| Psoriasis is a common,recurrent,chronic inflammatory skin disease affecting 3%of the general population and leads to significant morbidity.Psoriasis is characterized by the development of erythematous papules and overlying scaly plaques.The etiology of this disease is complex,the specific pathogenesis is not yet clear.It may include abnormal T cell function and cytokine mediators,reduced apoptosis,genetic factors,and more.Histologically,psoriasis is characterized by several unique features,such as epidermal hyperplasia with dysregulated keratinocyte differentiation,pronounced inflammatory cell infiltration and increased vascularization.It affects the psychological well-being of patients,leading to anger,embarrassment,low self-esteem and even depression.Currently,psoriasis is managed by topical treatment with steroids,immuno-suppressants and several other agents.Photochemotherapy and other systemic therapies,such as using methotrexate,cyclosporine and biological therapies,are also in practice.However,many psoriasis patients are still sufferring from frequent relapse,adverse drug effects,and other untoward reactions such as development of non-melanoma skin cancer.The etiopathogenesis of psoriasis remains unclear at present.It is urgently needed to explore the molecular mechanisms underlying the pathogenesis of psoriasis and to discover novel therapeutic strategy that is more effective and safer for this disease.Metformin(1,1-dimethylbiguanide hydrochloride)is an oral hypo-glycemic agent commonly used for the treatment of type 2 diabetes mellitus for its hypoglycemic effects and ability to reduce cardiovascular morbidity and mortality.There have been many studies in recent years evaluating the role of metformin in psoriasis.For example,it can inhibit T-cell proliferation and IFN-?,IL-17 secretion upon stimulation.It has been previously reported that diabetics receiving metformin treatment have a lower occurrence of psoriasis than other insulin sensitizing agents.It can cause significant improvement in psoriasis area and severity index scores.Previous studies have shown that metformin inhibits cell growth and proliferation of a number of types of cancer,including liver,colon and prostate cancer.Metformin has an excellent therapeutic index with few side effects.In recent years,many scholars have found that the drug has great potential in the treatment of acne,psoriasis,skin tumors and other skin diseases,especially its role in the study of psoriasis.Reactive oxidative species(ROS)are reactive molecules that can regulate cell signaling and homeostasis.Generally,elevated ROS under oxidative stress conditions are involved in the pathogenesis of various diseases,including cancer and inflammation,by damaging cellular components.However,accumulating evidence has also demonstrated that ROS have dual roles in tumors.Lower levels of ROS can promote cell survival and tumorigenesis,while elevated levels of ROS can induce tumor cell apoptosis or senescence and inhibit cancer cell growth.The anti-tumor potency of metformin has been linked to the generation of ROS.In mannan-induced inflammation,ROS deficiency promoted Ps and arthritis,diseases that were suppressed by monocyte/macrophage-derived ROS,similar to collagen-induced arthritis inflammation.Moreover,the generation of free radicals(ROS)plays a major role in the anti-psoriatic activity of anthralin,the most effective topical drug and in phototherapy of psoriasis.Recently,imiquimod-induced psoriatic dermatitis was shown to be prevented by ROS.The anti-tumor potency of metformin has been linked to the generation of ROS.Met decreased mitochondrial viability and increased the production of H2O2 in neuronal cell cultures.Nuclear factor erythroid-derived 2 related factor 2(Nrf2)is a transcription factor that plays a critical role in coordinated induction of genes encoding numerous phase ? detoxifying and antioxidant enzymes,such as superoxide dismutase(SOD),catalase,and heme oxygenase-1.An inducible antioxidant program modulated by Nrf2 and its repressor protein can tightly control ROS levels.A great number of malignant tumors,including colon,lung,breast,and pancreatic cancer,exhibit an increased activity of Nrf2.Recently,studies from several groups showed that the extension of Nrf2 target genes is actually much wider than previously thought and encompasses some additional genes that regulate both cell proliferation and differentiation.For instance,Nrf2 has been proven to enhance cancer cell proliferation through upregulation of metabolic genes.Previous studies have shown that Nrf2 can regulate the expression of key markers for