The protective effects of Fuzhengzhiyanghefuzhiyang decoction (FZHFZY) on imiquimod-induced psoriasis in mice through suppression of P38/Erk/NF-κB signaling

Psoriasis is a chronic immune-mediated skin disease affecting approximately 2–3% of world's population. Fuzhenghefuzhiyang decoction (FZHFZY), a Chinese medicine formula created by Prof. Lu Chuanjian, has been shown to have remarkable anti-psoriasis effect in clinical practice. However, the mechanism of action of FZHFZY is unknown. The purpose of this study was to investigate the protective effects of FZHFZY in psoriasis-like skin inammation both in vitro and in vivo and elucidate the mechanism of action of FZHFZY. we in Results indicated that can obviously decrease scores. FZHFZY also suppressed the mRNA levels of IL-6, TNF-α, IL-23 and IL-8 in the skin and its anti-inammatory activity may be related to its suppression of the P38/Erk/NF-κB signaling. In addition, immunohistochemistry (IHC) data showed that FZHFZY can suppress the expression of F4/80 which is the marker of macrophages in the psoriasis skin. Therefore, we designed to investigate the roles and underlying mechanisms of FZHFZY in LPS-stimulated RAW264.7 macrophages in vitro.


Abstract Background
Psoriasis is a chronic immune-mediated skin disease affecting approximately 2-3% of world's population. Fuzhenghefuzhiyang decoction (FZHFZY), a Chinese medicine formula created by Prof. Lu Chuanjian, has been shown to have remarkable anti-psoriasis effect in clinical practice. However, the mechanism of action of FZHFZY is unknown. The purpose of this study was to investigate the protective effects of FZHFZY in psoriasis-like skin in ammation both in vitro and in vivo and elucidate the mechanism of action of FZHFZY.

Methods
In vivo study, we evaluated the protective effect of FZHFZY in imiquimod-induced psoriasis-like mice model. Results indicated that FZHFZY can obviously decrease psoriasis area and severity index (PASI) scores. FZHFZY also suppressed the mRNA levels of IL-6, TNF-α, IL-23 and IL-8 in the skin and its antiin ammatory activity may be related to its suppression of the P38/Erk/NF-κB signaling. In addition, immunohistochemistry (IHC) data showed that FZHFZY can suppress the expression of F4/80 which is the marker of macrophages in the psoriasis skin. Therefore, we designed to investigate the roles and underlying mechanisms of FZHFZY in LPS-stimulated RAW264.7 macrophages in vitro.

Results
Our results revealed FZHFZY treatment could signi cantly inhibit in ammation by modulating the expression of mediators, such as IL-6, TNF-α, IL-23 and IL-8, which expression was increased remarkably in the activated RAW264.7 cells. Our results also showed that FZHFZY inhibited the P38/Erk/NF-κB signaling pathways in RAW264.7 cells induced by LPS.

Conclusions
Taken together, our present study demonstrates that FZHFZY alleviated in ammatory response by suppressing the P38/Erk/NF-κB signaling in imiquimod-induced psoriasis-like mice model and LPSstimulated RAW264.7.

