| Background:Psoriasis is a chronic inflammatory skin disease common in dermatology.Mainly manifested as scaly erythema or plaque,Psoriasis vulgaris is the commonest type of psoriasis.The exact etiology and pathogenesis of psoriasis are still unclear.It is believed that immunological disorders mediated by T cells,aberrantly activated keratinocytes and abnormal expressed keratinocytes are the crucial elements in the development of psoriasis.Keratin 17(K17)is specifically highly expressed in psoriatic keratinocytes,which is involved in the disease by promoting keratinocyte proliferation,inducing the release of inflammatory mediators,and stimulating T cell activation.It is considered a major marker of active keratinocyte proliferation.Acitretin is a first-line systemic therapy for thetreatment of moderate-to-severe chronic plaque psoriasis.Although the efficacy of acitretin in psoriasis treatment is well-established,previous studies have reported some potential side effects such anincreased incidence of liver toxicity.TCM has played a unique role in the treatment of psoriasis in China,especially excelling in improving curative effect,decreasing recurrence,reducing side effects and economical burden.However,the target of TCM and its active ingredients in the treatment of psoriasis remains unclear and the mechanism of action is not understood,which has become a major obstacle to its further development and wide application.Total glucosides of paeonia(TGP)is an active ingredient extracted from TCM herb Baishao(white paeony root),with main active component as paeoniflorin(PF).In recent years,pharmacologists have found that PF has many functions,such as anti-inflammation,analgesic,liver-protection,as well as regulating immune response and cell proliferation.Therefore,PF has been applied to treat immune-related chronic inflammatory skin diseases such as psoriasis and atopic dermatitis.There are many clinical reports of PF for psoriasis treatment.However,there are no standardized multi-center,randomized,double-blind,controlled clinical study data that fully confirmed its efficacy and safety of the treatment.To explore its mechanism of action in the treatment of psoriasis,literature reported that the PF can achieve therapeutic effects by inhibiting inflammatory infiltration,inflammatory factors and angiogenesis.However,the development of psoriasis is closely related to the activation of keratinocytes and the alteration of biological functions.It still needs further investigation in terms of whether PF works in psoriasis by affecting keratinocytes,if it affects the keratinocytes closely associated with cell proliferation and what is its specific regulation mechanism.To further verify the therapeutic effect of PF on psoriasis,the present study designed a standardized randomized,parallel-controlled clinical study to fully validate its efficacy and safety in the treatment of plaque psoriasis vulgaris in clinical applications.At the same time,based on the understanding that high expressed keratin 17 affects the active proliferation of keratinocytes and promotes the development of psoriasis,this study intends to proceed from the hypothesis that “PF inhibits keratinocyte proliferation by regulating K17”,and explore the mechanism of PF in the treatment of psoriasis.Objectives:1.To evaluate the efficacy and safety of PF in the treatment of plaque psoriasis vulgaris via clinical research;2.To clarify the mechanism of PF regulating the proliferation of keratinocytes and secretion of inflammatory factors by targeting and inhibiting K17 expression in the treatment of psoriasis via animal experiments,molecular biology,and cell biology,etc..Methods:1.Evaluation of the efficacy and safety of PF in the treatment of plaque psoriasis vulgaris: A multicenter,randomized,double-blind,controlled clinical study was designed.Three domestic clinical research centers were selected to enroll patients of 18-65 years old,who were clinically diagnosed with plaque psoriasis vulgaris.Enrolled patients were randomly and evenly divided into two groups: The experimental group received oral TGC + avi-A capsule combined treatment;the control group received oral avi-A capsule treatment + TGC simulant.After 12 weeks of treatment,efficacy of PF in the treatment of plaque psoriasis vulgaris was evaluated by comparing patients with a Psoriasis Area and Severity Index(PASI)improvement of 50%(PASI50),75%(PASI75),and 90%(PASI90)between the two groups.Safety of PF in the treatment of plaque psoriasis vulgaris was evaluated by comparing the changes in liver function indicators(alanine aminotransferase and aspartate aminotransferase)before and after treatment by collecting adverse event reports.2.Cell experiments in vitro: the role and mechanism of PF regulating K17 expression: A psoriasis-like cell model was constructed using the keratinocyte cell line HaCat stimulated with a quaternity including TNF-?,IFN-?,IL-17,and IL-23,as well as treated with different concentrations of PF(75-1200 ug/ml).Western Blot was used to verify if PF inhibits keratinocyte proliferation by down-regulating psoriasis-related keratins K6,K16 and K17.HaCat treated with cell counting kit Kit-8(CCK-8)for 0,24 and 48 hours was determined for the viability of cell proliferationReal-time PCR,Western-blot and immunofluorescence were used to observe the changes of K17 mRNA and protein expression in HaCat.Psoriasis and K17-associated inflammatory pathways were screened,HaCat cells were treated with PF at optimal concentration and time,and STAT1,STAT3,ERK,NFkB of paclitaxel inhibiting phosphorylation activity in HaCat cells were screened by Western-blot method.Immunofluorescence was used to determine the translocation changes of nuclear transcription factors,and the signal pathway of PF to regulate K17 expression was clarified.3 Animal experiment: the effect of topical PF on imiquimod(IMQ)-induced psoriasis-like mouse model: An IMQ-induced psoriasis-like mouse model was constructed.Imiquimod was applied in the morning after the back of the mouse was shaved,and PF was applied in the