The Study On Phenformin Inhibiting Pro-inflammatory Cytokine Expression In Skin And Gingival Keratinocytes By Regulating C-Myc Via MTOR Signaling.

Background and objectivesThe epithelial barrier is an important biological barrier in the human body,which divides multiple tissues and organs,such as the skin,respiratory tract,gastrointestinal tract,oral cavity,etc.,from the external environment.Its functional disruption is an important risk factor for tissue and organ dysfunction and disease occurrence.Once the skin,especially the maxillofacial skin,is sick or damaged,it will seriously affect people’s beauty and health.Many skin diseases and skin injuries are related to skin inflammation.Therefore,conducting in-depth research on skin inflammation can help us further understand various diseases,develop new treatment methods,and have great significance in the diagnosis and treatment of maxillofacial skin inflammation diseases and skin trauma.Although significant progress has been made in the development of new methods for treating skin inflammation in recent years,there are still significant difficulties in the treatment of many skin diseases that are clearly related to inflammation.In recent years,with the introduction of the concept of integrated medicine,more and more scholars have realized that the oral cavity is an important component of the human body,and many oral mucosal and soft tissue inflammations are local manifestations of systemic diseases,which are also prone to affecting the skin and causing inflammation.Oral mucosa,which has similar histological structure and cellular components to skin tissue,is the first line of defense against the external environment of the external body.The outermost oral mucosa epithelium and the epidermal layer of skin tissue(mainly composed of keratinocytes)are the pioneers of this defense line and play a key role in immune response.Epidemiological investigation shows that gingivitis is one of the most common oral mucosal inflammatory diseases,and further exploration of the mechanism of gingival keratinocyte inflammation will provide help for the research of periodontal related diseases.Studies have shown that inflammation of the skin and mucous membranes is usually caused by the secretion of pro-inflammatory cytokines by keratinocytes after external stimulation,which subsequently interacts with immune cells in tissue to induce an immune response.Keratinocytes play a leading role in inflammatory response and are key cells in the study of inflammatory response.Metformin has been the most commonlyused biguanide drug in the treatment of type Ⅱdiabetes.It has recently been found that metformin has a wide range of anti-cancer and anti-aging effects,and can inhibit the inflammatory response of different types of cells.However,there are few studies on its role in skin inflammation.Another biguanide drug,phenformin,is a derivative of metformin,and is significantly better than metformin in many types of cancer..The main reason is that metformin needs to bind to organic cation transporter(OCT)to enter the cell to exert its function,while phenformin has strong lipophilicity and does not need OCT to enter the cell directly.Since OCT transporter is not expressed in skin,we speculate that phenformin,rather than metformin,plays an important role in regulating the inflammatory response of keratinocytes.Therefore,the aim of this study was to investigate whether phenformin inhibits the inflammatory response of skin and gingival keratinocytes and elucidates the potential molecular mechanisms..Materials and methods1.The effects of phenformin on acute skin inflammation in vivoThe mouse ear edema model induced by 12-O-tetradecanoylphorbol-13-acetate(TPA)was used to determine the role of metformin and phenformin in skin inflammation by measuring the thickness of the ear,the thickness of subcutaneous edema,the degree of inflammatory cell infiltration and the proliferation of epidermal and dermal cells via H&E and IF staining.2.Phenformin modulation of isolated cultured skin tissues and skin and gingival keratinocytes to secret pro-inflammatory cytokine expression in vitroReal-time fluorescence quantitative polymerase chain reaction(quantity real-time polymerase chain reaction,qRT-PCR)was used to detect the expression of cytokines in skin tissue cultured in vitro after