1-O-β-(2 ,3-di-O-alka(e)noyl-6 -O-acetyl-D-mannopyranosyl)-D-erythritol

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

cell culture study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study follows generally accepted scientific standards and with detailed descriptions reported

Qualifier:

no guideline required

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

basic toxicokinetics

other: toxicodynamic i.e. in vitro proien binding in cultured human epidermal keratinocytes.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL:

- Source and lot/batch No.of test material: MELs used in the present study were produced and provided by DKBIO (Daejeon, Korea).

- Expiration date of the lot/batch: Not stated

- Purity test date: Not stated

RADIOLABELLING INFORMATION (if applicable): N/A

- Radiochemical purity:

- Specific activity:

- Locations of the label:

- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: Not stated
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
- Treatment of test material prior to testing: MELs were synthesized in a yeast strain of Pseudozyma sp., extracted from the culture broth with ethyl acetate.
- Preliminary purification step (if any): Purified through a fatty acid removal process. The purified product was predominant MEL-B with a purity of about 95% and their molecular weight range is 500 to 850.
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: Purified MELs were dissolved with dimethyl sulfoxide
and maximum concentration of 4 ug/ml was used in the experiment.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Dissolved with dimethyl sulfoxide

OTHER SPECIFICS: N/A

Species:

other: HaCaT keratinocytes (an immortalized cell line derived from normal human keratinocytes)

Strain:

other: human keratinocytes

Sex:

not specified

Details on test animals or test system and environmental conditions:

N/A - cell culture used: HaCaT keratinocytes (an immortalized cell line derived from normal human keratinocytes) cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 100 U/ml potassium penicillin, and 100 mg/ml streptomycin sulfate at 37°C in a humidified incubator containing 5% CO2. The cells were washed with phosphate-buffered saline, detached with Accutase , and plated to 60-mm dishes at 0.3 × 106 cells/well in fresh culture medium. Cells grown to 70%–80% confluence were serum-starved for 4 h and then treated with the indicated concentrations of reagents.

Route of administration:

other: cell culture

Vehicle:

unchanged (no vehicle)

Dose / conc.:

0.25 other: µg/mL

Dose / conc.:

1 other: µg/mL

Dose / conc.:

4 other: µg/mL

Details on results:

UVA-irradiation downregulates AQP3 expression at the protein and mRNA levels:
The AQP3 protein level was dose-dependently reduced under UVA irradiation, and significantly decreased to 50% of the level seen in the UVA non-irradiation control group under 3 J/cm2 of UVA irradiation (UVA 3 J/cm2). Similarly, qRT-PCR revealed that the mRNA expression of AQP3 was significantly reduced to 54% of the control level at 24 h after irradiation with UVA 3 J/cm2. A time-course experiment revealed that a significant decrease in AQP3 mRNA expression could be detected as early as 6 h after UVA irradiation, and this level continued to decrease gradually until 24 h post-irradiation. Since cell viability was not significantly different from the control group at 24 h after UVA 3 J/cm2 irradiation, subsequently UVA 3 J/cm2 was used to significantly reduce AQP3 expression in HaCaT keratinocytes.

MELs ameliorate the UVA-induced downregulation of AQP3:
UVA-induced downregulation of AQP3 protein expression was markedly and dose-dependently ameliorated by 1 and 4 µg/ml of MELs. These levels were increased by 124% and 142%, respectively, compared to the UVA 3 J/cm2-irradiated group. AQP3 mRNA expression was increased by 164% when UVA 3 J/cm2-irradiated cells were treated with 1 g/ ml MELs compared to those receiving UVA 3 J/cm2-irradiation alone. This indicates that MEL 1µg/ml is sufficient to alleviate the UVA-induced reduction of AQP3.

Phosphorylation of JNK is involved in the UVA- induced downregulation of AQP3:
Phosphorylation levels of the three MAPKs were significantly increased at both time points compared to those in non-irradiated cells. UVA-induced downregulation of AQP3 protein expression was markedly ameliorated by the JNK inhibitor, which brought this level up to 138% that of the UVA 3 J/cm2-irradiated group. Similarly, the mRNA expression level of AQP3 was 136% higher in the JNK inhibitor-treated group than in the UVA 3 J/cm2-irradiated group. In contrast, the inhibitors of ERK or p38 failed to rescue the UVA-induced decrease of AQP3 protein expression.

MELs ameliorate UVA-induced AQP3 downregulation by suppressing phosphorylation of JNK:
UVA-induced upregulation of phosphorylated JNK was suppressed by MEL pre-treatment, whereas the levels of phosphorylated ERK and p38 were unchanged. Particularly, JNK activation decreased PPAR-γ mRNA expression and subsequently led to AQP3 downregulation in hepatic stellate cells. These findings suggest the possibility that the MELs could up-regulate AQP3 expression through JNK inactivation and subsequently the increased PPAR-γ expression in UVA-irradiated HaCaT cells. The mRNA expression of PPAR-γ was downregulated by UVA 3 J/cm2 irradiation in HaCaT keratinocytes; however, this phenomenon was significantly mitigated by MELs or the JNK inhibitor. Taken together, MELs ameliorated UVA- induced AQP3 downregulation through JNK inactivation and PPAR-γ up-regulation in cultured human keratinocytes.

