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\begin{document}

\title{Allicin Ameliorates IMQ-induced psoriasis-like Skin Inflammation Via
Disturbing the Interaction of Keratinocytes with IL-17A}



\author[1]{Lu Zhang}%
\author[1]{Xuehong Ma}%
\author[1]{Rongmei Shi}%
\author[2]{Libo Zhang}%
\author[2]{Ruolin Zhao}%
\author[2]{Ran Duan}%
\author[2]{Yuanyuan Qin}%
\author[2]{Sijia Gao}%
\author[1]{Xinxia Li}%
\author[2]{Jingjing Duan}%
\author[1]{Jianguang Li}%
\affil[1]{Xin Jiang Medical University}%
\affil[2]{China Pharmaceutical University}%


\vspace{-1em}



  
  \date{June 09, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Psoriasis is an inflammatory skin disease of
chronic recurrence mediated by the interaction between IL-17 and
keratinocytes, which sustains a vicious circle of inflammation.
Currently, there is no safe and effective natural medicine for the
clinical treatment of psoriasis. Given its prominent anti-proliferative
and anti-inflammatory properties, we investigated the mechanism of
allicin improving psoriasis. Experimental Design: Pharmacodynamics and
toxicology experimental studies were estimated after topical
administration of allicin on the skin of mice. Changes in inflammatory
factors expression were analyzed by qPCR and immunohistochemistry after
topical treatment with allicin in mice with psoriasis-like lesions
induced by imiquimod. The impacts of allicin on proliferation and
apoptosis of keratinocytes were analyzed by CCK8 assay and flow
cytometry. The interaction between IL-17A and keratinocytes was studied
using HaCaT cells, and the mechanism of action of allicin was explored
by Western Blot. Transcriptomic changes following the action of allicin
were probed by RNA-seq. Key Results: Our study demonstrated that allicin
significantly improved the epidermal structure by inhibiting excessive
proliferation and evasion of apoptosis of keratinocytes. Furthermore,
allicin reduced the secretion of inflammatory cytokines (IL-17A/F,
IL-22, IL-12, IL-20), chemokines (CXCL2, CXCL5, CCL20), and
antibacterial peptides (S100A8/9). Mechanistically, allicin directly
inhibited the IL-17-induced TRAF6/MAPK/NF-\selectlanguage{greek}κ\selectlanguage{english}B and STAT3/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling
cascades in keratinocytes, thus breaking the positive inflammation
feedback and alleviating imiquimod-induced psoriasis-like dermatitis in
mice. Importantly, topical administration of allicin did not cause skin
allergy, and the safety and adaptability of long-term application were
verified.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Psoriasis is a chronic inflammatory mediated autoimmune skin disease in
which keratinocytes and immune cells play central roles. The
characteristic histological findings of psoriasis mainly include the
aberrant proliferation of keratinocytes and brisk immune cells
infiltration. Erythema and scaling appearance of psoriasis lesions
severely impact the patient's quality of life. At present, the most
advanced therapy currently includes neutralizing antibodies and
immunosuppressors for treating psoriasis, which is effective but has
many disadvantages, including high cost and systemic side effects such
as infection of the upper respiratory tract(Hawkes \emph{et al.} , 2017;
Armstrong \emph{et al.} , 2020). Therefore, there remains a need for
novel drugs with improved efficacy and less toxicity.

At present, the pathogenesis of psoriasis remains unclear. Many studies
have confirmed that the development and maintenance of psoriasis occur
through the interplay between inflammatory factors and
keratinocytes(Pasquali \emph{et al.} , 2019; Ghoreschi \emph{et al.} ,
2021). An increasing body of evidence suggests that IL-17A is a critical
inflammatory mediator that mainly targets keratinocytes and drives
changes within psoriatic lesions(Martin\emph{et al.} , 2013; Kirkham
\emph{et al.} , 2014; Blauvelt \emph{et al.} , 2018). In keratinocytes,
IL-17 activates a series of signal transduction factors to induce the
production of chemokines (CXCL1, CXCL8, and CCL20) and other
inflammatory factors such as antimicrobial peptides (AMPs, including
S100A8, S100A9), which recruit T cells and myeloid dendritic cells to
the psoriatic lesions(Blauvelt \emph{et al.} , 2018; Christmann \emph{et
al.} , 2020). The positive feedback loop of inflammation in the local
skin lesions amplifies and exacerbates the chronic inflammatory process
of psoriasis. Although monoclonal antibodies that block IL-17 are
effective for psoriasis, the benefits of reducing the interaction
between IL-17 and keratinocytes have not been widely studied.

It is widely acknowledged that inflammation response is initiated by
multiple signaling pathways, including the mitogen-activated protein
kinase (MAPK) and nuclear factor-kB (NF-\selectlanguage{greek}κ\selectlanguage{english}B) signaling(Zhao \emph{et al.}
, 2021). The critical contribution of the MAPK and NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways in
psoriasis has been documented in multiple studies(Zhang\emph{et al.} ,
2018; Wang \emph{et al.} , 2021). NF-\selectlanguage{greek}κ\selectlanguage{english}B is a critical transcription
factor of inflammatory genes, which responds to inflammation by binding
to specific promoter elements in target genes. Tumor necrosis factor
(TNF) receptor-associated factor 6 (TRAF6) has been reported to be a
signaling adaptor that can regulate the activation of MAPK and
NF-\selectlanguage{greek}κ\selectlanguage{english}B(Matsumoto\emph{et al.} , 2018). Ample evidence suggests that IL-17
promotes the expression of inflammatory chemokines and AMPs through
NF-\selectlanguage{greek}κ\selectlanguage{english}B in keratinocytes(Harper \emph{et al.} , 2009; Wang \emph{et al.} ,
2013). IL-17 can also induce the signal transducer and activator of
transcription 3 (STAT3) phosphorylation to activate the
NF-\selectlanguage{greek}κ\selectlanguage{english}B(Kim\emph{et al.} , 2017). Therefore, blocking the IL-17-induced
activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B is a promising way to alleviate psoriatic
dermatitis. The significance of blocking the downstream signal of IL-17
targeting keratinocytes and thus inhibiting the progression of psoriasis
warrants further studies.

