Effects of tyndallized lactic acid bacteria separated from Phellinus linteus on inflammation and skin barrier damage induced by DNCB

doi:10.21203/rs.3.rs-4702126/v1

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Effects of tyndallized lactic acid bacteria separated from Phellinus linteus on inflammation and skin barrier damage induced by DNCB

https://doi.org/10.21203/rs.3.rs-4702126/v1

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Background

The fruiting bodies of the Sanghwang mushroom (Phellinus linteus) have a long history of use in folk medicine throughout Asia, particularly in Korea, Japan, and China. However, research on the Sanghwang mushroom is still at an early stage. In this study, we investigated the therapeutic potential of tyndallized lactic acid bacteria separated from Phellinus linteus (PL-tLB) in the context of atopic dermatitis.

Methods

Keratinocytes, represented by HaCaT cells, were subjected to TNF-α/IFN-γ stimulation followed by treatment with PL-tLB. The results confirmed PL-tLB's concentration-dependent suppression of inflammatory cytokines and chemokines. Atopic dermatitis is a complex, chronic inflammatory skin condition characterized by the dysregulation of skin barrier function. We further validated the efficacy of PL-tLB using an atopic-like mouse model induced by 2,4-Dinitrochlorobenzene (DNCB).

Results

The experimental model mice exhibited revealed an increase in ear thickness and mast cell infiltration after DNCB stimulation, which were subsequently reduced following treatment with PL-tLB. Real-time PCR analysis of ear tissue demonstrated reduced downregulated expression of inflammatory cytokines and chemokines after PL-tLB administration. Additionally, we assessed the expression of skin barrier and tight junction proteins, revealing improvements upon PL-tLB treatment.

Conclusions

These findings suggest that PL-tLB holds promise as a potential treatment and functional material for managing atopic dermatitis.

Atopic dermatitis

skin barrier

tight junction

Phellinus linteus

Phellinus linteus, a type of medicinal mushroom with a rich history in traditional Asian medicine, has been revered for its potential health benefits [1]. This remarkable fungus, also known as "mesima" or "song gen," belongs to the Hymenochaetaceae family and is found predominantly in the forests of East Asia [2, 3]. The Phellinus linteus is a complex matrix brimming with a diverse array of bioactive constituents, including polysaccharides, phenolic compounds, terpenoids, and proteins [4]. These compounds have shown immunomodulatory, anti-inflammatory, and antioxidant properties in various studies [5, 6]. Researchers have identified and isolated several key compounds from phellinus linteus, such as hispolon, ergosterol, and various beta-glucans [4, 7]. Numerous studies have investigated the potential health-promoting properties of phellinus linteus. Its immunomodulatory effects have been of particular interest, as they may hold promise for conditions characterized by immune system dysregulation. Additionally, the mushroom's anti-inflammatory [8, 9], antioxidant [10], and anticancer properties [11] have been studied extensively, suggesting its potential as an adjunctive therapy in various chronic diseases, including cancer, cardiovascular disorders [12], and neurodegenerative conditions [13].

The human skin, an intricate interface between the body and its environment, serves as a formidable shield, maintaining homeostasis while warding off external threats [14]. The skin barrier, which encompasses the epidermis, stratum corneum, and lipid matrix, orchestrates a delicate balance between moisture retention, immune surveillance, and environmental defense [15]. The role of antioxidants becomes most important in skin health [16]. Antioxidants play an important role in neutralizing reactive oxygen species (ROS) generated by environmental factors or intrinsic processes [17]. However, in conditions such as atopic dermatitis (AD), where the skin barrier is damaged by genetic predisposition and external environmental factors, [16] the delicate balance of antioxidants can be disrupted [18]. Atopic dermatitis is a complex and multifactorial condition characterized by a predisposition to allergic hypersensitivity reactions that has become an increasingly prevalent global health concern. The rising prevalence of atopic disorders, such as allergic rhinitis, asthma, and eczema, has spurred intensive research into novel therapeutic approaches that can alleviate symptoms and improve the quality of life for affected individuals. AD development involves skin barrier dysfunction and abnormal immune responses [19]. In AD, destruction of the skin barrier impairs the effectiveness of the antioxidant defense system [20, 21]. Deficiency of key proteins such as filaggrin and changes in lipid composition, which are common in AD, may interfere with the cohesion of the skin barrier. This disruption allows for allergen invasion, pathogenic colonization, and proinflammatory cytokine signaling, providing an environment in which antioxidants can be overwhelmed [15, 22]. Key players in its pathogenesis include T helper type 2 (Th2) cells, elevated IgE levels, and the involvement of cytokines such as interleukins (IL) 4, 13, and 31 [23]. While symptomatic relief can often be achieved, there is a pressing need for targeted therapies that address the underlying molecular and cellular mechanisms [24]. Current treatments range from topical corticosteroids and immunomodulators to systemic therapies; however, their limitations in long-term efficacy and potential side effects necessitate the exploration of novel therapeutic avenues [25]. While inflammation's pivotal role in AD is well-documented, the intricate interplay between an impaired skin barrier and disease development is increasingly recognized as a crucial contributor [26].

