Berberine protects immortalized line of human melanocytes from H2O2-induced oxidative stress via activation of Nrf2 and Mitf signaling pathway

doi:10.1016/j.jdermsci.2019.03.007

Journal of Dermatological Science

Volume 94, Issue 1, April 2019, Pages 236-243

https://doi.org/10.1016/j.jdermsci.2019.03.007 Get rights and content

Abstract

Background

Oxidative stress plays important roles in the pathogenesis of vitiligo. The removal of hydrogen peroxided (H₂O₂) has been established to be beneficial to vitiligo patients. Berberine (BBR), a natural isoquinoline alkaloid, has antioxidant activity, however, whether BBR can defend human melanocytes against oxidative injury remains to be elucidated.

Objective

In the present study, we investigated the potential protective effect of BBR against oxidative stress on an immortalized normal human melanocyte cell line PIG1.

Methods

Generally, PIG1 cells were pretreated with various concentrations of BBR for 1 h followed by exposure to 1.0 mM H₂O₂ for 24 h. Cell apoptosis, intracellular reactive oxygen species (ROS) levels were assessed through flow cytometry. Cell apoptosis, melanogenesis and the activation of Nrf2-ARE and Mitf signaling pathway were assayed.

Results

Our results showed that cell viability rose and intracellular ROS generation, cell apoptosis of melanocytes decreased significantly in response to H₂O₂ through pretreatment with BBR. Furthermore, We found that BBR can dramatically induce Nrf2 nuclear translocation, increase total Nrf2 levels and enhance ARE activity. Besides, Nrf2-siRNA transfection can abrogate the protection of BBR in melanocytes against oxidative injury. At last, we verified that BBR could facilitate melanogenesis function via modulation of Mitf and its target proteins.

Conclusion

The results above suggest that BBR can protect melanocytes against oxidative stress via its anti-oxidative activity. Also, we found H₂O₂-induced activation of NFκB was inhibited by BBR. Therefore, it is worthy of investigation BBR as a potential drug for treatment of vitiligo.

Introduction

Vitiligo, affecting about 1% of the people worldwide, is a chronic depigmentation disorder of skin through the selective destruction of melanocytes [1], [2], [3]. Widely accepted evidence shows that oxidative injury mediates as an initial pathogenic trigger in melanocytes degeneration in patients with vitiligo [4], [5], [6]. Furthermore, it can inhibit melanin synthesis, initiate target injury of melanocytes by the immune system and compromise their survival [7]. Overproduction of ROS can also activate NFκB which could further induce melanocytes to secrete IL-6 and IL-8. Secretion IL-6 and IL-8 by melanocytes can attract of T cell infiltration which could magnify the inflammatory reaction and play a vital role in melanocyte death [8], [9]. All of these changes may inhibit melanogenesis and lead to depigmentation in vitiligo lesions.

The Nrf2 is a master transcription factor that plays a vital role in protecting cells from oxidative injury [10]. Our previous studies have verified that Nrf2-ARE pathway is critical for the protection of human melanocytes against H₂O₂-induced oxidative stress lesions, and its primary effector is HO-1 [10]. Thus, it is a pivotal approach for vitiligo treatment by targeting on the Nrf2-ARE pathway. The melanin synthesis capacity of the epidermis melanocytes was considered to be weakened in patients with vitiligo [11]. Particularly, suppression of Mitf could lead to further inhibition of numerous genes about pigmentation and melanocyte differentiation (e.g., TYR, TRP1 and DCT), as well as essential genes for keeping cellular environmental homeostasis, as well as maintaining genes encoding proteins such as the Bcl2 family that regulate apoptosis [12]. NFκB, a master regulator of proinflammatory responses, was recognized as a redox-regulated transcription factor which can be activated by H₂O₂ [8]. By catalyzing the phosphorylation and degradation of IκBs at specific amino acid residues, NFκB can translocate from cytoplasm to nucleus and further activate immune mediators such as IL-6 and IL-8 [13].

Given the above, the purpose of our study was to investigate whether BBR could exert protective effects on human melanocytes from H₂O₂-induced oxidative stress and apoptosis and to elucidate the underlying molecular mechanism involved. Here, we show that BBR could protect human melanocytes against H₂O₂-induced oxidative stress through activation of Nrf2-ARE, potentiating function of melanin synthesis of melanocytes and ameliorating activation of NFκB.