Natural medicine have become new therapeutic options for the treatment
of psoriasis given their good efficacy and few side effects. Garlic is a
dietary additive rich in organic sulfur-containing organosulfur
compounds, with specific pharmacological properties, such as
antioxidant(Zhang \emph{et al.} , 2006), anti-inflammatory(Wang \emph{et
al.} , 2017), anti-tumor(Sarvizadeh \emph{et al.} , 2021), immune
regulation, and anti-fungal{[}15{]}(Caporaso \emph{et al.} , 1983).
Allicin (diallylthiosulfinate) is the main active ingredient of garlic,
rapidly produced from the non-proteinogenic amino acid S-allyl cysteine
sulfoxide (alliin) catalyzed by the alliinase enzyme in the presence of
water (Borlinghaus \emph{et al.} , 2014; Rose \emph{et al.} , 2019).
Given that allicin is not chemically stable and has a short half-life,
we extracted stable precursors alliin and alliinase from garlic, which
can continuously produce allicin in situ when mixed with an
appropriative solvent on the skin tissue. However, the involved
mechanism of allicin on anti-inflammatory response in psoriasis has not
been documented.

The present study showed that allicin ameliorates imiquimod
(IMQ)-induced psoriatic lesions by inhibiting IL-17A expression and
abnormal proliferation of keratinocytes. Mechanistically, allicin
inhibits IL-17A/IMQ induced NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling transcriptional activation
and downregulates the expression of chemokines and AMPs in
keratinocytes. Our data substantiate the hitherto undocumented
therapeutic potential of allicin in alleviating psoriasis.

\textbf{Materials and methods}

\textbf{Reagents}

5\% Imiquimod Cream (3 g: 0.15 g, 40201001, Sichuan Med-shine
Pharmaceutical), Allicin ointments contains two tubes, tube A loads
alliin (1.7, 2.0, 3.4, 4.0, 5.0, 8.0 mg/g,) and alliinase, tube B is the
special solvent for tube A loaded water-soluble gel, mixed equal A and B
before use (Xinjiang Ailexin Pharmaceutical Co., Ltd). Calcipotriol
cream (0.005\%, 15 g: 0.75 mg, Chongqing Huapont Pharmaceutical Co.,
Ltd). Triamcinolone acetonide and econazole cream (10 g: econazole: 0.10
g, triamcinolone acetonide:10 mg, Hunan Dinuo Pharmaceutical Co., Ltd),
1-chloro-2, 4-dinitrobenzene (Tokyo Chemical Industry), Recombinant
Human Il-17A (PeproTech, Cat\#200-17).

\textbf{Antibodies}

TRAF6 (Cat\# 66498-1-Ig), ERK1/2 (Cat\# 11257-1-AP), p-ERK1/2
(Thr202/Tyr204) (Cat\# 80031-1-RR), JNK (Cat\# 24164-1-AP), Phospho-JNK
(Tyr185, Cat\# 80024-1-RR), STAT3 (Cat\# 10253-2-AP), Bax (Cat\#
50599-2-lg), Bcl-2 (Cat\# 26593-1-AP), Caspase3 (Cat\# 19677-1-AP), Ki67
(Cat\# 27309-1-AP), IL-17A (Cat\# 26163-1-AP), CD3 (Cat\# 17617-1-AP),
F4/80 (Cat\# 28463-1-AP) were purchased from ProteinTech.
Myeloperoxidase (Cat\# MPO, ab9535) was purchased from Abcam. P38 (Cat\#
WL00764) and Phospho-p38(Thr180/Tyr182) (Cat\# WLP1576) were purchased
from Wanleibio. Phospho-STAT3 (Tyr705, Cat\# 9145), NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 (Cat\#
8242), Phospho-NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 (Ser536, Cat\# 3033) were purchased from Cell
Signaling Technology.

\textbf{Preparation of Allicin}

Allicin is biosynthesized from alliin catalyzed by alliinase extracted
from garlic, as previously reported(Li\emph{et al.} , 2011). Alliin
(purity [?] 99\%, batch No. AL160615) was accurately weighed and dissolved
in DMEM medium to make the stock solutions (1.0 mg/mL). Alliinase
([?]10000U/g, batch No. 201801001) was freshly prepared and dissolved at
37 to ensure optimal enzyme activity. Equal volumes were mixed for
30min at room temperature to ensure that alliin sufficiently reacted
with alliinase. Centrifuged for 10 minutes at 4 \selectlanguage{ngerman}°C at 12,000 rpm, the
allicin stock solution was diluted immediately prior to experimentation.
The allicin and alliinase were provided by Xinjiang Ailexin
Pharmaceutical Co., Ltd (Xinjiang, China).

\textbf{Cell Culture}

The human immortalized epidermal keratinocyte cell line HaCaT was
purchased from the iCell Bioscience Inc (Shanghai, China, passages
20--22) and maintained in high-glucose Dulbecco's Modified Eagle Medium
(DMEM, BIOIND, Israel) contained 10\% fetal bovine serum (FBS, BIOIND,
Israel) and 1\% penicillin-streptomycin (PS, NCM Biotech, China) under
standard conditions (37°C with 95\% humidity and 5\%
CO\textsubscript{2}). Cells were digested by 0.25\% trypsin-EDTA and
terminated by DMEM containing 10\% FBS.

\textbf{Cell Viability Assay}

The cell viability was determined by Cell Counting Kit-8 (CCK8)
according to the manufacturer's protocol (Cat\#C0043, Beyotime, China).
HaCaT cells (1×104 cells/200\selectlanguage{greek}μ\selectlanguage{english}l) were seeded into 96-well plates
overnight and incubated with different concentrations of allicin for 48
h. After adding 20\selectlanguage{greek}μ\selectlanguage{english}l of CCK-8 solution to each well for 1 h at 37\selectlanguage{ngerman}°C, the
optical density (OD) was measured using a multifunctional microplate
reader (2300, PerkinElmerEnSpire) at 450 nm.

\textbf{Treatment of Psoriasis-like Skin in Mice}

A total of eight groups of BALB/c mice (male and female, 8-11 weeks,
20-25 g) were randomly selected (n = 6 per group) and purchased from
Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China),
containing normal control group (NC), IMQ group, vehicle control group,
allicin-low-dose group (AL, 1.7 mg/g, measured with alliin),
allicin-middle-dose group (AM, 3.4 mg/g, measured with alliin) and
allicin-high-dose group (AH, 5.0 mg/g, measured with alliin), positive
control groups, with the commonly used first-line treatment for
psoriasis, calcipotriol cream (15 g: 0.75 mg) or triamcinolone acetonide
and econazole cream (TAE). 5cm\textsuperscript{2} of the back skin of
mice was exposed by shaving dorsal hair. A daily topical dose of 62.5 mg
IMQ for inducing psoriasis-like skin was provided at 9:00 a.m. for six
consecutive days. Then mice were treated with allicin ointment twice, at
an interval of four hours. Vehicle control mice were topically treated
with a blank allicin-free gel. The experiment was terminated on the
seventh day, following sacrifice of the mice, the back skin was
dissected rapidly and flash frozen in liquid nitrogen and stored at 80°C
for subsequent analyses.

