RESUMO
Objective To evaluate the effect of isotretinoin on expression of ache-associated inflammatory genes induced by peptidoglycan in human SZ95 sebocytes,and to explore the molecular mechanism underlying the treatment of acne with isotretinoin.Methods Cultured SZ95 sebocytes were divided into 3 groups:control group receiving no treatment,peptidoglycan group treated with 20 mg/L peptidoglycan alone,and costimulation group treated with 20 mg/L peptidoglycan combined with 10-5 mol/L isotretinoin.After 3-hour treatment,real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of pro-inflammatory cytokines including interleukin (IL)-1α,IL-1β,IL-6,IL-8,tumor necrosis factor (TNF)-α,Toll-like receptor 2 (TLR2) and MyD88 (a downstream gene of TLR2) in SZ95 sebocytes in the above groups.After 24-hour treatment,enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of IL-1α,IL-1β,IL-6,IL-8 and TNF-α in the cell culture supernatant in the above groups.After 48-hour treatment,Western blot analysis was conducted to determine the protein expression of TLR2 and MyD88.Statistical analysis was carried out with SPSS 23 software by one-way analysis of variance (ANOVA) for the comparison among the 3 groups,and by Bonferroni method for multiple comparisons.Results The mRNA and protein expression of pro-inflammatory cytokines including IL-1α,IL-1β,IL-6,IL-8 and TNF-α all significantly differed among the 3 groups (all P ＜ 0.01),and was significantly higher in the peptidoglycan group than in the control group and costimulation group (both P ＜ 0.016 7).The mRNA expression of MyD88 also significantly differed among the control group,peptidoglycan group and costimulation group (6.707 ± 0.950,10.270 ± 0.477,7.892 ± 0.900 respectively,F =10.17,P ＜ 0.01),and was significantly higher in the peptidoglycan group than in the control group and costimulation group (t =4.740,3.298 respectively,both P ＜ 0.016 7).The mRNA and protein expression of TLR2 were markedly higher in the peptidoglycan group than in the control group,but did not differ between the peptidoglycan group and the costimulation group.Conclusion Isotretinoin can inhibit peptidoglycan-induced expression of inflammatory factors possibly associated with the occurrence of acne in human SZ95 sebocytes,likely by inhibiting the expression of MyD88,but not TLR2,in the innate immune response,which may be one of the mechanisms underlying the treatment of acne with isotretinoin.
RESUMO
Objective To estimate the effect of the enviromental pollutant 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD),a representative of the dioxin family,on the expression of cytochrome P4501A1 (CYP1A1) in cultured human immortalized SZ95 sebocytes in vitro,so as to improve understanding of the pathogenesis of chloracne.Methods SZ95 sebocytes were cultured with or without the presence of 10 nmol/L TCDD for two hours or three days.Real time fluorescence-based PCR was performed to quantify the mRNA expression of CYP1A1,immunohistochemistry and Western blot to determine the expression level of CYP1A1 protein,in the SZ95 cells.Chi-square test was done to compare the protein and mRNA expressions of CYP1A1 between untreated and treated SZ95 cells.Results Real time PCR showed that the mRNA expression of CYP1A1 was low in SZ95 sebocytes,and increased by 5.622 times after 2-hour treatment with TCDD(P ＜ 0.05).Immunohistochemistry revealed a weak expression of CYP1A1 protein in the cytoplasm and nuclei of untreated SZ95 sebocytes,which was also significantly enhanced by the TCDD treatment.Western blot results showed that the relative expression level of CYP1A1 protein was 4.233 ± 0.252 in SZ95 sebocytes treated by TCDD for three days,significantly higher than that in untreated sebocytes(0.123 ± 0.208,P ＜ 0.05).Conclusions There is a low expression of CYP1A1 mRNA and protein in SZ95 sebocytes,which can be upregulated by TCDD,suggesting that the CYP1A1 gene is a downstream target of the aryl hydrocarbon receptor responsible for the abnormal differentiation of human sebocytes.