This research aims to provide an in-depth exploration of the molecular mechanisms underpinning the potential role of Phellinus linteus in AD management. By delving into its intricate phytochemical composition and the mechanisms driving its bioactivity, we sought to elucidate how Phellinus linteus may exert a modulatory influence on the immune pathways implicated in AD disorders. Additionally, we examined existing preclinical and clinical studies that shed light on the effects of Phellinus linteus on AD conditions, addressing its impact on symptom severity, immune responses, and overall well-being.

Preparation of PL-tLB

Tyndallized lactic acid bacteria separated from Phellinus linteus (Parabiotics®), (PL-tLB) was provided by Vitech Co., Ltd (Jeonbuk, Korea), underwent the following processing step: Two strains, Lactobacillus plantarum VIOAP03 (KCTC14498BP) and Lactobacillus fermentum VIMPP04 (KCTC14499BP), were combined and processed. The VIMPP04 strain was prepared through a centrifugation process after undergoing pre-cultivation and primary culture in an industrial medium. Simultaneously, the VIOAP03 strain underwent pre-cultivation in an industrial medium, followed by main culture in a medium enriched with Phellinus linteus concentrate. After centrifugation, the concentrated VIMPP04 strain solution was blended with the primary VIOAP03 strain culture solution. To render the cells nonviable, a process known as tindilization (heat sterilization) was employed. This was followed by rapid freezing using a liquid nitrogen freezer and subsequent freeze-drying.

Cell culture

The human keratinocyte cell line (HaCaT) was subcultured using standard laboratory procedures at 37°C, 90–95% humidity, and 5% CO2. The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Cell viability assay

Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which was performed as described in previous studies [27–29].

Enzyme-linked immunosorbent assay (ELISA)

Quantify the levels of cytokines (specifically IL-1β, IL-6, and IL-8) and chemokines (CCL17/TARC, CCL22/MDC) in the cell culture medium using ELISA kits (BD Biosciences, San Diego, CA, USA, and R&D Systems, Minneapolis, MN, respectively). Additionally, we assessed serum immunoglobulin (Ig) E, G1, and G2a levels in mouse blood with ELISA kits (BD Biosciences, San Diego, CA, USA), following the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader.

Real-time polymerase chain reaction (real-time PCR)

Isolation of total RNA from cells and ear tissue using TRIzol reagent. Real-time PCR was conducted following a protocol outlined in a prior study [30], with eukaryotic 18S rRNA and mouse GAPDH utilized as internal reference controls for normalization. TaqMan primers and probes sourced from Applied Biosystems (Thermo Fisher Scientific, MA, USA) were employed for specific gene targets, including interleukin (IL)-1β (Hs01555410_m1, Mm00434228_m1), IL-4 (Mm00445259_m1), IL-6 (Hs00174131_m1, Mm00446190_m1), CCL17/TARC (Hs00171074_m1, Mm01244826_g1), CCL22/MDC (Hs01574247_m1, Mm00436439_m1), FLG (Mm01716522_m1), Involucrin (IVL, Mm01962650_s1), Loricrin (LOR, Mm01962650_s1), Claudin-4 (Cldn-4, Mm00515514_s1), Occludin (OCLN, Mm00500912_m1), and Tight Junction Protein-1 (TJP-1 or ZO-1, Mm00493699_m1).

