Modulation of psoriatic-like skin inflammation by traditional Indian medicine Divya-Kayakalp-Vati and Oil through attenuation of pro-inflammatory cytokines

Background and aim Psoriasis (Ps) is a chronic skin inflammatory disorder, that progresses to scaly-red dermal plaque formations associated with inflammation. Divya-Kayakalp-Vati (DKV) and Divya-Kayakalp-Oil (DKO) are traditional Ayurveda herbo-mineral formulations, that are prescribed for the treatment of inflammatory dermal ailments. In the present study, we evaluated the efficacy of Divya-Kayakalp-Vati and Divya-Kayakalp-Oil (DKV-O) combined treatment in ameliorating Ps-like skin inflammation under in-vitro and in-vivo conditions. Experimental procedure Efficacy of DKV-O was analyzed in λ-carrageenan-treated Wistar rats paw edema and 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated CD-1 mouse ear edema models through physiological and histopathological analysis. Mode of action for the DKV-O was studied in LPS-stimulated THP-1 cells through pro-inflammatory cytokine analysis. Observed effects were correlated to the phytochemicals constituents of DKV-O analyzed using the HPLC method. Result and conclusion DKV and DKO formulations were individually found to contain phytochemicals Gallic acid, Catechin, Berberine, Curcumin, Phenol and Benzoic acid. DKV-O treatment significantly reduced the paw volume and edema in Wistar rats stimulated through λ-carrageenan-treatment. Furthermore, DKV-O treatment significantly reduced the ear edema and enhanced biopsy weight, epidermal thickness, inflammatory lesions and influx of neutrophils stimulated by TPA-treatment in CD-1 mice. DKV alone ameliorated the LPS-stimulated release of Interleukin (IL)-6, IL-17A, IL-23, and Tumor Necrosis Factor-alpha cytokines in the THP-1 cells. Taken together, DKV-O showed good efficacy in ameliorating acute systemic inflammation stimulated by effectors such as, λ-carrageenan and TPA in animal models. Hence, Divya-Kayakalp-Vati and Divya-Kayakalp-Oil co-treatment can be further explored as an anti-inflammatory treatment against dermal diseases like psoriasis and atopic dermatitis.


Introduction
Psoriasis (Ps) is an innate and adaptive autoimmune disorder, which affects approximately 125 million people worldwide. 1 Ps is a systemic inflammatory disease represented by the activation of the innate and adaptive immune systems and increased release of proinflammatory cytokines. 2e6 Onset of the disease can cause longterm damage to multiple tissues and organs. Dermal Ps cause a variety of lesion phenotypes with Ps vulgaris being the common manifestation represented by the occurrence of scaly-red inflammatory plaques having enhanced hyperkeratosis, parakeratosis and reduced stratum granulosum, and pain with tenderness. 7,8 Currently, there is no permanent cure available for Ps; and antiinflammatory drugs are applied to quell the Ps flaring.
Recently, herbal formulations have gained popularity as 'cosmeceuticals' as they don't produce any side effects. 9 Divya-Kayakalp-Vati (DKV) and Divya-Kayakalp-Oil (DKO) have been formulated following method prescribed in ancient India Ayurvedic textbooks Bhavprakash Nighantu and Ayurved Saar Sangrah for treatment of dermal etiologies. 10 Table 1). Several of the phytocomponents have been reported to display antiinflammatory activities. 12e20 Animal models for l-carrageenan-stimulated Wistar rat paw edema and 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated CD-1 mouse ear edema represents acute systemic inflammation that is similar to Ps-like morbidities through the production of cytokine, reactive oxygen and nitrogen species. 21,22 l-carrageenan-treatment induces a bi-phasic inflammatory response, with the early response represented by an increase in vascular permeability, and the later-stage represented through an induction of edema. 23 TPA treatment stimulates the development of dermal inflammation including IL17/IL23 axis, edema, epidermal hyperplasia and has been used for studying the Ps-like disease-modulating efficacy of different medicines. 24e28 In the present study, using l-carrageenan-stimulated Wistar rats paw-edema and TPA-stimulated CD-1 mice ear edema models, we analyzed the anti-inflammatory activity for combined treatment of DKV (oral) and DKO (topical) (DKV-O) in ameliorating Pslike inflammation. We investigated the anti-inflammatory mode of action for DKV through modulation of cytokine release in bacterial lipopolysaccharide (LPS)-stimulated phorbol 12-myristate-13-acetate (PMA)-transformed THP-1 cells. We summed up this study with a chemical analysis of DKV and DKO; and appended those findings with observed biological efficacies.

