UVA Induce Pyroptosis In Fibroblasts By Activating Mitochondrial Pathway And The Protective Role Of Paeoniflorin Through This Mechanism

Background:Ultraviolet light in the atmosphere can cause a variety of skin problems.Prolonged exposure to the hot sun without protection can cause acute photodamage to the skin,characterized by erythema,blisters,and pain.The penetrating ability of UVA is strong,which can cause the damage of dermis cells.Fibroblasts,the cellular component of the dermis,secrete extracellular matrix and synthesize collagen and elastic fibers,and UVA can cause damage to fibroblasts resulting in imbalance of dermal matrix components,which in turn produces the manifestation of skin photoaging.Pyroptosis is a new regulatory cell death discovered in recent years,which is mediated by the gasdermin family of proteins.After the gasdermin proteins are cleaved,their N-terminal accumulate on the cell membrane and form pores,causing changes in intracellular osmotic pressure,which leads to cell swelling and death.This process is called pyroptosis.GSDME,as a member of the gasdermin protein family,has also been found to be involved in cell pyroptosis.GSDME can be cleaved by caspase-3 and form gasdermin E-N termimal which induces cell pyroptosis.Oxidative stress is the main factor of UVA-induced fibroblast damage.After oxidative damage of mitochondria,it activates apoptosis of the mitochondrial pathway,forming the Cyto c/Apaf-1/Caspase-9 apoptosis complex,and then cascading activates the apoptosis effector caspase-3,so caspase-3 as the upstream molecule of gasdermin E and the effector molecule of apoptosis,which links apoptosis with the pyroptosis.At present,it has been found that UVA can cause apoptosis and autophagy in fibroblasts,and whether it can cause pyroptosis and the involved pathway is unknown.Paeoniflorin is the main active ingredient of the Chinese medicine total glucosides of paeony,which is a monoterpenoid glycoside compound.As an antioxidant,paeoniflorin can reduce the oxidative damage of fibroblasts caused by UVA through activating Nrf2/HO-1 pathway.However,there is little research on paeoniflorin inhibits pyroptosis by reducing oxidative stress.In this study,we first explored whether UVA induces pyroptosis of fibroblasts and its pathway,then explored whether oxidative stress is involved,and then further explored whether paeoniflorin can play a protective role.Objective:The aim of this study was to find out the molecular mechanism of cell pyroptosis in skin fibroblast photodamage and explore the theoretical basis of paeoniflorin in skin photodamage protection,providing a new direction for skin photodamage protection and treatment.Methods:1.Define UVA causes pyroptosis of fibroblasts through oxidative stress.Immortalized human skin fibroblasts and dermal fibroblasts extracted from human foreskin tissue were used as research objects.Different doses of UVA(0,10,20,30J/cm~2)were irradiated into cells to establish a photodamage model.After irradiation,cell morphology was observed under the light microscope.Cell vitality,lactate dehydrogenase release and YO-PRO-1/PI fluorescence staining were used to detect cell damage,so as to select the appropriate dose to induce pyroptosis.The cells were treated with different concentrations of antioxidant NAC(0,1.25,2.5,5,10m M),and cell viability was detected to evaluate the drug toxicity of NAC and select the appropriate treatment concentration.Fibroblasts were treated with NAC after the UVA irradiation.After irradiation,cell morphology was observed under the light microscope.Cell vitality,lactate dehydrogenase release and YO-PRO-1/PI fluorescence staining were used to detect cell damage.2.Clarify the UVA-induced pyroptosis pathway and the effect of oxidative stress.After the cells were treated with appropriate dose of UVA,RT-q PCR and Western blot were used to detect partial molecules involved in pyroptosis at transcription level and protein level respectively,including NLRP3,caspase-1,caspase-3,caspase-4,GSDMD and GSDME.And then knocking down the exact molecule with plasmids was used to validate the activated pyroptosis pathway.Fibroblasts were treated with NAC after the UVA irradiation,and then PARP,caspase-3,GSDME were detected by Western blot to make sure the role of oxidative stress at the pyroptosis pathway.Determin the involvement of mitochondrial-mediated apoptosis pathway and oxidative stress.After the cells were treated with UVA irradiation,the changes of mitochondrial membrane potential were detected by JC-1,and molecules involved in mitochondrial-mediated apoptosis pathway were detected by Western blot,including Bax,Cyto c and caspase-9.The proteins of cells were extracted at different times after irradiation and then detected mitochondrial-mediated apoptosis pathway molecules and pyroptosis molecules respectively,to observe the order of their activation.To explore the effect of oxidative stress,fibroblasts were treated with NAC after the UVA irradiation.After irradiation,the changes of mitochondrial membrane potential were detected by JC-1.Bax,Bcl-2,Cyto c and caspase-9 were detected by Western blot.3.Clarify the inhibitory effect of paeoniflorin to pyroptosis.Cells were treated with different concentrations of paeoniflorin(0,100,200,400,800μM),and cell viability was detected to evaluate the toxicity of paeoniflorin and select the optimal concentration of paeoniflorin.For the drug treatment group,cells were treated with paeoniflorin for 3 hours,and then UVA irradiation was performed.After irradiation,paeoniflorin was added again and continue cell culture.Cell morphology was observed under the light microscope.Cell vitality,lactate dehydrogenase release and YO-PRO-1/PI fluorescence staining were used to detect cell damage.The changes of mitochondrial membrane potential were detected by JC-1.Bax,Bcl-2,Cyto c,caspase-9 and caspase-3,GSDME were detected by Western blot.Results:1.UVA irradiation caused pyroptosis of skin fibroblasts in a dose-dependent manner.UVA dose of 30J/cm~2was selected for subsequent experiments.When the fibroblast cells were treated with 5m M NAC to inhibit oxidative stress after light exposure,the proportion of pyroptosis was significantly reduced compared with those without NAC treated.2.When UVA dose was 30J/cm~2,the caspase-3/GSDME-dependent pyroptosis pathway was activated,but the NLRP3 inflammasome pathway of caspase-1/GSDMD was not activated.At the protein level,cleaved GSDME was found to be reduced in the knockdown of caspase-3 after the UVA irradiation.The activation of the caspase-3/GSDME pyroptosis pathway was inhibited in the NAC treated group.After UVA irradiation,the mitochondrial membrane potential decreased in a dose-dependent manner,while Bax and Cyto c,the related molecules of mitochondrial-mediated apoptosis pathway increased,and caspase-9 was activated through being cleaved.Moreover,the activation of mitochondrial-mediated apoptosis pathway was earlier than the occurrence of pyroptosis.Compared with untreated cells,the antioxidant NAC treated group showed that mitochondrial membrane potential increased,Bax,Cyto c and cleaved caspase-9expression decreased after UVA irradiation.3.After the cytotoxic test,400μM concentration of paeoniflorin was selected for subsequent experiments.Compared with untreated cells,the paeoniflorin treated group showed that mitochondrial membrane potential increased,Bax,Cyto c and cleaved caspase-9 expression decreased,caspase-3/GSDME pathway was inhibited,and the proportion of pyroptosis decreased after UVA irradiation.Conclusion:1.UVA induces pyroptosis in skin fibroblasts through oxidative stress.2.UVA induces mitochondrial-mediated apoptosis pathway activation,which activates the GSDME dependent pyroptosis pathway.3.Paeoniflorin can inhibit UVA-induced mitochondrial-mediated apoptosis pathway and pyroptosis by inhibiting oxidative stress.