\textbf{Measurement of Skin Inflammation Severity}

We used the cumulative Psoriasis Area and Severity Index (PASI) score to
assess the severity of skin lesions, which consists of erythema, scale,
and thickness. The score was graded independently from 0 to 4: 0, none;
1, slight; 2, moderate; 3, marked; 4, very marked. The PASI scores of
the mice were recorded on the first day of IMQ treatment.

\textbf{Skin Sensitization Test}

Guinea pigs (both sexes, 6-7 weeks, 330-410 g) were purchased from
Pizhou Orient Breeding (Jiangsu, China) and randomly assigned into three
groups (n=10). The back hair was removed before the skin sensitization
test. The Control group was treated with a blank solvent, and the
allicin-treated group received topical allicin treatment (4 mg/g,
measured with alliin). In the positive control group, 1-chloro-2,
4-dinitrobenzene (WT6CA-VO, Tokyo Chemical Industry) was used as a
contact-sensitizing agent; a 1\% sensitization concentration solution or
0.1\% excitation concentration was prepared with ethanol. On
sensitization day, guinea pigs were treated on the left skin, and on the
excitation day were treated on the right skin. The first sensitization
and administration day was defined as the first day of the test (D1).
Guinea pigs were sensitized on Days 1, 8 and 15), and all groups were
stimulated 14 days after the last sensitization (D29). The allergic
reactions of the skin were observed at 6 h, 24 h, 48 h, and 72 h. Skin
anaphylaxis includes erythema and edema. The evaluation criteria of
erythema: without erythema (0), barely visible erythema (1), visible
erythema (2), severe erythema (3), purplish-red erythema with mild
eschar (4); The evaluation criteria of edema: without edema (0), barely
visible edema (1), obviously higher than the edge of the surrounding
skin (2), skin bulge 1mm or clear contour (3), skin bulge more than 1mm
or blisters or ulceration (4).

\textbf{Toxicity Assay of Repeated Administration and Skin Irritation
Test}

New Zealand rabbits (both sexes, 14-16 weeks, 2.3-2.7 kg) were purchased
from Qindao Kangda Biological Technology (Shandong, China), randomly
assigned into four groups (n=10) contained control-group (blank
solvent), allicin low-dose group (2 mg/g, measured with alliin), allicin
middle-dose group (4 mg/g, measured with alliin), and allicin high-dose
group (8 mg/g, measured with alliin). After four consecutive months of
topical administration of allicin, body weight, organ weight,
hematologic and clinical chemistry analysis were evaluated. Skin
irritation included erythema and edema and was evaluated based on the
above criteria. Stimulus Scores = Total Cumulative Stimulus Scores /
Total Observation Times. Stimulation grade: without irritation (0-0.49),
mild irritation (0.5-2.99), moderate irritation (3.0-5.99), severe
irritation (6.0-8.00).

\textbf{Hematoxylin and Eosin Staining}

Collected back skin tissues from mice were conserved in 10\%
formaldehyde solution for 72 h, then dehydrated. Hematoxylin and eosin
(H\&E) staining was performed on 4 mm thick paraffin-embedded sections.
The tissue sections were examined under an OLYMPUS light microscope.
Histological scores were evaluated by a cumulative score containing four
independent scoring criteria (0-4): Epidermal thickness, inflammatory
cells infiltrate, capillaries dilatation, and parakeratosis. Three
different fields of view were used to measure the thickness of the
mice's epidermis.

\textbf{Immunohistochemistry}

Paraffin sections were deparaffinized with xylene and rehydrated in
decreasing ethanol concentrations; then, antigen retrieval was carried
out with the EDTA buffer (PH 9.0) for 25 min at 100°C. To neutralize the
endogenous peroxidase, sections were cooled down to room temperature and
incubated with 3\% H\textsubscript{2}O\textsubscript{2} for 10 min to
neutralize the endogenous peroxidase. After washing twice with PBST (1×
PBS with 0.05\% Tween 20), sections were blocked with 10\% goat serum
for 45 min and incubated in the specific primary antibodies against
Ki67, IL-17A, CD3, F4/80, and myeloperoxidase (MPO) with appropriately
diluted ratio overnight at 4°C. After washing with PBST, tissues were
incubated with HRP-conjugated goat anti-rabbit secondary antibody for 45
min at room temperature. All sections were stained using DAB for a few
seconds and lightly counterstained with hematoxylin. Images were
obtained with an Olympus BX41 microscope.

\textbf{TUNEL staining}

The apoptosis cells in the skin lesions of mice were detected using the
One Step TUNEL Apoptosis Assay Kit (C1086, Beyotime). After
deparaffinization and rehydration, skin slices were treated with
proteinase K (20\selectlanguage{english} \selectlanguage{greek}µ\selectlanguage{english}g/ml) for 20 min at 37 and then rinsed in PBS
three times. According to the manufacturer's instructions, sections were
stained with TUNEL staining reagents at 37\selectlanguage{ngerman}°C for 60 min. Following
several washes with PBS, nuclei were stained with DAPI Staining Solution
(C1005, Beyotime) for 3 min at room temperature. Images were obtained
using an inverted fluorescence microscope (Leica, DMI3000B), in which
TUNEL-positive cells produced green fluorescence, and DAPI yielded blue
fluorescence.

\textbf{Western Blotting Analysis}

Total protein was extracted from snap-frozen skin tissues or HaCaT cells
using RIPA buffer (P0013J, Beyotime) containing 1\% phosphatase and
protease inhibitors. After centrifugation, clarified lysates were
quantified using the BCA Protein Assay Kit (Thermo Scientific) and
boiled with SDS loading buffer at 95 \selectlanguage{english} for 15 min. Then, we separated
protein samples by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred them to polyvinylidene
fluoride (PVDF) membranes (Amersham International), where 5\% milk in
TBST (TBS with 0.05\% Tween 20) was used as the blocking buffer to block
the membrane at room temperature for 1 h. Next, primary antibodies were
diluted to a suitable concentration based on the manufacturer's
recommendations by dilution buffer (P0023A, Beyotime) and incubated
overnight at 4\selectlanguage{english} degC. After three washes in TBST, the protein samples were
incubated with horseradish peroxidase (HRP)-conjugated secondary
antibodies at room temperature for 2 h. The protein bands were detected
by the enhanced chemiluminescence (ECL) western-blotting substrate
(Fdbio science, FD8000) and quantified using Image Lab (Bio-Rad).