Chemicals and reagents used
DKV and DKO were procured from Divya Pharmacy, Haridwar, India. RPMI-1640 cell culture media, FBS, antibiotic/antimycotic were purchased from Gibco, USA. Bacterial origin endotoxin LPS (O111:B4), l-carrageenan, TPA, indomethacin (INDO), DEXA, and standard compounds (>95% by HPLC) such as Gallic acid, Catechin, Berberine, Phenol, Benzoic acid, and Curcumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits for TNF-a and IL-6 were purchased from BD Biosciences, USA. Primers for the study of IL-17A and IL-23 were purchased from Eurofins, India. HPLC grade acetonitrile, ortho-phosphoric acid, and diethylamine along with hematoxylin, potassium aluminum sulfate dodecahydrate, and mercury (II) oxide red were purchased from Merck India Pvt. Ltd, India. Eosin yellow and ferric chloride were purchased from HiMedia Laboratories, India. All other chemicals and reagents purchased were of the highest commercial grade.

Herbal sample preparation
20 g of DKV (Batch no. A-KKVE090) was pulverized and refluxed for 6 h at 88 C in 300 mL of 70% ethanol. The solution was filtered and dried at 45 C through evaporation under vacuum in a rotary film evaporator and stored until further use. 90 g of DKO (Batch no. BKKT056) was mixed in 200 mL of 90% methanol and stirred for 1 h. The prepared solution was stored at À20 C for 2 days. Following, separation of methanol from the frozen oil layer, it was removed manually. DKO was thawed, filtered, and dried using a Rotavapor. The dried DKO residue was resuspended in 90% methanol, and stored for further experiments.

High-performance-liquid-chromatography (HPLC) analysis
Chemical analysis of the DKV and DKO was performed using the binary HPLC system (Waters Corporation, Milford, MA, USA). Clear separations of DKV and DKO were done using an aqueous 0.1% ortho-phosphoric acid (v/v) (pH 2.5), diethylamine, and acetonitrile mobile-phase. A reversed-phase C18 analytical column of 4.60 Â 250 mm and 5 mm particle sizes (Sunfire, Waters, USA) set at 35 C was utilized for the analysis of Gallic acid, Phenol, Benzoic acid, and Catechin at 275 nm, and Berberine and Curcumin at 475 nm. A gradient-based program was used for the analysis and was repeated six times to confirm repeatability (relative standard deviation of <2.5%).

Experimental animals
Male Wistar rats (8e10 weeks) and CD-1 mice (6e8 weeks) were procured from Liveon Biolabs Pvt. Ltd, India, and from Hylasco Biotechnology Pvt. Ltd, India (licensed supplier for Charles River Laboratory), respectively. Animals were placed under a controlled environment with a relative humidity of 60e70% and 12 : 30 Briefly, 20 mL of TPA solution prepared in acetone was applied topically on the right ear of CD-1 mouse at the concentration of 2.5 mg/ear every second day till the 10th day. The left ear was reserved as vehicle control and treated with 20 mL of acetone alone during the study duration. Ear thickness was measured every day using a digital Vernier caliper (Mitutoyo, Tokyo, Japan) and changes were determined by subtracting the ear thickness of day 0 from the respective time points. Based on treatments, animal groups were divided as normal control, vehicle control, TPA only (disease control), 0.

