Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I

Abstract

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H₂O₂) production in the blood. Hochu-ekki-to significantly inhibited ROS and H₂O₂ generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Keywords: Hochu-ekki-to; NETosis; UV-B irradiation; histone citrullination; inflammation; neutrophil extracellular trap.

Figure 1

Effect of Hochu-ekki-to and DNase I on dorsal skin dermatitis 5 days after UVB irradiation. (a): Representative macroscopic images of the dorsal skin in mice from each group 5 days after the final UVB irradiation session. (b): Draize scores of dorsal skin samples from mice 5 days post-final UVB irradiation session. The data are expressed as the mean ± SD of six animals per group. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates; * p < 0.05, ** p < 0.01).

Figure 2

Histological analysis of dorsal skin dermatitis 5 days after UVB irradiation. (a): Histological analysis of dorsal skin samples from mice 5 days after UVB irradiation using hematoxylin and eosin staining. The data reflect one typical experiment with six animals per group. Scale bar = 100 μm. (b): At the end of the study, the dorsal skin thickness in mice was measured. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates; * p < 0.05, ** p < 0.01).

Figure 3

Effect of Hochu-ekki-to and DNase I on reactive oxygen species generation in blood after UVB irradiation. Generation of reactive oxygen species in the blood after UVB irradiation using the OxiSelect In Vitro ROS/RNS Assay Kit. (a): Total ROS, (b): H₂O₂. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates; * p < 0.05, ** p < 0.01).

Figure 4

Effect of Hochu-ekki-to and DNase I on the expression of Ly6G (a,b). Immunostaining was performed to examine Ly6G expression. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates; * p < 0.05, ** p < 0.01).

Figure 5

Effect of Hochu-ekki-to and DNase I on the expression of PAD4 (a,b). Immunostaining was performed to examine the expression of PAD4. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates; * p < 0.05, ** p < 0.01).

Figure 6

Effect of Hochu-ekki-to and DNase I on the expression of citH3 (a,b). Immunostaining was performed to examine the citH3 expression. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates; * p < 0.05, ** p < 0.01).

Figure 7

Hochu-ekki-to decreases A23187-induced neutrophil extracellular trap formation (NETosis). After treatment with or without 50, 500, and 1000 ug/mL Hochu-ekki-to for 30 min, NETosis levels in nHL-60 cells induced with 10 μM A23187 for 1 or 6 h were analyzed using a SYTOX green assay. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test using the SPSS (version 20) software program (biological replicates).