\textbf{Reverse Transcription and Real-time PCR Analysis}

Total RNA was extracted from snap-frozen skin tissues or HaCaT cells
using the Trizol Reagent (Thermo Fisher Scientific, 15596026) on ice in
an RNAse free environment, after which complementary DNAs were
synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA
wiper) (Vazyme Biotech, R312-01). The qRT-PCR was performed using the
Step One Plus Real-Time PCR system (Applied Biosystems). \selectlanguage{greek}β-\selectlanguage{english}Actin was
used as the reference gene for normalization. The fold changes of mRNA
relative expression were calculated using the comparative cycle method
(2-\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct). The primer sequences are shown in Supplementary Table S1.

\textbf{Cell cycle analysis}

HaCaT cells were seeded in six-well plates and allowed to adhere. Then
the cells were serum-starved for 24 h, followed by treatment with
10\%-FBS/DMEM containing various concentrations of allicin (6.4, 9.6,
12.8\selectlanguage{greek}μ\selectlanguage{english}g/mL) for 48 h. Later, the cells were collected by trypsinization
and fixed in 70\% ethanol overnight at -20\selectlanguage{ngerman}°C. After being washed with
PBS, HaCaT cells were resuspended and incubated with PI/RNase staining
buffer according to the manufacturer's instructions (C1052, Beyotime)
for 30 min at 37°C in the dark and then subjected to flow cytometric
analysis. The samples were acquired with the Attune NxT (Thermo Fisher
Science), and the cell cycle distribution was further analyzed using
ModFit software (Verity Software House).

\textbf{Cell apoptosis assay}

Annexin V-FITC Apoptosis Detection Kit (Beyotime, China) was used to
quantitatively evaluate allicin-induced apoptosis. The supernatant was
collected and mixed with HaCaT cells detached using EDTA-free trypsin.
After centrifugation, HaCaT cells were resuspended in PBS. Then cells
were incubated with 195\selectlanguage{greek}μ\selectlanguage{english}l binding buffer and incubated with 5\selectlanguage{greek}μ\selectlanguage{english}l Alexa
Fluor 488 and 10\selectlanguage{greek}μ\selectlanguage{english}l PI for 15 min at room temperature. The proportion of
cell apoptosis was measured by flow cytometry and analyzed with FlowJo
V10.

\textbf{RNA-seq analysis}

Total RNA was extracted from the back skin lesions of the IMQ-treated
and high-dose allicin-treated groups for transcriptome sequencing. RNA
was isolated and purified using Trizol reagent (Invitrogen, Carlsbad,
CA, USA). The RNA amount and purity of each sample were quantified using
NanoDrop ND-1000 (Wilmington, DE, USA). The final cDNA was sent for
library preparation and sequencing on an Illumina Novaseq 6000 at LC
Bio Technology (Hangzhou, China). With StringTie and Ballgown, we
estimate the expression levels for all transcripts and calculate the
expression levels for mRNAs with FPKM (FPKM = {[}total exon
fragments/mapped reads (millions) \selectlanguage{ngerman}× exon length (kB){]}). The
differentially expressed genes (DEGs) were selected by R package edgeR
or DESeq2 with fold change \textgreater{} 2 or fold change \textless{}
0.5 and p-value \textless{} 0.05. Following that, we conducted gene
ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes
(KEGG) enrichment analyses of the differentially expressed mRNAs in the
two groups.

\textbf{Statistical analysis}

All data are presented as mean ± SEM (standard error of the mean).
GraphPad Prism was used to compare two groups using Unpaired Student's
t-tests and one-way ANOVA followed by Tukey's post hoc test for
comparing multiple groups using one-way ANOVA (Version 8.0.1), and
statistically significant differences are indicated as follows: *p
\textless{} 0.05, **p \textless{} 0.01, ***p \textless{} 0.001, ****p
\textless{} 0.0001.

\textbf{Results}

\textbf{Allicin Ameliorated IMQ-Induced Psoriasis-like Skin Lesion in
Mice}

IMQ-treated mice skin exhibited typical psoriatic characteristics, such
as acanthosis, inflammatory cell infiltrates, and altered dermal
vascularity. Therefore, IMQ was applied to mouse skin to explore how
allicin interferes with the pathophysiology of psoriasis. To assess
allicin's effect on restoring the appearance of psoriatic lesions
induced by IMQ, we consecutively applied IMQ for six days after shaving
the back of mice. The treatment groups received mixed ointment with
different allicin contents four and eight hours after receiving IMQ
(Figure 1A). Compared with the normal control group, IMQ treatment
exhibited apparent erythema and scaling of the skin, whereas the
phenotypic changes in allicin-treated groups were significantly improved
(Figure 1B). Dorsal skin thickness, erythema and scaling were scored
daily from 0-4 according to the severity degree. Additionally, an
overall evaluation of skin lesions was depicted by a modified PASI score
(0-12), calculated by summing the three individual scores. Compared with
the IMQ-group, allicin-treated groups showed a significant alleviation
in erythema severity (Figure 1C), scaling (Figure 1D), and thickening
(Figure 1E). Figure 1F showed a better overall improvement with allicin
treatment than positive control groups. H\&E staining showed that the
epidermal layer of the IMQ-treated group was significantly incrassated,
and the corium layer was infiltrated by inflammatory cells accompanied
by vascular hyperplasia. However, the characteristic pathological
changes of the skin in the allicin-treated groups were significantly
improved compared with the model group (Figure 1G, H). These results
collectively indicated that allicin was efficient in relieving
IMQ-induced psoriasis-like lesions.

