Ta-Xi-San Suppresses Atopic Dermatitis Involved in Multitarget Mechanism Using Experimental and Network Pharmacology Analysis

2.1. Materials and Reagents

TXS was obtained from Shaanxi Academy of Traditional Chinese Medicine. Methanol and formic acid (LC-MS-grade) were purchased from Thermo Fisher Scientific Co., Ltd. (Waltham, MA, USA). 2,4-Dinitrofluorobenzene (DNCB) was purchased from Macklin Biochemical Technology Co., Ltd. Hydrocortisone Butyrate Cream was obtained from Hunan WZT Pharmaceutical Co., Ltd. (Xiangtan, China). The rat IL-4, TNF-α, and IgE ELISA kits were obtained from Nanjing Keygen Biotech (Nanjing, China). PTGS2 and HSP90AA1 ELISA kits were purchased from R&D (Minneapolis, MN, USA).

2.2. TXS Preparation

TXS (160 g, Shaanxi Academy of Traditional Chinese Medicine) was soaked with 1000 mL in water for 30 min, decocted for 5 min again after boiled, and then filtrated. The decoction filtrated was concentrated to 160 mL (1 g crude drug/mL). The solution was filtered using 0.22 mm membranes. The filtrate was used and stored at 4°C before its use for animal assays.

2.3. Building Atopic Dermatitis Mice Model and Treatment

BALB/c mice (male, 18–20 g) were purchased from Xi'an Jiao Tong University (Xian, China). All the mice were fed under the standard experimental conditions (−25 ± 2°C, humidity 60 ± 5%, and 12/12 h day/night cycle). The AD mice model was induced by DNCB (dissolved in acetone/olive oil (3 : 1)), which was applied to dorsal skin and ear. Mice were segmented into 6 groups (n = 6 per group): (1) normal group, (2) matrix group, (3) DNCB model group, (4) DNCB + matrix + 0.65 mg crude drug/mL TXS, (5) DNCB + matrix + 1.30 mg crude drug/mL TXS, and (6) Hydrocortisone Butyrate Cream (Hyd B). Firstly, the mice were shaved with an aesthetic state (ethyl carbamate) using an electric shaver. Then, 100 μL 0.5% DNCB or acetone/olive oil (3 : 1) was applied to the shaved dorsal skin for 2 days, and there was no treatment in the 3rd∼6th days . 70 μL 0.5% DNCB on dorsal skin and 20 μL 0.5% DNCB on ear every 2 days were applied from the 7th day. The AD model was finished on the 28th day for a total of 10 times (on days 1, 2, 7, 10, 13, 16, 19, 22, 25, and 28). DNCB was applied on the same site r. TXS was applied from the 7th day, 500 μL on dorsal skin and 200 μL on ears, twice a day until the 28th day. Hyd B was also applied from the 7th day, 300 mg on dorsal skin and 70 mg on ears, twice a day until the 28th day. The flowchart of the experimental program we studied is shown in Figure 1. Our animal study followed the guidelines (Care and Use of Laboratory in Xi'an Jiao Tong University Health Science Center, license approval no. SCXK (Shan) 2017-003).

2.4. Evaluation of Skin Lesion and Measurement of Ear Weight

The relative severity of AD was evaluated weekly via scoring the lesions of the dermal tissues with the scoring procedure [29]: (1) erythema and hemorrhage, (2) erosion (excoriation), (3) edema, and (4) scaling (dryness). In each score, 0 was defined as exhibiting no symptoms, 1 as mild symptoms, 2 as moderate symptoms, and 3 as severe symptoms. Only one researcher participated in scoring the apparent AD symptoms throughout the experiment to minimize technique variations. The ears of each mouse were harvested and weighted.

2.5. The Thymus Index and Spleen Index Measurement

The spleen and thymus of mice in each group were removed integrally. The thymus index and spleen index were defined as the organ/body ratio (organ mass (mg)/corresponding animal mass (g)). The organ and the body weight were calculated using an electronic balance.

2.6. Histological Analysis

The dorsal skin and ear samples were taken 24 h after final herbal administration and then fixed in 4% paraformaldehyde buffer, followed by embedding of the tissues in paraffin wax. The tissue sections were 2 μm thick, and then they were stained with H&E.

2.7. Cytokines Quantitation

Total IgE in serum was analyzed using an ELISA Quantitation Kit according to the manufacturer's protocol. The serum concentrations of the cytokines, including TNF-α and IL-4, were also quantified using a mouse cytokine enzyme immunoassay kit, respectively.