keratinocyte hyperproliferation,so the dysregulation of Nrf2 may contribute to the pathogenesis of psoriasis.The mitogen-activated protein(MAP)kinase superfamily,which consists of extracellular signal-regulated kinases(ERK)1/2 and three stress-responsive subfamilies,the c-Jun NH2-terminal kinases(JNKs),p38-kinases and ERK-5,is normally stimulated by growth factors and cellular stress or inflammatory cytokines.ERK1/2 is an important signaling pathway in the MAPK family that regulates cell growth and differentiation.The classical pathway whereby Erkl/2 types of MAPkinases are activated is through the Ras-Raf-Mek cascade,The ERK1/2 signaling pathway is closely associated with psoriasis.ERK activation is associated with cell survival,proliferation,and differentiation in response to mitogens and cell survival factors.In addition,activation of the Raf/ERK signaling cascade in human cancer cells has been demonstrated to be required for Nrf2 activation,which promotes Nrf2 nuclear translocation and binding to the specific DNA sequence.Metformin can reduce the expression of Nrf2 through the inactivation of Raf-ERK signaling in cancer cells.It could inhibit cancer cell growth by suppressing HO-1 expression through inhibition of Raf-ERK-Nrf2 signaling pathway.This study provides evidence that metformin may be involved in cancer prevention and identifies the mechanisms underlying the reduced cancer risk in diabetic patients treated with this drug.Metformin can regulate immune function from multiple levels.Our previous study showed that MET has anti-proliferative roles in human immortalized keratinocyte cells(HaCaT)via inhibition of the mTOR signaling pathway.The studies cited above found that MET is effective in psoriasis patients,but the underlying mechanism has not yet been fully elucidated.Studies have yet to establish whether MET promotes human keratinocyte apoptosis via ROS and whether the Raf-ERK-Nrf2 signaling pathway participates in the process.HaCaT cells with full epidermal differentiation capacity have been widely used as cellular models for the study of psoriasis.The present study explores the effects of MET on the proliferation and apoptosis of HaCaT cells,on the regulation of ROS,and the underlying mechanisms.In this study,the effect of metformin on the activity and apoptosis of HaCaT keratinocytes and the level of reactive oxygen species produced by metformin were detected by in vitro cell experiments.The signal transduction pathways involved in this study were preliminary studied by molecular biology methods.As a drug against the proliferation of keratinocytes,it lays the foundation for the clinical treatment of psoriasis.Part 1.The effect of Metformin on the viability of HaCaT cell by CCK-8 and flow cytometry in vitroObjective:The morphological changes of cells in the experimental group and the control group were observed after metformin treatment.The effects of metformin on the growth?apoptosis and ROS level of HaCaT keratinocytes were detected by CCK-8 and flow cytometry.Materials and Methods:1.The experiment was divided into different concentrations of metformin in the experimental group and the control group.HaCaT keratinocytes were seeded at a density of 2×105/ml.Incubate overnight at 37? in a 5%CO2 incubator.(1)HaCaT keratinocytes were treated with different concentrations of metformin at concentrations of 0,10,20,40,and 60 mmol/L,respectively.After 24 hours of stimulation,the activity of cells in each group was detected by CCK-8 assay.(2)HaCaT keratinocytes were treated with different concentrations of metformin at concentrations of 0,10,20,40,and 60 mmol/L,respectively.After 24 hours of stimulation,HaCaT cells were digested with 0.25%trypsin without EDTA.Collected in flow-tube,labeled with Annexin V-FITC,flow cytometry was used to detect the apoptosis of cells in each group.2.Under the pretreatment with active oxygen scavenger NAC,Reactive oxygen species(ROS)produced by metformin(final concentration was 40 mM)and its effects on proliferation and apoptosis of HaCaT cells were detected.They were divided into control group,metformin group,metformin + NAC group.The metformin+NAC group was pretreated with NAC solution at a final concentration of 10 mM for 1 hour.(1)After 24 h stimulation,effect of Reactive Oxygen Species(ROS)were detected.DCFH-DA probe was labeled,and HaCaT cells were trypsinized and collected in flow tubes.Flow cytometry was used to detect the level of reactive oxygen species in each group.(2)After 24 h stimulation,CCK-8 assay was used to detect the activity of cells in each group.