Histopathological analysis
CD-1 mice were humanely sacrificed on day 10, exactly 6 h after the last drug treatment. Ear biopsy samples were fixed in 10% formalin and embedded in paraffin. The paraffin-embedded samples were sectioned using microtome to 5 mm thickness. The tissue samples were stained with hematoxylin and eosin dyes. Epidermal thickness (from the basal layer to stratum corneum) of the ear tissue sample was measured using Magcam DC5 microscopic camera (Magnus Opto Systems India Pvt. Ltd., Noida, India) equipped with a stage micrometer and was analyzed using MagVision image analysis software (Magnus Opto Systems India Pvt. Ltd., Noida, India). Distribution of the lesions was recorded as focal, multifocal, and diffused. Severity of the observed lesions and and neutrophil influx was recorded as NAD¼ No abnormality detected, 1 ¼ minimal (<1%), 2 ¼ mild (1e25%), 3 ¼ moderate (26e50%), 4 ¼ moderately severe/marked (51e75%), and 5 ¼ severe (76e100%). Other parameters such as the extent of lesions, severity of hyperkeratosis, number and size of pustules, epidermal hyperplasia (measured in the interfollicular epidermis), the severity of inflammation in the dermis and soft tissue, and any other lesion(s) were also recorded and scored.

Real-time Polymerase Chain Reaction (RT-PCR) assay
THP-1 cells were seeded in 24 wells culture plates at the concentration of 5 x 10 5 cells/well and stimulated with 20 ng/ml of PMA overnight. The next day, old media containing PMA was replaced and the transformed THP-1 cells were washed with lukewarm sterile PBS. THP-1 cells were replenished with fresh culture media and incubated for one day for stabilization. PMAtransformed THP-1 cells were pre-exposed to varying concentrations of DKV for a period of 1 h. After incubation time was over, the THP-1 cells were stimulated with LPS (1 mg/ml) for 24 h following completion of exposure time, cell culture media was separated and stored at À80 C till further use. Cells were washed and total RNA was isolated using RNeasy mini kit (Qiagen, USA) including an oncolumn DNase digestion with the RNAse-free DNase set (Qiagen, USA) and quantified using Nabi microdigital spectrophotometer. cDNA synthesis was performed using the Verso cDNA synthesis kit (Thermo Scientific™) and stored at À80 C until further use.
RT-PCR was conducted using qTOWER3G RT-PCR machine (Analytik Jena, Germany). Primer sequences selected for the study were: IL-17A:

Enzyme-linked immunosorbent assay
Stored cell culture media from the LPS stimulated THP-1 cells, treated with DKV was used for the analysis of TNF-a and IL-6 using ELISA. Assays were performed following the manufacturer's protocol and absorbance was recorded at 450 nm using the Envision microplate reader (PerkinElmer, USA).

Statistical analysis
The data are expressed as Mean ± Standard Error of Mean (SEM) for each group. Statistical analysis was performed using GraphPad Prism version 7.03 (GraphPad Software, Inc., San Diego, CA, USA). A one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons post-hoc test was used to calculate the statistical significance in-ear edema, biopsy weights, epidermal thickness, lesion scores, cytokine(s), and myeloperoxidase analysis. All analyses and comparisons were evaluated at a 95% level of confidence (p < 0.05).

Anti-inflammatory effects of DKV-O in l-carrageenan stimulated wistar rats
Plantar injection of Wistar rats with l-carrageenan (0.1 ml of 1% solution in normal saline) stimulated an increase in the absolute paw volume and edema up to 5 h (Fig. 1A and B). Treatment of the l-carrageenan stimulated rats with 75 mg/kg of DKV (p.o.) þ 40 ml/ paw of DKO (T.A.) significantly reduced the absolute paw-volume and edema (Fig. 1A and B). Treatment of the l-carrageenan stimulated rats with positive control INDO (10 mg/kg) (p.o.) significantly reduced both the paw volume and edema compared to the vehicle control animal ( Fig. 1A and B).

