Enzyme-Treated Asparagus Extract Prevents Hydrogen Peroxide-Induced Pro-Inflammatory Responses by Suppressing p65 Nuclear Translocation in Skin L929 Fibroblasts

Abstract

We recently reported that enzyme-treated asparagus extract (ETAS) attenuates hydrogen peroxide (H 2 O 2)-stimulated matrix metalloproteinase-9 expression in skin fibroblast L929 cells. To further elucidate the anti-aging effects of ETAS on skin, we examined whether ETAS has preventive effects on H 2 O 2-induced pro-inflammatory responses of skin fibroblasts. H 2 O 2 induced Ser536 phosphorylation and nuclear accumulation of nuclear factor-κB (NF-κB) p65, and increased the mRNA levels of interleukin-12α (IL-12α) and inducible nitric oxide synthase (iNOS) in L929 cells. Pretreatment of the cells with JSH-23, an inhibitor of NF-κB nuclear translocation, abolished the H 2 O 2-induced expression of IL-12α and iNOS, indicating that the increased transcription is regulated by p65. The H 2 O 2-stimulated nuclear accumulation of p65 and induction of IL-12α and iNOS mRNA were significantly attenuated after pretreatment with ETAS for 3 h, and these responses were completely abolished when the duration was extended to 24 h. However, ETAS did not affect the H 2 O 2-stimulated degradation of IκBα and phosphorylation of p65. On the other hand, ETAS treatment for 24 h resulted in decreased protein levels of importin-α. These results suggest that ETAS prevents pro-inflammatory responses by suppressing the p65 nuclear translocation in skin fibroblasts induced by H 2 O 2 .

Anti‐Inflammatory Effect of ETAS®50 by Inhibiting Nuclear Factor‐κB p65 Nuclear Import in Ultraviolet‐B‐Irradiated Normal Human Dermal Fibroblasts

“…Although ETAS 50 significantly repressed the accumulation of p65 protein in the nuclear fraction after UV-B irradiation ( Figure 4(a) ), ETAS 50 treatment did not influence the reduced level of cytosolic I κ B α protein after UV-B irradiation ( Figure 4(b) ), suggesting that ETAS 50 inhibits nuclear translocation of p65 without affecting the machinery involved in its liberation from I κ B α . We also recently reported that ETAS 50 pretreatment attenuated hydrogen peroxide-induced IL-12 α and iNOS mRNA expression by suppressing p65 nuclear accumulation without restoring cytosolic I κ B α protein levels in murine L929 skin fibroblast cells [ 21 ], and ETAS 50 did not reduce hydrogen peroxide-induced protein carbonylation, an index of protein oxidation [ 20 ]. The present and previous findings suggest that ETAS 50 modulates the machinery for p65 nuclear import without affecting upstream events after UV irradiation, such as ROS generation and ROS-induced signal transduction, which mediates p65 liberation from I κ B α .…”

“…In the present study, the same batch of ETAS 50 (Amino Up Chemical Co. Ltd.) was used as in previous studies [ 20 , 21 ]. Methods for preparing ETAS 50 are briefly described as follows [ 14 , 15 , 22 ]: (1) fresh bottom parts of asparagus were boiled in water at 121°C for 45 min; (2) the boiled bottom parts of asparagus and the resulting extract were treated with cellulase and pectinase which are widely used in food industry; (3) after these enzymes were inactivated by incubation at 121°C for 20 min, the extract was centrifuged and the resultant supernatant was mixed with dextrin (Pinedex; Matsutani Chemical Industry, Hyogo, Japan) as a filler; (4) the mixture was then concentrated in vacuo at 105°C, sterilized at 121°C for 45 min, and then spray-dried to produce an ETAS 50 powder consisting of 52.6% ETAS 50 and 47.4% dextrin.…”

“…The dose is commonly utilized in in vitro experiments [ 10 , 12 , 23 , 24 ] and is within the range of natural environment because it is approximately one seventieth of the average daily accumulated amount in Japan. To assess its effects, ETAS 50 or dextrin (vehicle) was directly dissolved in complete medium to produce a final concentration of 1 mg/mL, and then the supplemented medium was sterilized using a Millex 0.22- μ m Syringe-Driven Filter Unit (Merck Millipore, Darmstadt, Germany) [ 20 , 21 ]. Immediately after UV-B irradiation, the cells were cultured in supplemented medium for different periods.…”

“…Moreover, we recently reported that ETAS 50 attenuates hydrogen peroxide-induced MMP-9 expression by suppressing phosphorylation of c-Jun N-terminal kinase and activator protein-1 c-Jun subunit [ 20 ], as well as hydrogen peroxide-induced IL-12 α and iNOS expressions by suppressing the nuclear translocation of NF- κ B p65 subunit in murine skin L929 fibroblasts without reversing the carbonylation level of proteins, an index of protein oxidation [ 21 ]. To determine the antiphotoaging potencies of ETAS 50, this study aimed to clarify whether and how ETAS 50 prevents UV-B-induced proinflammatory responses in normal human dermal fibroblasts (NHDFs).…”

ETAS®50 Attenuates Ultraviolet‐B‐Induced Interleukin‐6 Expression by Suppressing Akt Phosphorylation in Normal Human Dermal Fibroblasts

“…We recently reported that ETAS 50 suppresses hydrogen peroxide-induced interleukin-12 α (IL-12 α ) and iNOS mRNA expression in murine skin L929 fibroblasts and UV-B irradiation-induced IL-1 β mRNA expression in normal human dermal fibroblasts (NHDFs), by inhibiting nuclear import of p65 [ 19 , 20 ]. Moreover, ETAS 50 attenuated hydrogen peroxide-induced MMP-9 expression by suppressing the phosphorylation of JNK and its downstream transcription factor c-Jun [ 21 ].…”