An Emerging Role of Proanthocyanidins on Psoriasis: Evidence from a Psoriasis-Like Mouse Model

Background Psoriasis is an immune-mediated, chronic inflammatory disease, and genetic, immune, oxidative stress (OS), and environmental factors are all thought to contribute to its occurrence. Proanthocyanidins (PCs) are natural flavonoids consisting of catechins and epicatechins which have anti-inflammatory and anti-OS activities. PCs have been widely used to treat various diseases, but reports regarding psoriasis are rare. Objective To investigate the therapeutic effect and potential mechanisms of action of PCs in a psoriasis-like mouse model. Methods Thirty male BALB/c hairless mice were assigned to six groups (n = 5): normal, model, low-dose PCs, medium-dose PCs, high-dose PCs, and control groups. The final five groups were dorsally exposed to 5% imiquimod (IMQ) cream once a day for 6 consecutive days, while the normal group received no intervention. Following the first day of IMQ application, mice in the PC-treated group were dosed with different amounts of PCs daily by oral gavage for six days, whereas mice in the control group received normal saline in the same way. One week later, skin lesions were evaluated by the severity of scoring system based on psoriasis area and severity index (PASI), and pathological alterations were assessed by hematoxylin-eosin (HE) staining. Indicators of inflammation or OS, such as interleukin- (IL-) 17, IL-23, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), catalase (CAT), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and heme oxygenase-1 (HO-1), were determined by ELISA, RT-PCR, western blot, and immunohistochemistry (IHC) analysis. Results IMQ administration induced the formation of large dark red plaques with thickly layered scales on the dorsal skin of mice; nevertheless, the lesions were substantially alleviated by PC administration. Histopathological alterations were observed in both model and control groups with epidermal hyperkeratosis, granulosa layer thinning, acanthosis, downward extension of rete ridges, dermal papillae expansion, capillary hyperplasia, and infiltration by inflammatory cells around blood vessels. These pathological changes, however, were restored by a range of doses of PCs, high-dose PCs in particular. Different doses of PCs significantly lowered the spleen index, levels of inflammatory or oxidative proteins (IL-17, IL-23, MDA, ROS, p-PI3K, and p-STAT3), and the mRNA expression of Il-17, Il-23, Vegf, and iNos. Protein and mRNA levels of anti-OS and anti-inflammatory biomarkers, including SOD, CAT, GSH, and HO-1, greatly increased after PC treatment, especially at the highest dose. Conclusions Our findings reveal that PCs ameliorate psoriasis-like symptoms, suppressing the inflammatory response and mitigating OS damage in an IMQ-induced psoriasis-like mouse model. These effects are probably related to the inactivation of STAT3 and PI3K and activation of HO-1 signaling.


