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Investigation Of The Protective Effect And Mechanism Of Paeoniflorin Against Photodamage Induced By UVA Radiation In Skin

Posted on:2021-05-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y S LuFull Text:PDF
GTID:1364330611992147Subject:Dermatology and Venereology
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Objective:Ultraviolet A(UVA)radiation is a major environmental challenge to the skin.As the consequence of UVA over-exposure,skin cells produce reactive oxygen species(ROS)including singlet oxygen,superoxide anions and hydrogen peroxide.ROS interact with lipid-rich membranes,enzymes and cellular DNA,and may change their structures and interfere with their functions.Accumulated oxidative injuries in skin cells are major causes of photodamages,photoaging and photo-carcinogenesis.Present studies show that paeoniflorin(PF)has the properties of anti-inflammatory,anti-oxidative stress,anti-tumor,and immunomodulatory.Although there was evidence suggested PF could protect keratinocytes from Ultraviolet B(UVB)induced cell damages,no studies have revealed the interactions between PF,UVA and skin cells.Lipid-binding protein perilipin2(PLIN2)known as adipose differentiation-related protein,has been previously to be involved in the regulation of oxidative stress.In this study,we detected the protective property of PF against UVA radiation and the function of PLIN2 in vivo and in vitro,and detected the underlying mechanism.This study may provide potential antiphotodamage therapeutic strategy.Methods:1.In our study,primary human dermal fibroblasts were isolated from circumcised foreskins.Fibroblasts were exposed to different doses of UVA(0,20,22.5,25 J/cm~2),and MTS assay was used to detect the cell viability of fibroblasts at24,48,72 hours after exposed to UVA.Then the cytotoxicity of PF on human fibroblasts was examined by MTS.Cells were treated with different concentrations of PF(0,200,400,800μM)and cell viability was detected at 24,48,and 72 hours.The cytoprotective function of PF(0,200,400,800μM)against UVA radiation was then evaluated by MTS assay.Cellular morphology of fibroblasts visualized by inverted microscope and cells apoptosis detected by using flow cytometry were used to confirm the cytoprotective properties of PF against UVA on human dermal fibroblasts.The protective property of PF was also detected in vivo.C57BL6 mice were received PF(200 mg/kg in 0.9%NS daily)for 7 days before UVA(30 J/cm~2 once)exposure.Mice were euthanized 24 hours after UVA radiation,and dorsal skins were rapidly obtained.The histological properties of mouse skin were assessed using H&E staining.2.After the demonstration of the protective property of PF,we detect the anti-oxidant property of PF against UVA.The generation of intracellular ROS was detected by fluorescence microscopy.MDA levels and SOD activity were detected by a microplate reader.Western blot assay was used to detect the protein levels of Nrf2,HO-1 and NQ-O1.To detect whether the antioxidant property of PF was mediated by Nrf2,fibroblasts were transfected with Nrf2 siRNA.Then we detected the protein levels of Nrf2,HO-1 and NQ-O1,MDA and cell viability after UVA radiation and PF pre-treatment+UVA.3.We identified the expression level of PLIN2 under UVA radiation or PF pre-treatment+UVA by Western blot and RT-qPCR assay in fibroblasts.Immunohistochemical staining was used to detect PLIN2 in the skin of mice.Western blot was used to detect the expression of PLIN2 after Nrf2 knockdown to evaluate the relationship between PLIN2 and oxidative stress.Then we examined its function by overexpressing PLIN2 with lentiviral vector in fibroblasts.MDA production was examined to evaluate the level of oxidative stress.The cell proliferation was detected by MTS and EdU staining.The protective functions of PLIN2 overexpression and accompanied with PF against UVA were detected by MTS and MDA assay.Results:1.We found that PF had no cytotoxicity to the fibroblasts,UVA radiation could induce cell death in a dose-dependent manner.UVA at the dose of 22.5 J/cm2was ultimately explored in our study,pre-treatment of PF at a concentration of 800μM could significantly neutralize the cytotoxicity of UVA.PF pre-treatment could prominently alleviate the cells apoptotic of UVA radiation.Mice pre-treated with PF were likely to be protected from dermal impairments.2.PF could attenuate ROS formation induced by UVA in fibroblasts.Pre-treatment of PF has reversed the impact of UVA on MDA and SOD.Western blot assay revealed that the protein levels of Nrf2,HO-1 and NQ-O1 were increased in PF treated cells.However,Nrf2 knockdown abrogated the increase of NQ-O1 and HO-1 after UVA radiation and PF pre-treatment+UVA,and PF was unable to alter the impacts of UVA on MDA and cell viability in absence of Nrf2.In consistency with the in vitro work,we also found additive increases in Nrf2,HO-1 and NQ-O1expression in the skin of mice pre-treated with PF before the UVA exposure,when compared to UVA alone.3.Western blot and RT-qPCR showed significantly higher level of PLIN2 in the cells of UVA treated compared to untreated cells,while PF+UVA showed lower level of PLIN2 than the cells treated with UVA alone.The same expression of PLIN2 was observed in the mice skin by immunohistochemical staining.We have detected that Nrf2 silencing could lead to increase of PLIN2 expression.We found a significant increase in cell proliferation in PLIN2 overexpressed cells compared with the negative cells.PLIN2overexpressed cells have displayed significantly lower level of MDA compared with the negative control group.Furthermore,PLIN2 overexpressed cells displayed significantly higher levels of cell viability and lower level of MDA production compared with UVA radiation cells.More importantly,PLIN2 overexpression combined with PF pre-treatment were likely to enhance the cytoprotective function of single agent alone by showing even less reductions in cell viability and MDA generation induced by UVA radiation.Conclusion:1.PF inhibited UVA-induced injuries in human dermal fibroblasts and mice.2.PF inhibits UVA induced damage in fibroblasts and mouse skin through the Nrf2/HO-1/NQ-O1 pathway.3.PLIN2 was associated with the regulation of oxidative stress stimulated by UVA radiation and PF treatment.PLIN2 overexpression promoted cell proliferation and inhibited oxidative stress in human dermal fibroblasts.PLIN2 overexpression and the combination of PF and PLIN2 overexpression demonstrated protective effect against UVA related oxidative stress.
Keywords/Search Tags:Paeoniflorin, UVA, Oxidative stress, Nrf2, PLIN2
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