Section snippets

Cell culture and treatment

The PIG1 immortalized human melanocyte cell line (given as a present by Dr. Caroline Le Poole, Loyola University, Chicago, USA) were cultured in Medium 254 from Invitrogen (Portland, OR, USA) supplemented with Human Melanocyte Growth Supplement from Invitrogen, 5% fetal bovine serum from Invitrogen in a humid atmosphere of 5% CO₂ and maintain the temperature at 37 °C. Oxidative injury in PIG1 cells was inducted by treatment of 1.0 mM H₂O₂ from the Sigma- Aldrich Chemical Company (St. Louis, MO,

Berberine ameliorates H₂O₂-induced oxidative damage in human melanocytes

In our study, we first assessed the effect of BBR on PIG1 cell proliferation and viability. As shown in the Fig. 1A and B, melanocytes pretreated with 0.1 μM–5.0 μM BBR induced cell proliferation in a time-dependent mode. Nevertheless, melanocytes pretreated with 10.0 μM–40.0 μM BBR signally suppressed cell growth compared to untreated groups. Besides, there is no apparent morphologic change in melanocytes pretreated with 5.0 μM BBR (Fig. 1C). PIG1 cells are treated with 1.0 mM H₂O₂ with or

Discussion

In our study, we studied the effect of BBR on meliorating H₂O₂-induced oxidative injury in human melanocytes and clarified some of the mechanisms involved. We found that BBR exerts a cytoprotective influence on PIG1 cells by improving anti-oxidant enzymes, downregulating intracellular ROS, reducing Bax/Bcl-2 ratio and suppressing PARP cleavage. Furthermore, we found that BBR could protect melanocytes against oxidative stress by activating Nrf2-ARE, such as HO-1, NQO-1, and SOD. Besides, we

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81872521, 91742201 and 81703128), and the Project of Youth Science and Technology New Star in Shaanxi Province of China (2018KJXX-016). We thank Dr. Caroline Le Poole for providing the immortalized human epidermal melanocyte cell line PIG1.

