Introduction
The increasing interest in natural bioactive compounds for therapeutic and cosmetic applications has led to extensive research into various traditional medicinal plants and fungi. One such organism gaining significant attention is the Chaga mushroom (
Inonotus obliquus). This review focuses on a comprehensive
clinical study (PMC10454022) that investigates the role of Chaga mushroom extracts in reducing pro-inflammatory cytokines like TNF-alpha and IL-6 in vitro, utilizing human keratinocytes (HaCaT cells) as the primary model. The study's findings are pivotal in demonstrating the potential efficacy of Chaga extracts in managing inflammation and could pave the way for novel anti-inflammatory therapies.
Background
The Importance of Keratinocytes
Human skin is the body's largest organ and a complex structure crucial for multiple physiological functions, including protection against external stressors, temperature regulation, sensation, and immune surveillance. The primary cell type in the epidermis, keratinocytes, plays a significant role in these functions, especially in the context of the immune response. These cells originate from the basal layer of the epidermis and undergo systematic differentiation as they migrate toward the stratum corneum.
Pro-Inflammatory Cytokines
Pro-inflammatory cytokines are crucial mediators of various cells, including keratinocytes, macrophages, mast cells, and lymphocytes. These cytokines, such as TNF-alpha, IL-1β, IL-6, and IL-8, are involved in the initiation and progression of inflammatory responses and are linked to various diseases, including cardiovascular diseases, cancers, and age-related conditions. While these cytokines play essential roles in immune defense, their excessive production can lead to chronic inflammation and related pathologies.
Inflammaging
The concept of inflammation, introduced by Professor Claudio Franceschi, describes a chronic, low-grade inflammatory status that contributes to the aging process and tissue damage. In the skin, factors such as UV radiation, smoking, and air pollution can exacerbate inflammation, leading to conditions like cancer and skin diseases associated with aging. Therefore, targeting pathways that reduce pro-inflammatory cytokine production can mitigate inflammation and promote healthier aging.
Chaga Mushroom and Inotodiol
Chaga mushroom has been traditionally used in various regions for its medicinal properties. It contains bioactive triterpenes such as inotodiol, demonstrating multiple biological activities, including anti-tumor, anti-inflammatory, anti-allergic, antiviral, anti-aging, and antioxidant effects. This study aims to elaborate on the anti-inflammatory and anti-aging potential of inotodiol, particularly under conditions of oxidative stress induced by UV radiation and TNF-alpha.
Study Overview
Objectives
The primary objectives of the study were:
- To evaluate the effect of inotodiol on pro-inflammatory cytokine production in keratinocytes stimulated with UV or TNF-alpha.
- To assess how inotodiol affects hyaluronic acid synthesis in HaCaT cells, both with and without TNF-alpha stimulation.
Methodology
Preparation of Inotodiol Concentrate
A lanostane triterpenoids-rich concentrate containing 10% inotodiol was prepared from Chaga mushrooms. This concentrate was compared with pure inotodiol for its anti-inflammatory activities in HaCaT cells.
Cytotoxicity Assessment
The study assessed the cytotoxicity of pure inotodiol and inotodiol concentrate using the MTT assay on HaCaT cells. Cytotoxicity was determined based on ISO EN 10993-5 criteria, with cell viability under 70% considered toxic.
Pro-Inflammatory Cytokine Expression
The mRNA expression levels of IL-1β, IL-6, and IL-8 were evaluated by real-time qPCR in HaCaT cells treated with TNF-alpha. Additionally, the impact of UV-induced pro-inflammatory cytokine expression was assessed.
Hyaluronic Acid and Collagen Synthesis
The study also examined the influence of inotodiol on collagen (COL1A2) and hyaluronic acid (HAS2 and HAS3) synthesis. The degradation of hyaluronic acid by hyaluronidase (HYAL-3) under TNF-alpha stimulation was studied to understand the broader implications of inotodiol on skin aging.
