The Potential Of Diosgenin In Treating Psoriasis: Studies From HaCaT Keratinocytes And Imiquimod-induced Murine Model

Background and ObjectivePsoriasis is a chronic inflammatory disease affecting mostly the skin and joints with a tendency to relapse.Current treatments failed to fully satisfy the patients and own limitations respectively.Therefore,pursuing new agents which are safe and effective for psoriasis treatment is vital.Diosgenin,mainly derived from fenugreek,is a natural sapogenin which has been determined to possess an extensive range of pharmacological effects.The anti-cancer,immunomodulatory and anti-oxidant effects of Diosgenin have been broadly studied in various cell lines and disease models,however it has not been observed in psoriasis studies to the author’s knowledge.Thus,here we report the effects of Diosgenin observed from in vitro and in vivo studies of psoriasis and provide the sound theoretic basis of potentially developing it as a future therapeutic approach for psoriasis treatment.MethodsImmortalized HaCaT keratinocytes were utilized for in vitro studies.1.HaCaT cells were treated with different concentrations of Diosgenin for various time points.The cell viability was measured by CCK-8 assays,24h treatment of Diosgenin at2μg/ml（D2）and 10μg/ml（D10）were chosen for further studies.The alteration in HaCaT cellular morphology was observed and the DNA replication ability was determined by Ed U staining.Flow cytometry was performed to examine the cell cycle of HaCaT cells while the expression of NFκB-pathway-associated and cell-cycle-related proteins were determined by WB to explore the potential regulatory mechanism of the anti-proliferative effect.2.After being treated with D2 and D10,apoptosis in HaCaT cells and the apoptosis-related protein expressions were determined.3.To mimic the hyperproliferation in psoriatic keratinocytes,we stimulated HaCaT cells with LPS or IL-22 while co-treating with D2 or D10.After determined the cell viability,WB and Immunofluorescence were performed to detect the expression and location of proteins from NFκB pathway and IL-22RA1-depedent pathways.The HaCaT cell cycle and apoptosis along with the expression of the related proteins were further detected.4.To mimic the inflammation on psoriatic keratinocytes,we stimulated HaCaT cells with LPS or TNF-αwhile co-treating with D2.Several cytokine or chemokine expressions were detected via RT-PCR and ELISA.The regulatory effect of Diosgenin on TLR4 pathway was also observed.5.To mimic the parakeratosis in psoriatic skin,we cultured HaCaT cells in low Ca²⁺（0.03m M）medium while co-treating with D2 or D10.Cells cultured in high Ca²⁺（1.2m M）were seemed as the positive control.The expression of several differentiation markers and k17 was detected.5.Two kinds of endothelial cell lines namely HUVEC and HMEC-1 were treated with D2 or D10,and a tube formation assay was performed to determine several parameters such as the total length,branches and segments.The m RNA expression of VEGF-αin HaCaT cells was also determined.For in vivo studies,8-week-old female Balb/c mice at similar weight were utilized.The psoriasis-like dermatitis was established by 7 consecutive days of topically applying imiquimod（IMQ）on the shaved mice back.The condition of skin lesion and the body weight of each mouse was monitored throughout the whole experiment.Daily dosing of Diosgenin at 10mg/kg（DL）or 50mg/kg（DH）before the IMQ application was performed.On day 8,Mice were euthanized and the PASI scores were calculated.HE staining,IHC staining and TUNEL assays were performed to observe the epidermis proliferation and differentiation,inflammatory cell infiltration,cutaneous apoptosis and angiogenesis.The blood samples were obtained to detect the serum level of several cytokines.The spleens were weighed and harvested for flow cytometry to determine the proportion of CD3+CD4+and CD3+CD8+T lymphocytes.Finally,organs such as the heart,liver,spleen,lung and kidney of mice were harvested for HE staining.The liver/kidney function and blood routine examinations were detected for drug safety purpose.Results1.Diosgenin could inhibit HaCaT cell proliferation on a time and concentration dependent manner.On the one hand,HaCaT cells gained morphological changes after being treated with high concentration of Diosgenin（D10）,and the inhibitory effect might partially due to the cycle arrest at G0/G1 phase via down-regulating the NFκB pathway.Diosgenin could regulate the proportion of Bax and Bcl-2 expression and increase the cleaved Caspase-3 expression to induce apoptosis.In LPS-induced HaCaT cells,similar mechanisms were observed while in IL-22 induced HaCaT cells,D10 inhibited cell growth via downregulating the expression of IL-22RA1 which inhibited the phosphorylation of STAT3 and activation of the IL-22RA1-dependent pathways including AKT/m TOR signaling.D10 could also reduce angiogenesis via inhibiting the VEGF-αexpression in HaCaT cells.On the other hand,low concentration of Diosgenin（D2）could significantly reduce the expression of several cytokines and chemokines stimulated by LPS via TLR4 signaling regulation.In TNF-αinduced HaCaT cells,the reduction of IL-6 and IL-8 expression was also observed.Furthermore,D2 could regulate HaCaT cell differentiation by inducing the expression of both early-stage differentiation markers and terminal differentiation markers while D10 could reduce the expression of k17.2.For in vivo studies,compared to the IMQ group and the IMQ+DL group,Dosing DH could apparently optimize the PASI scores.HE staining revealed a reduced epidermal thickness.Meanwhile,DH gavage administration could inhibit the epidermal proliferation and inflammatory cell infiltration including T lymphocytes,dendritic cells and neutrophils,induce the epidermal differentiation and reduce angiogenesis.Besides the effect on the skin,DH could ameliorate spleen hypertrophy induced by IMQ application,both DL and DH seemed to increase the proportion of CD3+CD4+T lymphocytes in the spleen.For drug safety test,no obvious changes in body weight,major organ damage,blood routine test or liver/kidney function were observed,indicating it to be a potentially safe drug for therapeutic purpose.ConclusionWe investigated the effects of Diosgenin on the proliferation,differentiation and inflammation in HaCaT cells and the angiogenesis in HUVEC and HMEC-1.The results from the in vivo studies were corresponding to the in vitro studies.In conclusion,our research provided the sound theoretic basis and potentials for further studying Diosgenin as a therapeutic approach to psoriasis.