(3)After 24 h stimulation,HaCaT cells were digested with 0.25%trypsin without EDTA and collected in flow-tubes,labeled with Annexin V-FITC,and flow cytometry was used to detect the apoptosis of the cells.Results:(1)CCK-8 assay detected the effect of different concentrations of metformin on the activity of HaCaT cells.The results showed that the average survival rate of the control group,lOmM,20mM,40mM,60mM metformin group cells were 100.00±0.00%?9.27±0.46%?78.77±2.00%?70.47±1.43%? 29.57±2.31%,respectively.As can be seen,there was no significant change in the activity of the 10 mM group compared to the control group.When metformin concentration was ?20 mM,the growth of HaCaT cells could be effectively inhibited in a concentration-dependent manner.Compared with the control group,P<0.01,the difference was statistically significant.(2)The effect of metformin on the morphology of HaCaT keratinocytes.The growth status of HaCaT cells before and after metformin treatment was observed under an inverted microscope.The size of normal cultured cells was uniform,fuller,the boundaries of cells were clear,clusters were distributed,and adherent growth was observed.The connection is tighter.After intervention with ?20mM metformin for 24h,the morphology of the cells changed significantly,the cell size became smaller,the adhesion between cells decreased,and there was more single distribution,some cells were detached and floated,and adherent cells were growing inside the cells.We could see cells and cell debris floated in the culture fluid,and apoptic cells increased.With the increase of metformin concentration,this cell inhibitory effect is more pronounced.(3)The apoptosis of HaCaT cells was detected by flow cytometry with different concentrations of metformin.HaCaT cells were treated with metformin at concentrations of 0,10 mM,20 mM,40 mM,and 60 mM for 24 h.Flow cytometry was used to detect apoptosis.The mean apoptotic rate(the sum of early and late apoptotic cells)in the group,10 mM,20 mM,40 mM,60 mM metformin group cells was 6.15±0.55%,6.11 ±0.51%,11.53±0.57%,36.13±0.71%,55.41±2.93%,respectively.Compared with the control group,there was no significant change in the number of apoptotic cells in the 10 mM metformin group.When the concentration was ?20 mM,the number of apoptotic cells began to increase in a concentration-dependent manner.Compared with the control group,P<0.01,the difference was statistically significant.(4)After metformin was treated with different concentrations of metformin for 24 hours,the intracellular reactive oxygen species was detected by flow cytometry.The results showed that when the concentration was 20,40,and 60 mM,the average active oxygen species level was 1.81,3.59,7.84 times the control group.Metformin was shown to increase the production of reactive oxygen species(ROS)in HaCaT cells in a concentration-dependent manner.Compared with the control group,P<0.01,the difference was statistically significant.(5)Pretreatment with 10 mM active oxygen scavenger NAC for 1 hour before 40 mM metformin was applied to HaCaT cells for 24 hours.The growth of HaCaT cells was observed under an inverted microscope.The morphology of HaCaT cells pretreated with reactive oxygen species scavenger(NAC)was significantly improved compared with that of 40mM Metformin alone.The number of shedding and floating cells was significantly reduced(6)Pretreatment with NAC,reactive oxygen species(ROS)produced by metformin,cell activity and apoptosis in HaCaT cells were detected.The results showed that compared with the metformin alone group,the ROS decreased by about 44.67%,the average cell viability increased from 69.87 ±0.32%to 90.95 ±1.01%%and the average percentage of apoptotic cells decreased from 35.70 ± 0.97%to 18.32 ± 0.67%.P<0.01,the difference was statistically significant.Conclusion:Metformin can significantly increase the production of reactive oxygen species in HaCaT cells in a concentration-dependent manner.Reactive oxygen species scavenger NAC reversed this effect,suggesting that metformin exerts its effect of inhibiting cell proliferation and promoting apoptosis by increasing reactive oxygen species in HaCaT cells.Part 2.Effect of Metformin on the viability and intracellular ROS level of HaCaT by regulating Nrf2Objective:To detect the effects of different concentrations of metformin on the expression of Nrf2,p-Nrf2 protein and Nrf2 mRNA levels.Nrf2 agonist oltipraz(OPZ)and metformin were co-expressed in HaCaT cells and detected their production of reactive oxygen species(ROS),the levels of proliferation and apoptosis.To investigate the molecular mechanism of metformin-induced apoptosis in HaCaT cells.Materials and Methods:HaCaT keratinocytes in logarithmic growth phase were inoculated overnight at a density of 2×10 5/ml.1.Western blot and real-time fluorescence quantitative PCR were used to detect the effects of different concentrations of metformin on the expression of Nrf2,p-Nrf2 protein and Nrf2 mRNA levels.The experiment was divided into different concentrations of metformin in the experimental group and the control group.After 24 hours,the drug was stimulated with different concentrations of metformin at 0,10,20,40,and 60 mmol/L.