Cbd oil for acne inflammation pubmed

The ameliorative effect of hemp seed hexane extracts on the Propionibacterium acnes-induced inflammation and lipogenesis in sebocytes

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Abstract

In this study, we investigated the anti-microbial, anti-inflammatory, and anti-lipogenic effects of hemp (Cannabis sativa L.) seed hexane extracts, focusing on the Propionibacterium acnes-triggered inflammation and lipogenesis. Hemp seed hexane extracts (HSHE) showed anti-microbial activity against P. acnes. The expression of iNOS, COX-2, and the subsequent production of nitric oxide and prostaglandin increased after infection of P. acnes in HaCaT cells, however, upon treating with HSHE, their expressions were reduced. P. acnes-induced expressions of IL-1β and IL-8 were also reduced. HSHE exerted anti-inflammatory effects by regulating NF-κB and MAPKs signaling and blunting the translocation of p-NF-κB to the nucleus in P. acnes-stimulated HaCaT cells. Moreover, P. acnes-induced phosphorylation of ERK and JNK, and their downstream targets c-Fos and c-Jun, was also inhibited by HSHE. In addition, the transactivation of AP-1 induced by P. acnes infection was also downregulated by HSHE. Notably, HSHE regulated inflammation and lipid biosynthesis via regulating AMPK and AKT/FoxO1 signaling in IGF-1-induced inflammation and lipogenesis of sebocytes. In addition, HSHE inhibited 5-lipoxygenase level and P. acnes-induced MMP-9 activity, and promoted collagen biosynthesis in vitro. Thus, HSHE could be utilized to treat acne vulgaris, through its anti-microbial, anti-inflammatory, anti-lipogenic, and collagen-promoting properties.

Introduction

Hemp (Cannabis sativa L.), also known as cannabis, has been an important plant used in dietary supplements, clothing, cosmetics, and medicines [1,2]. Hemp has also been used as a medicament for the treatment of medical conditions such as rheumatic pain, intestinal constipation, disorders of the female reproductive system, and malaria [3]. However, due to its hallucinogenic effects, the use of cannabis has been legally restricted in almost all countries of the world, except for some countries, such as the US that partially allow it for medicinal use. In US, with a doctor’s recommendation, the medical use of cannabis is legal in 29 states, the District of Columbia, and the territories of Guam and Puerto Rico. Seventeen other states allow the medical use of cannabis with low THC/high CBD. Tetrahydrocannabinol (THC) is the major psychoactive component of cannabis, even small amounts of which could be highly hallucinogenic, while cannabidiol (CBD) is the non-psychoactive component of cannabis. THC binds cannabinoid CB1 and CB2 receptors with high affinity and activates them, while CBD has a very low affinity for both CB1 and CB2 receptors, and acts as an indirect antagonist of these receptors [1,4]. THC is effective for relieving pain, inhibiting inflammation, and relaxing muscle tension. Thus, it can be used for patients with multiple sclerosis, neurodegenerative disorders, and severe pain. However, overdose of THC may cause serious mental side effects of hallucinations [5,6]. On the contrary, hemp seed does not have any psychotropic chemical components. Moreover, hemp is the only seed plant without any saturated fatty acids, containing mostly polyunsaturated fatty acids including linoleic acid and gamma-linolenic acid [7].

Acne vulgaris is observed in 85% of adolescents and refers to a disease that causes inflammation of sebaceous glands attached to hairfollicles. Propionibacterium acnes (P. acnes), present on normal skin, activates an immune response and changes the lipid composition of the sebaceous glands [8,9]. P. acnes stimulates the inflammatory cytokines such as interleukin (IL)-1β, IL-8, and leukotrienes by activating Toll-like receptors (TLRs) and induces the expression of enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), causing chronic inflammation [10–12]. It also activates the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signals, and upregulates related genes to carry out innate immunity.

Keratinocytes and dermal fibroblasts secrete matrix metalloproteinases (MMPs) which mediate the digestion of many components of extracellular matrix (ECM) in response to external stimuli such as bacterial infection, oxidative stress, and UV radiation. MMPs are classified into five main subgroups, such as collagenases, gelatinases, stromelysins, matrilysins, and 5 membrane-type MMPs, based on their structure and substrate specificity. MMP-1 is a collagenase that digests fibrillar collagen via recognition of a hemopexin-like domain, while MMP-2 and MMP-9 are gelatinases that degrade a number of ECM components, such as type I and IV collagen [13,14].