Conclusions:

Based on the condition of the study in the publication, it can be concluded that MEL-B treatment significantly ameliorated effects of UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels. The mitogen activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. The possible mechanism concluded to involves suppression of JNK activation to block the decrease of peroxidase proliferator-activated receptor gamma (PPAR-γ), which acts as a potent transcription factor for AQP3 expression. Collectively, these findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Executive summary:

Bae et al. 2018: The ameliorating effects of MELs on UVA-induced downregulation of AQP3 was investigated in cultured human epidermal keratinocytes (HaCaT keratinocytes). Prior to defining the effect of MELs on UVA-induced AQP3 downregulation, the dose of UVA for significantly downregulating AQP3 expression in the experimental system was first determined. HaCaT keratinocytes were irradiated with various doses of UVA (0.5, 1, 2, or 3 J/cm²) and analysed the protein level of AQP3 after 24 h. Since cell viability was not significantly different from the control group at 24 h after UVA 3 J/cm² irradiation, non-cytotoxic concentrations of MELs (0.25, 1, and 4 µg/ml) were selected based on the results of our cell viability assay and applied to cells for 24 h following UVA 3 J/cm² irradiation.

Since the MAPK pathway is reportedly involved in modulating AQP3 expression (Coa C 2006 & 2006), UVA- induced AQP3 downregulation was investigated in associated with changes in the MAPKs, JNK, ERK, and p38 MAPK. Serum-starved HaCaT keratinocytes were harvested at 15 min and 30 min after UVA 3 J/ cm² irradiation. Next, serum-starved cells were treated with an inhibitor of each MAPK for 1 h followed by UVA 3 J/cm² irradiation, and collection of cell lysates 24 h later.

The AQP3 protein level was dose-dependently reduced under UVA irradiation, and significantly decreased to 50% of the level seen in the UVA non-irradiation control group under 3 J/cm² of UVA irradiation (UVA 3 J/cm²). Similarly, qRT-PCR revealed that the mRNA expression of AQP3 was significantly reduced to 54% of the control level at 24 h after irradiation with UVA 3 J/cm². A time-course experiment revealed that a significant decrease in AQP3 mRNA expression could be detected as early as 6 h after UVA irradiation, and this level continued to decrease gradually until 24 h post-irradiation. Since cell viability was not significantly different from the control group at 24 h after UVA 3 J/cm² irradiation, subsequently UVA 3 J/cm² was used to significantly reduce AQP3 expression in HaCaT keratinocytes.

UVA-induced downregulation of AQP3 protein expression was markedly and dose-dependently ameliorated by 1 and 4 µg/ml of MELs. These levels were increased by 124% and 142%, respectively, compared to the UVA 3 J/cm²-irradiated group.  AQP3 mRNA expression was increased by 164% when UVA 3 J/cm2-irradiated cells were treated with 1 g/ ml MELs compared to those receiving UVA 3 J/cm²-irradiation alone. This indicates that MEL µ1 g/ml is sufficient to alleviate the UVA-induced reduction of AQP3.

Phosphorylation levels of the three MAPKs were significantly increased at both time points compared to those in non-irradiated cells. UVA-induced downregulation of AQP3 protein expression was markedly ameliorated by the JNK inhibitor, which brought this level up to 138% that of the UVA 3 J/cm²-irradiated group. Similarly, the mRNA expression level of AQP3 was 136% higher in the JNK inhibitor-treated group than in the UVA 3 J/cm²-irradiated group. In contrast, the inhibitors of ERK or p38 failed to rescue the UVA-induced decrease of AQP3 protein expression.

UVA-induced upregulation of phosphorylated JNK was suppressed by MEL pre-treatment, whereas the levels of phosphorylated ERK and p38 were unchanged. Particularly, JNK activation decreased PPAR- γ mRNA expression and subsequently led to AQP3 downregulation in hepatic stellate cells. These findings suggest the possibility that the MELs could up-regulate AQP3 expression through JNK inactivation and subsequent the increased PPAR- γ expression in UVA- irradiated HaCaT cells. The mRNA expression of PPAR- γ was downregulated by UVA 3 J/cm² irradiation in HaCaT keratinocytes; however, this phenomenon was significantly mitigated by MELs or the JNK inhibitor. Taken together, MELs ameliorated UVA- induced AQP3 downregulation through JNK inactivation and PPAR-γ up-regulation in cultured human keratinocytes.

