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Report No. 011 Silibinin promotes the prevention and repair of skin cell damage caused by ultraviolet B wave irradiation
Report No. Silibinin promotes the prevention and repair of skin cell damage caused by ultraviolet B wave irradiation
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Overview

Silibinin is the main active constituent of silymarin, a biennial herb of the Asteraceae family native to Europe, extracted mainly from the seeds of Milk thistle, scientific name Silybum marianum. It is also known by the name silybin. Milk thistle is a herbal medicine that has been used in Europe and China for about 2000 years to treat liver diseases. In recent years, the purification method of the active ingredients and the mechanism of effects on cells have been elucidated, and it has been reported that it has various pharmacological effects such as protective effects not only on the liver but also on various organs, anti-inflammatory effects, and anti-oxidative effects [1-3].
Cells are affected by factors in the experimental system, i.e., soluble factors in the culture medium, pH of the medium, and the condition of the scaffold to which they adhere. The three-dimensional culture method using extracellular matrix components such as collagen or laminin as scaffolds for cell adhesion is considered to be an experimental condition closer to the living environment [4].
At Nippi Research Institute of Biomatrix, the effects on cells have been studied in the presence of collagen or laminin in cell culture experiments (Research Reports No. 002 , & No. 008 ; Refs. [5, 6]).For example, it is known that cells respond differently to drugs depending on the state of the scaffold to which they adhere (cell adhesion mediated drug resistance; CAM-DR, cell adhesion mediated drug resistance) [7].
Prof. Takashi Ikejima at Shenyang Pharmaceutical University, Shenyang, China, has long been studying anti-inflammatory drugs in many medical fields. Among them, he has focused on silibinin derived from natural products. About a dozen years ago, he became acquainted with Professor Emeritus Toshihiko Hayashi of the University of Tokyo and became interested in the collagen that Prof. Hayashi has been studying for many years. They began a joint research project with our laboratory, which had been conducting research on extracellular matrices, to examine in detail the effects of silibinin on cells cultured in the presence of collagen or gelatin. Through this joint research, we found that many of the effects of silibinin on cells are unique to the presence of collagen, and we also discovered a novel mechanism of effect for silibinin. We have jointly published papers and presented at conferences on these findings, and dozens of students from the Ikejima lab have gone on to earn doctoral and master's degrees.
Silymarin is a mixture of several flavonoids with four major components: silibinin, isosilibinin, silychristin, and silydianin(Fig. 1) [1-3].Silibinin is known to be a mixture of two diastereomers(Note 1),silibinin A and silibinin B in nearly equimolar proportions(Fig. 2) [8].



Fig.1 Silymarins extracted from the seeds of Milk thistle



Fig. 2 Reversed-phase HPLC separation and photodiode array spectra of silibinin A and B
Diastereomeric isomers show nearly identical spectra but are separated by HPLC columns.


Milk thistle has been empirically suggested to have a protective effect on liver from toxins, but as chemical studies have progressed, it has been found that its extract, silymarin, in particular, silibinin, has a strong effect [1-3]. In addition to its effect on the liver, it has been reported to exhibit anti-cancer effects such as inhibition of cell proliferation, epithelial-mesenchymal transition (EMT) induction inhibition, and apoptosis induction in some cases. Silibinin has been shown to inhibit cancer progression in many types of cancer cells, including human prostate cancer cells [9], human mammary gland cancer cells [9, 10], human colon cancer cells [11], human lung cancer cells [12], and human skin cancer cells [13]. In addition to its direct anti-cancer effects, silibinin may also be used for chemoprevention [11, 14]. I.e., suppressing the onset of cancer by its anti-inflammatory effects when taken before the onset of cancer, and for complementary and alternative medicine (CAM) [15] (Note 2). Furthermore, hepatoprotective properties of silibinin may also help reduce the side effects of anti-cancer drugs, making it an ingredient that deserves further and multifaceted research.
Our joint research with Prof. Ikejima on the effects of silibinin is still ongoing. We have shown that silibinin is effective not only in the treatment of liver disease and cancer, but also in increasing insulin production, which is important for maintaining blood glucose levels [16], and in treating mouse models of Alzheimer's disease [17] and Parkinson's disease [18, 19]. In addition to its effect on malignant tumor cells, silibinin has also been shown to prevent or restore cellular damage caused by excessive UV-B (ultraviolet light: wavelength 280-320 nm) irradiation of normal skin cells. Excessive UV-B irradiation induces damage to human skin cells (both fibroblasts and epidermal keratinocytes), such as cellular senescence (Note 3) or cell death (apoptosis) (Note 4), and skin inflammation. Addition of silibinin suppresses apoptosis when human skin fibroblasts are cultured under UV-B irradiation conditions that induce apoptosis. A similar phenomenon can be seen in epidermal keratinocyte cell line (HaCaT) culture. Recently, we reported two papers on the protective mechanism of silibinin against UV-B irradiation-dependent cellular damage. Silibinin promotes recovery from damage [20, 21].
Silibinin is a phytoestrogen, and its mechanism of action is thought to be by binding to the estrogen receptor (ER), exerting estrogen-like effects, and regulating the expression level and subcellular localization of the ER. Estrogen is a female hormone that depends on its concentration to affect skin homeostasis, physical characteristics of the human sex, and immune response [22]. Because estrogen usually acts on cells via the ER, ER expression levels significantly reflect estrogen activity. ERs are found in the nucleus and at the plasma membrane and act differently depending on their localization; there are two isoforms of ER, ERα and ERβ, which may have opposite effects on cell proliferation, either enhancing or inhibiting, but they share some common features in regulating cell function. However, they differ in terms of distribution in organs and cells (cell membrane or organelle) and biological and pathological functions [23].Detection of ERs expressed in fibroblasts and HaCaT under UV-B irradiation (apoptosis induction) conditions revealed increased expression and nuclear translocation of both ERα and ERβ upon silibinin addition. In addition, suppression of ER expression by siRNA method attenuated the apoptosis inhibitory effect of silibinin, suggesting that silibinin protects skin from UV-B by increasing ERs expression and nuclear translocation (Fig. 3) [20]. Although the physiological effects of estrogen are complex, it is known that the weakening of the effects of various female hormones, including estrogen, due to aging can cause loss of skin elasticity, increased susceptibility to osteoporosis, etc. Proper activation of ER is considered effective for anti-aging.



