Research In The Function And Mechanism Of PPARγ In Psoriatic Keratinocytes

Background:Psoriasis is a common immune-mediated chronic relapsing and inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes(KCs)along with the infiltration of inflammatory cells.The clinical manifestations of psoriasis are localized or widely-distributed well-defined scaly erythema or plaques,.The histological manifestations of psoriasis are hyperkeratosis,parakeratosis,thinner or absent of the granular layer,thickening of the spinous layer,downward extension of epidermal processes,and uplift of the dermal papilla.The pathogenesis of psoriasis is complex,which is generally believed to be caused by the interaction between genetic background,environmental triggers,abnormal immune response and other factors.In the recent years,it has been uncovered that patients with moderate-to-severe psoriasis can be combined with obesity,metabolic syndrome,cardiovascular disease,non-alcoholic fatty liver disease,inflammatory bowel disease,and uveitis,which also suggests that the pathogenesis of psoriasis may be related to metabolism disorders.Previous studies have found that multiple signaling pathways involved in inflammatory metabolism may be involved in the pathogenesis of psoriasis,including the nuclear hormone receptor peroxisome proliferators activate receptors(PPAR)pathway.PPARs are a class of ligand-activated transcription factors that belong to the nuclear receptor superfamily.After PPAR is activated by the corresponding ligand,it binds to the retinoid X receptor(RXR)in the nucleus to form a heterodimer,which interacts with the target gene peroxisome proliferator response element.Combined with PPAR response element(PPRE),it regulates different metabolic pathways by recruiting different transcription cofactors or coactivators,and plays an important role in glucose and lipid metabolism,cell proliferation and differentiation,inflammation and immune processes.PPAR is divided into three subtypes,PPARα,PPARβ/δ,and PPARγ.PPARγis the most widely and deeply studied subtype.It is expressed at low levels in the skin and is involved in the regulation of cell proliferation,differentiation and inflammatory response.The gene network expression,including Proliferation and differentiation of normal KCs.Domestic and foreign studies and the previous data of this research team all suggest that the expression level of PPARγ in the skin lesions of psoriasis patients is reduced.We speculate that PPARγ may be involved in the occurrence and development of psoriasis,but the specific target cells it affects and The regulatory mechanism has not yet been reported.Objective:1.To study the expression of PPARγ in the skin lesions of psoriasis patients,and to explore the effect of targeted biological agents on the expression of PPARγ.2.To investigate the effect of PPARγ agonist pioglitazone(PIO)on psoriasis-like skin lesions induced by imiquimod(IMQ)in mice.3.To investigate the effect of PPARγ agonist PIO on human immortalized keratinocyte Ha Ca T cells.4.To explore the effect of KCs-specific knockout of PPARγ on skin.Methods:1.Collect normal skin tissue and psoriatic skin lesions for RNA sequencing(RNA Sequencing,RNA-seq),and perform KEGG(Kyoto Encyclopedia of Genes and Genomes,KEGG)and GO(Gene Ontology,GO)analysis.At the same time,RNA-seq,quantitative real-time PCR(q RT-PCR)and immunofluorescence methods were used to detect the expression of PPARγ in skin lesions of patients with psoriasis and before and after targeted biologic therapy.2.After 8 weeks of intragastric administration of PPARγ agonist PIO,a mouse model of psoriasis-like dermatitis induced by IMQ was constructed to explore whether PIO could affect the occurrence of psoriatic-like dermatitis.3.Use Ha Ca T cells to explore the effect of PIO on Ha Ca T cells from the cellular level.4.Use conditional gene targeting technology(Cre/Lox P recombinase system)to construct a conditional gene knockout mouse model to achieve the purpose of knocking out target genes in specific tissue cells of mice.The effect of KCs-specific PPARγdeletion on skin was investigated by cross-breeding KCs conditional PPARγ knockout mice.Results:1.Expression of PPARγ in psoriatic skin lesions(1)Using KEGG to analyze the previous RNA-seq results of our team,we found that compared with normal skin tissue,the PPAR signaling pathway was significantly downregulated in psoriatic skin lesions,suggesting that the PPAR signaling pathway may play an important role in the pathogenesis of psoriasis.