Keywords
phospholipid - inflammation - skin disease - psoriasis - animal
Introduction
Psoriasis is a chronic and immune-mediated skin disease, which can be caused by
hereditary, environmental, and lifestyle factors or a combination of them [1]. The disease is characterized by the
presence of erythematous plaques covered with white or silvery scales, which may be
as localized or extensive on the skin [2].
It is established that abnormalities in keratinocyte proliferation and
differentiation contribute to the development of acanthosis, hypogranulosis,
parakeratinization, and proliferation of cutaneous blood vessels, all of which are
hallmarks of psoriatic plaques [3].
Furthermore, in psoriatic skin lesions, there is an elevation in the levels of
inflammatory cytokines, which exacerbates disease severity and promotes
neovascularization within the skin [4].
Based on the critical role of the IL-23/Th17 axis in the pathogenesis of psoriasis,
several IL-23 and IL-17 antagonists have been approved or are under investigation
[5]. However, it is noteworthy that not
all patients exhibit a favorable response to IL-17 and IL-23. Moreover, TNF-α and
IL-17 collaborate in a sustained inflammation within psoriatic lesions, hence the
simultaneous inhibition of both cytokines can represent a promising therapeutic
strategy [5]
[6].
Despite considerable research that has been conducted on the mechanisms underlying
the attenuation of psoriasis [7]
[8], no effective treatment has been
established that can completely eradicate the disease so far. Even the mildest form
of the disease, characterized by localized inflammatory lesions, has the potential
to rapidly progress into a more severe manifestation that affects over 10% of the
body surface area [9]
[10]. Typically, treatment options for
patients with psoriasis include local therapy, phototherapy, and systemic
treatments. However, these approaches have demonstrated certain limitations in
clinical practice [11]
[12]
[13], indicating a continued necessity for the exploration of new
therapeutic choices for psoriasis.
PS is a dietary supplement that has received approval from the Food and Drug
Administration (FDA) and is recommended for the prevention of memory impairment and
dysfunction in older adults [14]. A
clinical study involving patients with AD demonstrated that those receiving oral PS
exhibited significantly higher scores in semantic memory tests, vocabulary
assessments, and picture-matching tasks compared to the control group [15]. Moreover, experimental studies have
reported the anti-inflammatory and immunomodulatory effects of PS phospholipids
[16]
[17]
[18]
[19].
PS phospholipids are integral components of the cell membrane, with those located on
the outer membrane being recognized by the PS receptors of phagocytes [20]. Research also indicates that the
interaction between phagocytes and PS liposomes simulates the recognition of
apoptotic cells during the process of efferocytosis [20]. This interaction leads to
anti-inflammatory and immunosuppressive effects within the organism [16]
[17]
[19]. PS liposomes have been
shown to reduce the production of pro-inflammatory cytokines, including tumor
necrosis factor-alpha (TNF-α), interferon-gamma IL-1, interleukin-1 (IL-1), IL-6
interleukin-6 (IL-6), while also suppressing humoral and cellular immune systems
responses by antigen antigens in in vitro and in vivo studies [17]
[21]
[22]
[23]. The study’s results of Xuemei et al.
[16] determined that therapeutic stem
cells have immunomodulatory effects through the release of PS due to apoptotic stem
cells. The data indicate PS regulate the immune system by the increase of the
IL-10-production, decrease of neutrophil infiltration, inhibition of NK cell
activation, and induction of macrophage polarization towards M2 [16].
In this study, we aimed to investigate the anti-psoriatic properties of topical PS
using a murine model. To evaluate the severity of skin inflammation, we employed the
Psoriasis Area and Severity Index (PASI) score, with mice subjected to IMQ-induced
psoriasis. Moreover, the effect of topical PS on the other hallmarks of the
psoriasis model was evaluated through Baker’s scoring system, histopathological
parameters, real-time polymerase chain reaction (PCR), and immunohistochemistry
analysis.