Animal model

To obtain female BALB/c mice at 6 weeks of age from Samtako (Osan, Korea). Throughout the duration of the study, all animals were housed in accordance with the established conditions described in prior research [27]. The animal care and treatment procedures adhered strictly to the guidelines set forth in the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Furthermore, these protocols received formal approval from the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-21312).

Generation of DNCB-induced AD-like skin lesions

Thirty mice (n = 5/group) were divided into groups: PBS vehicle, DNCB vehicle (PBS), DNCB plus PL-tLB (at doses of 1×10⁹, 1×10¹⁰, and 1×10¹¹ cells/kg), and DNCB plus DX (1 mg/kg). In the first induction week, each ear was sensitized with DNCB (2%, 20 µL/ear) once. After 1 week, both ears of each BALB/c mouse received DNCB challenge (1%, 20 µL/ear, twice a week) for 3 weeks. PL-tLB or DX was orally administered by gavage on five consecutive days each week during DNCB challenge. DX served as the positive control, per established methods [27, 28]. After the experiment, mice were euthanized via cervical dislocation under isoflurane anesthesia.

Histological analysis

The mouse ear tissue was fixed using 10% (w/v) paraformaldehyde. Paraffin-embedded tissues were then sectioned to a thickness of 3 µm using a microtome. After paraffin removal, the sections were stained with hematoxylin and eosin (H&E) as well as toluidine blue.

Statistical analysis

Statistical analysis was conducted using Prism 5 software (GraphPad Software, San Diego, CA, USA). Data is expressed as the mean ± SD from nine individual experiments, and statistical significance was assessed through one-way ANOVA followed by Tukey’s multiple comparisons tests.

Effect of PL-tLB on the viability of HaCaT cells

To assess the viability of PL-tLB, human keratinocytes (HaCaT) were exposed to PL-tLB at concentrations of 0, 10, 30, 60, and 100 µg/ml for a duration of 24 hours, and cytotoxicity was subsequently confirmed. The assessment of cytotoxicity was carried out using the MTT assay after treatment with PL-tLB at varying concentrations. The results demonstrated that the group treated with high concentrations of Phellinus linteus exhibited pronounced toxicity, as depicted in Fig. 1. Consequently, subsequent experiments were conducted using PL-tLB at concentrations of 3, 10, and 30 µg/ml.

Effect of PL-tLB on chemokine and cytokine levels in HaCaT cells

We investigated the anti-inflammatory potential of PL-tLB by assessing RNA and protein expression using real-time PCR and ELISA assay. HaCaT cells were subjected to stimulation with proinflammatory mediator’s TNF-α/IFN-γ. In the stimulated group, we compared the expression levels of key chemokines, such as CCL17 and CCL22, recognized as biomarkers of atopic dermatitis, with those of the control group. Furthermore, we observed an upregulation in the expression of proinflammatory cytokines, namely IL-1β, IL-6, and IL-8, in the TNF-α/IFN-γ treated group as compared to the control group (Fig. 2). However, PL-tLB treatment effectively mitigated this increase in gene expression levels in a concentration-dependent manner. Notably, at a concentration of 30 µg/mL, PL-tLB exhibited gene-suppressive effects on par with those of cyclosporine A. Protein levels of chemokines and cytokines showed a similar trend to their gene expression patterns (Fig. 2). In conclusion, our findings demonstrate that PL-tLB treatment efficiently attenuates atopic dermatitis and suppresses inflammatory response mediators in keratinocytes.

PL-tLB effect on AD-like skin lesions in BALB/c mice

To evaluate the potential anti-AD effects of PL-tLB, we established an experimental animal model by administering DNCB to the ears of mice over a 4-week period. Consistent topical application of DNCB twice a week led to a noticeable increase in ear edema compared to the control group. In contrast, the group treated with PL-tLB exhibited a more significant reduction in ear edema when compared to the DNCB-exposed group (Fig. 3A). To assess the impact of PL-tLB on skin hypertrophy, we stained ear tissues with H&E and observed them under light microscopy. Continuous exposure to DNCB induced substantial inflammatory changes, such as dermal and epidermal thickening in the ear tissues of AD-afflicted mice when compared to the control group (Fig. 3A, middle panel). Mast cell infiltration is a common occurrence in AD skin lesions; thus, we examined mast cell infiltration through toluidine blue staining. Mast cell infiltration was elevated in the DNCB-treated group in comparison to the control group, and this increased mast cell invasion was mitigated by PL-tLB treatment (Fig. 3A, lower panel). Furthermore, ear thickness was notably increased due to DNCB exposure (Fig. 3B). However, when compared to the AD group, the PL-tLB treatment group displayed a significant reduction in both epidermal and dermal thickness (Fig. 3A, middle panel).

