The Effect Of Paeoniflorin On Melanocytes Under Oxidative Stress And Its Mechanism

Objective:Vitiligo is an acquired disorder,characterised by depigmented macules,which are caused by the loss of melanocyte function.Studies have shown that oxidative stress induced by hydrogen peroxide（H₂O₂）is a key factor in the occurrence and progression of vitiligo.The pathogenic role of oxidative damage in vitiligo is mainly manifested by the imbalance between the oxidative and antioxidant pathways,impaired activation of the nuclear factor E2-related factor 2-antioxidant response element（Nrf2-ARE）pathway,cell membrane lipid peroxidation,catalase（CAT）and superoxide dismutase（SOD）and other antioxidant enzyme activities decreased.Paeoniflorin（PF）is a monoterpenoid glycoside,the main active ingredient of total glucosides of paeony.It can be used as an antioxidant to promote melanin synthesis by activating the Nrf2/HO-1 pathway.Melanocytes are distributed in the basal layer of the epidermis,and transport melanin through dendritic cells to its adjacent keratinocytes.Changes in the cytoskeletal structure of melanocytes such as cell body size,dendritic length,and number are related to change of actin and the tube structure.RhoA and its downstream Rho-associated protein kinase 1（ROCK1）can regulate the actin cytoskeleton structure.Since the formation of new melanocyte dendrites requires the polymerization of actin,F-actin is involved in the regulation of the melanocyte cytoskeleton structure.Anti-oxidation response element（ARE）-mediated secondary antioxidant enzymes can prevent cellular damage caused by oxidative stress.Transcription factor Nrf2 plays a key role in this process.When the oxidative stress occurred,Nrf2 can dissociate from Kelch-like ECH-associated protein1（Keap1）and translocate to the nucleus,upregulate the expression of anti-oxidative stress genes,including NADPH quinone oxidoreductase（NQO1）and HO-1.Decreased Nrf2expression can lead to decreased ability of melanocytes to prevent oxidative damage in patients with vitiligo.So up-regulation of Nrf2 expression may be helpful for the treatment of vitiligo.However,it has not been reported whether paeoniflorin or total paeoniflorin can reduce oxidative damage to melanocytes in patients with vitiligo.In this study,the effect and mechanism of paeoniflorin on melanocytes induced by hydrogen peroxide were explored.Methods:The subjects of this study were immortalized normal human melanocyte cell line（PIG1）and immortalized vitiligo melanocyte cell line（PIG3V）.Paeoniflorin powder was dissolved in 250μl of paeoniflorin powder in a 250ul DMSO solution.The 200μM solution was stored frozen in a deep low-temperature refrigerator at-80°C,and diluted with 254 Medium to the required concentration for subsequent experiments.The effects of paeoniflorin（50μM,100μM,200μM,400μM）at different concentrations on melanocyte cell proliferation and cell activity were measured using the MTS method.Flow cytometry was used to measure cell cycle distribution and apoptosis,and oxidative stress markers,reactive oxygen species levels,were also measured using flow cytometry.The WST-8 method was used to determine the content of superoxide dismutase（SOD）,and the content of catalase（CAT）was determined using the catalase detection kit.The expression of oxidative stress-related genes was screened using the PCR array human oxidative stress kit.Differential gene protein expression levels were detected using Western Blot;the content and distribution of melanosomes and the cytoskeleton structure of F-actin were detection by the immunofluorescence staining.The expression of backbone-related proteins RhoA,ROCK1 and Nrf2 signaling pathway-related proteins were detected using Western Blot.At the same time,siRNA was used for gene knockout experiments,and sp600125 reagent was used for JNK gene suppression experiments.Data analysis was performed by GraphPad Prism version 7.0 software（GraphPad Software,San Diego,CA）.One-way ANOVA was used for multiple group comparisons.Data was presented as mean±SD（standard deviation）for at least three independent experiments.A P-value<0.05 was considered statistically significant.The rest of the pictures in this study were stitched using Photoshop 7.0 software.Results:Paeoniflorin promoted cell proliferation at both concentrations of 50μM and100μM,but cell proliferation was inhibited when the drug concentration was 200μM or400μM in PIG1.The drug concentrations at 50μM,100μM,200μM,400μM all promoted cell proliferation in PIG3V.Finally,Paeoniflorin concentration of 50μM was selected for subsequent experiments.Paeoniflorin pretreatment for 24h could not improve the reduction of cell viability after H₂O₂ treatment.However,paeoniflorin pretreatment for 48 h can significantly improve the cell viability of stressed cells（P<0.05）.In summary,the results of cell proliferation and cell viability showed that the paeoniflorin concentration was 50μM.Pretreatment not at 24 h but at 48 h could reduce H₂O₂-induced oxidative damage and increase cell viability.After 24 hours of H₂O₂ 1.0mM treated,PIG1 and PIG3V cells showed significant cell cycle arrest in the G2/M phase.The proportion of PIG1 cells in the G2/M phase increased from about 5%to 27%.The proportion increased from about 