Introduction
Psoriasis is an immune-mediated, chronic, relapsing inflammatory disease that primarily affects the skin and joints. It has a prevalence estimated at 0.51% to 11.43% worldwide [1]. In histopathology, psoriasis is characterized by hyperproliferation of keratinocytes (KC), epidermal hyperplasia, dermal angiogenesis, and infiltration of inflammatory cells [2,3]. Psoriasis does not just affect the skin but has been linked to the high incidence of systemic disorders, such as diabetes, cardiovascular disease, gastrointestinal dysfunction, and chronic nephropathy [4][5][6][7]. In addition, psoriasis has a strong impact on psychological health with an increased morbidity from depression, anxiety, and suicide [8,9]. Various treatment approaches have been developed, including immunosuppressive agents, retinoid derivatives, corticosteroids, vitamin D3 analogs, calcineurin inhibitors, biologics, and UVA/UVB irradiation. However, many of these therapies have transient curative effects and fail to prevent relapse. In combination with the frequent high cost of treatment and serious side effects, there are few satisfactory therapies for this condition. Accordingly, there is a pressing need for the exploration and development of a long-term safe and effective approach to psoriasis.
Proanthocyanidins (PCs) are a group of natural flavonoids derived from grapes, apples, cranberries, and black currants which contain catechin and epicatechin and possess anti-inflammatory, antioxidant, antiangiogenic, immunomodulatory, and antiproliferative properties [24][25][26]. Owing to these multifunctional capacities, PCs have been suggested to ameliorate many inflammation and OS-related complaints [25,27,28]. Previous work has confirmed that PCs activated HO-1 to reduce the production of ROS, NO, and MDA and promoted the expression of SOD and glutathione peroxidase (GSH-Px) [29,30]. The impact of these effects is to mitigate oxidative damage. PCs also reduce the secretion of inflammatory factors, such as IL-2, IL-6, IL-8, IL-17, IFN-γ, and TNF-α, to inhibit inflammatory responses [31,32]. In multiple-experimental studies, PCs have been demonstrated to scavenge ROS through blocking the Janus kinase (JAK)/STAT signaling pathway, thus inhibiting cell proliferation/migration and inflammation aggression [33,34]. An in vitro effect of PCs on suppressing psoriatic KC growth and proliferation has also been reported [35]. Besides, PCs have been found to have few side effects and to be generally safe, even for pregnant women, during clinical trials. However, the efficacy of PCs for treatment of psoriasis has rarely been studied. Since immune-mediated inflammation and OS are hallmarks of psoriasis, the antiinflammatory, antioxidant, antiproliferative, antiangiogenic, and immunomodulatory activities of PCs would make them ideal candidates for treatment of this disorder. We have previously speculated on the potential utility of PCs for psoriasis [21]. The current study used an animal model of psoriasis to investigate the potential for PCs administration to ameliorate psoriatic symptoms. After the first day, mice in the PC groups were given PCs of appropriate concentrations in 0.3 mL/mouse doses by gavage once a day for 6 days while external IMQ application continued. The control group received an equal volume of NS. The model group received only IMQ, and the normal group received no treatment. During this period, skin lesions were observed daily by the naked eye, and photographs were taken. On the seventh day, mice were sacrificed and specimens of dorsal skin, serum, and spleen were collected for histopathological examination, measurement of OS and inflammation-related factors, and analysis of the spleen index.

Materials and Methods
2.5. Visual Skin Evaluation. The psoriasis area and severity index (PASI) was used to score dorsal lesions based on the parameters of erythema, scaling, and thickness (Table 1). Each parameter was scored independently on a scale from 0 to 4, as follows: 0 = no clinical signs, 1 = slight clinical signs, 2 = moderate clinical signs, 3 = marked clinical signs, and 4 = very marked clinical signs. The total score indicated the severity of lesions.
2.6. Spleen Index Assay. Spleens were excised and weighed after sacrifice. The spleen index was calculated as follows: spleen index ðmg/gÞ = spleen weight ðmgÞ/mouse weight ðgÞ.

Skin Tissue Preparation for
Histopathology. Samples of dorsal skin were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 3 μm sections for hematoxylineosin (H&E) staining. Epidermal thickness from the granular to the basal layer was estimated at three randomly chosen sites on each slice. Two distinct fields were chosen and photographed under the microscope for each site. Mean values (± SD) of epidermal thickness, calculated by two investigators independently, are presented.

Analysis of OS and Inflammation-Related Indicators in
Mice Serum. SOD, GSH, MDA, ROS, CAT, IL-17, and IL-23 were measured by enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions. Optical density (OD) was read by Microplate Reader (Thermo Fisher Scientific, USA) and calculated from standard curves.
2.9. Western Blotting Analysis. Phosphorylated-PI3K (p-PI3K), p-STAT3, and HO-1 proteins were measured by western blotting. Samples of skin tissue were cut into small pieces and treated with lysate and protein loading buffers to extract total protein. Total protein concentration was Table 2: The primers of inflammation and OS-related genes.

