Gancao Nurish-Yin Decoction Ameliorates Imiquimod-Induced Psoriasis-Like Skin Lesions and Inammation in Mice

Psoriasis is an autoimmune skin disease with a high clinical prevalence, which is often poses treatment diculties. In this study, we tested whether Gancao nurish-Yin (GNY) decoction is effective for treating mice with imiquimod (IMQ)-induced psoriasis-like skin lesions. Mice were divided into 4 groups including healthy control group, model group (IMQ-induced psoriasis mice), and two psoriasis induced groups of mice treated with GNY-2.5g/kg and GNY-5g/kg given in the drinking water (n = 8/group). Psoriasis area and severity index (PASI) was used to monitor psoriatic symptoms. H&E staining of dermis and baker’s scores were used to evaluate disease severity. Inammatory cells positive for Gr-1, CD11c, RORγt and TGF-β were counted by using immunohistochemistry or immunouorescence staining methods. Flow cytometry was used to analyse CD4 + CD17 + Th17 cells, and CD25 + Foxp3 + Tregs within splenocyte cell population. Relative mRNA expression of IL-6, IL-10, IL-17A, IL-22, IL-23, TNF- (cid:0) , TGF-β and AMPK (cid:0) 1 in the dermis was was semi-quatied using qPCR. < p < 0.001 and NS, not Error depict

Yin. Although extracts from an herbal medicine would more easily be recognized by modern researchers, in clinical practice, its a basic principle to combine several herbals as an recognized formula to manage diseases [6] . Based on TCM prescription method and understanding on the e cacy of botanical ingredients, we designed a formula for psoriasis, and named it as Gancao nurish-Yin (GNY) decoction, which contain the ingredients, as described in Table   1. stabilizing the generation of free radicals and by decreasing thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) release from human whole blood samples [7] . Ginseng berry polysaccharide extract signi cantly inhibits secretion of interleukin-8 and differention of Th1 cells [8] .
In this study, we tested GNY decoction on imiquimod (IMQ)-induced psoriasiform in ammation in mice and subsequent changes in the in ammatory cells and cytokines, And the results could form basis for initiating clinical trials for using GNY decoction for psoriasis patients.

Animals
Eight weeks old BALB/c female inbred mouse strains were used in the experiments, which were purchased from Southern Medical University Experimental Animal Center and maintained in a pathogen-free animal house (approval numbers, CUMS-2018-0062-03). Mice were kept on environment with 12 hour light/dark cycles, water and food were taken randomly.

IMQ induced psoriasis
Mice were divided into 4 groups (control group, model group and two treatment groups viz., GNY-2.5 g/kg and GNY-5 g/kg groups) with 8 mice in each group. Each mouse from all the groups except the control group received a daily topical dose of 30 mg of imiquimod (5% aldara cream, Mingxinlidi Laboratory, China) on the shaved area in the back for 4 days to induce psoriasis lesions. All the mice applied with imiquimod developed skin in ammation.

Treatment and scoring protocols
Mice in the control group and model groups were given double distilled water (ddH 2 O), while mice in the GNY-2.5 g/kg and GNY-5 g/kg groups received different concentrations of GNY decoction in the drinking water. On day 10, when the manifestations of psoriasis-like lesions in all the groups were subsided, we applied IMQ for the second time and the treatment was continued for 3 more consecutive days (Fig. 1A). We used PASI (Psoriasis area and severity index) scored to monitor psoriasis symptoms as follows: erythema (0-4), scales (0-4) and thickness (0-4) were scored. 0, no symptoms; 1, mild; 2, moderate; 3, severe; 4, very severe. Skin thickness was measured in a speci ed area using Vernier calipers (Neill-Lavielle, Kentucky, USA).

Histology
At the ended of psoriasis period (day 14), mice were put into anesthesia by intramuscularly injection of Zoletil 50 (Virbac, France, 20 mg/kg) and sacri ced by neck broken method. The skin samples were collected for histopathological analysis. For para n embedding, tissues were xed in 4% methanol at room temperature overnight. Before performing standard dehydration and para n embedding procedures, tissues were washed in PBS for three times. Skin sample was prepared for para n sections (Leica, Solmas, Germany) and tissues were cut (8 µm) using a para n slicer. The sections were methanol-xed and processed for H&E staining by following the steps: depara nization, dehydration and staining with hematoxylin solution (Phygene, Shanghai, China) or 0.2% eosin. Images were acquired using an eclipse upright optical microscope digital camera (Nikon Ci-E, Tokyo, Japan).
Epidermal thickness was measured by selecting 10 random regions from skin sections of each mouse using 10 × magni cation. We used baker's scores to judge pathological manifestations of psoriasis [9] : Munro's small abscess was found in the stratum corneum, 2. 2.6 Immunohistochemistry and Immuno uorescence Skin samples were harvested at the end of psoriasis period (day 14) and 5 mice from each group were used. Tissues were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek Inc, California, USA), snap-frozen overnight at -80℃. Before embedding, skin samples were dehydrated with buffer containing 30% sucrose in PBS overnight and with 30% sucrose-PBS:OCT in a ratio of 1:1 gradually for 4 h. The tissues were sectioned (7 µm) using a cryostat and frozen at -20℃ until used. For immunohistochemistry, the frozen sections were dehydrated with different concentrations of ethanol and xed in acetone for 10 min and, 3% hydrogen peroxide and 5% goat serum were used as blocking agents. In ammatory cells were stained with biotin-anti-mouse Ly6C/6G, CD11c from The experiments were repeated three times to con rm the results.

