Effect Of Leptin On The Expression Of Proliferation And Differentiation Keratin In HaCaT Cell And Its Molecular Signal Pathway

Psoriasis is a chronic inflammatory skin disorder, which is mediated by Th cells andcharacterized with over proliferation of Keratinocyte. Its pathogenesis is not yet clarifiedat present. It is a disease of multifactorial inheritance that is likely to be caused by bothgenetic, immunologic, environmental and obesity factors.Recently, clinical observational study found that the proportion of psoriasis withobesity increased. For the treatments of obesity helps to improve psoriasis, it showed thatobesity is closely related to psoriasis. Leptin is a adipokines, which participates inmetabolism. Previous studies have shown that the high expression of leptin and leptinreceptor in psoriatic lesions, leptin may be associated with over hyperplasia of epidermis,and showed that leptin may be important molecules of connection between psoriasis andobesity.The keratin spectrum of keratinocyte (KC) in psoriatic lesions is changed, includingsignificant elevation of psoriasis-proliferation related keratin16(K16) and keratin17(K17), and significant reduction of psoriasis-differentiation related keratin1(K1) and keratin10(K10). Proliferation related keratin is no or lowly expressed in normal skin butis highly expressed in psoriatic plaques, with its expression level positively correlated withthe severity of the disease.Our preliminary studies found that leptin is over-expressed in psoriatic plaques andaffect the KC activity, cell cycle and cell proliferation, and it can stimulate KC to productthe inflammatory factor IL-6, IL-8and TNF-α. The above results indicate that leptinparticipated in the course of pathophysiology of psoriasis skin over-proliferation, but therole of leptin in the course of psoriasis pathophysiology is still not clear.Objectives:This study is to investigate the effect of the expression of leptin on KC keratin（K1,K10，K16, K17）and its molecular regulation, to establishes a basis for further study onleptin affect the mechanism of KC proliferation.Methods:HaCaT cells were cultured in vitro. Then we choosed Real-time PCR, Western blotand immunofluorescence staining to detect K1, K10, K16, K17mRNA and the changes ofprotein levels, and choosed Western blot to detect the possible changes of signal pathwaymolecules after leptin treatment, and we used molecular antagonists and siRNA to confirmthe signaling pathway of leptin on the regulation of the expression of keratinKeratinocytes.Results:1. The Effect of leptin on the regulation of HaCaT cells keratin.1) mRNA results: Compared with the HaCaT cell control group, K16and K17mRNA group showed varying degrees of increase (P <0.01), while down-regulation of K1and K10mRNA expression.2) Protein results: Compared with the HaCaT cell control group, the protein of K16and K17group increased in varying degrees, and the protein expression of K1and K10reduced.3) Immunofluorescence results: Compared with the HaCaT cell control group, the red fluorescence expression of K16and K17group was obviously increased, while the redfluorescence expression of K1and K10reduced obviously, and agree well with theRT-PCR and Western blot results.2.Regulation of leptin on HaCaT cells signal pathway molecules100ng/mL leptin was used to stimulate KC for5min,10min,20min,40min,80min,found that leptin can activate the signal transducers and activation of STAT3,and thepathway of ERK1/2.3. The effect of pathway inhibitors on the regulation of leptin on HaCaT cells keratin1) mRNA results: Compared with the pure leptin group, K1, K10, K16and K17mRNA of inhibitors and leptin combined treatment group decreased significantly (P <0.05).2) Protein results: Compared with the pure leptin group, K1, K10, K16and K17leptin of inhibitors and leptin combined treatment group significantly reduced.3) Immunofluorescence results: The red fluorescence expression of inhibitors andleptin combined treatment group K1, K10, K16and K17was reduced obviously, andagree well with the RT-PCR and Western blot results.4. Further verify the effect of pathway blockers on the regulation of leptin on HaCaTcells proliferation keratin1) mRNA results: Compared with the pure leptin group, K16and K17mRNA ofblockers and leptin combined treatment group decreased significantly (P <0.01).2) Protein results: siRNA was successful in blocking STAT3and ERK1/2molecularpathways, and K16and K17protein was significantly lower than leptin group.Conclusions:This study proves that leptin can induce high expression of K16and K17in KC, andlow expression of K1and K10. Its mechanism may be associated with STAT3andERK1/2signal transduction pathways, suggests that leptin can affect the proliferationactivity of keratinocyte in psoriasis, inhibition the differentiation of keratinocytes, andprompts that leptin may involved in the occurrence and development of obese psoriasis atthe keratin level.