Guggulsterone Suppresses Keratinocyte Proliferation And Inflammatory Function By Inhibiting JAK1-STAT3 Signaling

Psoriasis is an incurable chronic inflammatory skin disease that is influenced by a variety of genetic,environmental and immune factors.The main clinical manifestations of psoriasis are skin erythema,raised papules and a large number of white scales.The pathological changes in skin lesions include abnormal proliferation and differentiation of epidermal keratinocytes,infiltration of immune cells and angiogenesis.The disease has a long course,is difficult to cure,is prone to recurrence and often has multiple complications that seriously affect the quality of life of patients.Recently the methods for the treatment of psoriasis mainly include traditional drug therapy,physical therapy and biologic therapy,each of which has its own limitations,such as poor efficacy,drug resistance,toxic side effects and recurrent attacks after discontinuation of the drug.As expected,it is of great significance to find new treatments and new targets for psoriasis.The most important cells in the epidermis are keratinocytes,which make up more than 80%of epidermal cells.The tissue structure and function of the epidermis depend on the stable proliferation and normal differentiation of keratinocytes,and the excessive proliferation of keratinocytes in the skin of psoriasis patients suggests whether new targets can be found to target keratinocytes to achieve the purpose of treating psoriasis.Natural medicines refer to animal medicines,botanical medicines and mineral medicines that have been proven by the modern medical system to have certain pharmacological activities.With low toxic side effects,high biocompatibility,bioeffective diversity and multiple targets,they are an important basis for the development of new drugs.The study of the role of natural drugs in psoriasis is not yet indepth,therefore,in this paper,the following work is carried out around the mechanism of action of GS(Guggulsterone)in keratinocytes:(1)The results of MTT showed that GS inhibited the proliferation of primary keratinocytes cells,and the results of flow cytometry showed that GS caused G2/M phase block.The effects GS on the cell cycle-related proteins of HaCaT cells(human immortalized keratinocytes)were detected by Western blot,and the results showed that the levels of G2/M phase related proteins CDC25C and CDK1 protein were decreased,and the levels of p-CDK1 protein were increased.It is suggested that GS may block cells in the G2/M phase by inhibiting the expression of CDC25C,thereby reducing the dephosphorylation of CDK1 and thus inhibiting the activation of the CDK1/CyclinB complex.(2)GS significantly downregulated the protein expression levels of STAT3,p-STAT3 and p-p65,but the protein that played the main role was not clear.Western blot results showed that STAT3 inhibitor(STATTIC)was able to mimic the changes in CDC25C,CDK1 and p-CDK1 caused by GS,and STATTIC was able to cause a decrease in p-p65,but NFκB inhibitors(BAY)could not cause a decrease in p-STAT3.Combined with the MTT results STATTIC was able to mimic the proliferation inhibition caused by GS,suggesting that GS exerts its pharmacological effects by inhibiting STAT3 pathway.(3)By constructing STAT3 overexpressing HaCaT cells(STAT3OVE),STAT3 constitutively activated HaCaT cells(STAT3C)and HaCaT cells transfected with empty plasmid(NC).MTT assay,EdU incorporation assay,wound healing assay,qRT-PCR and Western blot showed that STAT3 overexpressing and STAT3 constitutively activated were able to partially rescue the changes in proliferation inhibition,migration inhibition,secretory phenotype and protein level caused by GS,and the effect of STAT3C cells was better.(4)GS was able to inhibit the activation of STAT3 by extracellular signals such as IL6 and TNFa.The typical upstream JAK kinase family of STAT3 mediated the action of external signals on STAT3.Western blot showed a significant decrease in the expression levels of JAK1 and p-JAK1 proteins,while JAK2 and p-JAK2 protein expression no significant changes were observed.(5)To investigate the reasons for the reduced expression levels of STAT3 and JAK1,we found that GS may repress STAT3 and JAK1 at post-transcriptional levels by qRT-PCR experiments.The online website TargetScan was used to predict miRNAs targeting both STAT3 and JAK1,and six candidate microRNAs of the microRNA17 family were identified,combined with published literature to identify miR-17-5p and miR-20a-5p,which are located on the same chromosome,were chosen as the main subjects.Western blot showed that miR17-5p mimics partially mimic the changes in protein levels caused by GS.However,miR-205p did not have this effect.Dual luciferase reporter gene assays showed that GS upregulated miR-17-5p directly bound to the 3’UTR of STAT3 and JAK1,thereby downregulating STAT3 and JAK1 expression.MTT assay,EdU incorporation assay,qRT-PCR and Dual luciferase reporter gene assays showed that miR-17-5p could partially mimic proliferation inhibition,DNA replication inhibition,secretion phenotype and STAT3 transcriptional activity inhibition caused by GS changes.miR-17-5p inhibitor partially rescues the protein changes caused by GS.It further confirmed that GS inhibited JAK1/STAT3 pathway by upregulating miR-17-5p.In summary,GS promoted the expression of miR-17-5p,which downregulated the expression of JAK1 and STAT3,and then inhibited the activation of STAT3,thus exerting its anti-proliferative as well as anti-inflammatory effects,indicating that the miR-17-5p-JAK1STAT3 axis is involved in regulating the anti-proliferative and anti-inflammatory activity of GS in keratinocytes.