Results
Body and spleen weights
In this study, the alterations in the spleen weight of the mice were assessed
independently of changes in the animals’ body weight. Consequently, the ratio of
spleen weight to body weight was calculated to facilitate comparisons between
the groups. According to our data ([Fig.
1a]), there is no statistical difference (P>0.05) in body
weight changes among the various groups over time. Furthermore, the spleen
weight in the IMQ group exhibited an increase, whereas a decrease was observed
in the treated CsA group ([Fig. 1b]).
The percentage of spleen weight/body weight reduced significantly
(P<0.05) in the IMQ+CsA group compared to the IMQ group ([Fig. 1c]). Furthermore, the percentage
of spleen weight to body weight did not demonstrate any significant difference
(P>0.05) in the IMQ+PS group compared to the IMQ group ([Fig. 1c]).
Fig. 1 Diagrams of a) the changes in body weight over six
consecutive days, b) the average spleen weight of the
experimental mice, and c) the percentage of spleen weight/ body
weight on the sixth day of the trial showed that significantly decreased
in the IMQ+CsA group. Data are presented with n=6 per group and
mean±SEM. ##P<0.01 is compared to the IMQ group.
Macroscopic observation and PASI score
As given in [Fig. 2a, b], the
inflammatory signs including erythema, scaling, thickness, and cumulative PASI
score, exhibited a significant reduction in the IMQ+PS and IMQ+CsA groups when
compared to the IMQ group. Moreover, the comparison between the Ctr group and
the IMQ group revealed a significant (P<0.05) in inflammatory signs
during the application of IMQ on the depilated dorsal skin of the mice. The
IMQ+PS group demonstrated a significant (P<0.05) in the PASI score on
the final day of treatment. No significant differences (P>0.05) were
observed between the IMQ+PS and IMQ+CsA groups regarding the inflammatory
indicators (see [Fig. 2b]). Additional
data detailing the results at various time points are presented in [Fig. 2b].
Fig. 2 The representative data about various experimental groups
of IMQ-induced psoriasis and their corresponding treatments include
a) The dorsal skin of the mice on the sixth day. b)
Diagrams of inflammatory signs score (numbered from 0 to 4) and
cumulative PASI score (numbered from 0 to 12) in 6 consecutive days.
Data are presented with n=6 and mean±SEM. The sign * is compared to the
Ctr group and the sign # is compared to the IMQ group:
*P<0.05, **P<0.01, ***P<0.001, and
****P<0.0001 #P<0.05.
Histopathology
As illustrated in [Fig. 3], hematoxylin
and eosin (H&E) staining, along with Baker’s score, were employed to assess
psoriatic-like reactions in murine models. The histopathological findings
indicated that IMQ induces psoriatic-like reactions characterized by
hyperkeratosis, parakeratosis, acanthosis, the presence of Munro
micro-abscesses, and a significant lymphocytic infiltrate (see [Fig. 3a]). Therefore, the IMQ group
showed a significant (P<0.01) increase in Baker’s score in comparison
with the Ctr group ([Fig. 3c]). The
data presented in [Fig. 3] indicate
that the inflammatory response was significantly reduced in the IMQ+PS and
IMQ+CsA groups, with p-values of<0.05 and<0.01, respectively, compared to
the IMQ group. IMQ+CsA improved Baker’s score rather than the IMQ+PS group but
we did not observe any significant (P>0.05) difference in the mean of
Baker’s score between them, as shown in [Fig. 3c].
Fig. 3
a) The representative H&E staining of the dorsal skin of mice
indicated that IMQ group has Munro’s microabscesses in the keratin
layer, lengthening and clubbing of rete ridges, and moderate-severe
dermal lymphocytic infiltration on the sixth day, even though PS similar
to CsA drug reversed the IMQ-induces pathologic changes. b) The
parameters of Baker’s scoring system [37] for pathological changes evaluation. c) Diagram of
Baker’s pathology scores reflected in the method section, was used for
histopathology evaluation. Data are presented with n=6 per group and
mean±SEM. The sign * is compared to the Ctr group, ** P<0.01,
and the sign # is compared to IMQ group, #P<0.05,
##P<0.01.
