Gypenosides Intervention Photoaging HaCaT Cells To HSF Cells In P38MAPK Signal Pathway

Purpose：In vitro simulated sunlight in the ultraviolet radiation, study on the effectivemethod in making cells photoaging model, research mechanism of HaCaT affect to HSF inthe process of skin photoaging, to explore the related inflammatory factors and p38MAPKsignal transduction pathway in the mechanism of two cells, and found the effect mechanismof gypenosides in prevention and treatment of skin photoaging, have a more comprehensiveunderstanding of the full.Material and method：Using UVA light on HSF cell to found cells photoaging model.The HSF recovery, passage to1×104/ml through, founding cells photoaging model byself-made ultraviolet irradiation device, HSF using UVA light (5lamps side byside,365nmUVA,15cm) according to the formula: UV dose=UV radiation intensity×time,dose of UVA is36J/cm2.The HaCaT were divided into blank control group(group A), UVB model group(group B),blank serum group(group C), serum group(group D), group A without UVB irradiation,group B, C, D were applied UVB irradiation(35mJ/cm2). Preparation of gypenosidescontaining serum, according to Meeh-Rubner’s formula in rats, gypenosides powder dosagewas160mg/(Kg d), according to the dose, gypenosides grain solution on rats were orallytreated with normal saline, once a day, intragastric administration for7days, seventh days ofnormal gastric perfusion,1H later, get abdominal aortic blood, put saponins of gypenosidesmedicated serum into group D, blank serum with normal saline was given to rats by gavagewith group C, serum collection method is same to group D. Using Elisa method to detect thegroup A-D in IL-1β, IL-6, TNF-level.HSF were divided into groupⅠ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ. GroupⅠwithout applying the UVAirradiation, groupⅡ, Ⅲ, Ⅳ, Ⅴ, Ⅵwere applied after UVA irradiation, the HaCaTsupernatant of cultured cells in A-D group were added to HSF group Ⅲ-Ⅵ. Using RT-PCRmethod and Western blotting method to detect p38MAPK, c-Jun, c-Fos, MMP-1in mRNA and protein level.Results：1. HSF morphological changesHSF cells showed typical characteristics photoaging changes after UVA irradiation. Cellfusion degree lower, cell edge became fuzzy and irregular, et al.2. Photoaging modelThe irradiation dose of HSF photoaging model have no gold standard, detected the OD value,determine the maximum efficiency ratio. The irradiation dose of UVA is36J/cm2. Self-madeultraviolet irradiation device can successfully establish HSF cells photoaging model, at thesame time, this method is economical, simple and practical.3. Elisa Results: low expression of IL-1β,IL-6and TNF-in normal HaCaT.(1)IL-1β level: compared with blank control group, expression of UVB model groupincreased significantly (P＜0.01); compared with the UVB model group, serum group weresignificantly lowered (P＜0.01); compared with blank serum group, serum group weresignificantly lowered (P＜0.01).(2)IL-6level: compared with blank control group, expression of UVB model group increasedsignificantly (P＜0.01); compared with UVB model group, serum group were significantlylowered (P＜0.01); compared with blank serum group, serum group were significantlylowered (P＜0.01).(3)TNF-level: compared with blank control group, expression of UVB model groupincreased significantly (P＜0.01); compared with UVB model group, serum group weresignificantly lowered (P＜0.01); compared with blank serum group, serum group weresignificantly lowered (P＜0.01).4. HSF p38MAPK results: low expression of HSF in both mRNA and protein level.Compared with groupⅠ, the expression of groupⅡincreased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅳ increased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅲ p38MAPK were significantly lowered inprotein level (P＜0.05) and not significantly lowered in mRNA level（P＞0.05）. Compared with group Ⅳ, the expression of group Ⅵ were significantly lowered (P＜0.01). Comparedwith group Ⅳ, the expression of group Ⅴ were not significantly lowered（P＞0.05）.5. HSF c-Jun results: low expression of HSF in both mRNA and protein level.Compared with groupⅠ, the expression of groupⅡincreased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅳ increased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅲ were not significantly lowered in mRNAand protein level（P＞0.05）. Compared with group Ⅳ, the expression of group Ⅵ weresignificantly lowered (P＜0.01). Compared with group Ⅳ, the expression of group Ⅴ werenot significantly lowered（P＞0.05）.6. HSF c-Fos results: low expression of HSF in both mRNA and protein level.Compared with groupⅠ, the expression of groupⅡincreased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅳ increased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅲ were not significantly lowered in mRNAand protein level（P＞0.05）. Compared with group Ⅳ, the expression of group Ⅵ weresignificantly lowered (P＜0.01). Compared with group Ⅳ, the expression of group Ⅴ werenot significantly lowered（P＞0.05）.7. HSF MMP-1results: low expression of HSF in both mRNA and protein level.Compared with groupⅠ, the expression of groupⅡincreased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅳ increased significantly (P＜0.01);compared with group Ⅱ, the expression of group Ⅲ MMP-1were significantly lowered inprotein level (P＜0.05) and not significantly lowered in mRNA level（P＞0.05）. Comparedwith group Ⅳ, the expression of group Ⅵ were significantly lowered (P＜0.01). Comparedwith group Ⅳ, the expression of group Ⅴ were not significantly lowered（P＞0.05）.Conclusion：1. In vitro study on cell photoaging, the self-made ultraviolet irradiation device for preparingphotoaging model is an economical and simple method. HSF dose is36J/cm2.2. UVB irradiation can make HaCaT to release inflammatory factors (IL-1β, IL-6, TNF-).3. UVA irradiation can activate HSF p38MAPK signal transduction pathway, increase c-Fosand c-Jun, form dimers, increase the expression of MMP-1, further exacerbat the p38MAPKsignal transduction pathway. 4. HaCaT release inflammatory factor to promote the activation of HSF p38MAPK signaltransduction pathway in a paracrine way. This might be one of mechanism of HaCaT affectto HSF in the process of skin photoaging.5. Gypenosides can decrease HaCaT to release inflammatory factors, so as to reduce theeffects of HaCaT on HSF p38MAPK signal transduction pathway related mRNA and proteinin paracrine way. This might be one mechanism of gypenosides in prevention and treatmentskin photoaging.6. Gypenosides can directly inhibit HSF p38MAPK signal transduction pathway relatedmRNA and protein. This might be another mechanism of gypenosides in prevention andtreatment skin photoaging.