Topical anti-inflammatory and anti-oxidative effects of porcine placenta extracts on 2,4-dinitrochlorobenzene-induced contact dermatitis
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The placenta is a reservoir enriched with growth factors, hormones, cytokines and minerals. While several beneficial effects of placenta extracts on wound healing, anti-aging and anti-inflammatory responses have been reported, relatively limited mechanistic exploration has been conducted to date. Here, we provide compelling evidence of anti-inflammatory and anti-oxidative activities of porcine placenta extracts (PPE) against contact dermatitis in vivo.
A contact dermatitis mouse model was established by sensitizing the dorsal skin of BALB/c mice using the contact allergen, 2,4-dinitrochlorobenzene (DNCB), and molecular consequences of topical application of PPE were investigated. PPEs were pre-sterilized via γ-irradiation, which is a milder but more effective way of sterilizing biomolecules relative to the conventional autoclaving method.
DNCB-induced skin lesions displayed clear contact dermatitis-like symptoms and topical application of PPE dramatically alleviated both local and systemic inflammatory responses. Inflammatory epidermal thickening was completely abrogated and allergen-specific serum IgE levels significantly reduced in the presence of PPE. Moreover, anti-oxidative activities of PPE were observed both in vitro and in vivo, which may lead to attenuation of inflammatory responses. Prolonged treatment with PPE strongly inhibited production of DNCB-induced reactive oxygen species (ROS) and subsequently prevented oxidative degradation of hyaluronic acid (HA), which triggers innate inflammatory responses.
Our findings supply valuable insights into the mechanisms underlying the anti-inflammatory effects of PPE and provide a functional basis for the clinical application of PPE in inflammatory diseases.
KeywordsPlacenta extract Contact dermatitis DNCB ROS Hyaluronic acid Anti-inflammatory effects
Allergic contact dermatitis
Antigen presenting cell
Enzyme-linked immunosorbent assay
Fetal bovine serum
Hematoxylin and eosin
Institutional animal care and use committee
Irritant contact dermatitis
Histopathological and immunohistochemical
Neutral buffered formalin
Phosphate buffered saline
Porcine placenta extract
Pattern recognition receptor
Reactive oxygen species
The placenta is a temporary organ in female mammals during gestation that connects the fetus to the mother’s uterine wall, providing oxygen, nutrients and fetal immunity to the developing fetus. The placenta serves a plethora of growth factors, hormones, minerals, vitamins and cytokines, and also provides uracil, tyrosine, phenylalanine and tryptophan that control oxidative stress [1, 2, 3]. Several studies to date have attempted to utilize placenta extracts for medical treatment purposes or as a cosmetic ingredient. The clinical efficacy of placenta extracts has been widely investigated and their activity in wound healing and anti-aging established [1, 4, 5]. Additionally, anti-inflammatory and anti-oxidative effects of human placenta extracts have been reported [2, 6, 7]. While the beneficial effects of human placenta extracts in relieving inflammatory skin disease have been widely investigated [8, 9, 10], little is known about the underlying molecular mechanisms.
One of the common inflammatory skin diseases is ‘contact dermatitis’ triggered by contact with foreign substances, such as natural or synthetic chemicals. These types of foreign molecules elicit allergies or skin irritations resulting in inflammatory symptoms, such as itchiness, red rash, blisters and burning skin. Contact dermatitis is clinically subdivided into two distinct types: irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD) [11, 12]. ICD is triggered by direct contact with toxic chemicals, such as solvents, latexes, and cosmetics. ACD is the more prevalent form triggered by low molecular weight allergens (< 500 Da), such as nickel, fragrances, dyes and preservatives.
In contrast to ICD, which induces instant local inflammation, ACD predominantly induces adapted immunity mediated by T or B cells or a subset of natural killer (NK) cells. ACD is triggered via two steps comprising a sensitization phase and an elicitation phase [11, 13]. In the sensitization phase, the contact allergen is recognized as a foreign substance by antigen presenting cells (APC), for instance, Langerhans cells or dermal dendritic cells. Stimulated APCs migrate to local lymph nodes where they present the contact allergen to naïve T cells, leading to the generation of allergen-specific effector T cells. In the elicitation phase, re-exposure to the same allergen triggers recruitment of neutrophils, monocytes and effector T cells to inflammation regions, inducing secondary immune responses.