treatment with Poly(I:C)and different concentrations of phenformin.Through ELISA kit and qRT-PCR technology to detect Poly(I:C)/TPA and the expression of proinflammatory cytokines in primary keratinocytes derived from human skin tissue after combined treatment with different concentrations of phenformin or phenformin alone.qRT-PCR technique was used to detect the expression of pro-inflammatory cytokines in primary keratinocytes derived from gingival tissue after treatment with Poly(I:C)and different concentrations of phenformin or phenformin alone.3.Analyzing the role of NF-κB and MAPK signaling pathways in the regulation of pro-inflammatory cytokine expression in skin keratinocytes by phenforminSkin keratinocytes cells treated with Poly(I:C)and different concentrations of phenformin were collected and the total and phosphorylated levels of JNK,p38,Erk,and NF-κB proteins were detected by Western blot technique.Skin keratinocytes were treated with 1.0 mM phenformin and an equal volume of PBS(control group),respectively,and cell samples were collected at different time points to analyze the expression of pro-inflammatory cytokines by qRT-PCR,and detection of total and phosphorylated levels of AMPK,Erk and NF-κB proteins by Western blot technique.4.Defining the molecular mechanism by which phenformin regulates the expression of pro-inflammatory factors in keratinocytes at an early time pointTranscriptome expression changes in skin keratinocytes of PBS group and phenformin group were detected using RNA-sequencing technology to find differential expression genes(DEGs),which were analyzed by cluster analysis.The Hallmarke pathway gene set was extracted from MSigDB database for gene set enrichment analysis of DEGs,and key genes were screened,and verified in vitro by qRT-PCR and Western blot.Knockdown of key gene expression levels by transfection of siRNAs or inhibitors of key genes in skin keratinocytes and overexpress key genes by viruses infection in keratinocytes.In vitro assay of whether down-expression or over-expression of key genes inhibits or promotes the regulation of cellular inflammatory response by phenformin using qRT-PCR,Western blot techniques.Subsequently,the above conclusions were further clarified by in vivo experiments by applying TPA alone or a combination of TPA and inhibitors of key genes to the ears of C57 mice.The potential upstream and downstream pathways of the key genes were explored by reviewing the literature combined with the results of RNA-seq,and the gene expresions was reduced by corresponding inhibitors,which were validated at the mRNA and protein levels by qRT-PCR and Western blot techniques.Results1.Phenformin suppresses acute skin inflammation induced by TPA in the SkinPhenformin significantly inhibited TPA-induced ear swelling,redness and other inflammatory reactions in mice.Histological analysis(H&E stain)confirmed the increased ear thickness caused by TPA treatment,which was significantly inhibited by phenformin,and we also observed that phenformin treatment reduced the thickness of subcutaneous edema and the degree of inflammatory cell infiltration.The pretreatment with metformin did not significantly inhibit TPA-induced ear thickness increase.The IF result of staining showed an increased infiltration of immune cells in the skin treated with TPA,which was suppressed by the addition of phenformin(TPA+Phen).Moreover,IF staining of Ki-67 revealed that the increased number of proliferative cells in the epidermis and dermis caused by TPA was also inhibited by phenformin treatment.2.Phenformin inhibits the expression of pro-inflammatory cytokines in skin tissue and in vitro cultured skin and gingival keratinocytesIn vitro skin organ culture system,phenformin significantly decreased the expression of inflammatory cytokines(IL-1β(IL-1B),IL-6,IL-8(CXCL8),IL-12β(IL-12B),IL-23β(IL-23B),CCL2,CXCL16 and TNF-α)induced by Poly(I:C).The results of qRT-PCR and ELISA experiments confirmed that phenformin significantly inhibited the expression of proinflammatory cytokines induced by Poly(I:C)or TPA in skin keratinocytes,and inhibited the basic expression of keratinocytes cytokines without inflammatory stimulation.In addition,qRT-PCR results also confirmed that phenformin could significantly inhibit the expression of proinflammatory cytokines in gingival keratinocytes in both