Baicalin inhibits ultraviolet A-induced oxidative damage and apoptosis in human fibroblasts

Abstract

Objective To investigate the inhibitory effect of baicalin on ultraviolet A (UVA)-induced oxidative damage and apoptosis in human diploid fibroblasts (HDFs) and its possible mechanism.Methods Diploid fibroblasts were isolated from healthy adult male foreskin tissue and subjected to primary culture and subculture.To determine the working concentrations of baicalin,CCK-8 assay was performed to evaluate the proliferation of HDFs treated with various concentrations (3.125-100 mg/L) of baicalin alone or in combination with UVA irradiation.Then,some HDFs were divided into 5 groups:the control group receiving no treatment,UVA group irradiated with UVA alone,3 combination groups irradiated with UVA followed by treatment with baicalin at 6.25 mg/L,12.5 mg/L and 25 mg/L,respectively.After additional culture for 24 hours,fluorescence microscopy and flow cytometry were performed to detect and quantify reactive oxygen species,superoxide anion and nitric oxide,flow cytometry to determine mitochondrial membrane potentials and cell apoptosis rate,and Western blot to measure the expression of caspase 3.One way analysis of variance was carried out to compare these parameters among these groups.Results Compared with the control group,the UVA group showed a significantly higher level of reactive oxygen species,superoxide anion and nitric oxide (813.1 ± 30.29 vs.442.5 ± 29.45,353.3 ± 19.13 vs.223.4 ± 10.67,107.1 ± 11.44 vs.42.17 ± 2.59,all P ＜ 0.05),together with elevated mitochondrial membrane potential (771.6 ± 68.27 vs.429.5 ± 27.14,P＜ 0.05) and apoptosis rate (18.14 ± 2.492 vs.2.90 ± 0.574,P＜0.05).There was a significant decrease in the level of reactive oxygen species,superoxide anion and nitric oxide,mitochondrial membrane potential and apoptosis rate in the 3 combination groups compared with the UVA group (all P ＜ 0.01).The relative expression level of caspase-3 was significantly higher in the UVA group than in the control group (1.2680 ± 0.0091 vs.0.3675 ± 0.1115,P ＜ 0.05),but statistically lower in the 3 combination groups irradiated with UVA followed by treatment with baicalin of 6.25,12.5 and 25 mg/L,respectively than in the UVA group (0.5588 ± 0.1705,0.5365 ± 0.1718 and 0.5454 ± 0.2083 vs.1.2680 ± 0.0091,all P ＜ 0.01).Conclusions Baicalin can attenuate UVA-induced oxidative damage and apoptosis in HDFs,likely by scavenging reactive radicals,inhibiting mitochondrial membrane depolarization,and blocking the activation and expression of the downstream effector of apoptosis,caspase-3. Key words: Ultraviolet rays; Fibroblasts; BAICALIN; Oxidative stress; Apoptosis