Recent studies on human cells or the mouse model for ACD have demonstrated that contact allergens also induce oxidative stress via production of ROS [14, 15, 16]. ROS depolymerize a component of the extracellular matrix (ECM) known as HA and further promote expression of hyaluronidase, an enzyme that can break down high molecular weight HA to a lower molecular weight. Fragmented HA is recognized as an endogenous danger signal to pattern recognition receptors (PRR), such as Toll-like receptors (TLR). Low molecular weight HA activates TLR2 and TLR4, consequently eliciting pro-inflammatory innate immune responses .
A previous report by our group demonstrated anti-inflammatory activity of PPE in vitro . Treatment of the lipopolysaccharide-stimulated murine macrophage cell line, RAW 264.7, with γ-irradiated PPE led to a dramatic reduction in nitric oxide and pro-inflammatory cytokine levels. In the present study, we further investigated the anti-inflammatory and anti-oxidative activities of PPE in vivo and the underlying molecular mechanisms using the contact dermatitis mouse model.
Female BALB/c mice were purchased from Orient Bio (Seongnam-si, Korea) and maintained in individual cages under specific pathogen-free conditions at 22 ± 1 °C with 12 h of light/dark cycle. Six-week-old mice with an average body weight of 18 g were employed for the study and euthanized by CO2 inhalation after the termination of each experiment. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University.
Preparation of PPE
Placenta extracts were isolated from pig placenta, kindly provided by KR Biotech (Seoul, Korea). Pig placenta were obtained immediately after vaginal deliveries, and the umbilical cord and amnion were discarded. Remaining tissue was exhaustively washed with ice-cold phosphate buffered saline (PBS) to remove all traces of blood. Porcine placenta tissues were disrupted using a tissue homogenizer (Tissue Tearor, Biospec Products Inc., Bartlesville, OK, USA) in cold PBS. Tissue homogenates were centrifuged at 6000 g for 15 min, and the supernatants were lyophilized. Sterilization of placenta extracts was performed via γ-irradiation in air using an isotope source of Cobalt-60 at Greenpia Technology Co. (Seoul, Korea). The absorbed γ-irradiation dose was 25 kGy at a rate of 1 kGy/h.
Induction of contact dermatitis with a DNCB sensitizer and topical PPE treatment
One day prior to sensitization, mice dorsal hairs were completely shaved with an electric trimmer and depilatory cream applied (total 30 mice). For sensitization, 0.7 cm2 gauze-attached patches (Tegaderm™, 3M™ Health Care, St. Paul, MN, USA) were treated with 150 μl of 1% DNCB and applied to the back skin of mice for 20 h on days 0, 3, and 6. For elicitation of responses, patches were treated with 100 μl of 0.5% DNCB and applied to back skin of mice for 20 h on days 9, 12, and 15. DNCB (99% purity, Sigma-Aldrich, St. Louis, MO, USA) was resolved in Acetone–olive oil (AOO) (Sigma Aldrich, St. Louis, MO, USA). Mice were randomly divided into 5 groups representing different experimental conditions (n = 6 per group). To examine the potential beneficial effects of PPE on contact dermatitis, back skin of mice were topically treated with PPE or dexamethasone (DEX, a positive control) on a daily basis after removal of DNCB patches. PPE (10 mg) was thoroughly mixed with 1 g of base cream and 3 mg DEX (0.3 g daily) with 1 kg of base cream using a homogenizer. PPE or DEX cream mixtures were applied to back skin of mice with until the end of the experiment (day 17). Mice were treated with cream alone as a negative control group to determine the effect of vehicle itself on inflammatory responses.
Histopathological and immunohistochemical (IHC) analyses
Back skin tissues of mice (1 cm2) were collected, fixed with neutral buffered formalin (NBF) for 24 h, and embedded in paraffin. Paraffin blocks were cut into 3 μM thick sections and stained with hematoxylin and eosin (H&E) using a standard protocol (Abion CRO, Seoul, Korea). Epidermal thickness was measured at the thickest part of five different dorsal epidermis specimens per mouse group, and significant differences between each pair of conditions were analyzed.