Key Findings
Triterpenoid Concentration and Composition
The analysis using LC/MS identified six major triterpenes in the inotodiol concentrate, with inotodiol being the most abundant, accounting for 69.1% of the total composition. Betulin was the second most abundant triterpene, comprising approximately 12%.
Cytotoxicity Results
The MTT assay demonstrated that pure inotodiol was non-toxic (cell viability >70%) except at the highest concentration (20 μg/mL). The inotodiol concentrate was non-toxic at all tested concentrations, indicating its safety for potential therapeutic use.
Reduction of Pro-Inflammatory Cytokines
Both pure inotodiol and inotodiol concentrate significantly reduced the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) after TNF-alpha stimulation. Inotodiol exhibited a dose-dependent reduction in cytokine expression, whereas the inotodiol concentrate showed effectiveness at lower concentrations.
Anti-Inflammatory Effects Under UV Radiation
UV radiation-induced the overexpression of IL-6 and TNF-alpha in keratinocytes, contributing to acute inflammation and photoaging. Adding inotodiol and inotodiol concentrate reduced the mRNA expression levels of these cytokines, highlighting their potential to mitigate UV-induced skin inflammation.
Enhancement of Hyaluronic Acid and Collagen Synthesis
Inotodiol and inotodiol concentrate upregulated gene expression in hyaluronic acid (HAS2 and HAS3) and collagen (COL1A2) synthesis. This effect suggests that inotodiol reduces inflammation and promotes extracellular matrix production, which is crucial for maintaining skin integrity and reducing aging signs.
Inhibition of Hyaluronic Acid Degradation
The study found that inotodiol significantly reduced HYAL-3 mRNA expression under TNF-alpha stimulation, preventing the degradation of hyaluronic acid. This effect further supports the anti-aging potential of inotodiol in preserving skin hydration and elasticity.
Discussion
Implications for Anti-Inflammatory Therapies
The findings of this study underscore the promising role of Chaga mushroom extracts, particularly inotodiol, in reducing pro-inflammatory cytokine levels in human keratinocytes. By decreasing the expression of cytokines like TNF-alpha, IL-6, and IL-8, inotodiol could be integrated into therapeutic strategies for managing inflammatory skin conditions and related systemic diseases.
Potential for Anti-Aging Applications
The dual action of inotodiol in reducing inflammation and enhancing extracellular matrix production points to its valuable application in anti-aging skincare products. By upregulating collagen and hyaluronic acid synthesis while inhibiting their degradation, inotodiol can help maintain skin firmness, elasticity, and hydration, addressing both the aesthetic and functional aspects of skin aging.
Safety and Efficacy
The cytotoxicity assessment confirms that inotodiol and its concentrate are safe for use at appropriate concentrations, with non-toxic effects observed at therapeutic doses. This safety profile, combined with the demonstrated efficacy, makes inotodiol a strong candidate for developing new natural remedies for inflammation and aging.
Future Research Directions
While the in vitro results are promising, translating these findings to clinical applications requires further research. Future studies should focus on:
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In Vivo Studies: Evaluating the effects of inotodiol in animal models and human clinical trials to confirm its safety and efficacy in real-world scenarios.
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Mechanistic Insights: Investigating the precise molecular mechanisms by which inotodiol exerts its anti-inflammatory and anti-aging effects.
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Formulation Development: Developing stable and effective formulations for topical and systemic administration of inotodiol.
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Comparative Studies: Comparing the efficacy of inotodiol with other known anti-inflammatory and anti-aging agents to position its use within the broader context of available treatments.
Conclusion
The clinical study on the effects of Chaga mushroom extracts, specifically inotodiol, on pro-inflammatory cytokine reduction presents compelling evidence of its anti-inflammatory and anti-aging potential. By effectively reducing cytokine production and enhancing extracellular matrix synthesis, inotodiol shows promise for therapeutic and cosmetic applications to manage inflammation and promote skin health. Further research and development could unlock new avenues for utilizing this natural bioactive compound in medical and skincare treatments, contributing to healthier aging and improved quality of life.
