ABSTRACT
Objective To estimate the effect of ginsenoside Rb1 on the production and clearance of cyclobutane pyrimidine dimer (CPD) as well as on the expression of two nucleotide excision repair-associated proteins,xeroderma pigmentosum group C (XPC) and excision repair cross-complementing group 1 (ERCC1),by ultraviolet B (UVB)-irradiated murine epidermal cells and human HaCaT keratinocytes.Methods Totally,42 BALB/c mice were shaved on the back and divided into four groups: untreated group (n =6),UVB group irradiated with UVB only (n =12),low-dose and high-dose Rb1 group (both n =12) treated with Rb1 of 0.5 g/L and 2g/L (100 μl/cm2) respectively two hours before UVB irradiation.The dose of UVB in the animal experiment was 180 mJ/cm2.Half of the mice in each group were killed at 0.5 and 16 hours respectively after the irradiation,then,the back skin was resected and subjected to the determination of CPD levels in the epidermis by immunohistochemical SP method.Some cultured HaCaT cells were divided into several groups to be treated with different concentrations (5,20,50 mg/L) of Rb1 before or after different doses (15 and 30 mnJ/cm2) of UVB irradiation,and cells were collected at 0.5 and 12 hours after the irradiation.Subsequently,genomic DNA was extracted and CPD was detected by dot blot hybridization.Some HaCaT cells were cultured with or without the presence of Rb1 (50 mg/L) and irradiated with UVB (30 mJ/cm2),then,the cells were collected immediately or at 0.5,2,4 and 12 hours after the irradiation,and total protein was extracted and subjected to immunoblot analysis for the quantification of XPC and ERCC1 proteins.Results There was a high level of CPD in the epidermis of mice at 0.5 hour after the irradiation,with no significant differences between these groups (P ＞ 0.05).The number of CPD-positive cells per high power field (× 400) in the murine epidermis at 16 hours was statistically lower in the low-and high-dose Rb1 group than in the UVB group (32.1 ± 8.5 and 14.6 ± 4.1 vs.67.3 ± 11.2,both P ＜0.01).The CPD level in HaCaT cells was similar between these groups at 0.5 hour after UVB irradiation,but was markedly decreased at 12 hours in Rb1-treated groups.After UVB irradiation,the protein expressions of XPC and ERCC1 decreased with time in untreated HaCaT cells but increased with time in Rb1 (50 mg/L)-treated HaCaT cells.In detail,the XPC/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein ratio in untreated HaCaT cells was 0.68 ± 0.11 immediately after the irradiation,significantly higher than that at 0.5 hour (0.47 ± 0.09,P＜0.05),2 hours (0.45 ± 0.08,P＜0.05),4 hours (0.37 ± 0.06,P＜0.01),and 12 hours (0.18 ± 0.03,P ＜0.01),and that in Rb1-treated HaCaT cells was 0.56 ± 0.07 immediately after the irradiation,compared to 0.48 ± 0.14 at 0.5 hour (P＞ 0.05),0.68 ± 0.15 at 2 hours (P＞ 0.05),0.97 ± 0.20 at 4 hours (P＜0.01),and 0.79 ± 0.12 at 12 hours (P ＜0.05).The ERCC1/GAPDH protein ratio in untreated HaCaT cells was 0.28 ± 0.03 immediately after the irradiation,higher than that at 0.5 hour (0.25 ± 0.03,P ＞ 0.05),2 hours (0.21 ± 0.02,P＜0.05),4 hours (0.14 ± 0.02,P＜0.01) and 12 hours (0.11 ± 0.01,P＜0.01),and that in Rb1-treated HaCaT cells was 0.27 ± 0.04 immediately after the irradiation,compared to 0.24 ± 0.04 at 0.5 hour (P＞ 0.05),0.29 ± 0.05 at 2 hours (P＞ 0.05),0.35 ± 0.05 at 4 hours (P＜0.05),0.39 ± 0.05 at 12 hours (P ＜0.01).Conclusions Ginsenoside Rb1 shows no obvious effect on the UVB-induced production of CPD,but markedly accelerates the clearance of CPD,which may be partly associated with the upregulation of XPC and ERCC1 protein expression.
ABSTRACT
Objective To investigate the production and removal of cyclobutane pyrimidine dimer (CPD) by HaCaT cells after UVB irradiation, and the effect of baicalin in this process. Methods HaCaT cells were cultured and irradiated with given dosages of UVB, and the production and removal of CPD by HaCaT cells at given time points after UVB irradiation were assessed by immunohistochemical method. In parallel studies, HaCaT cells were preincubated with baicalin, and the effect on CPD was evaluated. Results The damage to HaCaT cells was dependent on the dosage of UVB radiation. After irradiation with 30 mJ/cm2 of UVB, CPD formation peaked at 0.5 h. CPD was removed rapidly from HaCaT cells during the first 4 h; the rate of removal decreased thereafter, and the removal was almost complete by 24 h after the irradiation. The amount of CPD decreased significantly in HaCaT cells that were preincubated with baicalin solution before UVB irradiation than that in those without the preincubation (U = 2.324, P