16%to 44%in PIG3V.After paeoniflorin pretreatment for 24 hours,the G2/M phase of the PIG1 and PIG3V cell cycle increased（P<0.05）,but it did not increase at 48 hours in stressed cells.Cell cycle results showed that paeoniflorin regulates the cell cycle of G2/M phase in stressed cells.After treatment with 1.0 mM H₂O₂ for 24 hours,the average percentage of apoptotic cells in PIG1 and PIG3V cells increased significantly to about 35%.Paeoniflorin pretreatment for 24 h can inhibit the apoptosis rate of two melanocytes（P<0.05）,but paeoniflorin pretreatment for48h cannot reduce the number of apoptotic cells of melanocytes induced by H₂O₂.Apoptosis results indicated that paeoniflorin can improve the oxidative damage of melanocytes induced by H₂O₂.PIG1 or PIG3V cells were pretreated with paeoniflorin for24h,then cells were treated with H₂O₂ for 24h,and markers related to oxidative stress were detected.After 24h of hydrogen peroxide treatment,the levels of oxidative stress-specific markers,reactive oxygen species was significantly increased in H₂O₂treated cells（P<0.05）,and paeoniflorin pretreatment for 24 hours could significantly reduce the level of ROS（P<0.05）.In PIG1 and PIG3V cells treated with H₂O₂,the activities of antioxidant enzymes SOD and CAT were significantly reduced.Therefore,paeoniflorin can significantly enhance the antioxidant capacity of melanocytes.The results of the PCR array showed that compared to the group of paeoniflorin and hydrogen peroxide,MB expression in PIG1 cells was up-regulated by 2.61-fold,PTGR1gene expression was down-regulated by 2.38-fold,and PIG3V cells were down-regulated by 3.64-fold.Compared with the PF+H₂O₂ group and the H₂O₂ group,the MB gene expression was up-regulated in PIG1 cells,and there was no significant difference in expression in PIG3V cells.PTGR1 gene expression was up-regulated in PIG1 cells and down-regulated in PIG3V cells.The expression of PDLIM1 gene in PIG1 cells was up-regulated at the protein level,and PIG3V was down-regulated.Since PDLIM1 gene is involved in the regulation of cytoskeleton composition,Western blot were performed to further detect whether paeoniflorin affects the expression of related skeleton proteins.Paeoniflorin was pretreated for 24 hours and then treated with 1.0 mM hydrogen peroxide for 24 hours.The results showed that paeoniflorin pretreatment in PIG1 cells for 24 hours up-regulated the expression of H₂O₂-induced skeletal proteins RhoA,ROCK and F-actin,while PIG3V down-regulated the expression of ROCK1 protein.PDLIM1 gene knockout-related experiments show that paeoniflorin in PIG1 cells inhibits the expression of RhoA and its downstream gene ROCK1 through PDLIM1 under oxidative stress.PDLIM1 inhibits ROCK1 expression in PIG3V cells.Knockout ROCK does not change PDLIM1 expression,but it can regulate F-actin expression.Immunofluorescence results suggested that the number of melanosomes and the F-actin content of skeletal protein in H₂O₂-induced oxidative damage cells were reduced.Paeoniflorin pretreatment for 24 hours could inhibit the reduction of melanosomes and cells induced by H₂O₂.Paeoniflorin in PIG1 and PIG3V cells both increase JNK/Nrf2/HO-1 expression.JNK in PIG1 cells positively regulates Nrf2 expression,whereas JNK in PIG3V cells inhibits Nrf2 expression.Further results showed that PDLIM1 gene expression was up-regulated in PIG1 and PIG3V cells after JNK gene suppression.After PDLIM1 gene knockout,Nrf2 gene expression was up-regulated in PIG1 and PIG3V cells,indicating that the JNK/Nrf2/HO-1 signaling pathway interacts with the PDLIM1/RhoA/ROCK1 pathway to protect melanocytes from oxidative damage.Conclusion:1)Paeoniflorin concentration of 50μM for 24h can promote the activity of PIG1 and PIG3V cells.H₂O₂-induced oxidative damage leads to cell cycle arrest in the G2/M phase,Paeoniflorin influenced the G2/M phase of the cell cycle and inhibits H₂O₂-induced melanocyte apoptosis.Paeoniflorin inhibits the increasing of reactive oxygen species levels of PIG1 and PIG3V induced by H₂O₂,and increased the levels of antioxidant enzymes SOD and CAT.2)Comparing PF+H₂O₂ group with H₂O₂ group,MB expression in PIG1 cells was up-regulated and PTGR1 was down-regulated,and PIG1 cells in PIG3V cells were down-regulated.When PF+H₂O₂ group compared with the H₂O₂ group,the protein expression of MB,PTGR1 and PDLIM1was up-regulated,However,the expression of MB protein was not significantly changed,and the expression of PTGR1 and PDLIM1 protein was down-regulated in PIG3V.3)Paeoniflorin pretreatment in PIG1 cells for 24 h can up-regulate the expression of H₂O₂-induced cytoskeleton proteins RhoA,ROCK and F-actin,while PIG3V down-regulates the expression of ROCK1 protein.Paeoniflorin inhibits the expression of RhoA/ROCK1 through PDLIM1 in PIG1 cells under oxidative stress.PDLIM1 inhibited the expression of ROCK1.ROCK1 cannot regulate the expression of PDLIM1 gene,but positively regulates the expression of F-actin protein in PIG3V.The results of immunofluorescence showed that paeoniflorin inhibited the reduction of melanosomes and dendrites induced by H₂O₂.This study indicated that Paeoniflorin can activate the JNK/Nrf2/HO-1 signaling pathway and interact with the PDLIM1/RhoA/ROCK1pathway to protect melanocytes from oxidative damage.