Human sebocytes are specialized sebum-producing epithelial cells that produce and release lipid droplets. Sebum is an integral component of the epidermal barrier and its formation is involved in the skin immune system. Excess sebum production causes acne by inducing an inflammatory reaction under the proliferating skin microflora [15]. Peroxisome proliferator-activated receptor gamma (PPARγ) present in sebaceous gland cells regulates the lipid production and lipid metabolism via modulation of the AMP-activated protein kinase (AMPK) signal. AMPK is a serine/threonine kinase that plays a regulatory role in glucose and lipid metabolism [16]. Activation of AMPK by phosphorylation down-regulates the expression of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP-1), by inhibiting phosphorylation of mTOR [17,18]. Upon activation by insulin-like growth factor-1 (IGF-1), protein kinase B (AKT) phosphorylates fork head box protein O1 (FoxO1) to enhance lipogenesis. FoxO1 is reported to directly bind and modulate PPAR-γ function [19]. Additionally, 5-lipoxygenase induces the release of leukotriene (LT)-B4, a pro-inflammatory lipid, which promotes lipid synthesis in acne lesions. Thus, inhibition of 5-lipoxygenase may be an ideal target for downregulation of inflammation and lipid accumulation in sebocytes [20,21].

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Hemp seeds have been primarily used in nutraceutical and pharmaceutical industries, utilizing their health-promoting properties and ideal nutrient content. However, information on its anti-acne activity and underlying mechanism is limited. In this study, the ameliorative effects of hemp seed hexane extracts on P. acnes-induced inflammation in HaCaT cells and IGF-1-stimulated lipogenesis in sebocytes were examined at the molecular and cellular level.

Materials and methods

Preparation of hemp seed hexane extracts

Hemp seeds imported from Canada were purchased from an online Korean store called Nooriwon. The dried seeds were ground and soaked with three volumes of hexane for 24 h while stirring. The extract was serially filtered using a No. 2 filter and a nylon filter (0.45 μm) (both from Whatman International Ltd., UK). The extraction procedure was repeated twice with the residue. The solvent was evaporated using a rotary vacuum evaporator (Eyela, Japan) at 35°C. Residual solvent was re-evaporated twice at 70°C for 10 min. To increase the solubility of HSHE in the cell culture media, same volume of dimethyl sulfoxide (DMSO) was added to the hexane-extracted hemp seed oil and mixed by vortexing at room temperature. All experiments were performed under conditions that did not show toxicity by hemp seed hexane extract and DMSO [22].

Microbial cultivation and anti-acne activity determination

Propionibacterium acnes (P. acnes, KCTC) was obtained from the Korean Culture Center of Microorganisms, Seoul, Korea. P. acnes was grown anaerobically in solid and liquid Reinforced Clostridial Medium (RCM) at 37°C for 72 h. P. acnes were harvested via centrifugation of the cultures at 4,500 rpm for 15 min at 4°C. The resulting bacterial pellets were pooled, washed in cold 1× PBS, and centrifuged again. Finally, the pooled bacterial pellets were resuspended in serum free medium (bacterial concentration was 1.2X10 9 CFU/mL). The suspension was heated at 80°C and the heat-killed bacteria were used for the stimulation experiments reported previously by us [9].

To determine anti-acne activity of the HSHE, the culture of P.acnes was standardized using microbiological No. 0.5 McFarland Standard’s solution according to the recommendations of the Clinical Laboratory Standards Institute (CLSI) [23]. The anti-microbial effect of HSHE was measured on agar medium. 200 μL cell suspension (10 5 cells/mL) was mixed with 100 μL of HSHE diluted in RCM medium at 37°C for 30 min. The control was mixed with 100 μL medium and reacted under the same conditions. Then, 100 μL of each mixture was spread on the surface of RCM medium on a petri dish. Both control and experimental groups were incubated at 37°C for 72 h.

Cell culture

The human keratinocyte (HaCaT) and human fibroblast cell lines (Hs68) were obtained from the American Type Culture Collection (ATCC). The cells were incubated in complete Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) containing 100 U/mL penicillin, streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS) at 37°C [24,25]. Primary human sebocytes (Celprogen Inc., USA) were maintained in Human Sebocyte Complete Growth Media purchased from the same vendor. For HSHE treatment, cells were seeded and incubated overnight prior to the treatments. For the stimulation experiment, HaCaT and Hs68 cells were incubated with heat-killed P. acnes adjusted at the appropriate concentration in serum free media for 24 h at 37°C in 5% CO2. After stimulation, the HaCaT cells were treated with or without hemp seed hexane extracts for 24 h at 37°C in 5% CO2. Sebocytes were pre-treated with or without hemp seed hexane extracts for 2 h. Then, 120 ng/mL IGF-1 was added to each plate and the cells were incubated for 22 h [9].

NO production measurement

The nitrite concentration in conditioned medium was measured as an indicator of NO production according to the Griess reaction. Each supernatant (100 μL) was mixed with the same volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride in distilled water). The absorbance of the mixture was determined with an ELISA reader (Sunrise, Tecan, Switzerland) at 570 nm [9, 26].

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Enzyme-Linked Immunosorbent Assay (ELISA)

The effects of HSHE on IL-8 and prostaglandin E2 (PGE2) productions in heat-killed P. acnes-infected HaCaT cells and on the 5-lipoxygenase production in sebocytes, stimulated with IGF-1, were measured with ELISA [27]. The IL-8 (BD Science, USA), PGE2 (LSBio, USA) and 5-lipoxygenase (MyBioSource, USA) concentrations were calculated according to the standard curve using standard in the ELISA kit.