Measurement of DNCB-induced serum IgE and IgG
DNCB-induced serum IgE and IgG levels were assessed using an enzyme-linked immunosorbent assay (ELISA) kit. Briefly, blood samples of mice were obtained from facial veins on the last day of the experiment (day 17) (n = 3 per group). Collected samples were incubated at room temperature for 30 min and centrifuged at 3500 rpm for 25 min, and supernatants were stored at − 80 °C until use. The 96-well plates were pre-coated with 0.17 mg/ml DNP-ovalbumin (Santa Cruz, Dallas, TX, USA), and then blocked with 2% BSA for 1 h at 37 °C, followed by washing with PBS-T. Diluted serum samples and standards were added to each well and incubated for 2 h at room temperature. After washing with PBS-T, goat anti-mouse IgG (Santa Cruz, Dallas, TX, USA) and goat anti-mouse IgE (Southern Biotech, Birmingham, AL, USA) in 1% PBS were added at ratios of 1:4000 and 1:2000, respectively. Following several wash steps, TMB Peroxidase EIA Substrate (Bio-Rad, Hercules, CA, USA) was added to each well for colorimetric development, and the reaction was subsequently terminated with 1 N H2SO4. Absorbance was detected at 450 nm using a microplate reader (Epoch Microplate Spectrophotometer, BioTek, Winooski, VT, USA).
The human keratinocyte cell line, HaCaT, was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.
Cellular ROS detection assay using H2DCFDA
The DNCB-induced total ROS content in HaCaT cells was measured using 2′,7′-dichlorofluorescein diacetate (H2DCFDA), a ROS detection reagent (Thermo Fisher Scientific, Waltham, MA, USA). H2DCFDA, a cell-permeable non-fluorescent probe, is oxidized into a highly fluorescent compound in cells. Intracellular fluorescence intensity is proportional to the amount of ROS generated in cells. Prior to the experiment, HaCaT cells were pre-incubated with serum-free DMEM containing PPE or DEX for 24 h. Incubation medium was collected and stored at − 20 °C until use. For the ROS detection assay, HaCaT cells were plated on 96-well plates (Sigma Aldrich, St. Louis, MO, USA) at a density of 2 × 104 cells per well. The next day, PPE or DEX pre-incubated medium was gently mixed with 40 μM DNCB-treated medium, added to each well, and incubated for different time periods (3, 6, 12, 24, and 48 h). After washing with PBS, cells were treated with 5 μM H2DCFDA for 30 min at 37 °C in the dark and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in mounting solution for 30 min at RT. Following several washes, DCF fluorescence and DAPI staining were visualized under a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, Japan), and the representative images were captured using a NIS-Elements image browser.
Ex vivo ROS detection assay using dihydroethidium (DHE)
Ears of mice were topically pre-treated with PPE or DEX mixed with cream for 15 min and subsequently irritated with 5% DNCB to trigger ROS production. After 15 min, mice were euthanized and 0.5 cm2 ear sections were obtained for the ROS detection assay. Ear sections were incubated ex vivo with 5 mM DHE (Sigma Aldrich, St. Louis, MO, USA) in DMSO for 30 min in the dark. DHE is an ethidium-based fluorescent dye that intercalates within DNA of cells upon oxidization, staining the nucleus a bright fluorescent red. After washing with PBS, ear tissues were placed on glass slides and ROS production were monitored by visualization under a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, Japan).
Significant differences between pairs of groups were analyzed by two-tailed Student’s t-tests or one-way ANOVA followed by Tukey’s multiple comparisons tests. P < 0.05 was considered statistically significant.
Topical PPE application attenuates DNCB-induced inflammatory responses in mice
PPE treatment alleviates epidermal hyperplasia and reduces serum IgE levels
DNCB sensitization is commonly employed as a method to elicit cell-mediated immunity [17, 18, 19, 20, 21]. Recent reports have shown that DCNB is dispensable for inducing humoral immune responses through type I hypersensitivity, which produces allergen-specific antibodies [8, 22, 23]. To further establish the suppressive effects of PPE on immune responses, serum immunoglobulin levels were measured using ELISA and compared between each mouse group. Allergen-specific serum IgE levels were significantly increased in mice treated with DNCB alone (~ 6-fold), compared to the normal group (Fig. 3b, Additional file 1: Figure S2). Administration of PPE resulted in a significant decrease in serum IgE levels by 60% relative to the DNCB-treated group, while cream (vehicle) alone did not exert an apparent effect. Serum IgG levels were additionally elevated in response to DNCB treatment but remained unaffected by PPE. Our findings support anti-inflammatory effects of PPE on allergen-specific immune responses.
