Protective Effect And Mechanism Of Ellagic Acid On Human Keratinocytes Damage Caused By Zinc Oxide Nanoparticels

Object: Zinc oxide nanoparticles(ZnO NPs),one of the metallic oxide nanoparticles,are widely used in daily life.It is often added to cosmetics and sunscreens products due to its good anti-UV resistance.Studies have shown that ZnO NPs can enter the body through respiratory tract and dermal tract,thereby inducing body damage.Studies have shown that the skin toxic effects caused by ZnO NPs are caused by oxidative stress,and the NRF2 pathway and autophagy play an important role in this.Ellagic acid(EA)is a naturally occurring polyphenol chemical found in many fruits and vegetables,which has various pharmacological properties such as anti-oxidant,anti-inflammatory,anti-tumor and anti-depressant,and has been widely used in medicine and food fields.The antioxidant effect of EA is obvious,and it has been found that EA can reduce skin damage caused by UVA,but the role and molecular mechanism of EA in skin toxicity induced by ZnO NPs is still unclear.In this study,the protective effect of EA on skin damage caused by ZnO NPs is investigated by examining the role and mechanism of EA in the induction of skin cytotoxicity by ZnO NPs.Methods: 1.The hydrodynamic diameter and Zeta potential of ZnO NPs were tested using dynamic light scattering instrument.2.Human keratinocytes cell was selected as the research model,cell viability assay using the CCK-8 kit to assess the skin toxicity properties of ZnO NPs and the effect of EA on ZnO NPs-induced skin toxicity;fluorescent enzyme markers to detect the effect of EA intervention following ZnO NPs on the mitochondrial membrane potential(MMP)of cells,The m RNA of autophagy-related genes P62 and LC3 B and the protein expression of P62 and LC3BⅡ/Ⅰwere determined in the cells by RT-q PCR technique and Western Blot technique.3.The expression of mitochondrial fusion division factor MFN1,MFN2,DRP1 and mitophagy-related pathway PINK1 and PARKIN in HaCaT cells pretreated with EA,followed by treatment with ZnO NPs was detected by RT-q PCR,and protein results also indicated that the expression of mitochondrial autophagy proteins PINK1 and PARKIN increased.4.RT-q PCR and Western Blot techniques were applied to detect the gene and protein expression levels of NRF2 and its downstream antioxidant factors in HaCaT cells before and after EA treatment.5.The knockdown efficiency of HaCaT cells(NRF2-KD)with established stable trans-silencing NRF2 was verified using RT-q PCR technique.The effect of ZnO NPs on the survival rate of Scramble and NRF2-KD cells before and after EA treatment was detected using the CCK-8 kit;RT-q PCR and Western blot techniques were applied to detect the expression of NRF2 pathway and autophagy,mitochondrial autophagy-related genes and proteins in Scramble and NRF2-KD cells.Results: 1.HaCaT cells were exposed to different concentrations of ZnO NPs and EA and stimulated for 24 h.ZnO NPs decreased cell viability in a dose-dependent and time-dependent manner,and EA had no significant effect on cell viability,EA pretreatment followed reduced MMP loss compared with the ZnO NPs group.2.Compared with the control group,the expression of NRF2 and its downstream related protein HO-1 was upregulated in the ZnO NPs group,activating NRF2 and the downstream antioxidant genes HO-1,GCLC and GCLM;compared with the ZnO NPs group,the expression of NRF2 and HO-1 was significantly upregulated and the m RNA expression of NRF2,HO-1,GCLC and GCLM was increased after EA intervention;Western Blot assay results showed that compared with the control group,the expression of protein levels of autophagy proteins P62 and LC3 B II/Ⅰ were elevated in the ZnO NPs group,while the m RNA levels of P62 and LC3 B were also increased;compared with the ZnO NPs group,the protein levels of P62 and LC3 B II/Ⅰ were significantly elevated after EA intervention,and the m RNA levels of P62 and LC3 B also increased significantly.3.RT-qPCR results indicated that ZnO NPs treatment significantly decreased m RNA expression levels of mitochondrial fusion genes MFN1 and MFN2 and increased expression of mitochondrial mitotic gene DRP1.Compared with the ZnO NPs group,EA pretreatment significantly increased the m RNA expression levels of MFN1 and MFN2,decreased the expression of mitochondrial division genes DRP1,and up-regulated the m RNA and protein expression levels of mitophagy factors PINK1 and PARKIN.4.The cell viability of NRF2-KD cells was significantly reduced after EA pretreatment compared with Scarmble cells;the m RNA level expression of NRF2 downstream antioxidant factors were significantly inhibited in NRF2-KD cells after EA intervention compared with Scarmble cells;P62 protein and m RNA expression was increased and LC3BII/Ⅰ protein and m RNA expression was decreased;the m RNA expression levels of MFN1,MFN2 and DRP1 were significantly decreased,and the expression of mitophagy proteins PINK1 and PARKIN were significantly inhibited.Conclusion : 1.EA can reduce the toxic effects of ZnO NPs-induced HaCaT cells,reduction of mitochondrial damage,and enhance the ZnO NPs-induced NRF2 signaling pathway,cellular autophagy and mitophagy response.2.NRF2-Knockdown attenuates the protective effect of EA against ZnO NPs-induced HaCaT toxicity and inhibits the NRF2 signaling pathway and mitophagy.3.EA exerts a protective effect against ZnO NPs-induced HaCaT toxicity by activating mitophagy in dependence on NRF2.