Statistical analysis
The data were analyzed using GraphPad Prism 8 software and the results are presented as mean ± SEM. Two-tailed unpaired Student's t test was used for comparisons between 2 groups, and one-way analysis of variance with Bonferroni or Newman-Keuls correction was used for multiple comparisons. Probability values ≤ 0.05 were considered as signi cant. *, p < 0.05; **, p < 0.01; ***, p < 0.001 and NS, not signi cant. Error bars depict SEM with a 95% con dence interval.

GNY decoction improved clinical manifestations and alleviated hyperproliferation of keratinocytes in IMQ-induced psoriasis
Experimental scheme was shown in Fig. 1A. After 4 days of applying IMQ on the shaved back of BALB/c mice, the skin showed typical erythema, scaling, and increased thickness. The average PASI score increased over the course of IMQ treatment, suggesting progression of skin lesions in the model group, and also in GNY decoction treatment groups. However, the erythema and scale scores in both GNY-2.5 g/kg and GNY-5 g/kg groups were less than model group and subsided earlier. After second IMQ stimulation, GNY decoction treament effects were more obvious with more smooth skin, shallow erythema and sparse scales. Treatment effects were more obvious in GNY-5 g/kg group than GNY-2.5 g/kg group (Fig. 1B-C). H&E staining of the skin lesions in both the treatment (GNY-2.5 g/kg and GNY-5 g/kg) groups showed a signi cant decrease in pathological parameters as evidenced by less epidermal thickening, acanthosis, parakeratosis, residual condensation of nuclei in the stratum corneum. The epidermal thickness and pathological scores in the model group were higher than control and GNY decoction treated groups (Fig. 1D -F).

GNY decoction decreased the expression of Gr-1 and CD11c positive cells
Gr-1 (granulocyte receptor 1) is a myeloid differentiation antigen, a glycosylphosphatidylinositol (GPI)-linked protein expressed on granulocytes and macrophages [10] , which participates in the pathogenesis of autoimmune diseases [11] . Expressed on monocytes and macrophages, CD11c is characteristically co-expressed with CD1c (BDCA-1). CD1c + dermal myeloid DCs were shown to have a capacity for priming and activating CD4 + T cells, and further initiating the are of psoriasis in ammation [12] . In this study, we observed that the model mice expressed higher levels of Gr-1 and CD11c than mice in the control group, while GNY decoction reduced their expression in a dose dependant manner ( Fig. 2A & B).

GNY decoction decreased the expression of RORγt and TGF-β in the dermis
Studies have elucidated the role of IL-17A-producing helper T cells (Th17) in psoriasis, which could be speci cally marked by RORγt [12] . Immuno uorescence was used to detect RORγt positive cells. In the dermis of IMQ modeled mice presence of RORγt + cells was observed (1-3 cells/section), while they were expressed in both control and GNY decoction treated mice at negligible levels (Fig. 2C). TGF-β promotes Th17 cell development [13] . Compared to healthy mice, psoriatic mice expressed higher number of TGF-β + cells. GNY decoction at both the concentrations signi cantly decreased the expression of TGF-β + cells (Fig. 2C).

GNY decoction regulated Treg/Th17 cells
Flow cytometry analysis of T cell subsets of splenocytes showed increased percentage of CD4 + CD17 + Th17 cells, with decreased CD25 + Foxp3 + Tregs in psoriasis mice. Th17 cells are pomotors of autoimmunity and in ammation, while Treg cells inhibit these phenomena and maintain immune homeostasis [14] . At both the concentrations of GNY decoction differentiated CD4 + CD17 + Th17 cells were signi cantly decreased, while CD25 + Foxp3 + Tregs in spleen were found to be increased only with the lower dose of GNY decoction (Fig. 3. A & B).
3.5 GNY decoction decreased several in ammatory cytokines and AMPK 1 in the psoriasis mice.
Complex network of cytokines, cytokine receptors and their signal transduction pathways is elevated in the serum of psoriasis patients [15] . Among them, we semi-quali ed relative mRNA expression level of IL-6, IL-10, IL-17A, IL-22, IL-23, TNF-, TGF-β and AMPK 1 in the dermis samples by qPCR. All these genes were elevated in psoriasis mice, and except IL-10 gene, expression of all the other genes were signi cantly decreased by higher concentration of GNY decoction (Fig. 4).