inflammatory and non-inflammatory conditions.3.Phenformin inhibits Erk and NF-κB activity in skin keratinocytesThe activation of Erk and NF-κB activity was inhibited after 24 hours of treatment for keratinocytes with phenformin.Phenformin can activate the AMPK as early as 2 h after treatment but suppresses the NF-κB and MAPK-Erk activity at least 4 h after treatment.qRT-PCR analysis revealed that the expression of all pro-inflammatory cytokines began to decrease as early as 2 hours after phenformin treatment,which was earlier than the down-regulation of Erk and NF-κB activity.4.Phenformin down regulates pro-inflammatory cytokine expression in skin and gingival keratinocytes by controlling c-Myc expression through inhibition of the mTOR pathway at earlier time points237 genes were up-regulated and 360 genes were down-regulated by phenformin treatment after 10 h treatment,,including CXCL1,CXCL2 and CXCL8(IL-8),which were among the down-regulated differentially expressed genes(DEGs).AMPK downstream targeted pathways,including glycolysis,metabolism,cell polarity(apical surface/apical junction)and p53,were among the list of up-regulated pathways.The inflammatory response and interferon response pathways appeared in the list of negatively regulated pathways.MYC-targets version 1 and 2(VI/V2)were in the list of down-regulated pathways and were ranked the top two pathways according to the analysis of gene ratio,defined as the percentage of significant genes over the total genes in a given pathway.The calculated enrichment scores further revealed that MYC-targets,inflammatory signaling,cytokine signaling as well as chemokine-signaling-related genes were mainly down-regulated in phenformin-treated cells.C-Myc target genes,CDK4,DDX19,APEX1,CAD and NOP16,together with c-Myc were validated by qRT-PCR analysis to be inhibited by phenformin treatment.The analysis of qRT-PCR showed a decreased expression of c-Myc that appeared as early as 2 h after phenformin treatment,which was further supported by the Western blot analysis of c-Myc protein expression in human keratinocytes treated with 1 mM phenformin.Furthermore,both Poly(I:C)and TPA could induce c-Myc expression and that induction was down-regulated by phenformin treatment.AICAR was used to treat human keratinocytes and showed that AICAR also significantly suppressed c-Myc expression,as well as c-Myc targets.We found that following the knockdown of c-Myc with a c-Myc siRNA or JQ1(a c-Myc inhibitor),the expression of pro-inflammatory cytokines,including IL-1β,IL-6 and IL-8,was significantly decreased compared to the control group.The overexpression of c-Myc induced the expression of pro-inflammatory cytokines and could counteract the down-regulation of cytokine expression induced by treatment with phenformin.the c-Myc inhibitor JQ1 treatment significantly reduced the ear swelling and reddish induced by TPA in vivo.Histological analysis(H&E stain)confirmed that treatment with JQ1 reduced the ear thickness and the infiltration of immune cells.The reduced infiltration of immune cells in JQ1-treated ears was verified by immunostaining of the immune cell markers Gr-1 and F4/80.We found that phenformin could suppress the activation of the mTOR pathway,indicated by the reduced phosphorylated level of mTOR,as well as its downstream targets p70S6K and 4E-BP1,as early as 2 h after treatment,which was associated with the decreased expression of c-Myc in keratinocytes.Then,the activation of the mTOR pathway by MHY1485 resulted in an increased expression of c-Myc,which counteracted the phenformin-suppressing effect in keratinocytes.We found that the mTOR activator MHY1485 enhanced the expression of cytokines,such as IL-1β,IL-6 and IL-8，to rescue the suppression of cytokine expression by phenformin.ConclusionsThe present study showed that phenformin(AMPK activator)suppressed acute skin inflammation and inhibited the expression of pro-inflammatory cytokines in isolated cultured skin tissues,skin keratinocytes,and gingival keratinocytes,and this effect was further reduced by down-regulation the expression of c-Myc via mTOR,followed by blocking Erk and NF-κB pathways to further reduce inflammatory cytokine expression in inflammatory cells,This study provides a basis for the development of phenformin for the clinical treatment of skin and oral mucosa inflammation.