Moreover, while there were no significant (P>0.05) changes in dermal
volume between the IMQ+CsA and IMQ+PS groups, our findings indicated that 5% CsA
reduced the number of infiltrating lymphocytes in the dermal layer more
effectively than 10% PS. Additionally, pathological changes, including Munro’s
microabscesses in the keratin layer and elongation of the epidermis, observed in
the PS-treated group demonstrated an improvement in psoriatic signs similar to
that seen in mice receiving CsA ([Fig.
3a]).
Epidermal thickness
Histological examination of the epidermal thickness of the back skin in mice was
conducted using Masson’s trichrome staining on the sixth day of the experiment.
As illustrated in [Fig. 4a, b], the
volume of hyperkeratosis and epidermal thickness was significantly reduced in
the IMQ+PS group (P<0.01) and the IMQ+CsA group (P<0.0001)
compared to the IMQ group In contrast, the IMQ group exhibited a significant
increase (P<0.0001) in epidermal thickness and hyperkeratosis when
compared to the Ctr group. Although CsA treatment resulted in a more reduction
of hyperkeratosis compared to PS, there was no significant difference
(P>0.05) between the two treatments in IMQ-induced psoriasis, as
shown in [Fig. 4a, b].
Fig. 4
a) The histological examination utilizing Masson’s trichrome
staining was conducted on the dorsal skin of mice from the respective
groups on the sixth day. The application of topical PS resulted in a
reduction of epidermal thickness, which was evaluated for each
experiment based on four smooth regions delineated by red rectangles.
b) Diagram of epidermal thickness measurement (by ImageJ
software) on the sixth day. Data are presented with n=6 and mean±SEM.
The sign * is compared to Ctr group, ****P<0.0001, and the
sign # is compared to IMQ group, ##P<0.01,
####P<0.0001.
Immunohistochemistry
As shown in [Fig. 5], TNF-α marker in
IHC analysis of the IMQ+PS and IMQ+CsA groups were in lower levels (mild
expression) with a moderate cytokine expression compared to the IMQ group.
IMQ-induced psoriasis resulted in an increase of the TNF-α inflammatory cytokine
in the back skin of mice compared to the control group on the sixth day ([Fig. 5]).
Fig. 5 IHC analysis was conducted on the dorsal skin of the mice
on the sixth day. The scores of the TNF-α marker were given
qualitatively with n=3 per each group as follows: negative (no
expression), weak (mild expression), moderate (moderately expression),
and severe (highly expression).
Gene expression
In this study, the gene expression of key inflammatory mediators including IL-17,
IL-23, and TNF-α cytokines was analyzed, which are involved in the pathogenesis
of psoriasis-like inflammation. The levels of IL-17, IL-23, and TNF-α cytokines
were reduced significantly (P<0.01) in the IMQ+CsA treated group
compared to the IMQ group. Moreover, the IMQ+PS treatment significantly
decreased the levels of IL-17 (P<0.01), IL-23 (P<0.05), and
TNF-α (P<0.05) cytokines compared to the IMQ group, as illustrated in
[Fig. 6a–c]. Additionally, topical
CsA treatment reduced the levels of inflammatory mediators to a greater extent
than the PS drug in psoriatic mice, with no significant difference
(P>0.05) observed between the two groups ([Fig. 6a–c]).
Fig. 6
a, b & c) A diagram illustrating the levels of
IL-17, IL-23, and TNF-α in the dorsal skin of mice on the sixth day, as
measured by real-time PCR. Data are presented with n=6, mean±SEM. The
sign # is compared to IMQ group: #P<0.05,
##P<0.01.