PL-tLB affect serum immunoglobulin levels in BALB/c mice with AD-like skin lesions.

In AD, Th2 cytokines stimulate B cells, resulting in IgE secretion and influencing IgG1 and IgG2a levels. [31, 32]. ELISA measured IgE, IgG1, and IgG2a to evaluate PL-tLB's impact on immunoglobulin levels in the AD model. DNCB-induced mice exhibited elevated IgE, IgG1, and IgG2a levels, all of which were significantly reduced by PL-tLB treatment (Fig. 4). Importantly, the administration of PL-tLB at a dose of 1×1011 cells/kg exhibited a superior inhibitory effect on IgE levels compared to DX. In conclusion, our findings demonstrate that PL-tLB effectively alleviates AD lesions.

PL-tLB impact the mRNA expression of chemokines and cytokines in the ear tissues of DNCB-induced BALB/c mice.

To comprehend how PL-tLB alleviates AD lesions, we examined the expression of AD-associated chemokines (CCL17 and CCL22) and pro-inflammatory cytokines in ear tissues using real-time PCR. The DNCB-exposed group showed increased expression of these chemokines compared to the control group. However, PL-tLB treatment reversed this upregulation (Fig. 5). Similarly, pro-inflammatory cytokines (IL-1β, IL-4, IL-6, and TNF-α) showed increased expression in the DNCB-treated group, which was effectively countered by dose-dependent PL-tLB treatment. Treatment with PL-tLB at 1×10¹¹ cells/kg resulted in gene expression downregulation comparable to DX (Fig. 5). In summary, our findings show that PL-tLB reduces gene expression of chemokines and cytokines in a DNCB-induced AD-like mouse model.

PL-tLB impact the mRNA expression of skin barrier and tight junction proteins in the ear tissues of DNCB-induced BALB/c mice

We investigated the impact of PL-tLB on the expression of major skin barrier proteins in an AD-like mouse model. As shown in Fig. 6A, we observed a significant decrease in the gene expression levels of FLG, IVL, and LOR, which are important skin barrier proteins, in the DNCB-induced group when compared to the control group. However, it was confirmed that the PL-tLB treatment group exhibited a concentration-dependent recovery. This finding suggests that PL-tLB possesses the capability to restore skin barrier proteins, and we subsequently analyzed the primary factors responsible for this restorative effect. It has been reported that tight junctions perform complementary functions as part of the skin barrier. In this study, we investigated the impact of PL-tLB on the expression of Claudin-4, Occludin, and ZO-1 proteins, which constitute these tight junctions. The analysis of Cldn-4, Ocln, and ZO-1 revealed a significant decrease in their expression levels in the DNCB-induced group compared to the control group, with a concentration-dependent recovery observed in the PL-tLB treatment group (Fig. 6B).

Atopic dermatitis (AD) is a common and enigmatic inflammatory skin disorder, that impacts individuals across all age groups. The multifactorial nature of AD involves a compromised skin barrier, allowing for increased penetration of allergens and microbes, triggering immune responses [33]. Phellinus linteus, a medicinal mushroom, has increased research value as a source of bioactive compounds that exhibit diverse biological activities ranging from immunomodulatory to anticancer properties [1, 34]. Therefore, we investigated the anti-inflammatory effect of phellinus linteus on atopic dermatitis. The levels of inflammatory chemokines, including CCL17/TARC and CCL22/MDC, along with cytokines such as IL-1β, IL-6, and IL-8, increased following TNF-α/IFN-γ treatment but were suppressed upon PL-tLB treatment, as confirmed by our experiments. These findings suggest that PL-tLB has the potential to modulate inflammatory mediators.