\textbf{Preclinical Safety Evaluation of Allicin on Topical Application}

Further evaluation of skin irritability and irritation induced by
allicin was performed to assess the suitability and safety of allicin
for topical applications. Skin changes were observed after allicin
treatment daily, including erythema and edema. As shown in Figures 2A
and 2B, no allergic reactions were observed in the allicin
administration group. After four months of continuous application,
compared with the blank solvent control group, allicin led to slight
skin irritation in the first three months and yielded no irritation in
the fourth month due to gradual development of skin tolerance (figure
2C). Next, we inspected the toxicity of allicin for long-term
application, and there was no significant influence on weight (Figure
2D). The organ index was calculated after the major organs were resected
and weighed, using the formula Organ index = organ weight (mg) / body
weight (kg) ×100\%. The heart, liver, spleen, and thymus indexes showed
that allicin yielded no significant difference in body weight and major
organs compared with the control group (Figure 2E). Hematology (Figure
2F, Table S2) and biochemistry (Table S3) indicators were analyzed
before and after continuous topical application of allicin, with no
significant changes. Our data demonstrated that allicin was safe and
well-tolerated for long-term external use.

\textbf{Allicin Inhibits Keratinocytes Proliferation in HaCaT and
psoriatic lesions}

Sustained abnormal proliferation is the hallmark of keratinocytes in
psoriatic lesions; thus, we investigated whether allicin inhibits the
proliferation of keratinocytes in IMQ-induced psoriatic skin. HaCaT
cells were treated with different concentrations of allicin (3.2, 6.4,
9.6, 12.8, 25.6, 51.2 \selectlanguage{greek}μ\selectlanguage{english}g/mL), and the cell viability was assessed by
CCK8 assays after 48h (figure 3A). The effect of allicin treatment was
concentration-dependent and significantly reduced cell viability. We
next analyzed the influence of allicin on the cell distribution in the
different cell cycle phases with flow cytometry. Allicin significantly
decreased the HaCaT cell numbers in the G0/G1 phase, whereas the number
of cells in the G2/M phase was increased compared with the control group
(Figure 3B). The percentage of cells in the G2/M increased from 21.62\%
to 58.33\%, indicating that allicin inhibited the proliferation of
keratinocytes by inducing G2/M cell cycle arrest (Figure 3C). We used
immunohistochemistry analysis to examine the expression of Ki67 in skin
lesions, which is a marker expressed in proliferating cells. Figure 3D
showed that the Ki67 expression of allicin-treatment was significantly
fewer than with the IMQ-treated group. These results demonstrate that
allicin reduced epidermal thickness by inhibiting keratinocyte
hyperproliferation.

\textbf{Allicin Induces Keratinocytes Apoptosis in vivo and vitro}

The resistance of keratinocytes to apoptosis is well-established to
contribute to psoriatic epidermal thickening. We examined the effect of
allicin on inducing apoptosis in keratinocytes. An analysis of flow
cytometry with Annexin V/PI staining was carried out to determine the
percentage of apoptotic cells. As shown in Figures 4A and 4B, the
proportion of apoptotic cells was 8.74\%, 21.90\%, and 29.40\% after
treatment with 6.4, 9.6, and 12.8\selectlanguage{greek}μ\selectlanguage{english}g/mL of allicin, respectively, which
was significantly higher than the control group (5.39\%). Overall,
treatment with various concentrations of allicin dose-dependently
induced keratinocyte apoptosis. The molecular mechanism of
allicin-mediated apoptosis was explored using Western blot. Allicin
induced the levels of pro-apoptotic proteins Bax and cleaved-caspase3
and inhibited the expression of the anti-apoptotic protein Bcl-2 in
HaCaT cells (Figures 4C) and skin lesions (Figures 4D). In addition,
TUNEL-DAPI staining was performed to examine keratinocyte apoptosis in
skin lesions sections. Figure 4E showed that allicin treatment could
induce keratinocyte apoptosis in psoriatic skin. These data corroborated
the pro-apoptotic role of allicin in keratinocytes of psoriasis skin
lesions.

\textbf{Identification of IL-17 as a Key Target Regulated by Allicin
through RNA-seq}

To further uncover the molecular mechanism of underlying disruption of
IMQ-induced psoriasis development by allicin, we used RNA-sequencing
(RNA-seq) to compare gene alternations between IMQ-treated and
allicin-treated groups. A Volcano plot depicting changes in
differentially expressed genes (DEGs) after Allicin treatment is shown
in Figure 5A. Compared with the IMQ-treated group, 1285 genes were
significantly altered, including 344 downregulated and 941 upregulated
genes in the allicin-treated group (fold change[?]2 and q\textless{}0.05).
Among these DEGs, the downregulated genes were closely associated with
inflammation (Figure 5B). KEGG analysis showed that the DEGs were
involved in various signaling pathways, including cytokine-cytokine
receptor interaction, basal cell carcinoma, Wnt signaling pathway, and
IL-17 signaling pathway (Figure 5C). The gene expression level of IL-17A
and IL-17F was significantly different, which further confirmed that
allicin could alleviate IMQ-induced psoriasis-like skin inflammation in
mice through the IL-17-mediated signaling pathway. GO enrichment
analysis indicated that the downregulated DEGs were mainly enriched in
biological processes associated with signal transduction and innate
immune response (Figure 5D). Taken together, these results indicated
IL-17-associated inflammatory genes and IL-17 signal pathways were
regulatory targets of allicin in IMQ-induced psoriasis-like inflammatory
skin.

\textbf{Allicin Reduced the Secretion of Inflammatory Factors and Immune
Cells Infiltration in IMQ-Induced Mouse Model}

Psoriasis and other inflammatory skin diseases are widely thought to
result from abnormal cytokine expression. IL-17 mediates inflammatory
cell recruitment via inducing inflammatory cytokines and chemokines by
keratinocytes in psoriatic lesions(Nograles \emph{et al.} , 2008; Harper
\emph{et al.} , 2009). We examined the mRNA expression levels of
inflammatory cytokines in IMQ-induced psoriasis skin lesions. As seen in
Figures 6A and 6C, allicin significantly suppressed the mRNA levels of
IL-17A. Then the expression of IL-17A was further validated by IHC
(Figure 6C). In addition, allicin reduced IL-12, IL-20, and IL-22
levels, which are involved in the immune response of psoriasis (Figure
6B). Various immune cell subsets create a self-sustaining cycle of
inflammation during the development of psoriasis, including T cells,
neutrophils, and macrophages(Singh \emph{et al.} , 2013). Current
evidence suggests that the number of CD3\textsuperscript{+}cells is
increased in the skin tissue of patients with psoriasis (He \emph{et
al.} , 2021). We next determined whether allicin affected the
accumulation of immune cells in IMQ-induced psoriasis-like skin,
including T cells, macrophages, and neutrophils. The
immunohistochemistry results showed that allicin reduced the
infiltration of CD3\textsuperscript{+}, F4/80\textsuperscript{+}, and
MPO\textsuperscript{+} cells compared to the IMQ-treated group (Figure
6D, E, F). As a result, allicin treatment alleviated immune cell
infiltration and pro-inflammatory factor secretion substantially.