Discussion
The present study aimed to investigate the potential therapeutic effects of topical
PS in the improvement of IMQ-induced psoriasis in mice. Our data indicated that PS
improved tissue parameters, including erythematous plaques, skin thickness, and
hyperkeratosis, which were observed in IMQ- induced psoriasis in mice. The
histopathological analysis also revealed a reduction in skin inflammation, as
indicated by Baker’s score, in the PS treatment group. Furthermore, PS significantly
suppressed the gene expression of inflammatory cytokines IL-17, IL-23, and TNF-α in
psoriatic lesions. The anti-psoriatic effects of PS application align with
Al-Harbis’ study, which demonstrated that lymphocyte-specific protein tyrosine
kinase has an inhibitory effect on IMQ-induced psoriatic inflammation in a mouse
model [24]. The investigation revealed that
psoriatic inflammation is directly influenced by cytokine expression such as NFkB
and STAT3, TNF-α, IL-17A, etc. and clinical features like skin thickness, acanthosis
and leukocytic infiltration [24].
PS phospholipid plays a crucial role in cell signaling and exhibits immunomodulatory
and anti-inflammatory effects [17]
[21]
[22]
[23]. It is an FDA-approved
dietary supplement that is currently on the market and has not been associated with
any significant side effects to date [25].
Our recent study demonstrated that topical PS enhances wound healing and reduces
necrosis by inhibiting inflammatory cell infiltration and activating growth factors
in animal models [26].
IMQ-induced psoriasis demonstrated significant alterations in the expression of
inflammatory cytokine genes, characterized by erythematous plaques, overlying
scales, skin thickening, and hyperkeratosis at the psoriatic site. In contrast,
topical PS, similar to the drug CsA, exhibited comprehensive properties in
alleviating skin complaints and exerting anti-inflammatory effects associated with
cytokine gene expression in psoriatic mice. The CsA drug has been utilized in
clinical settings for severe skin disorders with immuno-inflammatory origins, such
as psoriasis and atopic dermatitis [27]. On
the other hand, side effects and poor absorption of CsA present challenges for its
short- and long-term topical use. [28].
In our study, a 5-day topical treatment with PS significantly improved the PASI score
and reduced skin thickness and hyperkeratosis in psoriatic mice. The PASI score is
utilized to assess the severity of psoriasis by calculating the values of thickness,
redness, and scaling in experimental models. [29]. Moreover, histopathological examinations indicated that skin
thickness and hyperkeratosis were significantly reduced following topical therapy
with PS and CsA in psoriatic mice. Furthermore, the expression of inflammatory
cytokines, including IL-17, IL-23, and TNF-α, was suppressed after a 5-day treatment
with topical PS therapy on the dorsal skin of psoriatic mice. Therefore, these
findings suggest that the anti-psoriatic properties of PS may be attributed to the
suppression of IL-17, IL-23, and TNF-α expression, which are associated with skin
inflammation [30]. It is known that the
IL-23/IL-17 axis has a key role in inflammation promotion of psoriasis. Furthermore,
the strategy of blocking TNF-α, in conjunction with monoclonal antibodies targeting
IL-17 and IL-23 cytokines, has proven effective in the treatment of patients with
psoriasis [31]
[32]
[33]. These pro-inflammatory cytokines contribute to the proliferation of
epithelial cells and hyperkeratosis, as well as the recruitment of additional
inflammatory immune cells in psoriatic lesions [33]. Skin-resident dendritic cells (DCs) and macrophages are one of the
main primary sources of IL-23 [34], and
activated plasmacytoid dendritic cells (pDCs) in the dermis secrete type I
interferon and TNF-α. These DCs will produce IL-12 and IL-23, and then lead immature
T cells into T helper (Th)1, Th17, and Th22 cells [33].