Anti-Ps-like-inflammation activity of DKV-O in TPA-Stimulated CD-1 mice
Stimulation of the CD-1 mice right ear with TPA (2.5 mg/ear)  induced Ps-like inflammation through an increase in ear edema, biopsy weight, and epidermal thickness (Figs. 2 and 3). Treatment of the stimulated CD-1 mice with 150 mg/kg DKV (p.o.) and 20 mL DKO (T.A.) significantly (p-value <0.01) reduced the ear edema with an inhibition percentage of 34.2 ± 19.5% ( Fig. 2A and B). Treatment of TPA-stimulated ear with DKV-O also significantly (p-value <0.01) reduced the elevated ear biopsy-weight with an inhibition percentage of 25.3 ± 12.3% and the epidermal layer thickness (Fig. 3AeC). DEXA treatment of the stimulated ear also significantly (p-value <0.01) reduced the TPA-stimulated ear edema, biopsy weight and epidermal thickness (Figs. 2 and 3).

Effect of DKV-O treatment on ear tissue histopathology in CD-1 mice
TPA-stimulation of the right ear in CD-1 mice induced epidermal hyperkeratosis, hyperplasticity, pustule formation, and neutrophils influx in the dermis region (Fig. 4AeC). Treatment of the TPAstimulated mice with DKV-O, or DEXA significantly (p-value <0.01) reduced the incidences of inflammatory lesion formation in the epithelial region, pustule formation, and an influx of the neutrophils ( Fig. 4D and E).
Quantification of the observed histopathological effects through lesion score analysis showed DKV-O treatment to significantly (pvalue <0.01) reduce the TPA-induced inflammatory lesions with a percent inhibition of 37.05 ± 10.52% (p-value <0.01) (Fig. 5A and B). Individually, DKV-O treatment ameliorated TPA-induced hyperkeratosis, and hyperplasia (p-value <0.05) in the epidermis, and neutrophil influx (p-value <0.05) in the dermal region of the stimulated ear of mice (Fig. 5C, D and 5F). No effect was observed on TPA-induced pustule formation from the DKV-O treatment (Fig. 5E). DEXA treatment also showed high anti-inflammatory efficacy through inhibition for all histopathological parameters observed having a percent inhibition of 84.47 ± 4.83% (p-value <0.01) (Fig. 5A).

In-vitro anti-inflammatory activity of DKV
LPS-stimulation of the PMA-transformed THP-1 cells showed an upregulation of pro-inflammatory cytokines-IL-17A, IL-23, TNF-a, and IL-6 ( Figs. 6 and 7). Pre-treatment of the THP-1 cells with varying concentrations of DKV showed a significant (p-value <0.01) reduction in the mRNA expression of IL-17A and IL-23 cytokines following LPS-stimulation ( Fig. 6A and B). Similarly, DKV pretreatment also reduced the LPS-stimulated release of TNF-a and IL-6 cytokines in the THP-1 cells ( Fig. 2A and B). Statistically significant (p-value <0.01) reduction of TNF-a release from the LPSstimulated THP-1 cells was only detectable at the DKV treatment concentrations of 3 mg/ml (47%) and 5 mg/ml (15%) (Fig. 7A). Similarly, a statistically significant (p-value <0.01) reduction in IL-6 cytokine release from the THP-1 cells was observed following pretreatment with DKV at the concentrations 0.3e5 mg/ml (Fig. 7B).
Based on the obtained results, DKV-O treatment showed antiinflammatory activity in Ps-like systemic inflammation through modulation of neutrophils influx, pro-inflammatory cytokine release and lesion formation.