Fig.3 Silibinin suppresses apoptosis induced in UV-B irradiated skin cells via regulation of ER (estrogen receptor) expression and subcellular localization.


Another mechanism which we found is the regulation via binding of silibinin to the transcriptional coactivator YAP (Yes-associated protein) directly, which acts downstream of the Hippo signaling pathway. Hippo signaling pathway is involved in the regulation of organ size, cell proliferation, tumor suppression, and cell differentiation. Hippo signaling pathway has important consequences for morphogenesis and functional maintenance in a wide range of organisms. YAP is known to interact with many molecules. The regulation of YAP activity is unique and complex, regulated by phosphorylation and subcellular localization, as well as by interacting factors. YAP interacts with the transcription factor p73 in the nucleus to induce apoptosis in cells when cells are under stress, such as DNA damage. On the other hand, when it interacts with transcription factors, such as TEAD, it may also engage in conflicting regulation depending on the interacting molecules, such as by activating transcription of genes involved in cell proliferation, thereby contributing to the suppression of apoptosis. p73 is a homologous molecule of the tumor suppressor gene product p53 and functions as an apoptosis inducer [24]. We found that pretreatment in HaCaT and fibroblasts with silibinin under UV-B irradiation (apoptosis induction) conditions, inhibited cell apoptosis induction by directly interacting with YAP and blocking YAP nuclear translocation. On the other hand, the activity of the YAP-TEAD system was not affected by UV-B irradiation. Based on these results, we believe that the protective effect of silibinin on skin cells is exerted by affecting the balance between YAP and p73 expression and nuclear translocation, accompanied by the inhibition of apoptosis (Fig. 4) [21].



Fig.4 Silibinin circumvents apoptosis induced in UV-B irradiated skin cells by regulating YAP expression levels, subcellular localization and interaction with p73.

The two mechanisms of action via the ER or YAP-p73 pathway also suggest that silibinin regulates multiple defense mechanisms that skin cells have to cope with UV-B. Collaborative research is underway to elucidate the inhibitory effect of silibinin on iron ion-dependent cell death (ferroptosis) caused by UV-B irradiation.

Silibinin is an interesting phytochemical (Note 5) that exhibits anti-inflammatory and antioxidant effects on various cell types and is expected to have anti-aging effects regardless of gender. More interestingly, animal studies have shown that it can also reduce damage to the hippocampal region of the brain (aging and apoptosis) caused by excessive exercise such as marathon running [25].
Milk thistle is a member of the Asteraceae family, the same as ragweed and wormwood, and is generally considered safe, although allergies should be considered (Note 6). Milk thistle can be used both internally and externally. We hope to make use of the results of this joint research and contribute to the creation of a wellness (Note 7) society by utilizing the medicinal properties of silibinin.

Note 1
Diastereomer: One of the isomers of a molecule. A stereoisomer that is not a mirror isomer (enantiomer).
Note 2
Complementary and Alternative Medicine: The National Center for Complementary and Alternative Medicine in the United States defines this medicine as “a variety of medical and health care systems, procedures, and products that are not currently considered conventional medicine”. In other words, complementary and alternative medicine is what is called folk medicine, and refers to medical treatment that is performed at one's own discretion and responsibility.
Excerpts from “the Shikoku Cancer Center website”.
https://shikoku-cc.hosp.go.jp/cam/camwhat/index.html
Note 3
Cellular senescence: the gradual decline (aging) of the ability of a cell to divide.
Excerpts by Toshinori Ide, “Cell Fate IV: Cellular Aging,” Science Co.ISBN：978-4-7819-1127-4
Note 4 Apoptosis：a type of cell death in the body of multicellular organisms, a controlled and regulated cell suicide, or programmed cell death (narrowly defined, the caspase-dependent form of it), actively triggered to keep the individual in a better state. Synonym for necrosis.
Apoptosis from
https://en.wikipedia.org/wiki/Apoptosis
Note 5 Phytochemicals：are compounds that are said to be present in plants.The term is generally used to mean “plant-derived compounds that are not required for the maintenance of bodily functions but may affect health”.
[Phytochemicals] (version of Sunday, May 15, 2022 05:20 UTC), excerpted from Wikipedia, the free encyclopedia
https://ja.wikipedia.org/wiki/Phytochemical
Note 6 See Ministry of Health, Labour and Welfare website, “Project for Promotion of Information Dissemination on 'Integrated Medicine',” eJIM.
https://www.ejim.ncgg.go.jp/pro/overseas/c04/35.html
Note 7
Wellness: An internationally accepted definition of “health” by the World Health Organization (WHO) that takes a more in-depth and expansive view of health. The term wellness was coined in 1961 by American medical scientist Halbert L. Dunn, M.D. In simpler terms, it is a concept proposed to promote healthy living by incorporating exercise into daily life as appropriate, as a life science.
“Wellness” (version of Thursday, March 18, 2021 16:46 UTC), excerpted from Wikipedia, Japanese edition, partially modified.
https://en.wikipedia.org/wiki/Wellness_(alternative_medicine)

References

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