The previous reports have shown that parenteral and oral administrations of PS reduce
Ag-specific CD4+T cells [22] and
delayed-type hypersensitivity reaction (DTH) [17]
[18] that are results of
inhibiting productively activated T lymphocytes [22]. Furthermore, PS liposomes reduced the levels of inflammatory
cytokines, including TNF-α, IFN-γ, IL-1β, IL-2, IL-6, and IL-8, in various
experimental studies [16]
[17]
[18]
[19]
[23] as well as the other biological
activities [35]. PS phospholipids on
apoptotic cells or within liposomal structures interact with PS receptors expressed
on phagocytes. This interaction triggers the production of anti-inflammatory
cytokines while reducing pro-inflammatory cytokines. [20]
[21]. Furthermore, our results demonstrated a significant reduction in
spleen mass percentage following a 5-day topical treatment with CsA in psoriatic
mice. The reports indicated that an increase in spleen weight occurs as a result of
the topical application of IMQ in the experiments. This application leads to the
activation of T cells and systemic inflammation, which play a significant role in
the pathogenic process of human psoriasis [36]
[37]. Therefore, changes in
spleen weight may serve as a marker in IMQ-induced psoriasis models.
Our data proposed that the topical application of PS interacts with individual
leukocytes in skin tissues, such as lymphocytes and dendritic cells, thereby
inhibiting inflammatory responses in psoriatic skin. Nevertheless, further studies
are necessary to elucidate the role of inflammatory cytokines, such as IL-17A, by
administering PS in IL-17a knockout mice or through pretreatment with an anti-IL-17A
receptor to confirm its anti-inflammatory pathways. Given the multiple interactions
between PS and the immune system, other signaling pathways, including NFATc1, NF-κB,
and STAT3, should be considered for investigation in psoriatic conditions in future
studies.
Conclusion
Our data indicate that PS hydrogel, similar to the CsA drug, has beneficial effects
on psoriatic inflammation in a mouse model. PS therapy reduced plaque-type psoriasis
indices, including skin thickness, hyperkeratosis, and scores associated with
dermatitis and inflammation. The expression of the cytokines IL-17, IL-23, and TNF-α
was suppressed during the 5-day treatment with PS in psoriatic mice. Furthermore,
further investigation is recommended to explore other signaling pathways of PS in
the future, which may be involved in the control of psoriasis.
Method
Drug and preparation
Carboxymethyl cellulose (CMC) and cyclosporine A (CsA) powder were purchased from
Sigma (USA). Phosphatidylserine (PS) and Imiquimod 5% cream were obtained from
Pharmin USA, LLC (USA) and Pharmaceuticals Meda (UK), respectively.
Phosphatidylserine 10% (w/v) [19] as
well as cyclosporine 5% (w/v) were prepared with ethanol 10% (v/v). The CMC (1%)
was added to the suspensions and stirred at warm conditions. To prepare the
vehicle, 1 ml of pure ethanol was mixed with 9 ml of distilled water and CMC 1%
without the drug.
Animal
Male BALB/c mice with an average weight of 30 gr were obtained from the School of
Medicine, Tehran University of Medical Sciences (Tehran, Iran). Their health was
checked and they were randomly placed in separate cages with suitable numbers to
adapt before the experiment. They were provided with unrestricted access to
water and food and were subjected to a 12-hour light and 12-hour dark cycle,
with a temperature maintained at 21±2°C. The cages were cleaned and maintained
regularly to ensure hygiene and a suitable living environment for the mice. The
experimental methods were deeply in line with the experiment and ethics followed
the guidelines set out by the Canadian Council for Animal Care and approved by
the Institutional Animal Care and use of committee IR.TUMS.MEDICINE.REC.
1403.247.
Experimental design
In this study, the experimental groups were divided into 4 groups with 6 mice
which were selected randomly as follows: Vehicle (Ctr): Vehicle administration
with no intervention of imiquimod. Imiquimod (IMQ): Intervention of imiquimod
and no administration (negative control); Imiquimod/cyclosporine (IMQ+CsA):
Intervention of imiquimod and CsA administration (positive control);
Imiquimod/phosphatidylserine (IMQ+PS): Intervention of imiquimod and PS
administration.