(2)Analysis of the diseaserelated differentially expressed genes of our team’s RNA-seq and psoriasis dataset(GSE13355)in the GEO database,found that the m RNA expression level of PPARγ in the PPAR signaling pathway was significantly down-regulated in psoriatic skin lesions.(3)qRT-PCR and immunofluorescence methods confirmed that the m RNA and protein levels of PPARγ in skin lesions of psoriasis patients were significantly reduced.The results of immunofluorescence showed that PPARγ was expressed in both the epidermis and dermis of normal human skin tissue,and in the epidermis,it was mainly expressed in the nucleus of keratinocytes on the basal layer;while the expression of PPARγ in the epidermis of psoriasis patients was significantly down-regulated.(4)After treatment with the interleukin-17A(Interleukin-17 A,IL-17A)inhibitor secukinumab or the tumor necrosis factor-alpha(tumor necrosis factor-alpha,TNF-α)inhibitor infliximab,the expression level of PPARγ increased in the skin lesions of patients with psoriasis compared with that before treatment.2.The effect of PPARγ agonist PIO on IMQ-induced psoriasis-like skin lesions in mice(1)PIO can significantly improve IMQ-induced psoriasis-like skin lesions in mice.(2)H&E staining and immunofluorescence detection showed that the epidermal thickness of mice in the PIO treatment group was significantly reduced,and the proportion of KCs(ki-67+)with active proliferation was significantly reduced.(3)qRT-PCR detection showed that the m RNA expression levels of the inflammatory factors IL-17 A,IL-22,IL-23,chemokine 20(cysteine-cysteine chemokine ligand 20,CCL20),S100 calcium-binding protein A8(S100 calcium binding protein A8,S100A8)and S100A9 in the skin lesions of the mice in the PIO treatment group were significantly down-regulated.(4)Immunochemistry showed that PIO could down-regulate the expression level of phosphorylated signal tranducers and activators of transcription(p-STAT3)in psoriasislike skin lesions.3.The effect of PPARγ agonist PIO on Ha Ca T cells in vitro(1)PIO can inhibit the abnormal proliferation of Ha Ca T cells induced by IL-17 A,and promote the up-regulation of KCs differentiation markers keratin 1(K1)and K10.(2)PIO reduced the expression of IL-17A-induced inflammation-related factors TNF-α,IL-6,Chemokine(C-X-C motif)ligand 1,CXCL1,CXCL2,CXCL8 and CCL20 in Ha Ca T cells.(3)PIO can down-regulate the expression of p-STAT3 in Ha Ca T cells induced by IL-17 A.4.The effect of KCs conditional PPARγ knockout on skin(1)Compared with wild-type mice at the same age,the KCs conditional PPARγknockout mice were smaller and lighter,and appeared progressive hair loss and spontaneous skin lesions.(2)H&E staining of tissue sections showed that there was no significant difference in epidermal thickness,dermis and inflammatory infiltration around the hair follicle KCs conditional PPARγ knockout mice and wild-type mice,but the hair follicle growth cycle was different.(3)Immunofluorescence showed that the expression level of KCs proliferation marker ki67 was up-regulated in the skin tissue of KCs conditional PPARγ knockout mice.(4)qRT-PCR detection showed that the expression levels of inflammatory factors IL-17 A and IL-6 were increased in the skin tissue of KCs conditional PPARγ knockout mice.Conclusion:This study confirmed that the expression level of PPARγ was significantly reduced in the skin lesions of psoriasis patients.The down-regulation of PPARγ expression may be related to the occurrence and development of psoriasis,and IL-17 A or TNF-α may affect the pathogenesis of psoriasis by regulating the expression of PPARγ in KCs.PPARγ agonist PIO can significantly alleviate IMQ-induced psoriatic dermatitis,inhibit the excessive proliferation of KCs,and reduce the expression of inflammatory factors.In vitro studies have confirmed that PIO can inhibit the abnormal proliferation of KCs induced by IL-17 A,down-regulate the expression of inflammatory factors,and induce the normal differentiation of KCs.Moreover,PIO may achieve the above effects by inhibiting the STAT3 signaling pathway.Studies in KCs conditional knockout of PPARγmice suggest that KCs-specific PPARγ deletion can affect hair follicles and skin inflammation.By exploring the expression,role and possible mechanism of PPARγ in psoriatic KCs,our findings will provide theoretical and experimental basis for the clinical application of PPARγ agonists.