Based on the condition of the study in the publication, it can be concluded that MEL treatment significantly ameliorated effects of UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels. The mitogen activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. The possible mechanism concluded to involves suppression of JNK activation to block the decrease of peroxidase proliferator-activated receptor gamma (PPAR- γ), which acts as a potent transcription factor for AQP3 expression. Collectively, these findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Description of key information

It can be concluded that MEL treatment significantly ameliorated effects of UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels. The mitogen activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. The possible mechanism concluded to involves suppression of JNK activation to block the decrease of peroxidase proliferator-activated receptor gamma (PPAR- γ), which acts as a potent transcription factor for AQP3 expression. Collectively, these findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Additional information

Bae et al 2018: The ameliorating effects of MELs on UVA-induced downregulation of AQP3 was investigated in cultured human epidermal keratinocytes (HaCaT keratinocytes). Prior to defining the effect of MELs on UVA-induced AQP3 downregulation, the dose of UVA for significantly downregulating AQP3 expression in the experimental system was first determined. HaCaT keratinocytes were irradiated with various doses of UVA (0.5, 1, 2, or 3 J/cm²) and analysed the protein level of AQP3 after 24 h. Since cell viability was not significantly different from the control group at 24 h after UVA 3 J/cm² irradiation, non-cytotoxic concentrations of MELs (0.25, 1, and 4 µg/ml) were selected based on the results of our cell viability assay and applied to cells for 24 h following UVA 3 J/cm² irradiation.

Since the MAPK pathway is reportedly involved in modulating AQP3 expression (Coa C 2006 & 2006), UVA- induced AQP3 downregulation was investigated in associated with changes in the MAPKs, JNK, ERK, and p38 MAPK. Serum-starved HaCaT keratinocytes were harvested at 15 min and 30 min after UVA 3 J/ cm² irradiation. Next, serum-starved cells were treated with an inhibitor of each MAPK for 1 h followed by UVA 3 J/cm² irradiation, and collection of cell lysates 24 h later.

The AQP3 protein level was dose-dependently reduced under UVA irradiation, and significantly decreased to 50% of the level seen in the UVA non-irradiation control group under 3 J/cm² of UVA irradiation (UVA 3 J/cm²). Similarly, qRT-PCR revealed that the mRNA expression of AQP3 was significantly reduced to 54% of the control level at 24 h after irradiation with UVA 3 J/cm². A time-course experiment revealed that a significant decrease in AQP3 mRNA expression could be detected as early as 6 h after UVA irradiation, and this level continued to decrease gradually until 24 h post-irradiation. Since cell viability was not significantly different from the control group at 24 h after UVA 3 J/cm² irradiation, subsequently UVA 3 J/cm² was used to significantly reduce AQP3 expression in HaCaT keratinocytes.

UVA-induced downregulation of AQP3 protein expression was markedly and dose-dependently ameliorated by 1 and 4 µg/ml of MELs. These levels were increased by 124% and 142%, respectively, compared to the UVA 3 J/cm²-irradiated group.  AQP3 mRNA expression was increased by 164% when UVA 3 J/cm²-irradiated cells were treated with 1 µg/ml MELs compared to those receiving UVA 3 J/cm²-irradiation alone. This indicates that MEL 1 µg/ml is sufficient to alleviate the UVA-induced reduction of AQP3.

Phosphorylation levels of the three MAPKs were significantly increased at both time points compared to those in non-irradiated cells. UVA-induced downregulation of AQP3 protein expression was markedly ameliorated by the JNK inhibitor, which brought this level up to 138% that of the UVA 3 J/cm²-irradiated group. Similarly, the mRNA expression level of AQP3 was 136% higher in the JNK inhibitor-treated group than in the UVA 3 J/cm²-irradiated group. In contrast, the inhibitors of ERK or p38 failed to rescue the UVA-induced decrease of AQP3 protein expression.

UVA-induced upregulation of phosphorylated JNK was suppressed by MEL pre-treatment, whereas the levels of phosphorylated ERK and p38 were unchanged. Particularly, JNK activation decreased PPAR- γ mRNA expression and subsequently led to AQP3 downregulation in hepatic stellate cells. These findings suggest the possibility that the MELs could up-regulate AQP3 expression through JNK inactivation and subsequent the increased PPAR- γ expression in UVA- irradiated HaCaT cells. The mRNA expression of PPAR- γ was downregulated by UVA 3 J/cm² irradiation in HaCaT keratinocytes; however, this phenomenon was significantly mitigated by MELs or the JNK inhibitor. Taken together, MELs ameliorated UVA- induced AQP3 downregulation through JNK inactivation and PPAR- γ up-regulation in cultured human keratinocytes.

Based on the condition of the study in the publication, it can be concluded that MEL treatment significantly ameliorated effects of UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels. The mitogen activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. The possible mechanism concluded to involves suppression of JNK activation to block the decrease of peroxidase proliferator-activated receptor gamma (PPAR- γ), which acts as a potent transcription factor for AQP3 expression. Collectively, these findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.