In the context of a DNCB-induced AD-like mouse model, the effectiveness of PL-tLB in mitigating atopy was rigorously verified. Notably, both the positive control group and the PL-tLB-administered group displayed visible improvements in keratinization compared to the control group, indicating a promising therapeutic impact. Furthermore, histological examinations, which encompassed H&E and toluidine blue staining, were conducted to validate the efficacy of PL-tLB. These assessments revealed noteworthy reductions in ear thickness and mast cell infiltration within the PL-tLB-treated groups. These findings underscore the potential of PL-tLB as a viable candidate for ameliorating atopy.

In AD, Th2 cells primarily secrete IL-4 and IL-5, which stimulate B cells to produce IgE, influencing the levels of IgG1 and IgG2a, as well as the production of various inflammatory cytokines [35]. IgE levels also play a role in sensitizing allergens [36, 37]. In this study, we found that oral administration of PL-tLB reduced IgE, IgG1, and IgG2a levels in AD-like models.

In the initial stages of AD, environmental triggers induce the release of cytokines from keratinocytes, such as IL-1β, CCL17/TARC, CCL22/MDC, and TSLP, which activate dendritic cells. These activated dendritic cells then produce CCL17/TARC and CCL22/MDC, facilitating the invasion and differentiation of Th2 cells [38, 39]. This research demonstrates that PL-tLB had a significant impact on reducing the mRNA expression of key cytokines like IL-1β, IL-4, IL-6, TNF-α, CCL17/TARC, and CCL22/MDC in the ear tissues of DNCB-induced mice. These findings provide compelling support for considering PL-tLB as a promising candidate for ameliorating atopy.

In conclusion, the use of steroid drugs, commonly employed for AD treatment, is linked to various long-term side effects. Therefore, there is a critical need to investigate natural alternatives as substitutes for steroids. Additionally, further comprehensive research is necessary to elucidate interactions among the compounds within PL-tLB. Considering the results derived from both in vitro and in vivo AD models, PL-tLB holds promise as an effective therapeutic agent. Consequently, the PL-tLB used in this study holds promise as a natural substance for alleviating skin diseases by strengthening the skin barrier. Nevertheless, additional research employing artificial skin and mechanistic studies in cell models are required to further substantiate these findings.

Declaration of Competing Interest

The authors declare no conflict of interest.

Author Contribution

Seon Gyeong Bak, and Hyung Jin Lim: Formal analysis, Writing - original draft. Yeong Seon Won, and Eun Hyun Park: Formal analysis, Investigation. Nisansala Chandimali, and Hyuck Se Kwon: Investigation, Methodology. Nayong Lee, Hyunjeong Oh, and Soon-Il Yun: Methodology. Sang-Ik Park and Seung Jae Lee: Conceptualization, Writing – review & editing, Supervision.

Acknowledgements

This work was supported by the Technology development of new products subject to purchase conditions (S3138172) funded by the Ministry of SMEs and Startups (MSS, Korea). This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022R1A6A3A01087055). This research was supported by several grants: the KRIBB Research Initiative Program (KGM5242423), the National Research Council of Science & Technology (NST) grant from the Korea government (MSIT) (No. CCL23251-100, KRIBB-NTM2672311), the “Regional Innovation Strategy (RIS)” program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (MOE) (2021RIS-002), and the Basic Science Research Program through the NRF, funded by the Ministry of Education, Science, and Technology (No. NRF-2022R1A2C1011742).

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No competing interests reported.

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Effects of tyndallized lactic acid bacteria separated from Phellinus linteus on inflammation and skin barrier damage induced by DNCB

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Materials and Methods

Preparation of PL-tLB

Cell culture

Cell viability assay

Enzyme-linked immunosorbent assay (ELISA)

Real-time polymerase chain reaction (real-time PCR)

Animal model

Generation of DNCB-induced AD-like skin lesions

Histological analysis

Statistical analysis

Results

Effect of PL-tLB on the viability of HaCaT cells

Effect of PL-tLB on chemokine and cytokine levels in HaCaT cells

PL-tLB effect on AD-like skin lesions in BALB/c mice

Discussion

Declarations

Declaration of Competing Interest

Author Contribution

Acknowledgements

References

Additional Declarations

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