Title: Trichosanthes kirilowii extract enhances repair of UVB radiation-induced DNA damage by regulating BMAL1 and miR-142-3p in human keratinocytes
Author: JI-HYE JOO, IN-KEE HONG, NAM KYOUNG KIM, EUNMI CHOI
Published journal name: MOLECULAR MEDICINE REPORTS (February, 2018)
Product name: Derma-CLOCK
INCI name: Trichosanthes Kirilowii Root Extract
Application: Skin(skin rejuvation cream, sensitive skin, etc), body, etc
Related information: Cosmetic composition for restoring UVB skin damage through regulation of miR-142-3p activity (10-1828056)
Ultraviolet B(UVB) radiation induces DNA damage, oxidative stress, inflammation, and suppresses the immune system in the skin, which collectively contribute to skin aging and carcinogenesis. The DNA damage response, including DNA repair can be regulated by the circadian clock and microRNA(miRNA) expression. The aim of this study was to evaluate the reparative action of Trichosanthes kirilowii extract(TKE) against UVB irradiation-induced DNA damage in human keratinocytes. TKE significantly upregulated the expression of the core clock protein brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1(BMAL1), while it downregulated miR-142-3p. Furthermore, the suppression of miR-142-3p by a specific inhibitor positively correlated with the repair activity. Consequently, TKE can be considered a potential candidate for the treatment of skin diseases related to UVB-induced damage. We have called this TKE a Derma-CLOCK® and decided to use it as a cosmetic ingredient.
Ultraviolet(UV) radiation leads to DNA damage, cell senescence, and apoptosis. It also results in the formation of the two most common lesions, cyclobutane pyrimidine dimers(CPDs) and 6-4 photoproducts(6-4 PPs). These lesions impede DNA replication and transcription and are repaired by nucleotide excision repair(NER). NER has a critical role in the repair of UV-induced DNA lesions and its actions involve endonucleolytic cleavage of two phosphodiester bonds followed by excision of the damaged DNA. The excised oligonucleotide is replaced by DNA repair synthesis, and the continuity of the DNA strand is re-established by DNA ligase.
The circadian rhythm is responsible for regulating various physiological processes such as hormone production, temperature, and the sleep pattern. It is found in most living beings including animals, plants, and fungi. The mammalian circadian clock system is organized as a central oscillator. The suprachiasmatic nucleus(SCN) and the peripheral oscillators are at the top of this system. The core clock oscillator includes the two heterodimeric transcriptional factors, CLOCK and brain and muscle aryl hydrocarbon receptor nuclear translocator(ARNT)-like protein-1 (BMAL1), which activates the transcription of their transcriptional repressors, Period (Per) and Cryptochrome(Cry).
The DNA damage response pathways include DNA repair and damage checkpoints and cell cycle arrest. Current evidence indicates that these pathways are related to the circadian clock. Furthermore, there is evidence that the normal circadian rhythm and gene expression levels in the skin are suppressed by UVB radiation as was demonstrated for the expression levels of Bmal1, Per, and Clock genes in human keratinocytes.
MicroRNAs(miRNAs) are small non-coding RNA molecules that are implicated in the regulation of gene expression by binding to the 3ʹ-untranslated regions(UTRs) of target mRNA and, thereby, interrupt protein translation. Numerous studies have shown that miRNAs play important roles in biological processes including cell proliferation, apoptosis, development, metabolism, and differentiation.
Recent studies have implicated miRNAs in the modulation of the circadian clock, revealing that miR-219 regulates the circadian period length, whereas miR-132 modulates Per gene transcription and protein stability. Although miR-142-3p is present in other cells, it plays a role in the post-translational modulation of BMAL1 in mouse SCN, NIH3T3 and human 293ET cells. However, the molecular mechanisms by which miR-142-3p mediates post-transcriptional regulation of BMAL1 in skin cells have not been elucidated. Several studies have reported an association between UV radiation exposure and changes in miRNA expression in keratinocytes. UVB radiation-exposed keratinocytes exhibit several specific patterns of miRNA response.