2.10.
qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) Analysis. Total RNA was extracted from skin samples by TRIzol RNA extraction kit, and mRNAs encoding OS-related genes, Il-17, Il-23, Vegf, iNos, Ho-1, Cat, Sod, and Gsh were detected according to the manufacturer's instructions using primers shown in Table 2. Target gene expression was determined relative to that of the internal control, Gapdh.
2.11. Immunohistochemistry (IHC) Analysis. Paraffin sections were dried at 65°C for 2 h, dewaxed, and subjected to microwave repair in EDTA buffer. Sections were incubated in 3% hydrogen peroxide solution at room temperature in the dark and blocked with 5% BSA. The BSA solution was   Oxidative Medicine and Cellular Longevity   with comparisons between two groups made by Student's t -test and multiple group comparisons by least significant difference (LSD) analysis of variance (ANOVA). A value of p < 0:05 was considered to indicate statistical significance. Orange 8.5 and GraphPad Prism 8.0 software were used to analyze data plots.

PCs Ameliorated Skin Appearance in the Psoriasis-Like
Mouse Model. After seven days of IMQ application, psoriasis-like lesions, including swelling and extensive dark red plaques overlaid with silvery-white thick scales, could be visualized on dorsal skin, with severity scores of 8-10 (p < 0:05; Figures 1(a) and 1(b)). Lesions were relieved by PC treatment and showed ameliorated severity scores and visual appearance compared with the model group (p < 0:05; Figures 1 and 2). No significant improvements were observed in the control mice, regardless of skin manifestations or the severity scores (p > 0:05; Figures 1 and 2). Furthermore, all PC-treated groups had lower PASI scores than the control, especially in the high-dose group (p < 0:05; Figure 2).

PCs Alleviated Skin Histopathology in the Psoriasis-Like
Mouse Model. Histological examination of normal mice showed a thin, 2-3-layer, epidermis without inflammatory cell infiltration of the dermis. After 7 days of IMQ administration, the epidermis thickened to 7-8 layers with parakeratosis/hyperkeratosis, granular layer deficiency, spinous layer hypertrophy and downward extension of epidermal rete ridges, dermal papilla stretching into the epidermis, telangiectasia, and perivascular inflammatory cell infiltration of the dermis. Such histopathological changes were similar to those observed in human psoriasis. PC treatment, however, notably ameliorated these changes, the high-dose PCs in particular. Little improvement was seen in the control group which was histopathologically similar to the model group ( Figure 3).

PCs Reduced the Spleen Index in the Psoriasis-Like
Mouse Model. IMQ application produced an increased spleen index, which was apparently reduced by PC administration relative to both the control and model groups, among which the high-dose group was the most prominent (p < 0:05; Table 3). No reduction of spleen index was observed in the control group (Table 3).  Table 4).   Oxidative Medicine and Cellular Longevity

PCs Affected Expression of Inflammation and OS-Related
Genes in the Psoriasis-Like Mouse Model. In comparison with the normal group, the model group showed elevated expression of Il-17, Il-23, Vegf, and iNos mRNA (p < 0:05) and suppressed expression of Ho-1, Sod, Cat, and Gsh mRNA (p < 0:05; Figure 4). By contrast, administration of PCs resulted in lower mRNA levels of Il-17, Il-23, Vegf, and iNos and higher mRNA levels of Ho-1, Sod, Cat, and Gsh (p < 0:05; Figure 4). The control group exhibited few changes relative to the model group (p > 0:05; Figure 4).