Background
Psoriasis is a chronic in ammatory skin disease. According to global epidemiological survey, the incidence of psoriasis ranges from 1-3% in the world's population [1]. In recent years, biological preparations have shown good safety and e cacy in the treatment of psoriasis, however, they have the disadvantage of recurrence in the short term after stopping the drug. Psoriasis is a chronic and recurrent disease with a very long treatment cycle. Evidence of long-term use of the preparation is not su cient, and the current high cost of drug purchase is not conducive to its promotion [2]. Traditional Chinese medicine has been used clinically for a long time due to its low price and good therapeutic effect. At present, researches on the pathogenesis of psoriasis mainly believe that it is an in ammatory response involving the immune system.
According to literature survey results, the nuclear factor κB (NF-κB) signaling pathway is involved in the pathological process of imiquimod-induced psoriasis-like dermatitis in mice [3]. And genome-wide association studies have shown that the activation of the NF-κB pathway is closely related to psoriasis [4]. NF-κB p65 is an important member of the NF-κB/Rel family of proteins [5]. The phosphorylation of NF-κB p65 protein can activate the NF-κB signaling pathway and cause in ammation. Actually, in the skin lesions of imiquimod-induced psoriasis model mice, it was found that the expression of NF-κB p65 phosphorylated protein was signi cantly increased [6].
Mitogen-activated protein kinase (MAPK) is a group of protein kinases that can be activated by different extracellular stimuli, such as cytokines, neurotransmitters and cell stress. Importantly, MAPKs have recently been associated with many autoimmune diseases (such as psoriasis), most prominently the p38 MAPK and Erk1/2 MAPK pathways [7]. The p38 MAPK signaling pathway is the key to regulating cellular and autoimmune responses [8]. It plays a critical role in the pathogenesis of psoriasis. The expression of phosphorylated p38 MAPK and Erk1/2 MAPK have been signi cantly increased in the epidermis of psoriasis patients [9].
Chinese medicine formula (CMF) is a personalized treatment carried out by clinicians based on the patient's individual situation, and it has attracted worldwide attention [10]. CMF is usually a combination of several Chinese herbal medicines, and its therapeutic effect is mainly the comprehensive activity of multiple active ingredients. In China, CMF is widely used in the clinical treatment of psoriasis, which is characterized by personalized treatment, low cost and low toxicity. Prof. Lu Chuanjian is a doctor of traditional Chinese medicine, and Fuzhenghefuzhiyang decoction (FZHFZY) is a combination of drugs that she summarized through clinical experience. Our previous study showed that the target cell shing combined with LC-MS analysis is a useful tool for screening bioactive compounds from complicated FZHFZY and the active components would contribute to the anti-psoriasis effects [11]. In our research, we used LPS-stimulated RW264.7 cells as an in vitro model and imiquimod-modeled mice as an in vivo model to study the mechanism of FZHFZY in reducing in ammation symptoms in psoriasis model.

Animals
Male BALB/c mice (6-8 weeks old, weighing 20 ± 2 g) were purchased from the Center of Laboratory Animals of Southern Medical University (Guangzhou, China). Mice were housed in a standard housing room under controlled conditions (maintained at 22 ± 2 o C, 45 to 55% relative humidity) and provided free access to food and water under a speci c pathogen-free (SPF) environment. The animal protocols were approved by the Animal Experimental Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine.  Table 1. All these herbs had been cut into small pieces and soaked with water for 30 min, and then kept boiling for 1 h. After boiling twice, the water extracts was pooled together and then concentrated to the concentration of 0.5 g/mL by electric heating volatilization.

Chemicals and reagents
The main ingredients of FZHFZY were identi ed by LC-ESI-MS n analysis and the ESI-MS spectra of samples and reference compounds were acquired in both negative and positive ionization mode.
Cell culture RAW 264.7 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). RAW 264.7 cells were cultured in DMEM medium containing 10% FBS, 100 μg/mL streptomycin and 100 IU/mL penicillin at 37 o C in a humidi ed 5% CO 2 atmosphere.
RAW 264.7 cell proliferation assay in vitro RAW 264.7 cell proliferation was measured using MTT assay. Brie y, Raw 264.7 cells in logarithmic growth were seeded into a 96-well microplate in triplicate. After 24-h incubation, various concentrations (150, 300, 600, 1,200, 2,400 and 4,800 µg/mL, respectively) of FZHFZY was added to each well. After further incubation for 24 h, 10 mL MTT (5 mg/mL) was added to each well. After 4-h incubation at 37 o C, the supernatant then was removed and 100 µL of DMSO was added into each well. Then the supernatants were collected and the absorbance (A value) value at 490 nm was measured using a Microplate Reader and the cell viability was indicated.