Discussion
Skin is the outermost physical protective barrier of the body providing protection and maintaining physiological homeostasis. Initiation of Ps-like inflammation is represented by infiltration of the innate immune cells such as macrophages, neutrophils, TH17 and dendritic cells in the dermal region of the affected site. This is accompanied by the production of cytokines and chemokines, and proliferation of the keratinocytes leading to thickening of the epidermal region. 31 Herbal formulations play an important role in the treatment of human diseases without any known side effects. 32 Their therapeutic properties have been attributed to the presence of a wide variety of phytochemicals acting together in synergy and through a multicentric approach towards diseases. 32,33 Herbal components of DKV such as Phyllanthus emblica L., Curcuma longa L. contain phytochemicals that have anti-inflammatory and antioxidant activities against skin inflammation. 34e36 In our study, phytochemicals such as Gallic acid, Catechin, Berberine, Curcumin, Phenol, and Benzoic acid were found to be present in both DKV and DKO. Gallic acid can modulate the NADPH-oxidase activity in polymorphonuclear leukocytes associated with induction of oxidative stress through reactive oxygen species generation. 37 Curcumin has been wellknown for ages to have antioxidant and anti-inflammatory activity. 38 Similarly, Catechin, Berberine, and Curcumin have been found to have anti-inflammatory and antioxidant abilities through modulation of inflammatory cells and mediators. 39e46 Macrophages play an important role in the pathogenicity of Ps through the release of pro-inflammatory cytokines and modulating the IL-23/IL-17 pathway. 6 During the onset of Ps lesions, macrophages are present in large quantities and involved in the release of pro-inflammatory cytokines. 6 In our study, LPS-stimulation of the PMA-transformed THP-1 cells showed an increase in the levels of IL-17A, IL-23, TNF-a and IL-6 that was ameliorated by DKV treatment. These soluble mediators are considered biomarkers of Ps disease as they process the inter-and intracellular signaling between the innate cells and affected dermal keratinocytes. 47,48 IL-17 and IL-23 are produced by the innate and adaptive immune cells, and keratinocytes lead to the formation of epidermal hyperplasia. In our study, pro-inflammatory macrophage activity modulation by DKV indicated the anti-inflammatory contribution of the phytochemicals present in the herbal formulations. 49,50 In-vivo efficacy of the DKV-O was observed in systemic acute inflammation models of l-carrageenan stimulated Wistar rat paw edema and TPA-stimulated ear edema. The biphasic response of lcarrageenan in stimulating rat paw-edema and TPA-modulated ear edema is mediated through the release of several proinflammatory soluble mediators, such as cytokine, chemokines and growth factors. 22,51 In our study, the DKV-O combination efficiently reduced the l-carrageenan and TPA stimulator-induced edema, hyperkeratosis, hyperplasia in the epidermal region, and inflammatory cell influx in the dermal region in the treated animals. TPA is known to induce Ps-like inflammation through the influx of inflammatory cells and modulation of IL-17 cytokine that eventually leads to keratinocyte growth promotion. 26 Similarly, carrageenan is known to trigger the release of pro-inflammatory cytokines including IL-6, TNF-a and IL-17 inducing the inflammatory cascade. 52

Conclusion
Taken together, DKV showed good efficacy in the modulation of pro-inflammatory cytokines in LPS-stimulated macrophages. In the l-carrageenan and TPA-stimulated acute inflammation animal models, DKV-O treatment reduced paw and ear edema, epidermal thickness, and Ps-like inflammatory lesions. Phytochemicals present in the DKV and DKO play an important role through a possible multicentric synergistic effect in the modulation of Ps-like inflammatory response that needs further exploration. Hence, combined treatment of Divya-Kayakalp-Vati and Divya-Kayakalp-Oil can be explored further for the treatment of inflammatory dermal diseases like Psoriasis.

Author contributions
AB provided a broad direction for the study, identified the test formulation, generated resources, and gave final approval for the manuscript. SS conducted the in-vivo study, analyzed the data, and performed report writing. KJ assisted in animal handling and in performing in-vivo studies. RS and KB performed in-vitro experiments and RT-PCR analysis. SV and PN conducted analytical experiments. KB performed data curing and wrote the manuscript. AV supervised overall research project planning, generated resources, and reviewed and finally approved the manuscript.

Funding
This research work was funded internally by Patanjali Research Foundation Trust, Haridwar, India.

Declaration of competing interest
The test article was sourced from Divya Pharmacy, Haridwar, Uttarakhand, India. Acharya Balkrishna is an honorary trustee in Divya Yog Mandir Trust, Haridwar, India. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd., Haridwar, India. Besides, providing the test article, Divya Pharmacy was not involved in any aspect of this study. All other authors have no conflict of interest to declare.