On day 1, mice were anesthetized by intraperitoneal injection of ketamine
(80 mg/kg) and xylazine (5 mg/kg), and then the hair of the fixed space between
their shoulder blades was completely shaved. A quarter of each sachet of IMQ
cream (62.5 mg) was applied with a spatula to the mentioned area (except Ctr
group) and this action was repeated for 5 days. Two hours later, the three
specified compounds were administered topically in a volume of 200 µl on the
backs of the mice, with the treatment repeated for five consecutive days.
Animals were weighed regularly once a day for 6 days. Photos were taken from the
back of the animals for PASI score evaluation, and this action was repeated for
6 days (before the intervention of IMQ).
On day 6, the mice were euthanized using carbon dioxide (CO2). Half of the dorsal
skin samples were preserved in 10% formalin, while the other half was stored at
−80°C for molecular analysis. Additionally, the spleen tissues were rinsed in
saline and subsequently weighed. Changes in spleen weight are regarded as a
hallmark of T cell activation and systemic inflammation in the IMQ-induced
psoriasis model [37].
Macroscopic observation and PASI score
For psoriasis area and severity index (PASI) in 6 days, evaluated inflammatory
signs that including erythema, scaling, and thickness, with these scores :0)
none; 1) slight; 2) moderate; 3) marked; 4) very marked, and this cumulative
scores (numbered from 0 to 12). The evaluation was done independently by two
researchers (n=6 for each parameter) and the mean of values was then calculated
[29].
Histopathology
For histological parameters characteristic of psoriasis, an expert pathologist
evaluated hematoxylin-eosin-stained skin sections (3–4 μm) using the Baker
scoring system. The scores included keratinosis, development of abscesses,
acanthosis, and dermal lymphocyte infiltrations which were graded by the
histopathological scores on a scale of 0 –11 [38]. To evaluate the precise histological characteristics of the
tissues, the prepared samples were stained with Masson’s trichrome dye, and the
thickness of the epidermis was measured using the length measurement tool in
ImageJ software. Briefly, the skin samples were deparaffinized and stained with
a hematoxylin solution, followed by a mixture of Orange G, Ponceau S, and a
light green staining agent.
Immunohistochemistry
This method was employed to assess TNF-α cytokine expression in the dorsal skin
of mice. Consistent with our previous study [39], we utilized specific primary and secondary antibodies, and the
analysis was conducted by an unbiased pathologist. The presence of brown-stained
cells in the sections indicates relative cytokine expression in the tissue.
Gene expression
For each mouse, a proper skin sample was also harvested sterilely and immediately
flash-frozen in liquid nitrogen and then stored at −80°C. The mRNA expressions
of IL-17, IL-23, and TNF-α factors were measured by real-time quantitative PCR
analysis. Total RNA was isolated from all tissues using an RNA X-Plus kit (Cinna
Gen, Tehran, Iran) and was quantified by NanoDrop spectrophotometer (Thermo
Scientific Nanodrop 2000). In the next step, cDNA was synthesized (Bio fact,
South Korea), and Real-time PCR was performed with the Cyber Green method (Yekta
Tajhiz Co, Cat No: YT2551) with the following program; 40 cycles of denaturation
(15 seconds at 95°C), annealing (20 seconds at 60°C) and elongation process
(30 seconds at 72°C). β-actin was used as the housekeeping gene and positive
control. The relative expression of transcript level of each case was calculated
according to 2-ΔΔct
[40].
Statistical analysis
We used Prism version 8 software for statistical analysis. The data were analyzed
by one-way analysis of variance (ANOVA) and presented as mean±standard error of
the mean (SEM). P<0.05 was considered as significant.
Data availability
Research data are not shared.
Disclosure statement
All authors declare that they have no conflict of interest.
Authors’ contributions
Bahareh Farasati Far: Writing and editing.
Partow Mirzaee Saffari: Animal study, Data analysis.
Razieh Mohammad Jafari: Methodology.
Ramin Goudarzi: Reviewing and editing.
Ahmad Reza Dehpour: Methodology.
Alireza Partoazar: Supervision, Conceptualization, Reviewing and editing.
All authors reviewed the manuscript.