Administration of drugs
The BALB/c mice were randomly divided into the following 5 groups (n = 6): the control, vehicle, DXA (1 mg/kg/day) and FZHFZY (0.125 and 0.25 g/mL respectively). The control group was normal mice that were totally untreated. DXA and FZHFZY were topically administered to mice in the DXA and FZHFZY group respectively, while distilled water was given to the control and vehicle cream to induce psoriasis, respectively. Moreover, FZHFZY group was treated with CMF three days before using imiquimod cream and the topical cream treatment administration was applied for 7 consecutive days.

Imiquimod-induced psoriasis-like mouse model
In accordance with our preliminary study [12], mice were topically administrated with a dose of 62.5 mg of 5% imiquimod cream applied to a shaved area (3 cm × 2.5 cm) on their back for 7 consecutive days.
The Psoriasis Area and Severity Index (PASI) had three parameters, namely skin erythema, scaling and thickness [13], which served as a measure to assess the severity of the psoriasis-like lesion severity. Parameters were scored independently on a scale ranging from 0 to 4 as shown in Table 2.

Histological analysis and immunohistochemistry
The skin lesions samples of the mice were xed in 4% paraformaldehyde and embedded in para n. Sections (5 μm) were then made and stained with hematoxylin and eosin (H&E) for histological analysis.
For immunohistochemical staining, antigen retrieval was conducted with citrate buffer (pH 6.0) followed by treatment with 3.0% H 2 O 2 to quench endogenous peroxidase activity. The sections were incubated overnight at 4 °C with speci c primary antibodies against F4/80. The sections were then incubated with biotinylated secondary antibodies for 1 h at room temperature followed by diaminobenzidine staining and hematoxylin counter staining.

Measurements of mRNA expression of in ammatory cytokines via RT-PCR
The mRNA levels of TNF-α, IL-6, IL-8, IL-17, IL-23 and IL-1β were determined by RT-PCR. Total mRNA was isolated from mouse skin tissue or cells using Trizol reagents and mRNA was then reversed transcription to cDNA. The primer sequences were shown in Table 3. The relative mRNA expression levels of cytokines versus GAPDH were measured using an ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scienti c, USA).

Western blotting analysis
Total protein from mouse skin samples was acquired with RIPA lysis buffer followed by centrifugation (12, 000 rpm and 15 min) at 4 °C. Equal amounts of proteins from each treatment group were subjected to fractionation by SDS-PAGE and electro-transferred to PVD membranes. The membranes were then blocked with 5% (w/v) skim milk in TBS-T containing 0.1% Tween-20 at room temperature for 2 h and subsequently incubated with primary antibody at 4 °C overnight. Then, the membranes were washed with TBS-T and blotted with the appropriate secondary antibody for 1 h. Finally, the protein bands were detected using the enhanced chemiluminescence (ECL). The band intensity was quanti ed using Image J software (NIH Image, Bethesda, MD, USA), and GAPDH was used as the loading control.

Statistical analysis
The data were statistically evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's test, and denoted as means ± standard deviation (SD). Statistically signi cant differences were identi ed as either P < 0.05 or P < 0.01. All analyses were carried out by GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA).

Chemical analysis of FZHFZY
The FZHFZY sample and reference substances were analyzed by using the optimized LC-ESI-MS n method. The TIC chromatogram of FZHFZY sample in the negative and positive ESI mode was shown in Fig. 1 and 18 main components were observed in the sample (Table 4).

In vivo evaluation FZHFZY ameliorates imiquimod-induced psoriatic skin lesion in mice
In order to assess the anti-psoriatic effects of FZHFZY, we established the psoriasis-like mouse model which was topically treated with imiquimod. The marked phenotypic changes and PASI scores were observed in Fig. 2. After treatment with imiquimod, the vehicle group showed marked psoriasis-like lesions, including skin erythema, scaling and thickness, while topical administration with FZHFZY in BALB/c mice at 0.125 and 0.25 g/mL elicited a remarkable decrease in the phenotypic changes and PASI scores induced by imiquimod application on the skin.

