 mice were used in this experiment. The mice were housed in plastic cages under specific pathogen-free conditions of 24–26°C temperature, 55% relative humidity on 12 h light/dark cycles with abundant food and water. All of them were acclimatized for 1 week and kept in good health condition before use. Animal handling and institutional approval were supported and approved by the Institutional Animal Care and Use Committee at Tongji Medical College of Huazhong University of Science and Technology (approval number: 2,520). All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (the seventh edition, United States).

Backs of the mice were shaved and depilated with depilatory cream to expose the back skin. Sixty mice were randomly divided into six groups (n = 10): the normal control (NC) group, the model group (IMQ group), the methotrexate (MTX) treated group, and three groups treated with low, middle and high dosage of DB. Five groups except for NC group received application of 80 mg IMQ cream daily on their back skins for 14 consecutive days. For the last 7 days, the low-dosage DB group (DB-L), middle-dosage DB group (DB-M) and high-dosage DB group (DB-H) received orally 1.43, 2.86, 5.72 g/kg DB (calculated from the human dosage) after IMQ application daily, while the MTX group received 1 mg/kg MTX daily, IMQ and NC groups received distilled water. On day 15, the mice were sacrificed by cervical dislocation after narcotized by inhalation of diethyl ether, the blood samples and back skin tissues were collected. The animal experimental procedure was shown in Figure 1.

During the experiment, body weight of the mice was recorded every day. Behaviors, such as activity, mental state, daily intake of water and food, defecation and urination of the mice were also observed. PASI was used to evaluate the severity of the inflammation in skin. Degrees of erythema, scaling and thickening were scored independently on a scale of 0–4: 0 for none; one for slight; two for moderate; three for severe; four for very severe. The severity of the inflammation was measured by the cumulative score (erythema plus scaling plus thickening) on a scale of 0–12 (van der Fits et al., 2009).

On day 15, the back skin samples were collected and fixed in 10% buffered formalin and embedded in paraffin. Then the samples were cut into 3 µm sections and stained with hematoxylin and eosin (H and E). The samples were examined with an optical microscope (Olympus BX53, Tokyo, Japan). Epidermal thickness was measured in three randomly selected spots.

The serum samples were separated by centrifugation from the blood samples at 12000 g, 4°C for 15 min. Levels of the cytokines IL-17A, IL-23, IL-22, IL-6, TNF-α, IL-1β in serum samples were measured using ELISA kits (Invitrogen, Carlsbad, CA, United States) according to manufacturer’s instructions.

The skin samples were extracted with Trizol reagent (Invitrogen, Carlsbad, CA, United States) to obtain the total RNA. Then the RNAs were reverse transcribed into cDNA with M-MLV reverse transcriptase (Promega, Madison, United States). Quantitative PCR was performed with SYBR Green qPCR Supermix (Invitrogen, Carlsbad, CA, United States) on an ABI 7500 real-time quantitative PCR system (Applied Biosystems, Foster City, United States). The PCR cycle parameters were as follows: denature at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s (denature) and 60°C for 32 s (annealing). Data were analyzed with the 2^−ΔΔCT method. Primers used were listed in Table 2.

Total protein was extracted from skin samples with RIPA buffer (Beyotime Biotechnology, Shanghai, China). After determination of the protein concentration by BCA kit (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), the protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (0.45 mm, Millipore, Boston, MA, United States). After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies against NF-κB p65, IκB, p38 MAPK, JNK, ERK, p-p38 MAPK, p-JNK, p-ERK and STAT3 (dilution ratio of 1:1,000, Cell Signaling Technology, Danvers, United States) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies with dilution ratio of 1:4,000 at 37°C for 1 h. Then the membranes were washed for 3 times with Tris buffer saline containing 0.1% (v/v) Tweens 20, and incubated with enhanced chemiluminescence reagent (Millipore, Boston, MA, United States). The signals were captured with X-ray films and quantified with the ImageJ software (NIH, Bethesda, MD, United States). GADPH was used as internal reference protein.

All data were expressed as mean ± standard deviation (SD). Data were analyzed with SPSS 20.0. Statistical significance was assessed using the one-way analysis of variance (ANOVA). Differences were considered statistically significant at p < 0.05.