(1)After 24 hours of drug treatment,total cellular protein was extracted and BCA method was used to determine protein concentration.After SDS polyacrylamide gel electrophoresis and transfer membrane,blocked non-specific antigen with 5%skimmed milk powder,added Nrf2,p-Nrf2 primary antibody overnight at 4?.After 3 times of TBST washing,goat anti-rabbit secondary antibody labeled with horseradish peroxidase was added and reacted for 1 hour.After the coloration,the chemiluminescence system was used to collect images.The experiment used GAPDH as an internal reference.(2)After 24 hours of drug action,total cellular RNA was extracted,then quantified,reverse transcribed into cDNA,and placed in a fluorescence quantitative PCR machine.2.Nrf2 agonist oltipraz(OPZ)and metformin were co-acted on HaCaT cells.And detected their effects on proliferation,apoptosis,and production of reactive oxygen species(ROS).They were divided into control group,metformin group and metformin + OPZ group.(1)The effect of reactive oxygen species(ROS)after 24h stimulation was detected by DCFH-DA probe.HaCaT cells were trypsinized and collected in flow-tubes.Flow cytometry was used to detect the level of reactive oxygen species in each group.(2)After t 24 h stimulation,CCK-8 assay was used to detect the activity of cells in each group.2.3 After t 24 h stimulation,HaCaT cells were digested with 0.25%trypsin without EDTA and collected in flow-tubes,labeled with Annexin V-FITC,and flow cytometry was used to detect the apoptosis of the cells.Results:(1)HaCaT cells were treated with different concentrations of metformin for 24 hours and the protein levels of Nrf2 and p-Nrf2 were detected by Western-blot.The results showed that compared with the control group,metformin at concentrations of 20,40,and 60 mmol/L,the Nrf2 protein levels decreased by 32.73%,54.17%,and 60.24%,respectively;the p-Nrf2 protein levels decreased by 26.39%,35.51%,49.51%.P<0.01,the difference was statistically significant.(2)Metformin was treated with different concentrations of metformin for 24 hours.The expression of Nrf2 mRNA in HaCaT cells was detected by qRT-PCR.The results showed that compared with the control group,metformin could decrease the expression level of Nrf2 mRNA by 17.64%,30.30%,and 43.40%at concentrations of 20,40,and 60 mmol/L,respectively.P<0.01,the difference was statistically significant.(3)HaCaT cells were treated with Nrf2 agonist oltipraz(OPZ)and metformin for 24 h.The growth status of HaCaT cells was observed under an inverted microscope.Compared with the metformin alone group(40 mM),the cell growth status was significantly improved and the cell size was consistent.The shedding and floating cells are significantly reduced.(4)After the combination of Nrf2 agonist oltipraz(OPZ)and metformin in HaCaT cells for 24 hours,compared with the metformin alone group,the average ROS decreased by 42.46%,the average cell viability increased from 71.07±1.39%%to 86.57±0.96%%,and the average percentage of apoptotic cells dropped from 35.70±0.97%to 24.13±0.95%.P<0.01,the difference was statistically significant.Conclusion:The results of this part of experiment confirmed that metformin could inhibit Nrf2 and p-Nrf2 protein levels and Nrf2 mRNA levels in a dose-dependent manner,and confirmed that metformin can inhibit cell proliferation and promote apoptosis by reducing the expression level of Nrf2.In conclusion,metformin exerts its apoptotic effect by inhibiting the activity Nrf2 in HaCaT cells.Part 3.Effect of Metformin on the viability of Raf-1-ERK1/2-Nrf2 signaling Pathway in HaCaT cellObjective:To detect the effects of different concentrations of metformin on the expression of of RAF-1,ERK1/2,p-RAF-1,and p-ERK1/2 protein levels;To detect the protein expression levels of p-ERKl/2,Nrf2,and p-Nrf2 with MEK inhibitor U0126.The molecular mechanism of metformin-induced apoptosis in HaCaT cells was further investigated.Materials and Methods:HaCaT keratinocytes in logarithmic growth phase were inoculated overnight at a density of 2×105/ml.1.Western-blot was used to detect the effect of different concentrations of metformin on the expression