Western blot analysis

Proteins were separated by 10% SDS-PAGE, and transferred onto polyvinylidene fluoride membrane (Bio-Rad Laboratories, CA) [28]. After blocking for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against iNOS, IL-1β, COX-2, IKKα, IKKβ, p-IKKα/β, IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, AKT, p-AKT, mTOR, p-mTOR, PPARγ, FAS, AMPKα, p-AMPKα, FoxO1, and p-FoxO1 (Cell Signaling Technology, USA), SREBP1 (Novus Biologicals, Canada) and β-actin (Santa Cruz Biotechnology, USA) which were diluted using manufacturers’ recommendations [25,29]. The membranes were then washed in 1× TBST and incubated with the appropriate secondary antibody HRP-conjugated (1:5000) at room temperature for 1 h. Protein bands were visualized using the Sensi-Q 2000 (Lugen, South Korea). The intensity of the bands was analyzed using ImageJ and normalized against β-actin [30].

Transient transfection and luciferase assay

The effects of HSHE on AP-1 activity were assayed in a Luciferase Reporter Assay System (Promega, Madison, WI, US) [8]. Upon reaching 60–70% confluency, the HaCaT cells were washed in PBS and the cells were then transfected with AP-1-Luc reporter vector (Affymetrix, Santa Clara, CA, US) using Fugene 6 (Promega, Madison, WI,US), according to the manufacturer’s protocol. After the 24 h transfection, cells were infected with P. acnes, medium was changed, and treated with various concentrations of hemp seed hexane extracts. The processed cells were cultured for an additional 24 h. Cells were washed with PBS, lysed with lysis reagent, and treated with the luciferase assay substrate. Luciferase activity was determined using the luminometer (Infinite F200 PRO, Tecan, Männedorf, Switzerland).

Confocal microscope analysis and gelatin zymography

Confocal microscopic analysis for NF-κB translocation and gelatin zymographic analysis of MMP activity on Hs68 cells was performed as described in our previous report [9].

Collagen synthesis-promoting assay

Collagen contents in the ECM of Hs68 cells were determined by Sircol collagen assay (Biocolor, UK) according to the manufacturer’s protocols. Collagen dye complexes formed in cell supernatants were added to the Sircol staining reagent and precipitated in soluble non-binding dyes. After centrifugation, the pellet was washed with ice-cold acid-salt wash reagent and reacted with alkali reagent. Samples were dispensed into a 96-well plate and the absorbance was read at 570 nm. The amount of collagen was calculated based on a standard curve obtained with the standard bovine type I collagen supplied with the kit [31].

Data analysis

Data was analyzed using the IBM Statistical Package for Social Sciences (SPSS, version 20). All the data were presented as mean ± standard deviation (SD) of triplicate experiments. One-way analysis of variance (ANOVA) with a Duncan multiple-comparison test was utilized to determine the statistical differences among groups. P-values

Results

Anti-microbial effect of hemp seed hexane extracts on P. acnes

At first, we investigated the anti-microbial effect of HSHE against P. acnes ( Fig 1 ). P. acnes was treated with 0, 15, 20 and 25% HSHE and the number of colonies was counted to examine anti-microbial activity against P. acnes of HSHE. 15 and 20% HSHE showed approximately 59% and 99% of anti-microbial activity compared to control. At 20% HSHE, complete inactivation of P.acnes was observed. Erythromycin (3 ppm), a conventional anti-microbial agent for acne, showed approximately 67% anti-microbial activity (Data not shown). These results suggest that HSHE is able to inactivate the growth of P. acnes.

The Therapeutic Potential of Cannabinoids in Dermatology

Cannabinoids have demonstrated utility in the management of cancer, obesity, and neurologic disease. More recently, their immunosuppressive and anti-inflammatory properties have been identified for the treatment of several dermatologic conditions. This review thus assesses the therapeutic potential of phytocannabinoids, endoocannabinoids, and chemically synthetic cannabinoids in the management of cutaneous disease. The PubMed® and Scopus® databases were subsequently reviewed in December 2017 using MeSH and keywords, such as cannabinoid, THC, dermatitis, pruritus, and skin cancer. The search yielded reports on the therapeutic role of cannabinoids in the management of skin cancer, acne vulgaris, pruritus, atopic and allergic contact dermatitis, and systemic sclerosis. While cannabinoids have exhibited efficacy in the treatment of inflammatory and neoplastic skin conditions, several reports suggest pro-inflammatory and pro-neoplastic properties. Further investigation is necessary to understand the complexities of cannabinoids and their therapeutic potential in dermatology.

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Keywords: acne; cannabinoid; cannabis; dermatitis; endocannabinoid; fibrosis; palmitoylethanolamide; inflammatory skin disease; pruritus; skin cancer; sclerosis; THC; tetrahydrocannabinol.

Conflict of interest statement

Adam Friedman is currently developing a nanoparticle encapsulated cannabinoid with Zylo Therapeutics – this work is not referenced in the manuscript. Dustin Marks has no conflicts of interest to report for this work. Funding: The George Washington Department of Dermatology received no funding in support of this manuscript.

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