In TCM theory, ingredients in the formula could be divided into four categories, as monarch, minister, assistant and guide drugs. Salvia miltiorrhizae radix et rhizome was the monarch drug of the formula. Phellodendri amurensis cortex and Sophorae flavescentis radix were the minister drug, Saposhnikoviae radix was the assistant drug, and Glycyrrhizae radix et rhizome was the guide drug. These ingredients are the representatives of the four categories in the DB formula, so reference compounds of these ingredients were chosen in the analysis of DB. MRM chromatograms of both DB and referential substances were shown in Figure 2. The retention times of salvianolic acid B, tanshinone IIA, berberine hydrochloride, prim-O-glucosylcimifugin, sophocarpidine, and ammonium glycyrrhetate were 23.2, 32.7, 16.6, 11.6, 1.8 and 27.2 min, respectively. The concentrations of these six components in DB were 12.3, 0.13, 6.71, 3.04, 49.5, 5.96 μg/mg.

During the experiment, no significant differences in body weight, activity, mental state, daily intake of water and food, defecation and urination were observed between the mice in different groups. Typical signs of erythema, scaling and epidermal thickening appeared after 7 days of IMQ application compared with NC group. From day 8 to day 14, DB treatment significantly attenuated the symptoms of psoriasis compared with IMQ-group (Figure 3A).

PASI score was determined for assessing the severity of skin inflammation for all mice. In Figures 3B–E, the PASI scores continually increased after IMQ application from day 1 to day 7. However, the PASI scores decreased gradually after MTX or DB treatment from day 8 to day 14. The PASI scores of DB and MTX-treated groups were significantly lower compared to IMQ group (p < 0.01).

Histological changes in the skin tissues after DB treatment were evaluated with H and E staining. In Figure 4, the IMQ-treated group displayed remarkable elevation in epidermal hyperplasia, hyperkeratosis, acanthosis and inflammatory cells’ infiltration. After application of DB, the psoriatic symptoms were alleviated significantly, especially in the high-dosage DB treated group. The epidermal thickness of the mice was measured in three randomly selected spots. In Figure 3F, the IMQ-treated group showed significant thickening of epidermis compared to the NC group (p < 0.01), while epidermal thickness was significantly reduced in DB and MTX-treated groups (p < 0.01).

ELISA assays were used to evaluate the effects of DB on the levels of the cytokines in serum. According to Figures 5A–F, TNF-α, IL-17A, IL-23, IL-22, IL-1β, IL-6 levels were significantly increased in the IMQ group compared to NC group (p < 0.01). DB treatment significantly decreased levels of these cytokines compared to IMQ group (p < 0.01). Levels of TNF-α, IL-23 and IL-6 were decreased with a dose-dependent manner.

The mRNA expressions of inflammatory cytokines in skin tissues after DB treatment were evaluated by real-time PCR. In Figures 6A–D, IMQ treatment significantly increased the mRNA expressions of IL-17, IL-23, IL-22 and IL-6 compared with NC group (p < 0.01). However, the mRNA levels of these cytokines dose-dependently declined in the DB-treated groups (p < 0.01).

Western blotting analysis were performed to evaluate the expression levels of the proteins related to NF-κB, MAPKs and STAT3 signaling pathways in the skin tissues after DB treatment. As shown in Figures 7A,B, the expressions of NF-κB p65 were up-regulated and the degradation of IκB was increased after IMQ application compared to the NC group (p < 0.01). DB significantly suppressed the expressions of NF-κB p65 and the degradation of IκB in a dose-dependent manner (p < 0.01).

In Figures 7C–E, IMQ treatment increased the expressions of p38, JNK and ERK significantly (p < 0.01), and these IMQ-induced enhancements in the expressions were markedly prevented by DB (p < 0.01). Meanwhile, DB suppressed the phosphorylation of p38 and ERK (p < 0.01), though the phosphorylation of JNK were not suppressed by DB (Figures 7F–H).

In Figure 7I, IMQ up-regulated the expression level of STAT3 (p < 0.01) while the expression of STAT3 was suppressed significantly after DB treatment in a dose-dependent manner (p < 0.01).