Histological evaluations and immunohistochemistryof F4/80
The histological examinations via H&E staining (Fig. 3A) from the control group of normal mice showed normal smoother epidermis without any in ammation or lesion. While the vehicle group treated with imiquimod showed that increased epidermal hyperplasia, acanthosis, parakeratosis, elongated rete-like ridges and abundant in ammatory in ltrates were found in the skin. However, treatment with either FZHFZY or DXA treatment groups exhibited much smoother epidermis with less parakeratosis and reduced epidermal thickening.
The recruitment and activation of macrophages in psoriatic skin have been deemed to be important pathogenic factors [14,15] and the use of F4/80 expression associated with markers can de nitively distinguish macrophages from other cells [16]. Therefore, we detected the expression of F4/80 in the skin. As can be seen from Fig. 3B, the expression of F4/80 was signi cantly up-regulated in the vehicle group compared to the control group, and then down-regulated to different degrees by treatment with FZHFZY.

FZHFZY suppresses mRNA expressions of pro-in ammatory cytokines in imiquimod-treated psoriatic mice
To analyze the effect of FZHFZY on in ammation in the skin, the mRNA expressions of TNF-α, IL-17, IL-23 and IL-1β in skin tissue were determined using RT-PCR. As shown in Fig. 4, the mRNA expressions of TNF-α, IL-17, IL-23 and IL-1β after treatment with imiquimod were signi cantly enhanced as compared with those in other groups. While, the mRNA levels of these cytokines after administration with FZHFZY were obviously lowered in imiquimod-treated group.
FZHFZY inhibits P38/Erk/NF-κB signaling in imiquimod-treated psoriatic mice Previous data have shown that FZHFZY ameliorates psoriatic skin lesion and suppresses proin ammatory cytokines in imiquimod-induced mice. To investigate whether FZHFZY protected imiquimod-treated psoriatic mice through inhibiting P38/Erk/NF-κB signaling, we determined the expression levels of P38, p-P38, Erk, p-Erk, NF-κB and p-NF-κB in imiquimod-induced mice skin by Western blot. As shown in Fig. 5, the results showed that FZHFZY treatment had no obvious effect on the expression of P38, Erk and NF-κB, while remarkably reduced the proportion of p-P38/P38, p-Erk/Erk and p-NF-κB/NF-κB, respectively (all P < 0.01).

In vitro evaluation
In vitro cytotoxicity of FZHFZY onRAW264.7 cells To evaluate the effect of FZHFZY on the viability of RAW 264.7 cells, RAW 264.7 cells treated with 0 to 4800 μg/mL FZHFZY for 24 h and then MTT assay was performed. The results shown in Fig.6 suggested that FZHFZY (0-2400 μg/mL) was not toxic towards RAW 264.7 cells. Therefore, four concentrations of FZHFZY (150, 300, 600 and 1200 μg/mL) were selected for the next experiment.

FZHFZY suppresses themRNA expressions of pro-in ammatory cytokines in LPS-induced RAW264.7 cells
To verify the effect of FZHFZY on LPS-induced RAW264.7 cells, RT-qPCR analysis was used to determine the mRNA expressions of pro-in ammatory cytokines. As shown in Fig.7, IL-6, TNF-α, IL-23 and IL-8 mRNA was signi cantly enhanced after LPS stimulation for 24 h. However, treatment with FZHFZY prior to the LPS challenge notably attenuated the enhancement of mRNA of these cytokines. The extent of the inhibition was likely to be dependent on the concentration of FZHFZY.

FZHFZY inhibits P38/Erk/NF-κB signaling in LPS-induced RAW264.7 cells
Since FZHFZY restrained P38/Erk/NF-κB signaling in imiquimod-induced psoriasis-like mice, we further explored the mechanisms underlying its anti-in ammatory effects in LPS-induced RAW264.7 cells. Therefore, Western blot analysis was performed to evaluate the effects of FZHFZY on P38/Erk/NF-κB signaling pathway involving protein expression of phosphorylated-and total P38, Erk and NF-κB. As shown in Fig. 8, the expression of p-P38, p-Erk and p-NF-κB markedly increased in RAW 264.7 cells after treatment with LPS compared to the control group. Nevertheless, treatment with FZHFZY markedly suppressed the expression of p-P38, p-Erk and p-NF-κB compared to the vehicle group in a dosedependent manner.