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Isoquinoline alkaloids are an important class of natural products that are abundant in the plant kingdom and exhibit a wide range of structural diversity and biological activities. With the deepening of research in recent years, more and more isoquinoline alkaloids have been isolated and identified and proved to contain a variety of biological activities and pharmacological effects. In this review, we introduce the research progress of isoquinoline alkaloids from 2019 to 2022, mainly in the part of biological activities, including antitumor, antimicrobial, antidiabetic, antiviral, anti-inflammatory, antioxidant, neuroprotective, hepatoprotective, analgesic, and other activities. This study provides a clear direction for the rational development and utilization of isoquinoline alkaloids, suggesting that these alkaloids have great potential in the field of drug research.
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Vitiligo is a pigmentary disorder, in which oxidative stress has been evidenced as part of the pathogenesis. Pathways responsible for protecting melanocytes from damage caused by reactive oxygen species are known as the nuclear factor erythroid factor 2 (Nrf2) pathway.
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A search for articles in Spanish and English was carried out in the PubMed, Ovid, Scopus and Web of Science Clarivate databases, using the keywords “Vitiligo AND nuclear factor erythroid derived 2 like 2 OR NRF2” without time restriction. All in vitro studies, narrative reviews, case series, cohort studies, and randomized and non-randomized clinical trials that specifically addressed the issue of Nrf2 associated with vitiligo were included.
El vitíligo es un trastorno pigmentario en el que se ha evidenciado el estrés oxidativo como parte de la patogenia. Se conocen vías encargadas de proteger a los melanocitos del daño causado por las especies reactivas de oxígeno, como por ejemplo la vía del factor nuclear eritroide similar al factor 2 (Nrf2).
El Nrf2 es un factor de transcripción que cuando el organismo se encuentra en homeostasis permanece inhibido, pero en presencia de estrés oxidativo permite la codificación de enzimas antioxidantes de fase II.
En el vitíligo se evidencian anomalías en la localización y función del Nrf2, así como polimorfismos que aumentan el riesgo de esta enfermedad. Así mismo, se han investigado múltiples moléculas que actúan en el Nrf2 buscando encontrar tratamientos emergentes útiles para el vitíligo.
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Novel berberine-loaded hyalurosomes as a promising nanodermatological treatment for vitiligo: Biochemical, biological and gene expression studies
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Therefore, BRB could be used safely as melanogenic agent for the treatment of vitiligo. ( Ali and Naaz 2015, Jiang et al. 2019). Unfortunately, the presence of skin cuticle barrier causes poor drug accumulation, hence, insufficient therapeutic outcomes (Sun et al. 2020).
Vitiligo is a depigmentation disorder that affects 0.5–2% of the world population. It has a severe impact on a patient's quality of life and even causes suicidal attempts. Up to date, no curative therapy is available which have created a substantial demand for novel vitiligo treatments. Berberine (BRB) is an isoquinoline alkaloid with promising pharmacological effects. However, it suffers from poor membrane permeability hindering its topical application. The current work is the first to design and assess topical BRB-loaded hyalurosomes for targeted vitiligo treatment. BRB-hyalurosomes are hyaluronan-immobilized phospholipid nanovesicles that showed promising invitro physicochemical properties. Novel ex vivo studies were performed using full-thickness human skin to mimic its dermal application. Furthermore, in-vivo studies were conducted using a vitiligo-induced mouse model followed by biochemical, histological and immunohistochemical investigations. In addition, gene expression of skin inflammatory markers was assessed using quantitative reverse-transcription PCR. Biological studies showed significant improvement of the biochemical markers in BRB-hyalurosomes group compared to the vitiligo-model group and BRB conventional gel. It is worthy to mention that placebo hyalurosomes demonstrated significant enhancement in the biological activity confirming its intrinsic activity. Conclusively, BRB-hyalurosomes is considered a novel nanodermatological tool that paving the way for its clinical application for vitiligo treatment.
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Berberine (Ber) is a primary active ingredient isolated from the root and bark of Coptidis rhizoma, a traditional Chinese herb recorded in the ancient book of traditional Chinese medicine, The Divine Farmer’s Classic of Materia Medica (Shen Nong Ben Cao Jing). It has been used for decades in China as a nonprescription drug to treat gastrointestinal infections, such as bacillary dysentery. Due to its multipharmacological effects on a wide spectrum of aging-related diseases, such as hypertension, hyperlipidemia, and Alzheimer's disease, Ber has been proposed as an attractive “elixir of eternal life” for anti-aging. In various model systems of aging/senescence, such as fetal lung diploid fibroblasts, Drosophila, and mice, Ber has readily demonstrated an anti-senescence or life span–prolonging function. Mitochondria are at the metabolic core of energy production and participate in the aging process as signaling organelles. This chapter discusses the metabolic control of Ber by mitochondria and caloric restriction as an anti-aging pharmacological mechanism.
SFRP5 inhibits melanin synthesis of melanocytes in vitiligo by suppressing the Wnt/β-catenin signaling
