Effects of sirolimus and 3-methyladenine on the autophagy in, and MMP-1 and MMP-3 secretion by, human skin fibroblasts / 中华皮肤科杂志

ABSTRACT

Objective To investigate the effects of sirolimus and 3-methyladenine (3-MA) on the autophagy in,as well as matrix matalloproteinase (MMP)-1 and MMP-3 secretion by,human skin fibroblasts (HSFs).Methods HSFs were isolated from the circumcised foreskin of a healthy male,and subjected to primary culture.After 3 to 10 passages,HSFs were incubated with sirolimus of 20,50,100,250 nmol/L and 3-MA of 0.5,2.0,5.0,10.0 mmol/L respectively for 4 hours followed by the observation of autophagy and detection of MMP-1 and MMP-3 levels in the supernatant by enzyme linked immunosorbent assay (ELISA).Those HSFs remaining untreated or treated with dimethyl sulfoxide served as the control.Results The percentage of autophagy-positive cells was 59.075％ ± 6.884％,76.350％ ± 5.226％,85.063％ ± 6.002％,86.288％ ± 5.558％ and 96.825％ ± 1.500％ respectively in HSFs treated with sirolimus of 0,20,50,100 and 250 nmol/L; significant differences were observed between the 5 groups (P ＜ 0.01 ) but not between the cells treated with sirolimus of 50 and 100 nmol/L (P ＞ 0.05).After being treated with 3-MA of 0,0.5,2.0,5.0 and 10.0 mmol/L,the percentage of autophagy-positive cells in HSFs was 63.037％ ± 5.876％,34.425％ ± 5.183％,19.700％ ± 3.028％,12.900％ ± 3.334％ and 7.775％ ± 2.293％ respectively with a significant difference between these groups (all P ＜ 0.01 ).Elevated levels of MMP-1 and MMP-3 were observed in the supernatant of HSFs treated with sirolimus of 250 nmol/L and 3-MA of 10.0 mmol/L (all P ＜ 0.05).Conclusions The autophagy in HSFs can be upregulated by sirolimus,but downregulated by 3-MA.For the secretion of MMPs by HSFs,3-MA and high concentrations of sirolimus exert a promotive effect,and the effect of 3-MA is in a concentration-related manner,but the influences of sirolimus at lower concentrations remain unclear.