The Effect Of Aps On NF-?B Signaling Pathway And Cytokine IL-1??IL-8 And TNF-? Induced By P.acnes

Object: Our study aims to discuss whether the L-ascorbic acid-2-phosphate(APS)could suppress nuclear factor-kappa B(NF-?B)signaling pathway induced by(propionibacterium acnes,P.acnes)and thereby reduced cytokine interleukin-1?(IL-1?),interleukin-8(IL-8)and tumor necrosis factor-?(TNF-?)secreted from sebaceous gland cells.And it can help us further understand the inflammatory reaction of acne vulgaris so as to provide some new methods for the treatment of acne vulgaris.Methods: 1.The cultivation of SZ95 human sebaceous gland cells: we recovered and cultured the SZ95 human sebaceous gland cells,when the cell grew up to a stable state,it could be frozen.2.We activated and cultivated propionibacterium acnes according to the instruction,then we picked propionibacterium acnes which had identified as the propionibacterium acnes and prepared bacterial suspension with APS solution at different con-centrations.Every time it should be prepared when using.3.Detection of the proliferation of SZ95 human sebaceous gland cells by cell counting kit-8 method:We firstly added different concentrations of APS solution,and then we detected the proliferation of SZ95 human sebaceous gland cells after 24 hours.4.After propionibacterium acnes suspensions stimulated SZ95 human sebaceous gland cells 24 hours later,we detected the expression of cytokine interleukin-1?(IL-1?),interleukin-8(IL-8)and tumor necrosis factor-?(TNF-?)by the quantitative real-time PCR detecting system(RT-PCR)and enzyme-linked immunosorbent assay(ELISA),and detected the expression of p-I?B? in the cytoplasm and NF-?B p65 in the cytoplasm and nucleus by western blot(WB).5.We firstly added different concentrations of APS solution with propionibacterium acnes,then we detected the expression of cytokine IL-1?,IL-8 and TNF-? by the RT-PCR and ELISA,detected the expression of p-I?B? in the cytoplasm and NF-?B p65 in the cytoplasm and nucleus by WB after 24 hours.Results: 1.We could observe SZ95 human sebaceous gland cells to overspread on the culture bottom and grow along the wall after 4-days inoculation,we passaged cells every 2-3 days and the cells were overcrowding after passaged 3 days.2.After cultivation in 37? anaerobic environment for 48 hours,propionibacterium acnes could grow grayish white bacterial colonies with consistent forms on blood plates.3.APS solution within 1.5625mmol/L –12.5mmol/L concentration,were co-cultured with SZ95 human sebaceous gland cells for 24 hours,which could facilitate the proliferation of the SZ95 human sebaceous gland cells.However,APS solution within 25mmol/L –200mmol/L concentration,could refrained the proliferation of the SZ95 human sebaceous gland cells,having significantly statistic difference(P<0.01).So,we screened out appropriate concentration of APS with 2.5mmol/L,5 mmol/L and 10 mmol/L,and the higher of APS concentration was,the bigger of proliferation rate was,having significantly statistic difference(P<0.01).4.We detected the expression of cytokine interleukin-1?(IL-1?),interleukin-8(IL-8)and tumor necrosis factor-?(TNF-?)by the quantitative real-time PCR detecting system(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)after 24 hours,which had increased with a different extent as compared with control group and it had significantly statistic difference(P<0.05).At the same time,we find out that p-I?B? protein in the cytoplasm and NF-?B p65 protein in the nucleus was higher than that of control group,while NF-?B p65 in the cytoplasm was lower than blank group.5.We firstly added different concentrations of APS solution with propionibacterium acnes,and then we detected the expression of cytokine IL-1?,IL-8 and TNF-? by the RT-PCR and ELISA,which reduced to some extent as compared with control group in a concentration dependent manner,and the higher APS concentration was,the greater decrease of IL-1??IL-8?TNF-? was,having significantly statistic difference(P<0.05).We also found out that the expression of p-I?B? protein in the cytoplasm and NF-?B p65 protein in the nucleus was reduced than that of P.acnes group,and the higher concentration of APS was,the weaker expression of protein was.However,the expression of NF-?B p65 protein in the cytoplasm was higher than P.acnes group,and the higher concentration of APS was,the higher expression of protein became.Concsusion: 1.propionibacterium acnes could induce the expression of cytokine IL-1?,IL-8 and TNF-? secreted from SZ95 human sebaceous gland cells.2.propionibacterium acnes could influence NF-?B signaling pathway and induce the expression of cytokine IL-1?,IL-8 and TNF-? secreted from SZ95 human sebaceous gland cells.3.APS could suppress NF-?B signaling pathway induced by P.acnes and thereby reduced cytokine IL-1?,IL-8 and TNF-? secreted from SZ95 human sebaceous gland cells.