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Moreover, vitiligo often affects the face and other visible areas of the body, resulting in a severe psychological burden and decreased quality of life in patients.8–10 Although several etiological theories have been proposed, which indicate that genetic predisposition,11 immune responses,12,13 melanocytorrhagy14 and metabolic abnormalities15 might be involved in the pathogenesis of vitiligo, the exact mechanism of loss of melanocytes in depigmented lesions is still unclear. Wnt signaling is known to play a crucial role in melanocyte stem cell differentiation.16,17
Secreted frizzled-related protein 5 (SFRP5) plays a pivotal role in regulating the development of many tissues and organs, however, as an inhibitor of Wnt signaling, the role of SFRP5 in vitiligo remains unknown. Hence, we speculated that SFRP5 might be associated with melanogenesis in melanocytes by regulating Wnt signaling in vitiligo. In this study, we found that SFRP5 was overexpressed in the skin lesions of patients with vitiligo. Compared with that in normal epidermal melanocytes (PIG1), the expression of SFRP5 was increased in vitiligo melanocytes (PIG3V). To investigate the effect of SFRP5 on melanin synthesis, PIG1 cells were infected with recombinant SFRP5 adenovirus (AdSFRP5), and PIG3V cells were infected with recombinant siSFRP5 adenovirus (AdsiSFRP5). The results showed that SFRP5 overexpression inhibited melanin synthesis in PIG1 cells through downregulation of microphthalmia-associated transcription factor (MITF) and its target proteins via suppression of the Wnt/β-catenin signaling pathway. Accordingly, SFRP5 silencing increased melanin synthesis and activated the Wnt signaling pathway in PIG3V cells. Moreover, SFRP5 overexpression also downregulated the transcriptional activity of T cell factor/lymphoid enhancer factor (TCF/LEF) in PIG1 cells. Furthermore, this inhibitory effect of SFRP5 on melanin synthesis was reversed by treatment with the β-catenin agonist, SKL2001. The inhibitory action of SFRP5 in pigmentation was further confirmed in vivo using a nude mouse model. Hence, our results indicate that SFRP5 can inhibit melanogenesis in melanocytes. Additionally, our findings showed that SFRP5 plays a vital role in the development of vitiligo, and thus may serve as a potential therapeutic target for vitiligo.
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For example, the Nuclear Factor kappaB (NF-kB) is a known transcription factor that plays a complex and essential role in the regulation of host antiviral immune response (Tilborghs et al., 2017). Most recently, berberine was shown to protect melanocytes against oxidative stress via its anti-oxidative activity by inhibiting H2O2-induced activation of NFkB via stimulating Nrf2 and Mitf signaling pathway (Jiang et al., 2019). ROS (reactive oxygen species) are viewed as a protection mechanism of the host cell that contributes to its apoptosis during fighting infections; however, this host defense-associated pathway could be hijacked to facilitate viral infection.
Cyprinid herpesvirus 2 (CyHV-2), which infects silver crucian carp including goldfish (Carassius auratus auratus) and Crucian carp (Carassius auratus gibelio) with high mortality, is an emerging viral pathogen worldwide. Previous studies showed that berberine (BBR), a bioactive plant-derived alkaloid, demonstrated potential antiviral actions against many different viruses. Here, we assessed the effect of berberine hydrochloride (BBH) on the replication of CyHV-2 in vitro and in vivo. Cytotoxicity assay indicated that 5–25 μg/mL BBH was non-toxic to the RyuF-2 cells. In viral inhibition assays, real time PCR was employed to titrate the genomic copy number of progeny virus, real time RT-PCR was applied to monitor the transcriptional levels of viral genes, and Western blot analysis was performed to detect the synthetic levels of viral proteins. The results demonstrated that BBH systematically impedes the viral gene transcription and suppressed the replication of CyHV-2 in RyuF-2 cells. In animal challenge test, BBH was confirmed to protect Crucian carps from CyHV-2 infection in a dose-dependent manner, which was supported by suppressed viral replication levels, reduced viral pathogenesis and higher survival rates. Furthermore, pharmacokinetics data of BBH in Crucian carp revealed its rapid absorption (T_max of 1.5 h), suitable plasma half-life (t_1/2z/h of 7–12 h depending on oral dosage), and dose-dependent drug exposure properties following oral administration (revealed by AUC_0-t values). These findings shed light on repurposing BBH to treat CyHV-2 infections in silver crucian carp.

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¹: The authors equally contributed to this work.

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Original artcleBerberine protects immortalized line of human melanocytes from H2O2-induced oxidative stress via activation of Nrf2 and Mitf signaling pathway

Abstract

Background

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Cell culture and treatment

Berberine ameliorates H2O2-induced oxidative damage in human melanocytes

Discussion

Conflict of interest

Acknowledgments

Lancet

J. Dermatol. Sci.

J. Invest. Dermatol.

J. Invest. Dermatol.

Cell

Cell

J. Invest. Dermatol.

Free Radic. Biol. Med.

J. Invest. Dermatol.

FEBS Lett.

Redox Biol.

Biochem. Pharmacol.

Int. J. Cardiol.

J. Dermatol. Sci.

Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress

Physiol. Rev.

L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions

Pigment Cell Melanoma Res.

Original artcle
Berberine protects immortalized line of human melanocytes from H₂O₂-induced oxidative stress via activation of Nrf2 and Mitf signaling pathway

Berberine ameliorates H₂O₂-induced oxidative damage in human melanocytes