\textbf{Allicin Reduced the mRNA Expression of Chemokines and
Antimicrobial Peptides (AMPs) both in IMQ-induced psoriatic lesions and
IL-17A Stimulated HaCaT Cells}

IL-17A is a potent stimulator of multiple immune-related proteins such
as chemokines and AMPs, which lead to further inflammatory responses in
keratinocytes of psoriasis lesions(Furue\emph{et al.} , 2020). Excessive
expression of AMPs in psoriatic lesions is involved in the pathogenesis
of dermatoses via host inflammatory reactions, such as cathelicidin,
\selectlanguage{greek}β-\selectlanguage{english}defensins, and S100 proteins(Wang \emph{et al.} , 2018). To
investigate whether allicin could reduce the production of chemokines
and AMPs by keratinocytes upon IL-17A stimulation, we measured the mRNA
expression of CXCL8, CCL20, S100A8, and S100A9 by RT-qPCR. The data
showed IL-17A remarkedly upregulated levels of CXCL8, CCL20, S100A8, and
S100A9, which were notably reduced in the allicin-treated group (Figure
7A). Similarly, compared with the IMQ-group, allicin significantly
decreased the mRNA expression levels of CXCL2, CXCL5, CCL20, S100A8, and
S100A9 in psoriasis-like lesions (Figure 7B).

\selectlanguage{greek}\textbf{Allicin Inhibited IL-17A-activated TRAF6/MAPK and STAT3/NF-κB
Signaling Pathways in Vitro and in Vivo}\selectlanguage{english}

It has been confirmed that activated IL-17A signaling recruits TRAF6,
which leads to simultaneous activation of the NF-\selectlanguage{greek}κ\selectlanguage{english}B by MAPK
phosphorylation(Kobayashi \emph{et al.} , 2003). Topical application of
IMQ induces IL-17-mediated psoriatic dermatitis via TRAF6 signaling. In
addition, IL-17-enhanced NF-\selectlanguage{greek}κ\selectlanguage{english}B transcriptional activity in keratinocytes
was stimulated by the activation of STAT3 and NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways. We next
co-cultured keratinocytes with IL-17A with or without allicin, then
further detected the activation of TRAF6, ERK1/2, P38, JNK, STAT3, and
P65. Figure 8A showed that IL-17A stimulation for 30 min effectively
upregulated the phosphorylation expression of ERK1/2, P38, JNK, and P65
in HaCaT cells, while treatment with allicin significantly inhibited the
protein expression of TRAF6 and MAPK and restrained the phosphorylation
of MAPK, STAT3, and P65 (Figure 8B, 8C). To investigate whether allicin
alleviates IMQ-induced psoriatic lesions by inhibiting downstream
pathways activated by IL-17A, we analyzed the protein expressions of
TRAF6/MAPK/STAT3/NF-\selectlanguage{greek}κ\selectlanguage{english}B, which were significantly repressed in
IMQ-induced psoriatic lesions (Figure 8D, 8E). Overall, the results
showed that allicin could inhibit inflammation by suppressing the
activation of IL-17A downstream signaling pathways (Figure 8F).

\textbf{Discussion}

The dynamic interplay between keratinocytes and T cell-derived cytokines
is crucial in the initiation and maintenance phases of psoriatic
alterations, perpetuating inflammation in a vicious cycle(Jiang \emph{et
al.} , 2020). Keratinocytes play multiple roles in psoriatic skin
lesions as the target cell of IL-17 and the producer of cytokines,
leading to the infiltration of inflammatory cells. More importantly,
keratinocytes are the main initiator of pathological changes in
psoriatic skin due to uncontrolled proliferation and differentiation.
Our study demonstrates that blocking the feedback between keratinocytes
and inflammatory factors is a promising approach for treating psoriasis.

Although traditional treatment therapies are effective, psoriasis is not
cured and is often recurrent, emphasizing the importance of long-term
safe and effective drugs. Accordingly, nontoxic, widely accessible, and
inexpensive natural products have gained momentum as a novel strategy in
recent years. Diallyl trisulfide, a sulfur compound found in garlic, has
been developed and approved to treat fungal and bacterial infections.
The anti-inflammatory, antiviral, and anticancer properties of allicin
prompt us to explore its potential in psoriasis. Based on our in vitro
and in vivo results, allicin alleviates psoriasis by suppressing
inflammation and hyperproliferation. Indeed, the influence of the
topical application of allicin on the systemic immune system was
negligible compared to systemic administration. Our data identified that
allicin treatment did not induce significant changes in body weight and
immune organ index values, indicating that allicin treatment was safe,
with no significant side effects on mice. The present study
substantiates that allicin is a promising natural drug candidate for
psoriasis.

We used the first-line drugs corticosteroids (TAE) and topical vitamin D
corticosteroid (calcitriol) in the present study as positive control
drugs. We demonstrated that allicin treatment has therapeutic effects on
inflammation comparable to the positive groups; moreover, improvement of
erythema and scales showed higher therapeutic efficacy with allicin than
with positive drugs. This finding may be attributed to the fact that
current first-line drugs merely inhibit inflammation and suppress
proliferation and differentiation of keratinocytes, while the
anti-psoriasis effect of allicin is multifaceted. Our results showed
that allicin not only decreased IL-17 secretion but also triggered
inhibition of keratinocyte viability and cell cycle progression and
promoted apoptosis, which directly relieved epidermal swelling.