2.8. UPLC-Q-Exactive-MS^E Analysis

The TXS aqueous extract was analyzed with a 150 mm × 2.1 mm Accucore AQ C18 column (2.6 μm). The mass spectrometer used in the UPLC-Q-Exactive-MS^E analysis was a quadrupole-electrostatic field orbital with an ESI mode and data treating used Mass Lynx 4.1 software. Column temperature was 30°C. The mobile phase consisted of 2 solvents: (A) 0.1% formic acid aqueous solution and (B) methyl alcohol (for a gradient elution program: 0–10 min, 95% solvent A; 20–30 min, 75% solvent A; 35 min, 30% solvent A; 45–60 min, 5% solvent A; 60–65 min, 95% solvent A). The flow rate was kept at 0.3 mL/min. The injection volume was set as 1 μL. The ion source of thermoelectric spray (HESI) MS was performed to collect data in full switch ion mode. Other parameters were as follows: capillary temperature: 300°C, auxiliary gas heater: 300°C, sheath gas flow rate: 30 L/min, spray voltage: 3.50 kV, full MS resolution: 7000, ddms2 resolution: 175000, and scan range: 100 to 1 200 Da. S-lens RF level was set as 55 V.

2.9. Network Pharmacology Study

2.10. Targets Verification

To identify the targets of TXS on AD treatment, we performed the ELISA assay. The protein expressions of PTGS2 and HSP90AA1 were quantitated. In brief, the ears and skin were fully ground with RIPA buffer adding 0.1 M PMSF and phosphatase inhibitors. Then, the supernatant was collected via centrifugation at 10000 rpm for 15 min at 4°C. The PTGS2 and HSP90AA1 expressions in supernatant were quantified using a mouse ELISA kit (R&D Systems, Minneapolis, MN, USA).

2.11. Statistical Analysis

Our data are shown as the mean ± standard deviation (SD). Statistical comparison among groups was analyzed with GraphPad Prism 5.01 (California, USA). ANOVA was firstly used followed by Student's t-test. P < 0.05 indicated significant difference between groups.

3.1. TXS Alleviates the Lesions in Ear and Back

The skin inflammation of the back and ear of mice in each group was observed by visual method (Figure 2(a)). The skin of the ear and back of normal mice was smooth and healthy. Compared with the normal mice, the ear of mice in DNCB group was obviously swollen, rough, desquamate, and auricle with scab. Meanwhile, the back skin of mice in model group was thickened, rough, erythema, desquamate, and scab, which showed a typical dermatitis feature. However, TXS dramatically decreased the excoriation, striking hemorrhage, and erosion and suppressed the scratching behavior compared to the DNCB-induced mice. In addition, in HYB treatment group, the inflammatory response was lighter, the damage and scratches just existed in edge of the ear, the back skin was thinner, and the vascular lines were clear.

At the same time, the lesion of back skin was scored according to the “inflammatory score standard.” Compared with the normal mice, the back inflammatory score of mice in DNCB group was significantly enhanced, and TXS (1.3 mg crude drug/mL) significantly reduced the inflammatory score (Figure 2(b)). There was a significant increase in ear weight of mice in DNCB group due to swelling of the ear compared with the mice in the normal group (Figure 2(c)). TXS and HBY administration decreased the ear weight and back of mice, respectively. In conclusion, both TXS and HYB promoted inflammation subsidence and skin healing of mice.

3.2. TXS Improved the Thymus Index and Spleen Index

As Figures 3(a) and 3(b) show, compared with the normal group, the thymus index of mice in DNCB group was memorably reduced; TXS administration could improve the thymus index of mice. The spleen index of DNCB-stimulated mice was significantly higher than that in the normal mice. High-dose TXS administration effectively reduced the spleen index.

3.3. Histological Analysis Results

Hematoxylin study was used to recognize whether TXS regulates pathological symptoms of AD-like skin lesions and ear injury in DNFB-induced AD mouse model. In model group, skin tissues of mice showed typical inflammatory pathological changes including parakeratosis, thickening of the spinous layer, telangiectasia, and infiltration of inflammatory cells in the dermis (Figure 4(a)). In mice in TXS and HYB treatment group, there was no parakeratosis in skin tissue, and the spinous layer was thickened; however, a trifling infiltration of inflammatory cell was still in the dermis (Figure 4(a)). Meanwhile, ear histological analysis showed that TXS decreased the epidermal thickening and inflammatory cells accumulation in the lesions (Figure 4(b)).