of RAF-1,ERK1/2,p-RAF-1,and p-ERK1/2 protein levels.After 24 hours of drug treatment,total cellular protein was extracted and BCA method was used to determine the protein level.After SDS polyacrylamide gel electrophoresis and transfer membrane,then blocked non-specific antigen with 5%non-fat dry milk,added RAF-1,ERK1/2,p-RAF-1 and p-ERK1/2 primary antibody overnight at 4?.After 3 times of TBST washing,goat anti-rabbit secondary antibody labeled with horseradish peroxidase was added and reacted for 1 hour.After the coloration,the chemiluminescence system was used to collect images.The experiment used GAPDH as an internal reference.2.Western-blot was used to detect the effect of MEK inhibitor U0126 on the expression of ERK1/2,p-ERK1/2,Nrf2 and p-Nrf2 protein levels.The experiment was divided into 0.1%DMSO group,U0126 group and the control group.HaCaT cells were incubated overnight.After 24 hours of drug treatment,total cellular protein was extracted and protein concentration was determined by BCA method.After SDS polyacrylamide gel electrophoresis and transfer membrane,then blocked non-specific antigen with 5%non-fat dry milk,ERK1/2,p-ERKl/2,Nrf2 and p-Nrf2 primary antibody were added and incubated overnight at 4?.After 3 times of TBST washing,goat anti-rabbit secondary antibody labeled with horseradish peroxidase was added and reacted for 1 hour.After the coloration,the chemiluminescence system was used to collect images.The experiment used GAPDH as an internal reference.Results:(1)Metformin was treated with different concentrations of metformin for 24 hours.The protein levels of RAF-1,ERK1/2,p-RAF-1 and p-ERK1/2 in HaCaT cells were detected by Western-blot.The results showed that the average levels of RAF-1 protein decreased by 16.90%,56.35%,and 79.45%when metformin at the concentrations of 20,40,and 60 mmol/L,respectively.The average levels of p-RAF-1 protein decreased by 28.76%,60.10%,and 81.97%,respectively.The ERK1/2 protein levels decreased by 29.87%and 36.46%,63.37%,respectively;p-ERKl/2 protein levels decreased by an average of 17.34%,35.46%,49.48%,respectively.Compared with the control group,P<0.01,the difference was statistically significant.(2)HaCaT cells were treated with MEK inhibitor U0126(10uM)for 1 h,the expression of ERK1/2,p-ERKl/2,Nrf2 and p-Nrf2 protein levels was detected by Western-blot.The results showed that compared with the control group,the ERK1/2,p-ERK1/2,Nrf2,and p-Nrf2 protein levels all decreased,with an average decrease of 26.94%,28.06%,21.78%,and 33.04%,respectively.Compared with the control group,P<0.01,the difference was statistically significant.Conclusion:The results of this part of experiment confirmed that metformin could inhibit the expression of RAF-1,p-RAF-1,ERK1/2,and p-ERK1/2 in a dose-dependent manner,and the MEK inhibitor U0126 was used to further confirm that Nrf2 is a downstream gene of ERK.In conclusion,metformin exerts its apoptotic effect by inhibiting the activity of Raf-1-ERK1/2-Nrf2 signaling pathway in HaCaT cells.Compared with the existing reports,the innovations in this paper mainly focus on the following aspects:(1)To investigate the effect of metformin on the expression of reactive oxygen species in HaCaT cells and its effect on the proliferation and apoptosis of HaCaT cells for the first time.To prove that metformin exerts its effect of inhibiting cell proliferation by increasing the reactive oxygen species in HaCaT cells.(2)To investigate the effect of Metformin on the expression of Nrf2 in HaCaT cells for the first time,and its effect on the proliferation and apoptosis of HaCaT cells,and to verify that metformin can inhibit Nrf2 and p-Nrf2 protein levels and Nrf2 mRNA levels in a dose-dependent manner.(3)The relationship among metformin,reactive oxygen species(ROS)levels in HaCaT cells,Raf-1-ERK1/2-Nrf2 signaling pathway,and HaCaT cell activity was first proposed.Metformin was confirmed by inhibiting Raf-1-ERK1/2-Nrf2 signaling pathway activity in HaCaT cells to increase reactive oxygen species levels and further exerts its pro-apoptotic effects.In summary,the present study investigated the relationship among metformin,reactive oxygen species(ROS)levels in HaCaT keratinocytes,Raf-1-ERK1/2-Nrf2 signaling pathway,and HaCaT cell activity in a new way.It provided experimental and theoretical basis for their new target for the treatment of psoriasis.To explore the specific molecular mechanism of the action of metformin on keratinocytes will help us to understand the drug further and provide more powerful and direct evidence for the treatment of psoriasis. |
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