Keratinocytes are actively involved in the pathogenesis and maintenance
of psoriasis by producing various pro-inflammatory factors and
chemokines. S100A8/9 and CCL20 are additional factors during
IL-17-driven dermal inflammation, which are strongly induced
preferentially during the early maturation of keratinocytes(Christmann
\emph{et al.} , 2020; Elnabawi \emph{et al.} , 2021). Overwhelming
evidence substantiates that S100A8/9 and CCL20 are overexpressed in
human psoriatic lesions, whereas serum concentrations of S100A8/S100A9
and CCL20 are reliable biomarkers for monitoring disease activity in
psoriasis, including psoriatic arthritis or infections, underlining
their clinical relevance(Benoit \emph{et al.} , 2006; Schonthaler
\emph{et al.} , 2013; Austermann \emph{et al.} , 2017; Dey \emph{et al.}
, 2017; Freise \emph{et al.} , 2019). Consistent with previous reports,
we identified upregulated expression of IL-17-dependent CCL20 and
S100a8/9, both in HaCaT cells and in mice psoriatic skin lesions. Our
study corroborated that allicin inhibited immune cell infiltration in
skin lesions; however, we did not investigate the effect of allicin on
systemic immune cells in depth. Mechanistically, allicin inhibited
transcription of NF-\selectlanguage{greek}κ\selectlanguage{english}B induced by IL-17, which triggered the expression
of psoriasis-relevant target genes encoding for CCL20 and S100A8/9.
Overall, our data suggest that interfering with IL-17 signaling in
keratinocytes is a promising strategy for treating psoriasis. Targeting
the IL-17 signaling pathway may improve treatment response and prevent
treatment resistance.

Interestingly, it has been reported that allicin inhibits the activation
of the MAPK/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway and NLRP3 inflammasome to improve
acrylamide-induced hepatotoxicity(Li \emph{et al.} , 2020). Furthermore,
allicin alleviated reticuloendotheliosis virus infection-induced
inflammation and oxidative damage by blocking the ERK/MAPK pathway(Wang
\emph{et al.} , 2017). In addition, alliin ameliorated gut inflammation
by suppressing MAPKs-PPAR-\selectlanguage{greek}γ/\selectlanguage{english}NF-\selectlanguage{greek}κ\selectlanguage{english}B/AP-1/STAT-1 signaling pathways(Shi
\emph{et al.} , 2017). These studies corroborated that allicin yields
superior anti-inflammatory effects by regulating MAPKs and NF-\selectlanguage{greek}κ\selectlanguage{english}B. During
the transcriptome analysis of allicin-treated and IMQ-treated mice
lesions, we observed dramatic changes in IL-17-dependent genes
expression following IL-17A/F gene expression down-graduation, and
substantial enrichment in IL-17 signaling pathways, confirming the in
vivo molecular mechanisms of allicin are related to IL-17 signaling in
psoriasis. Hyperactivity of the IL-17 signaling pathway in psoriatic
lesions further exacerbates the inflammatory response. Importantly, the
inhibition of IL-17 and IL-17 signaling blocks various signaling
pathways in keratinocytes. In this study, we documented the pivotal role
of allicin in blocking the IL-17 signaling pathway, including TRAF6,
MAPKs (ERK1/2, P38, JNK), STAT3, and NF-\selectlanguage{greek}κ\selectlanguage{english}B (P65).

\textbf{Conclusion}

This study elucidated the molecular mechanism of allicin in improving
IMQ-induced psoriatic lesions in mice. In this regard, allicin inhibits
the innate immune response to external stimuli in psoriatic lesions,
resulting in reduced expression of pro-inflammatory cytokines and
chemokines. Furthermore, allicin treatment mitigates psoriasis
progression by inhibiting the TRAF6/MAPK/NF-\selectlanguage{greek}κ\selectlanguage{english}B and STAT3/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling
pathways and activating the apoptotic signaling cascade in
keratinocytes. Overall, our data substantiated that interfering with
IL-17 signaling in keratinocytes with allicin is a promising strategy
for treating psoriasis, given its safety and effectiveness.

\textbf{Ethics statement:}

The animal study was reviewed and approved by the Institutional Animal
Care and Use Committee of the Center for New Drug Safety Evaluation and
Research, China Pharmaceutical University.

\textbf{Declaration of transparency and scientific rigor}

According to the BJP guidelines for Design and Analysis, this report
adheres to the principles for transparent reporting and scientific rigor
of preclinical research.

\textbf{Conflict of interest statement:}

The authors state no conflict of interest.

\textbf{Funding}

This work was supported by the Science Fund for Distinguished Young
Scholars of Xinjiang Autonomous Region (No. 2022D01E17) and the Natural
Science Foundation of Xinjiang Uygur Autonomous Region (No.
2021D01C255), Major science and technology special project of Xinjiang
Uygur Autonomous Region: research and development of the active
ingredient of medicinal garlic for external use against difficult skin
diseases in Xinjiang.

\textbf{Authors contribution}

Jianguang Li, Jingjing Duan, and Xinxia Li conceived and designed the
project. Lu Zhang, Libo Zhang, Ruolin Zhao, Ran Duan, Yuanyuan Qin,
Sijia Gao participated in the experiments. Xuehong Ma and Rongmei Shi
contributed to the preparation and the quality control of reagent
(allicin). Lu Zhang analyzed data and drafted the article. Jingjing Duan
edited and reviewed it. Jianguang Li and Xinxia Li critically read the
manuscript and gave helpful suggestions. All authors read and approved
the final manuscript.