PPE abrogates DNCB-induced ROS production in vitro and in vivo
To assess the anti-oxidative activity of PPE in vivo, PPE, DEX or cream alone was administered to ears of mice, and ROS production was induced via topical application of DNCB. ROS produced by DNCB treatment was stained with DHE fluorescent dye ex vivo after mice were sacrificed. Ear sections stimulated with DNCB exhibited strong fluorescence relative to the normal group, indicative of substantial ROS production (Fig. 4b). Cream alone did not exert a noticeable effect on ROS generation while DEX exerted a modest inhibitory effect. Conversely, PPE blocked ROS production significantly in vivo and completely suppressed fluorescence. Data from both in vitro and in vivo studies collectively demonstrate that PPE exerts strong anti-oxidative effects by suppressing DNCB-induced ROS production.
PPE inhibits ROS-induced HA degradation
Contact sensitizers induce skin inflammation through ROS production with concomitant breakdown of the ECM component, HA [16, 24]. To ascertain whether PPE suppresses DNCB-induced ROS production and skin inflammation, we examined its effect on ROS-induced HA degradation. We employed the Cu(II)/SO4/H2O2 system commonly used to generate ROS in vitro  as a source of ROS production. ROS-inducing compounds were mixed with high molecular weight HA with or without PPE. Combination of CuSO4 with 100 mM or 200 mM H2O2 resulted in clear HA fragmentation while CuSO4 or H2O2 alone had no effect (Additional file 1: Figure S4a). DEX did not exert an inhibitory effect on HA degradation. In contrast, both 0.1 and 1% PPE unequivocally blocked ROS-mediated HA fragmentation.
HA can be degraded not only through non-enzymatic reactions but also a family of enzymes known as hyaluronidases [16, 25, 26]. The potential involvement of PPE in hyaluronidase activity was evaluated using zymography. Briefly, back skin tissues were isolated from DNCB-sensitized mice, and hyaluronidase activity was measured in tissue lysates. DNCB treatment enhanced hyaluronidase activity by ~ 1.5-fold, compared to the normal group (Additional file 1: Figure S4b), which was not affected by either cream or DEX alone. However, PPE treatment clearly reduced hyaluronidase activity by ~ 30% relative to that in DNCB-treated mice, which was even lower than that of the normal control group. Our results support the theory that PPE prevents HA degradation by suppressing hyaluronidase activity.
While placenta extracts are reported to exert multiple beneficial effects on wound healing, anti-aging and anti-inflammatory responses, their in vivo efficacy in animal models and underlying molecular mechanisms remain to be established. Data from the current study present strong evidence that γ-irradiated PPE exerts anti-inflammatory and anti-oxidative effects in a contact dermatitis mouse model in vivo. Placental sterilization is an essential step in removing infectious agents to ensure the safety of PPE application for clinical use. The conventional method of sterilization is simple autoclaving [7, 8]. However, since autoclaving employs extremely high temperatures and pressures to sterilize microbes, the process may simultaneously inactivate beneficial bioactive peptides present in placental extracts in addition to killing pathogens. To protect the activities of potent placenta components, we used a milder sterilization method of γ-irradiation. This technique has been used to purify various medical sources and the standard protocol and intensity of irradiation for clinical purposes are well established . Previous studies by our group demonstrated anti-inflammatory effects of γ-irradiated PPE in vitro. Here, we provide evidence of anti-inflammatory and anti-oxidative activities of PPE in vivo .
DNCB is a cycloheximide benzene commonly used to trigger both innate and adaptive immunity that mimics contact dermatitis-like symptoms in mice [17, 22, 23, 28, 29]. Upon repeated application of DNCB-treated patches to mouse dorsal skin, contact dermatitis-like symptoms became evident, including red rashes, scabs and edema of immune organs. Topical application of PPE or DEX relieved inflammation of skin and prevented the increase in size and weight of immune organs, including lymph nodes and spleen. Notably, topical PPE treatment had a significantly stronger alleviatory effect on epidermal hyperplasia and hyperkeratosis in DNCB-induced inflammatory skin lesions than DEX.