afternoon to observe the IMQ-induced psoriasis phenotype in mice.After 7 days,changes were phenotypic erythema and scales were observed and epidermis was taken for HE staining to determine the effect of PF to the proliferation of psoriatic epidermis.Furthermore,Real-time PCR,Western-blot and immunofluorescence were used to observe the influence of PF to inflammatory factors and K17 expression in psoriatic epidermis.Results:1.Clinical study results: A total of 108 patients with moderate to severe plaque psoriasis were enrolled in 3 research centers.At the end of study,84 completed all follow-ups were analyzed for effectiveness.Among the 108 patients receiving oral meditation,11 were excluded for incomplete date and the other 97 went on for safely analysis.1)Improvement of PASI scores: PASI50 improvement is significantly higher in treatment group.After 12 weeks of treatment,compared with the baseline,the experimental groups were associated with better improvement in PASI scores: 40 patients in the experimental group had a 90% improvement in PASI50,52.5% in PASI75 improvement,12.5% in PASI90 improvement.Among the 44 in the control group,70.5% had PASI50 improvement,40.9% had PASI75 improvement,and 9.1% had PASI90 improvement.Chi-square test was used to compare the difference between the two group,.PASI50 improvement was statistically significant,but PASI75 and PASI90 improvement was not statistically significantly.2)Adverse reactions such as changes in liver function: liver impairment is significantly lower in the experiment group.Compared with the baseline,among the 48 patients in the experimental group,3 had elevated ALT,and the incidence of liver impairment was 6.25%.Among 49 patients in the control group,10 had elevated ALT,and the incidence of liver impairment was 20.4%.The increase in AST was not significant in both groups.Compared by chi-square test,two groups had a statistically significant difference in the incidence of total liver impairment.Other adverse reactions included 23 cases of elevated triglyceride,4 cases of elevatedtotal cholesterol,14 cases of dry mouth,6 cases of dry skin,itching,desquamation,2 cases of hair loss,6 cases of diarrhea.There was no statistical difference between the two groups.2.Cell test in vitro: PF regulating K17 expression:1)PF can inhibit the proliferation of HaCat: Different concentrations of PF(75-1200 ug/ml)were added to stimulate the cells,and cell proliferation was observed by CCK8 at 0,24 and 48 h.The results show that PF can inhibit quaternity-induced keratinocyte proliferation in a dose-dependent manner after PF stimulation 48.2)PF can inhibit the expression of K17 in HaCat: Western Blot assay showed that PF worked best on regulating K17 expression after PF treatment.Further study at the mRNA level revealed that PF can significantly reduce the expression of K17 in HaCat after quaternity stimulation in a concentration-dependent manner.Immunofluorescence results also showed that PF can effectively inhibit the expression of K17.3)PF can inhibit NFkB-related pathways: Western blotting was used to screen inflammation-related pathways.It was found that phosphorylation of NF-kB pathway was inhibited in the PF-treated group.By setting the time gradient,it was found that the phosphorylation levels of NF-kB and IkB? were the lowest after 4 h of treatment.At the same time,the immunofluorescence was detected at the 2h time point,and it was further confirmed that PF inhibited the phosphorylation levels of NF-kB and IkB?.3.Animal experiment: the effect of topical PF on imiquimod(IMQ)-induced psoriasis-like mouse model:1)Topical PF can alleviate the phenotypeof IMQ-induced psoriasis in mice: in the IMQ-treated group,the erythema and scales on the back increased significantly,while the psoriasis phenotype was significantly relieved in the mice treated with topical PF.2)Topical PF reduces the epidermal thickness in mice with IMQ-induced psoriasis: HE staining results showed that the epidermis of the topical PF group was significantly thinner than the imiquimod group.3)Topical PF reduces IMQ-induced inflammatory cell infiltration and down-regulations inflammatory factors in mice: Immunofluorescence detected that topical PF can significantly reduce neutrophil infiltration and RT-PCR revealed the expression of inflammatory factors was high in IMQ-treated group.The inflammatory factors were down regulated in topical PF group,among which the inhibitory effects of IL-17 and IL-22 were statistically different.4)Topical PF inhibits K17 expression in the epithelium of IMQ-induced mice: The expression of K17 in mouse epidermal tissue was examined.It was found by RT-PCR that the expression of K17 in the epidermal tissue of mice was inhibited at the mRNA level.At the same time,Western Blot and immunofluorescence results further confirmed that K17 expression was also inhibited at the protein level.Conclusion:Firstly,via scientific,normative and ethical multi-center,randomized,double-blind,parallel-controlled clinical studies,it was confirmed that total glucosides of paeony combined with acitretin in the treatment of moderate to severe plaque psoriasis associated with better efficacy,and the combination therapy can reduce the incidence of liver impairment caused by acitretin treatment alone,and thus is more suitable for long-term clinical application for moderate to severe plaque psoriasis.Study on the mechanism of action of PF in the treatment of psoriasis verified thatPF inhibits K17 expression via NF-kB pathway,and it inhibits the proliferation of keratinocytes,affects the abnormal immune response of psoriasis,and exerts a therapeutic effect.At the same time,animal experiments have found that the topical use of PF can significantly improve the psoriasis phenotype of animal models manifested as erythema and scale.This study provides a new therapeutic strategy for PF treatment of psoriasis,as a theoretic basis to further develop and apply TCM active ingredient paeoniflorin as a new and efficient topical treatment for psoriasis. |
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