| BackgroundPsoriasis is a chronic inflammatory skin disease induced by polygenic inheritance and costimulation of environmental factors.Immune disorders promote the pathogenesis of psoriasis.T cells are the main immune response cells in psoriatic lesions.Activated Th17 cells can secrete interleukin(IL),such as IL-17A,IL-17F,IL-22 and other cytokines.IL-17 stimulates keratinocytes to activate and proliferate.The activated keratinocytes will secrete inflammatory cytokines,chemokines and antimicrobial peptides,and further recruit concentrated granulocytes to migrate to the inflammatory site,resulting in microabscess.At the same time,these cytokines can also stimulate keratinocytes to produce inflammatory factors and chemokines,and form a vicious circle in psoriasis lesions,further aggravate the symptoms of psoriasis and promote the course of psoriasis.Therefore,it is important to clarify the expression level of inflammatory related factors in psoriasis and their role in pathogenesis in the treatment of psoriasis.The mitotic rate of keratinocytes in the basal layer of epidermis is higher than that of normal skin,which is one of the most characteristic pathophysiological manifestations of psoriasis.It can lead to epidermal thickening(acanthosis),lengthening of epidermal crest,abnormal differentiation and maturation of keratinocytes with excessive and incomplete keratinization,and then lead to abnormal accumulation of keratinocytes and formation of scales.During the occurrence and development of psoriasis,inflammation related factors stimulate the activation of keratinocytes,and the activated keratinocytes can also secrete and produce inflammatory factors and anti-apoptotic factors,which further promote the proliferation,inflammation and anti-apoptotic effects of keratinocytes and promote the progress of psoriasis.Therefore,inhibiting the proliferation or anti-apoptosis of keratinocytes is an effective way to treat psoriasis.White peony is a classic traditional Chinese medicine with a long history of medication.Modern pharmacological studies have shown that it has pharmacological effects such as cardiovascular protection,liver protection,analgesia,and anti-inflammatory.TGP is an extract from the dried roots of the traditional Chinese medicine,Paeoniflorin,which is the main component of TGP.TGP has a good effect in the treatment of psoriasis,but its drug mechanism in the immune regulation and anti-inflammatory reaction of the body is still unclear.MicroRNA(miRNA)is a small non-coding RNA,which plays the role of RNA silencing by binding with the target gene mRNA3’UTR region in the cell,and regulates gene expression at the post-transcriptional and translational levels of gene expression.Studies have shown that many traditional Chinese medicines play a therapeutic role by regulating the expression of miRNA.However,in the study of TGP in the treatment of psoriasis,the study of miRNA regulation has not been reported.Part Ⅰ Clinical observation of total glucosides of paeony in the treatment of plaque psoriasisObjectiveTo clinically observe the clinical curative effect and safety of total glucosides of paeony in the treatment of plaque psoriasis.Methods1.Clinical plaque psoriasis patients were collected.Then the patients were randomly divided into two groups,who were treated with combined treatment of total glucosides of paeony combined with calcipotriol and narrow-band UVB and control treatment with calcipotriol and narrow-band UVB.2.Collect and analyze the information of the two groups of patients,including gender,age and course of disease.3.The psoriasis area and severity index(PASI),dermatological life quality index(DLQI),PASI at the end of the 4th and 8th week of treatment and DLQI at the end of the 8th week of treatment were observed in both groups.PASI improvement rate,response rate of PASI 50,PASI 75 and PASI 90 were calculated and compared.4.Record adverse reactions during treatment.5.Use the SPSS26.0 software for data analysis.The measurement data were expressed by mean ± standard deviation(x±s)and the counting data were expressed by frequency(%).t test was applied,P<0.05 was considered statistically significant.Results1.There were no significant differences between the two groups in gender,age,course of disease,PASI and DLQI scores before treatment(P>0.05).2.After 4 weeks of treatment,PASI score of observation group was significantly lower than control group(P<0.05),the PASI 50 and 75 response rates in observation group were significantly higher than those in control group(PASI 50:63.33%vs 16.67%,PASI 75:3.33%vs 0,P<0.01);The effective rate of observation group was better than control group(63.33%vs 16.67%,P<0.01).After 8 weeks of treatment,PASI score and DLQI score of observation group were significantly lower than control group(P<0.05),the PASI 50,75,90 response rates in observation group were significantly higher than those in control group(PASI 50:100.00%vs 86.67%,PASI 75:76.67%vs 33.33%,PASI 90:36.67%vs 3.33%,P<0.05);The effective rate of observation group was better than control group(100.00%vs 86.70%,P<0.05).3.During treatment,no serious adverse reactions were observed in both groups,and the recurrence rate of PASI 90 patients in the observation group was significantly lower than that in the control group(P<0.01).ConclusionTotal glycosides of paeony has a definite effect on plaque psoriasis with few