PCs Upregulated HO-1 and Downregulated p-PI3K and p-STAT3 Protein Expression in the Psoriasis-Like Mouse
Model. Normal mice showed little expression of HO-1, p-PI3K, and p-STAT3 proteins in the skin. However, application of IMQ raised p-PI3K and p-STAT3 protein levels while decreasing that of HO-1 (p < 0:05; Figure 5). These changes were reversed by PC administration: HO-1 expression was enhanced, whereas that of p-PI3K and p-STAT3 declined, especially at the highest dose (p < 0:05; Figure 5). Similarly, insignificant differences were observed between the control group and the model group ( Figure 5). IHC analysis showed that p-PI3K and p-STAT3 were rare in the epidermal cells of the normal group as brown granular nuclear/cytoplasmic staining, but p-PI3K-positive (p-PI3K+) cells and p-STAT3-positive (p-STAT3+) cells were apparently observed in the epidermis of the model group after IMQ application. However, PCs significantly lessened the p-PI3K+ cells and p-STAT3+ cells along with the enhancement of drug concentration (p < 0:05). The p-PI3K+ cells and p-STAT3+ cells in the control group were slightly different from those in the model group, but no statistical significance existed (p > 0:05 ). By contrary, HO-1 was evident in the epidermis as brownstained sediments in the cytoplasm of the normal group; nevertheless, IMQ application greatly cut down the HO-1positive (HO-1+) cells. However, the positive cells gradually went up with the increase of PC concentration. In similar results to those with the model group, minimal HO-1+ cells could be found in the control group ( Figure 6).