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gut inflammation through MAPK-NF-\selectlanguage{greek}κ\selectlanguage{english}B/AP-1/STAT-1 inactivation and PPAR-\selectlanguage{greek}γ
\selectlanguage{english}activation. Mol Nutr Food Res, 61 (9) (2017) doi:10.1002/mnfr.201601013.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure1.} Allicin attenuates symptoms of IMQ-induced
psoriasis-like lesions in mice. (A) Treatment scheme in the IMQ-induced
mice model. (B) Representative images of IMQ-induced psoriatic symptoms
on mice dorsal skin treated with or without allicin for 6 days (AL,
allicin low-dose; AM, allicin middle-dose; AH, allicin high-dose; TAE,
triamcinolone acetonide and econazole cream). The severity of the
IMQ-induced dorsal lesions was evaluated by PASI scores (n=6), and the
cumulative scores of redness (C), scale (D), and thickness (E) were
measured daily (F). Representative images of H\&E staining (G) and
respective histopathological scores (H) of IMQ-induced psoriatic lesions
in each group (n=3). Scale bar = 50 \selectlanguage{greek}μ\selectlanguage{english}m. Data are expressed as means \selectlanguage{ngerman}±
SD. *p \textless{} 0.05, **p \textless{} 0.01, ***p \textless{} 0.001,
****p \textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure2.} Safety and adaptability evaluation of allicin topical
application on guinea pig and rabbit skin. Edema (A) and erythema (B)
scores were recorded at 6h, 24h, 48h, and 72h during the stimulation
phase of the anaphylaxis test (n=12). (C) Cumulative scores of skin
irritation after allicin topical application with different
concentrations in rabbits for 4 months. Effects on body weight (D) and
major organ index (E) of different concentrations of allicin applied to
rabbit skin for 4 months. (F) Hematologic studies before 3 days and
after 92 days of allicin topical administration (n=6). Data are
expressed as means \selectlanguage{ngerman}± SD. *p \textless{} 0.05, **p \textless{} 0.01, ***p
\textless{} 0.001, ****p \textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Allicin inhibits hyperproliferation in the
keratinocytes. (A) Cell viability of IL-17A-stimulated HaCaT cells
treated with different concentrations of allicin was measured by CCK-8
assay (n=6). (B) The cell cycle distribution of PI labeling in different
phases with different concentrations of allicin after 48h. (C)
Percentages of G0/G1, S, and G2/M-phase cells with different
concentrations of allicin after 48h (n=3). (D) Representative
immunohistochemical images and the relative expression of Ki67 in
IMQ-induced psoriasis-like lesions treated with or without allicin (AH,
allicin high-dose). Scale bar = 50 \selectlanguage{greek}μ\selectlanguage{english}m. Data are expressed as means \selectlanguage{ngerman}± SD.
*p \textless{} 0.05, **p \textless{} 0.01, ***p \textless{} 0.001, ****p
\textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Allicin promotes keratinocytes apoptosis in vivo and
vitro. (A) Apoptosis was measured by staining with annexin V/PI staining
followed by flow cytometry. (B) Allicin significantly increased the
percentage of early apoptotic cells (AV+/PI-) and late apoptotic cells
(AV+/PI+) in a dose-dependent manner after 48h (n=3). (C) Western blot
analysis of Bax, Bcl-2, and cleaved-caspase3/caspase3 by pretreating the
HaCaT cells with different concentrations of allicin for 48 h. (D)
Western blot analysis for Bax, Bcl-2, and cleaved-caspase3/caspase3 in
IMQ-induced psoriatic lesions with allicin-treated. \selectlanguage{greek}β-\selectlanguage{english}Actin was used as
the endogenous loading control. (E) Representative images of skin
sections stained with TUNEL (green) to detect apoptotic cells and nuclei
were treated with DAPI (blue) in IMQ-induced psoriasis-like lesions
treated with or without allicin. Scale bar = 50 \selectlanguage{greek}μ\selectlanguage{english}m. Data are expressed
means \selectlanguage{ngerman}± SD. *p \textless{} 0.05, **p \textless{} 0.01, ***p \textless{}
0.001, ****p \textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} RNA-seq reveals the mechanism of allicin ameliorating
IMQ-induced psoriasis-like lesions by repressing the IL-17 signal
pathway. (A) Volcano plot of downregulated (blue) and upregulated (red)
DEGs between allicin treated/untreated IMQ-induced psoriatic lesions.
(B) Heatmap of the top 50 downregulated DEGs with or without allicin.
(C) KEGG pathway analysis of DEGs (FDR \textless{} 0.1). (D) The
downregulated DEGs were classified based on their gene ontology (GO)
terms for biological process (BP), cellular component (CC), and
molecular function (MF).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6.} Allicin reduces the overproduction of inflammatory
cytokines. (A) Quantitative PCR analysis of mRNA level of the IL-17A
genes in back skin treated with IMQ in all groups (n=6). (B)
Quantitative PCR analysis of mRNA level of the IL-17F, IL-12, IL-20, and
IL-22 genes in IMQ-treated lesions with or without allicin (n=6). (C)
Representative immunohistochemical images and the relative expression of
IL-17A in each group (AL, allicin low-dose; AM, allicin middle-dose; AH,
allicin high-dose; TAE, triamcinolone acetonide and econazole cream).
Immunohistochemical staining of CD3+ cells (D), MPO+ cells (E), and
F4/80+ cells (F) in IMQ-induced psoriasis-like lesions treated with or
without allicin. Scale bar = 50 \selectlanguage{greek}μ\selectlanguage{english}m. Data are expressed as means \selectlanguage{ngerman}± SD. *p
\textless{} 0.05, **p \textless{} 0.01, ***p \textless{} 0.001, ****p
\textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 7.} Allicin suppresses the over-expression of chemokines
and AMPs. (A) Quantitative PCR analysis of mRNA level of the CXCL8,
CCL20, S100a8, and S100a9 genes in IL-17A-stimulated HaCaT cells with or
without allicin (n=3). (B) Quantitative PCR analysis of mRNA level of
the CXCL2, CXCL5, CCL20, S100a8, and S100a9 genes in IMQ-induced
psoriatic lesions treated with or without allicin (n=6). Data are
expressed as means \selectlanguage{ngerman}± SD. *p \textless{} 0.05, **p \textless{} 0.01, ***p
\textless{} 0.001, ****p \textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 8.} Allicin inhibits the activation of TRAF6/MAPK/NF-\selectlanguage{greek}κ\selectlanguage{english}B
and STAT3/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathways in the IL-17A-stimulated HaCaT cells.
(A) The protein expression of TRAF6, p38, phospho-p38, JNK, phospho-JNK,
Erk1/2, phospho-Erk1/2, STAT3, phospho-STAT3, p65, and phospho-p65 in
HaCaT cells stimulated with IL17A at indicated time points were measured
by western blotting. \selectlanguage{greek}β-\selectlanguage{english}actin was used as a loading control. (B, C) HaCaT
cells were pretreated with 12.8ug/ml allicin for 7.5h and subsequently
stimulated with IL-17A (25 ng/ml) for an additional 0.5h. Western blot
showing the protein expression of TRAF6, P38, phospho-P38, JNK,
phospho-JNK, ERK1/2, phospho-ERK1/2, STAT3, phospho-STAT3, P65, and
phospho-P65 in HaCaT cells (n=3). (D, E) Western blot showing the
protein expression of TRAF6, P38, phospho-P38, JNK, phospho-JNK, ERK1/2,
phospho-ERK1/2, STAT3, phospho-STAT3, P65, and phospho-P65 in
IMQ-induced psoriasis-like lesions treated with or without allicin
(n=5). (F) Putative pathway for allicin-mediated regulation of
inflammation and hyperproliferation in IMQ-induced psoriatic lesions.
Data are expressed as means \selectlanguage{ngerman}± SD. *p \textless{} 0.05, **p \textless{}
0.01, ***p \textless{} 0.001, ****p \textless{} 0.0001.

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