Study On Matrix Metalloproteinase-2 Expression In Human Dermal Fibroblasts By Propionibacterium Acnes

Objective:To investigate whether Propionibacterium acnes (P. acnes) stimulates the expression of matrix metalloproteinases-2 (MMP-2) in human dermal fibroblasts (HDF), and the role of human tumor necrosis factor-alpha (TNF-a) in the process, in order to learn more about the mechanism of acne inflammation and matrix remodeling after inflammation so that we can provide some new ideas for the treatment of acne vugaris. Methods: 1. Specimens from normal persons of circumcision in our laser chamber, we washed their foreskin and removed the epidermis and subcutaneous tissue, then cut dermis into small pieces, we cultured the separated cells into the medium in 37℃,5%CO2 incubator after digestion by trypsin. We observed the morphology of the cultured cells and determined them to be HDF by using the immunofluorescence identification. We cultured and passaged cells, the fifth to eighteenth passages of HDF were used for the experiments.2. We conducted anaerobic cultivation of P.acnes at 37℃ for 48 hours, the colonies of P.acnes were selected out and suspended in phosphate buffered saline (PBS) and washed by centrifugation, after that we lysed P.acnes by sonication on ice, the bacterial lysates was filtered with a microfiltration membrane of 0.22μm.3. We detected the expression of TNF-a in HDF which was stimulated by P.acnes lysates at the concentration of 1mg/ml、0.1mg/ml for 3h,6h,12h respectively by enzyme-linked immunosorbent assay (ELISA).4. P.acnes lysates at the concentration of 1mg/ml and TNF-a at the concentration of lOng/ml were used to stimulate HDF for 24hours respectively, we observed the expression of MMP-2 in HDF by using a confocal laser scanning microscope.5. HDF were stimulated by P.acnes lysates at the concentration of 1mg/ml, 0.1mg/ml, by TNF-α at the concentration of lOng/ml, lng/ml, and were pretreated overnight with anti-TNF-α antibody at the concentration of 1ng/ml then were treated with P.acnes lysates at the concentration of lmg/ml for different hours respectively. We evaluated the protein level of MMP-2 by western blot and the messenger ribonucleic acid (mRNA) expression level of MMP-2 by real-time quantitative polymerase chain reaction (real-time PCR) in HDF. Results:1. Scanning inverted microcope showed that cells presented with long spindle-shaped adherent growth, cells were passage after 7-10 days and coverd about 5 days, cultured cells could be long-term passaged. Cells were identified as HDF due to the Vimentin-positive rate was up to 99% by immunofluorescence.2. P.acnes colonies grew significantly under anaerobic cultivation for 48hours.3. P.acnes lysates at the concentration of lmg/ml、 0.1mg/ml stimulated HDF in time gradient (3h,6h,12h), the value of TNF-a in HDF was increased in different degrees with statistically significant (P<0.05) when compared with the control group by ELISA, the level of TNF-a increased from 3 hours after treatment, reached the highest level at 6 hours, and decreased gradually after 12 hours. The value of TNF-a stimulated by P.acnes lysates at the concentration of 1mg/ml was much higher than 0.1mg/ml, with statistically significant differences (P<0.01).4. The fluorescence intensity of MMP-2 was enhanced in HDF and the level of MMP-2 expressed in the cytoplasm of HDF was increased by P.acnes lysates or TNF-a compared with the control group.5. P.acnes lysates at the concentration of 1mg/ml and TNF-a at the concentration of lOng/ml stimulated HDF in time gradient (3h,6h,12h), the mRNA and protein level of MMP-2 in HDF was upregulated in different degrees when compared with the control group by real-time PCR and western blot with statistically significant differences (P<0.05), MMP-2 expression increased from 6 hours and reached the maximum value at 24 hours in a time-dependent manner.6. P.acnes lysates at the concentration of lmg/ml and 0.1mg/ml stimulated HDF for 24hours, the expression of MMP-2 was significant differences compared with the control group (P<0.05), and MMP-2 was upregulated in a dose-dependent manner in the presence of P.acnes lysates, a statistically significant difference was obtained at the concentration of 1mg/ml and 0.1mg/ml (P<0.05).7. TNF-a at the concentration of lOng/ml and lng/ml stimulated HDF for 24 hours, the expression of MMP-2 was significant differences compared with the control group (P<0.05), and MMP-2 was increased in a dose-dependent manner in the presence of TNF-α, a statistically significant difference was obtained at the concentration of lOng/ml and lng/ml (P<0.01).8. HDF were pretreated overnight with anti-TNF-α antibody at the concentration of 1ng/ml, then treated with P.acnes lysates at the concentration of lmg/ml for 24 hours, MMP-2 expression appeared to reduce, differences between anti-TNF-α antibody group and control group were statistically significant (P<0.01). Conclusion:1. P.acnes lysates could stimulate HDF to produce a number of TNF-α.2. P.acnes lysates and TNF-α could upregulate the mRNA and protein expression level of MMP-2 in time-dependent or dose-dependent manner in HDF.3. The anti-TNF-α antibody could inhibit the expression of MMP-2 that induced by P.acnes lysates, so we speculate that P.acnes lysates induced MMP-2 production may be mediated by TNF-α in HDF.