Discussion
Psoriasis is a common chronic in ammatory skin disorder, characterized by clearly delineated erythematous plaques which may be painful and itching with an overall prevalence of 2-3% of population worldwide and a substantial negative impact on the quality of patients' life [17][18][19]. The etiology and pathogenesis are thought to involve a hereditary component and environmental factors which trigger an in ammatory response, leading to hyperproliferation of keratinocytes [19,20]. It is widely accepted that the etiology of psoriasis involves genetic susceptibility, environmental, as well as sex and age-related factors [21]. Although conventional treatments are effective, unwanted side effects can impact on the long-term management of psoriasis [22]. Thus, there is an urgent need to investigate and develop novel strategies for psoriasis with minimal side effects. Chinese herbal medicine has long history of treating psoriasis in China, and numerous herbs have been used for psoriasis [22,23].
FZHFZY is a novel formulated Chinese medicine formula containing Dictamni Cortex, Angelica sinensis Radix, Rehmanniae radix Preparata, Cnidii Fructus, Granati Pericarpium, Smilax glabra Rhizoma and Cynanchum paniculatum Radix et Rhizoma. FZHFZY, a Chinese medicine formula proposed by Prof. Lu Chuanjian, has been shown to have remarkable anti-psoriasis effect in clinical practice with the effect of strengthening the body resistance and dispelling dampness, easing the skin and relieving itching. However, the mechanism of action of FZHFZY is unknown. In our research, we investigated the protective effects and the mechanism of action of FZHFZY in psoriasis-like skin in ammation both in vitro and in vivo. It is observed that FZHFZY markedly decreased the PASI scores, decreased the epidermal hyperplasia and epidermal thickening, which revealed that FZHFZY effectively ameliorated imiquimodinduced murine psoriasis.
The psoriasis biologics recommended by The Lancet mainly target TNF-α, IL-12, IL-23and IL-17, and have achieved high clinical effects [2]. In ammatory cytokines are small molecular peptides or glycoproteins synthesized and secreted by lymphocytes and monocytes, which participate in and mediate in ammation. Small molecule targeted drugs, such as methotrexate, apremilast and dimethyl fumarate, are accelerating the development. These small molecule drugs mainly target some in ammatory pathways and cytokine factors, such as TNF-α, IL-23 and NF-κB [24]. Similarly, our research results show that FZHFZY can inhibit the expression of NF-κB p65 protein (Figs. 5 & 8) and reduce the mRNA levels of in ammatory mediators TNF-α and IL-23 (Figs. 4 & 7). Furthermore, studies have found that P38 MAPK and ERK1/2 MAPK are widely involved in the pathogenesis of psoriasis [25], especially the role of P38 MAPK in psoriasis is well documented [26]. Phosphorylation of P38 MAPK in human keratinocytes cells stimulated by particular matters is critically important for the increase in the expression of in ammatory factors such as IL-1α and IL-1β [27]. Moreover, the mRNA and protein expression of IL-6 and IL-8 induced by TNF-α is dependent on p38 MAPK [28]. Similarly, our research results show that FZHFZY can inhibit the expression of p38 MAPK and Erk1/2 MAPK and reduce the mRNA levels of in ammatory factors IL-1α, IL-1β, IL-6 and IL-8.

Conclusions
Taken together, our data demonstrated FZHFZYF alleviated in ammatory response by suppressing of the P38/Erk/NF-κB signaling in imiquimod-induced psoriasis-like mice model and LPS-stimulated RAW264.7.         were determined using RT-PCR. Data shown are the means ± SD (n = 6). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with vehicle group.