Although both PPE and DEX exerted anti-inflammatory effects in DNCB-induced contact dermatitis mice, we observed a number of differences between their actions. For example, whereas PPE specifically reduced allergen-specific IgE, DEX induced reductions in both IgE and IgG levels and was slightly better at reducing inflamed spleen size. PPE also showed anti-oxidative activity against DNCB-induced ROS generation. Notably, prolonged treatment with PPE strongly inhibited ROS generation by DNCB in both HaCaT cells and a mouse model of contact dermatitis, whereas DEX did not prevent ROS production, either in vitro or in vivo. These findings suggested that the anti-inflammatory activities of PPE and DEX are regulated via slightly different mechanisms, which require further elucidation in future studies.
To further clarify the mechanisms underlying the anti-oxidative activity of PPE, we investigated its potential involvement in oxidative degradation of HA, a major component of the ECM that exists as high molecular mass polymers (> 106 Da). High molecular weight HA exerts anti-inflammatory, anti-angiogenic and immunosuppressive activities while low molecular weight HA (< 50 Da) elicits an opposite response . Fragmented HA is recognized by TLR2 and TLR4 and induces proinflammatory responses [31, 32]. In our experiments, pre-treatment with PPE clearly inhibited HA degradation in the Cu(II)/H2O2 system whereas DEX had no effect, in accordance with the results of the ROS scavenging assay. We have previously identified major active components in the aqueous fraction of PPE, including α-fetoprotein precursor, prefoldin subunit 5, heat shock factor protein 4, vimentin, lebercilin-like protein, precursors of cholecystokinins and lung and nasal epithelium carcinoma-associated protein 1, glycogen phosphorylase, odorant binding proteins, and vinculin . Our previous study also suggested that α-fetoprotein may act as a major anti-oxidant factor. To obtain reproducible results, we used consistent levels of active PPE components for each experiment. We measured the total protein amount of PPE using a protein quantification assay and generated each PPE batch with the same protein concentration. Although it would be ideal to measure the concentration of individual active PPE components or a single key component, such as α-fetoprotein, to evaluate the consistency of each batch, it is technically challenging to measure the levels of every component each time and would not be appropriate to select a single component as a standard molecule because PPE is a mixture of many different kinds of active components having both known and unknown functions. Based on the collective data, we propose that PPE alleviates inflammatory skin disease through two distinct routes: (1) preventing cellular and humoral immunity and (2) inhibition of innate inflammatory responses through prevention of oxidative stress. Further research is warranted to determine whether these distinct routes act synergistically or independently.
Contact dermatitis can generally be prevented by avoiding allergens and irritants but complete isolation from all environmental chemicals is not possible. Topical application of Calamine lotion or barrier cream is used to relieve itchiness or mild symptoms to a slight extent. However, in cases where symptoms do not improve and itches and pains become severe, corticosteroids (such as hydrocortisone and dexamethasone) should be prescribed by dermatologists. Unfortunately, prolonged treatment with steroids associated with various side-effects. Data from the current study suggest that PPE has distinct anti-inflammatory and stronger anti-oxidative activities compared to DEX. The synergistic anti-inflammatory and anti-oxidative effects of PPE may be effectively utilized to develop potent clinical therapeutics for contact dermatitis.
Topical treatment of γ-irradiated porcine placenta extracts on the skin lesion dramatically reduced both local and systemic inflammatory responses. Moreover, it also showed the anti-oxidative activity by inhibiting the generation of reactive oxygen species, which results in the prevention of the hyaluronic acids degradation that can trigger inflammatory responses. Our findings provide the fundamental understanding of the action of placenta extracts and further provide a new insight into using placental extracts on the treatment of inflammatory skin diseases.
We thank KR Biotech for the supply of pig placenta.
This work was supported by a grant of the Korean Health Technology R&D project, Ministry of Health & Welfare, Republic of Korea (HI15C2842, HI15C1685).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
J.H.H. performed experiments, analyzed data, and wrote a draft of manuscript. Y. H. performed experiments, analyzed data, performed statistical analyses, and revised the manuscript. H.J.L. designed, conducted and supervised experiments. M.J.K. performed statistical analyses and interpreted data. H.Y.S. conceived the study, analyzed data, and wrote and finalized the manuscript. All authors reviewed and approved the submitted version.
Ethics approval and consent to participate
All the animal experiments were approved by the Konkuk University Institutional Animal Care and Use Committee (IACUC).
Consent for publication
The authors have declared no conflicting interests.
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