Discussion
The current study reveals a novel impact of PCs on a mouse model of psoriasis in vivo. First, a psoriasis-like mouse model was successfully constructed through external use of IMQ. Then, skin symptoms, spleen index, and histopathological alterations were relieved by PC treatment, especially by the highest dose. Furthermore, PCs inhibited production of inflammatory mediators and regulated redox balance. Lastly, levels of PI3K/STAT3 were also reduced and those of HO-1 elevated in the presence of PCs. The above findings imply the capacity of PCs in mitigating inflammation and OS via inactivation of the PI3K/STAT3 pathway and activation of the HO-1 signal in order to alleviate experimental psoriasis. Animal models of psoriasis have been constructed by a variety of approaches, including spontaneous production, genetic engineering, xenotransplantation, and direct induction, among which the last is frequently employed. The current study, therefore, adopted a direct induction method to establish a psoriasis-like mouse model through the application of IMQ. Since 2009, the IMQ-induced mouse model has been used extensively in the preclinical research of psoriasis [36]. Typical psoriasis-like lesions appeared in the dorsal region after IMQ administration, manifesting as thickened/swollen skin and dark red plaques covered with thick scales. The severity index based on PASI scores gradually increased, reaching 8-10 on the 6-7th day. In histopathology, it was presented as KC hyperproliferation, epidermal thickening with parakeratosis/hyperkeratosis, granular layer deficiency, spinous layer hypertrophy, dermal telangiectasia, and inflammatory cell infiltration. All these manifestations are analogous to those observed in human psoriasis. Spleen index increased as did proinflammatory marker expression (IL-17, IL-23, MDA, and ROS), whereas expression of redox regulators (SOD, CAT, and GSH) decreased in the psoriasis-like model. These changes were consistent with those previously reported in the literature [37][38][39]. Therefore, the successful generation of the psoriasis-like mouse model was confirmed.
PC doses of 12.5 mg/kg, 25 mg/kg, and 50 mg/kg by gavage were adopted, based on reports of the previous outcomes [40][41][42] and current preliminary experiments. Such a range of dosage is considered to reduce the likelihood of toxic side reactions. Skin lesions, erythema/scales, epidermal thickness, dyskeratosis/hyperkeratosis, vascular proliferation, and inflammatory cell infiltration were reduced in varying degrees by PC administration, and this was especially obvious in the high-dose group, as was the reduction of the spleen index. The control mice, which were given saline, showed few improvements and were similar to the model group. These findings were in agreement with those of Rauf et al., Katiyar, Long et al., Q. Li et al., and Jia et al. who found that PCs inhibited KC proliferation promoting normal epidermal keratinization and reduced angiogenesis and infiltration of inflammatory cells [25,27,28,43,44]. Thus, the initial observations from the current study indicate a potential role for PCs in the amelioration of psoriatic symptoms. Mechanisms of PC action were next addressed.
Many studies have shown an effect of PCs in a variety of diseases through downregulating expression of inflammatory and OS-related indicators and upregulating 9 Oxidative Medicine and Cellular Longevity antioxidants. As the chief contributors to psoriasis pathogenesis, OS and the inflammatory response may be implicated in the mechanism of PC control of psoriasis. The current study found that IMQ produced increased levels of inflammatory and oxidative biomarkers (IL-17, IL-23, MDA, ROS, and VEGF) in skin and serum, while antioxidants (SOD, CAT, HO-1, and GSH) decreased. These findings indicate that IMQ application provoked a psoriasis-like inflammatory response and OS state. PC administration, however, reversed the above phenomena.  [29-31, 45, 46] who observed that grape seed PCs lowered expression of MDA, IL-23, and TNF-α and heightened the activities of SOD, GSH, and HO-1. In summary, we conclude that PCs are likely to be efficacious in managing IMQ-induced psoriasis-like symptoms through preventing the inflamma-tory response and fighting OS damage. However, further mechanistic clarification is required.
Mounting evidence suggests the abnormal activation of many signaling pathways in psoriasis. Candidate pathways include MAPK (mitogen-activated protein kinase)/NF-κB (the nuclear factor κB) and PI3K/AKT and JAK/STAT [22,47,48] with the latter having particular significance for psoriasis [49][50][51]. Activation of PI3K and STAT3 promotes the excessive proliferation and abnormal differentiation of KC, infiltration of inflammatory cells, and secretion of cytokines (IL-6, IL-17, IL-22, and IL-23) in psoriasis [52][53][54]. These events culminate in the formation of inflammatory lesions in an IMQ-induced psoriasis-like model [55]. Thus, regulation of PI3K and STAT3 signals would inhibit the inflammatory response, heighten antioxidant activity, and reduce oxidative damage to tissues and cells [56,57]. Suppression of PI3K/AKT/mTOR (mammalian target of rapamycin) signaling has also been suggested in the treatment of psoriasis [58] and the antioxidant protein, HO-1, may mitigate IMQ-induced psoriasis-like inflammation by blocking the STAT3 signal [59]. The aforementioned studies confirm the significance of PI3K, STAT3, and HO-1 in mediating the psoriatic state. The results from the current study show the stimulation of p-PI3K and p-STAT3 and suppression of HO-1 by IMQ application, changes which were reversed by administration of PCs and scarcely occurred in their absence. The indications are that PCs may alleviate psoriasis by blocking PI3K and STAT3-dominated inflammation/OSrelated signals and stimulating HO-1 antioxidant signals to reduce inflammation and mitigate OS. Previous scholars have reported compatible findings. Bashir et al. found that inhibition of the PI3K/AKT-dependent pathway by PCs prevented DNA damage and enhanced HO-1 expression [46]. PCs have also been reported to activate HO-1 via prohibiting MAPK/NF-κB signaling pathways to reduce the production of ROS, NO, and MDA, upregulate antioxidant activities (e.g., CAT, SOD, and GSH-Px), and arrest oxidative damage [31,45]. Moreover, Zhang et al. and Song et al. demonstrated that PCs block JAK/STAT signaling to deplete ROS and reduce cell proliferation and migration as well as the progression of the inflammatory response [33,34].

Conclusion
In summary, PCs relieve psoriasis-like symptoms, inhibiting the inflammatory response and attenuating oxidative damage, probably via inactivation of STAT3/PI3K and activation of HO-1. Further in vitro studies are planned to clarify mechanisms of PC action, including use of PI3K, STAT3, and HO-1 inhibitors. Our findings illuminate a promising psoriasis treatment and provide an experimental basis for mechanistic research into the antipsoriatic actions of PCs.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.