3.4. TXS Regulated the Level of Cytokines

Elevated levels of inflammatory cytokines and inflammatory cell infiltration are a typical characteristic in AD [30]. Thus, we estimated the cytokines level in the serum of DNFB-sensitized mice. Subsequently, we observed that the TXS has an effect on the release of inflammatory factors. The levels of IL-4, TNF-α, and IgE in serum were significantly increased in the model group's mice induced by DNFB compared to the normal group. However, production of IL-4, TNF-α, and IgE was inhibited by TXS and Hyd B treatment (Figures 5(a)–5(c)).

3.5. The Ingredients Analysis of TXS Extracts

The herb components of TXS extracts were detected via aligning each detectable compound mass data for each detectable compound with structure and confirmed by MS^E substructure data. The Base Peak Chromatogram (BPC) of TXS extracts in positive ion mode and negative ion mode is shown in Figures 6(a) and 6(b). TXS mainly contained alkaloids and flavonoid glycosides, and these ingredients presented a better MS response in positive than in the positive ion mode. In this research, 65 compounds extracted in TXS were identified through comparing and analyzing the primary and secondary mass spectrometry information, combining with databases (Metlin, Massbank, and Human Metabolome Database) and combining with retention behavior, database, and related literature reports. The 65 compounds were confirmed as the principal compounds for the following assay and are detailedly displayed in Table 1.

3.6. Potential Target Genes and Network Analysis

The 337 targets were collected form the 65 compounds in TXS based on UPLC-Q-Exactive-MS^E analysis after eliminating the duplicate targets. Simultaneously, 1371 AD-related targets were collected via searching CTD, GeneCards, and OMIM databases, and 1241 targets were decontaminated after removing duplicates. Then the compound targets and disease-related targets were merged, and we got the compound-target gene network.

The ingredient-target gene network is presented in Figure 7. This network consisted of 190 nodes (1 TXS formula node, 52 compounds, and 137 target genes) and 522 edges. Compounds in the network were classified as catechin class, flavonoids, coumarin, isoflavone, and alkaloids. Particularly, as shown in Tables 2 and 3, the top five ingredients of TXS which have maximum degrees are (−)-epigallocatechin gallate, apigenin, esculetin, wogonin, and epicatechin. Additionally, 27 genes (ADH1C, ADRA1B, IL-4, RELA, TP53, TOP2A, TNF, RXRA, PTGS2, PTGS1, PRKACA, PPARG, PIK3CG, NOS3, NOS2, NFKBIA, MAOA, JUN, HSP90AA1, ESR1, CDKN1A, CCND1, CASP3, BCL2, AR, AKT1, and ADRB2) were recognized due to their interactions with more than five ingredients. The ingredient-target gene network contributed to understanding the possible effects of TXS.

3.7. The Results of KEGG and GO Analysis

KEGG pathway enrichment showed that 199 pathways were harvested and the remarkable 30 related pathways are presented in Figure 8(a). Pathways in PI3K-Akt signaling pathway, MAPK signaling pathway, and TNF signaling pathway may be the latent mechanism to exert effects of TXS against AD. Subsequently, the results of GO evaluation of the top 10 enriched results are shown in Figure 8(b). The enriched biological process ontologies were dominated by positive regulation of transcription from RNA polymerase II promoter, negative regulation of apoptotic process, response to drug, inflammatory response, and positive regulation of transcription. The molecular function of targets was mainly enriched in ontologies including protein binding, identical protein binding, protein homodimerization activity, and enzyme binding. Meanwhile, cell component result showed that cytoplasm is the largest constitution.

3.8. TXS Regulated the PTGS2 and HSP90AA1 Expressions in Mouse Model

PTGS2 and HSP90AA1 are major regulatory targets for inflammatory response in AD and are core targets screened via network pharmacology. Thus, we investigate the regulatory effect of TXS on activation of PTGS2 and HSP90AA1. In our study, we found that the protein expressions of PTGS2 and HSP90AA1 in ears and skin of model mice were significantly enhanced compared to the normal mice; however, TXS decreased the elevated expressions of PTGS2 and HSP90AA1 (Figures 9(a)–9(d)). Our results revealed that TSX might be a useful drug for AD therapy via regulating PTGS2 and HSP90AA1 expressions.