A Pivotal Role Of GPX4 Deletion-mediated Lipid Peroxidation In Skin Cell Senescence

Research background:Oxidative stress is considered to be one of the important factors causing skin aging.Skin aging caused by both internal and external factors is closely related to oxidative stress damage to skin cells.In addition to general free radicals,some studies have reported that non-enzymatic reactions induce lipid-active free radicals involved in the process of skin aging.In recent years,with the discovery of the novel form of cell death,ferroptosis,enzyme-mediated lipid peroxidation has begun to attract researchers'attention.This form of lipid peroxidation,which is distinct from the non-enzymatic reaction-mediated lipid peroxidation,plays a pivotal role in the physiological and pathological processes.However,it is still unclear whether and how enzyme-mediated specific lipid peroxidation,especially the oxidation of phospholipids,participates in skin cell senescence.Glutathione peroxidase 4(GPX4)is the only enzyme that directly targets lipid reactive oxygen species on biofilms,and its deletion can cause accumulation of lipid peroxidation and destruction of structure and function of cell bio membrane.Therefore,this study will explore the role and mechanism of GPX4 and its mediated lipid peroxidation on skin aging.It aims to reveal the mechanism of skin aging and provide a new target for the prevention and treatment of skin aging and skin related diseases.Research methods and results:First,aging phenotypes,lipid peroxidation level,and GPX4 expression were examined in human skin at different ages,mice skin at different months,and ultraviolet(UV)-irradiated mice skin,respectively.(1)The skin tissues of normal human eyes were collected and divided into young adult skin group and old adult skin group according to age,and the aging phenotypes,lipid peroxidation level and GPX4 expression were detected.The results of Sirius Red and Masson staining showed significant collagen loss in the skin of old adult.Western blot analysis showed that phosphorylation level of p53 and the protein expression of p21 were up-regulated in the skin of old adult,lipid peroxidation product 4-HNE levels were also increased.The results of q RT-PCR,Western blot and immunofluorescence showed that the expression of GPX4 was significantly down-regulated in the skin of old adult.(2)the back skin of young adult and old adult mice were collected,the results of H&E,Sirius Red and Masson staining showed that the dermis of the old adult mice was thinner and the collagen fibers were significantly lost,compared to young adult mice skin.The result of senescence-associated?-galactosidase(SA?-Gal)staining showed that the expression of SA?-Gal was significantly increased in the old adult mice skin.Western blot results showed that the phosphorylation level of p53 was increased and the protein expression of p21 was up-regulated in the old adult mice skin.Further,LC-MS,assay kit and Western blot were used to detect the levels of GSH,MDA and 4-HNE in skin tissue.GSH content was significantly decreased and the levels of lipid peroxidation products MDA and 4-HNE were significantly increased in the old adult mice skin.The expression of GPX4in mice skin was detected by q RT-PCR,Western blot and immunofluorescence and the results showed that expression of GPX4 was decreased in the old adult mice skin.The above results indicated that the aging phenotypes of mice skin were appeared,lipid peroxidation was increased and GPX4 was down-regulated,with the increase of months.(3)The mice skin photoaging model was established by UV irradiation,and lipid peroxidation level and GPX4 expression were detected on the model.The results showed that the expression of SA?-Gal was increased,the level of collagen fibers was decreased,the level of lipid peroxidation was increased and the expression of GPX4was decreased in the skin of UV-irradiated mice.The results of the above three human and animal skin aging models indicated that aging skin was accumulated with lipid peroxidation,with the down-regulation of GPX4 expression,suggesting that GPX4may be involved in the accumulation of lipid peroxidation in aging skin.Secondly,GPX4 specific inhibitor RSL3 and GPX4 knockdown were used in human dermal fibroblasts(HDFs)to further investigate the role of GPX4 in skin cell senescence.(1)By morphological observation,it was found that HDFs treated with RSL3 become larger and flatter compared to the normal HDFs.Cell growth curve experiment showed that RSL3 inhibited the growth rate of HDFs.We further detected the cell proliferation by using the Ed U labeling assay and the results showed that RSL3reduced the number of Ed U positive cells,furtherly confirming the growth inhibition effect of RSL3 on HDFs.Subsequently,SA?-Gal and immunofluorescence staining were performed to detect senescence indicators,and the results found that RSL3significantly increased the ratio of SA?-Gal positive cells and promoted the formation of senescence-associated heterochromatin focis(SAHF).(2)GPX4 was depleted by si RNA targeting GPX4 in HDFs,and we found that HDFs whose GPX4 were low expression displayed decrease of proliferative ability,increase of SA?-Gal positive cell rate and SAHF formation,which indicating that GPX4 knockdown induced senescence of HDFs.The results of the above in vitro experiments indicated that GPX4 deletion can induce senescence of HDFs.Finally,we investigated whether GPX4 deletion mediated lipid peroxidation is involved in skin cell senescence and explored its regulatory mechanisms.Lipid peroxidation was measured by immunofluorescence and flow cytometry.RSL3significantly increased the level of oxidized C11-BODIPY and Liperfluo(a specific lipid peroxidation marker)in HDFs.Similarly,GPX4 knockdown also increased the oxidized C11-BODIPY level.Lipidomic based on LC-MS analysis showed that RSL3significantly increased the content of oxidative phospholipids(ox-PL),especially ox-PE and ox-PC in HDFs treated with RSL3.Moreover,significant lipid peroxidation was not detected in the H₂O₂-induced cell senescence model.We also found that the lipid peroxidation scavenger Ferrostatin-1(Fer-1)significantly inhibited RSL3-induced cell senescence and lipid peroxidation levels,while the conventional free radical scavenger NAC did not show the same effect.The above results indicated that GPX4 deletion triggers specific lipid peroxidation,rather than general ROS to induce senescence of HDFs.Furthermore,we detected the expression of DNA damage marker?-H2AX by immunofluorescence,phosphorylation level of Chk-1 and the expression of p53-p21senescence-related pathway by Western blot.The results showed that there was no obvious DNA damage in HDFs treated with RSL3,but the phosphorylation of p53 and the protein expression of p21 were up-regulated,and this change was effectively reversed by Fer-1,indicating lipid peroxidation mediated by GPX4 deletion activated the p53-p21 senescence-related pathway.Studies have found that promyelocytic leukemia-nuclear bodies(PML NBs)play an important role in cell senescence and regulation of p53.Therefore,immunofluorescence,Western blot and q RT-PCR were used to detect the formation of PML NBs and the expression of PML.RSL3 treatment or GPX4 knockdown significantly increased the expression of PML,and promote the formation of PML NBs,while Fer-1 could inhibit the formation of PML NBs in HDFs treated with RSL3.In addition,we also found that RSL3 could not induce cell senescence after PML knockdown.These results indicated that GPX4 deletion-mediated lipid peroxidation increased the protein expression of PML and promoted its formation in the nucleus,which in turn activated the p53-p21 pathway to induce cell senescence.At the same time,we also found that RSL3 could promote skin aging in mice in vivo,more fully demonstrating the role of GPX4 in skin aging.Research conclusions:In this study,we found that skin aging was associated with accumulation of lipid peroxidation and low expression of GPX4 by using different in vivo aging models.We elucidated the role of GPX4 on skin cell senescence by using specific inhibitors RSL3and GPX4 knockdown and further demonstrated that skin cell senescence induced by GPX4 depletion-mediated lipid peroxidation is associated with activation of the p53-p21 pathway by promoting the formation of PML NBs.