Discussion
The GNY decoction is a TCM formula modi ed from Gancao Xiexin Decoction (Licorice heat-draining decoction) recorded in Synopsis of Golden Chamber, which was composed by a legendary medical sage called Zhang Zhongjing more than 2,000 years ago. The formula of Gancao Xiexin Decoction was used to treat Huhuo (disease terminology of TCM), which is regarded as the autoimmune disease namely Behcet's disease nowadays [16] . Apart from Behcet's disease, Gancao Xiexin Decoction was applied and investigated with broad range of indications in China, like rheumatoid arthritis, digestive system diseases (e.g. ulcerative colitis, peptic ulcer), nervous system (e.g. neurosis) and endocrine system-related gynecological disorders [17] . However, the original formula contains coptis (Coptis Salisb.), which causes the decoction extremely bitter in taste and Pinellia ternate (Pinellia ternata (Thunb.) Breit.), which was recorded for mild toxicity in TCM as well as in modern research [18] . So, we changed the two herbs to dandelion (Taraxacum mongolicum Hand.-Mazz.) and Solomon's seal (Polygonatum odoratum (Mill.) Druce) as they are having similar nature of avor, property and functions according to TCM theory.
The GNY decoction showed ameliorating effects on clinical manifestations and keratinocyte proliferation in IMQinduced psoriasis in this study. Contribution of both non-speci c and speci c immune reactions in the pathogenesis of psoriasis is well known [19,20] . Subsequently. We investigated the therapeutic mechanisms of GNY decoction, involving both non-speci c and speci c immune reactions and found them to be decreased after treatment. Gr-1, CD11c and ROR-t positive cells were lower in the treating groups compared to model group. Granulocytes including neutrophils, eosinophils, and basophilsis have granules with enzymes released during infections, allergic reactions, asthma and psoriasis [21] , and Gr-1 is a speci c marker expressed on granulocytes and macrophages. CD11c is another surface marker mostly expressed on non-speci c immune cells like monocytes, dendritic cells, granulocytes, NK cells, and also subsets of T and B cells [22] . The best known surface antigen for the identi cation of dermal myeloid DCs is CD11c. Psoriatic skin was reported to have increased numbers of CD11c + myeloid DCs in the dermis,which has an important role in the psoriasis pathogenesis [23] . Retinoic acid receptor-related orphan receptor gamma-t (RORγt) has critical role in the differentiation, maintenance and function of IL-17-producing cells and is a highly attractive target for the treatment of IL-17-mediated autoimmune diseases, particularly psoriasis [24] . In this study, we observed presence of RORγt positive cells only in the dermis of model group, while in health controls and the GNY decoction treated groups negligible levels of RORγt positive cells were present.
Deregulation of TGF-β signalling was reported in human psoriasis [25] , and TGF-β promotes Th17 cell development [13] . The expression of TGF-β in IMQ-induced psoriasis was reduced by GNY decoction. Thus, Th17 cells could be one of the important effective targets in treating psoriasis by GNY decoction. Next, we analyzed the percentage of Th17 cells by ow cytometry, which showed signi cant reduction in this population of cells after treatment with GNY decoction. We also analyzed percentage of CD25 + Foxp3 + Tregs which were proposed to control in ammation in psoriasis [26] . In our study CD25 + Foxp3 + Tregs were reduced in the model group, and only low but not high concentration of GNY decoction treated mice had signi cantly increased levels of these cells, which suggests differential effects of components of GNY decoction on this cell population.
Next we focused on IL-17 signalling pathway. We investigated expression changes of the genes at mRNA level for IL-17A and IL-6, IL-10, IL-22, IL-23, TGF-β and TNF-, which interacted with IL-17 [27][28][29] . Except for IL-10, all these cytokines were decreased after administration of GNY decoction at higher concentration. We have also tested AMPK/IL-17 pathway. AMPK 1 was reported to be increased in auto-in ammatory condition [30] . In this study, the elevated AMPK 1 mRNA level in psoriatic mice model was decreased after treatment with GNY decoction, and its implications need to be explored further.

Conclusions
Using IMQ induced psoriasis model, we analyzed therapeutic effects of a TCM formula named GNY decoction. Decreased PASI scores demonstrated the effectiveness of GNY decoction, which improved clinical manifestations and alleviated keratinocyte hyperproliferation. IHC, IF, Flow cytometry and qPCR analysis further revealed its All data generated or analyzed during this study are included in this published article.

Competing Interests
All authors declare that they have no conflict of interest.

Funding
This study was supported by grants from Guangdong Medical Research (201810101430) given to YC and Southern Medical University, Guangzhou, China (C1034211, C1051004) given to KSN. Both were principle investigators in the grants.
Authors' contributions YC conceived the idea and design of this study, HMW and YC conducted the experiments, analyzed the data and composed the manuscript. YC and HMW list as rst co-authors. CHX and KSN reviewed and edited the paper. All authors have read and approved the manuscript.