Trichosanthes kirilowii is a traditional medicine used in East Asia for treating patients with diabetes, cancer-related symptoms, coughing, and breast abscesses. However, the effects of T. kirilowii on skin cells and its influence on DNA damage repair have not been fully investigated. Therefore, the aim of this study was to investigate the effects of T. kirilowii extract(TKE) against UVB-induced DNA damage in skin cells. In addition, the role of miR-142-3p and BMAL1 in the T. kirilowii extract-mediated repair of UVB-induced DNA damage was investigated.
TKE promotes repair of UVB-mediated DNA damage in HaCaT cells. DNA damage is caused by UV radiation. Keratinocytes are influenced by UV radiation due to outermost layer of the skin. HaCaT cells are spontaneously immortal keratinocyte cell line that has been widely used for skin research. We first examined the effect of TKE at concentrations of 25, 50, 100, and 200 µg/ml on the viability of HaCaT cells treated for 24 h using the MTT assay. As shown in Figure 1A, TKE at 200 but not up to 100 µg/ml was significantly cytotoxicity and based on these results, we selected 50-100 µg/ml for the subsequent experiments. We determine the reparative and protective effects of 8 h treatment with TKE against DNA damage induced by UVB radiation in HaCaT cells using the comet assay.
Exposure of HaCaT cells to UVB radiation(12.5 mJ/cm2) induced extensive DNA damage as reflected in the difference in tail lengths between the comets of cells exposed to UVB radiation and those that were not (Figure 1B). However, treatment of UVB-exposed cells with TKE(100 µg/ml) reduced the DNA damage or fragmentation compared to that of the untreated UVB-exposed cells (Figure 1B). These findings demonstrate that TKE may not only have a role in DNA damage repair but may also protect against UVB-induced DNA damage.
TKE modulates BMAL1 mRNA and protein expression levels in UVB-irradiated HaCaT cells. Several reports have indicated that NER, the DNA repair system, is dependent on the circadian rhythm.
To further investigate the molecular mechanism by which TKE modulates DNA damage repair, we examined the changes in the expression of BMAL1, which plays a pivotal role in the circadian rhythm. UVB radiation decreased the mRNA and protein expression of BMAL1 compared with that of the unstimulated controls, which was consistent with previous studies reports using normal human keratinocytes. However, TKE(100 µg/ml) treatment markedly increased the expression of BMAL1 mRNA and protein (Figure 2A and B). Overall, TKE treatment influenced BMAL1 expression levels, suggesting that specific cellular response mechanisms may be involved in the TKE-mediated DNA damage repair in keratinocytes.
TKE upregulates BMAL1 expression via inhibition of miR-142-3p. It has been reported that miRNAs are closely associated with the regulation of DNA damage as well as the circadian rhythms. To investigate whether miR-142-3p is involved in the regulation of BMAL1 in human keratinocytes, HaCaT cells were transfected for 24 h with the miR-142-3p mimic and a mimic control. The qPCR analysis demonstrated that the miR-142-3p mimic dramatically increased the expression of miR-142-3p in HaCaT cells compared with cells transfected with the mimic control (Figure 3A). Furthermore, western blot analysis demonstrated that the miR-142-3p mimic dramatically inhibited BMAL1 protein expression (Figure 3B).
In summary, the downregulation of BMAL1 mRNA and protein expression in HaCaT cells suggests that miR-142-3p may be involved in the molecular mechanisms of the repair and protection of UVB-induced DNA damage.
Based on these findings, we examined the effects of TKE on miRNA expression levels in HaCaT
cells. To investigate whether the expression of BMAL1 is regulated by TKE through miR-142-3p modulation, RT-qPCR was performed in HaCaT cells treated with or without TKE. We found that in HaCaT cells exposed to UVB radiation(12.5 mJ/cm2), the expression levels of miR-142-3p increased after 24h, but in similarly exposed cells treated with TKE(100 µg/ml) for 24 h, a decrease in miR-142-3p levels was observed (Figure 3C). Taken together, these observations suggest that TKE-mediated DNA damage repair in HaCaT cells might be correlated with the suppression of miR-142-3p expression.
In addition to the experiments mentioned above, collagen synthesis and collagenase(MMP) inhibition were confirmed. Clinical experiments using creams with TKE showed increased elasticity and increased dermal density. Asaresult, it was confirmed that the anti-aging effect of Derma-CLOCK® was due to the ability to DNA damage repair using the circadian rhythm and miRNA.
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