adverse reactions and high compliance of patients.Part Ⅱ Total glycosides of paeony inhibit the expression of inflammatory and antiapoptotic factors by regulating the expression and phosphorylation of STAT3 in keratinocytesObjectiveTo explore the relevant molecular targets and therapeutic mechanism of total glucosides of paeony in the treatment of psoriasis.Method1.Collect and analyze the transcriptome expression(RNA-Seq)sequencing results and gene chip analysis(RNA Array Assay)results of clinical psoriasis patients,and analyze the main signaling pathways and molecular targets related to the pathogenesis of psoriasis.According to the research in the first chapter,the blood of the patients was collected at the same time,and the content of IL-17 in the blood of the observation group and the control group was analyzed by ELISA technology.2.Different concentrations of IL-17 were used to stimulate HaCaT cells to construct apsoriasis cell model,and the CCK8 method was used to detect changes in cell activity after different periods of time,and the optimal concentration and time of action of IL-17 were determined.After treatment with different concentrations of total glucosides of paeony combined with drugs,the changes in the activity of keratinocytes were detected,and the optimal concentration and optimal action time of total glucosides of paeony were determined.3.According to the results of RNA-seq and RNA Array analysis,qRT-PCR was used to detect the expression of major inflammatory related factors and anti-apoptotic factor in HaCat cells induced by IL-17,and the expression of inflammatory related factors in HaCaT cells induced by IL-17 after treatment with total glucosides of paeony comparing.4.Transcription factors associated with IL-17-induced inflammation and anti-apoptotic factors were predicted using bioinformatics and bioinformatics database GTRD(gtrd(biouml.org)).After knocking out transcription factor genes,qRT-PCR,Western Blot and immunofluorescence staining techniques were used to detect the expression of inflammatory and anti-apoptotic factors in HaCaT cells treated with IL-17,and IL-17 induced by combined treatment with total glucosides of paeony HaCaT cells were compared to verify the expression regulation of related transcription factors and analyze the therapeutic mechanism of total glucosides of paeony.5.Random grouping was used in the experiment,and Graphpad-Prism 6 software was used to analyze the results byt test.The results of qRT-PCR and WB were compared in the Control group treated with PBS,and ACTIN was used as the internal reference.Result1.Analysis of whole-gene transcriptome expression sequencing results and gene chip results showed that IL-17-related signaling pathways play a dominant role in the development of psoriasis,and its downstream molecular targets IL-1B,CXCL1/2/10,CCL20,etc.are involved in the development of psoriasis pathological process of the disease.2.The analysis of the patient’s blood ELSIA results showed that the content of IL-17 in the blood of patients with psoriasis was significantly higher than that of normal people.And after treatment with total glucosides of paeony compound therapy(observation group),compared with the control group,the content of IL-17 was significantly reduced.3.The results of cell viability showed that the optimal concentration of IL-17 to induce HaCaT cells was 12.5 mmol/L,and the optimal treatment time was 24 h.Using different concentrations of total glucosides of paeony in combination,it was found that the concentration of 20 μg/mL had the best effect,and the treatment time was 24 h.After treatment with total glucosides of paeony,the activity of HaCaT cells induced by IL-17 was significantly reduced.The expression levels of inflammatory related factors IL-1B,CXCL1/2/10,and CCL20 mRNA levels were significantly reduced.4.Bioinformatics prediction found that STAT3 is a potential transcriptional regulator of IL-1B,CXCL1/2/10,and CCL20,and STAT3 is also the expression of the anti-apoptotic factor BCL-XL.After knocking out STAT3,the expression of inflammation-related factors were significantly reduced,and the expression of anti-apoptotic factor BCL-XL was also significantly reduced.5.After treating the cell model with total glucosides of paeony,the expression and phosphorylation level of STAT3 protein level were significantly reduced,and the expression of cell anti-apoptotic molecule BCL-XL was also significantly reduced in both mRNA level and protein level.The results of immunofluorescence staining were consistent with the results of WB,even after the cells were treated with total glucosides of paeony,the signal intensity of STAT3 and BCL-XL decreased significantly.But the mRNA level of STAT3 was not changed.ConclusionTotal glucosides of paeony can reduce the expression of inflammatory related factors and anti-apoptotic signals in keratinocytes by inhibiting the expression and phosphorylation of STAT3.Part III Total glucosides of paeony inhibit the inflammation and anti-apoptosis of psoriatic cells by reducing the expression of STAT3 through miR-125a/bObjectiveTo study the drug mechanism of total glucosides of paeony inhibiting the expression of STAT3.Method1.According to literature reports,qRT-PCR was used to detect the expression of miRNAs related to psoriasis treatment.2.The miRNA database(Target ScanHuman 8.0)was used to analyze the relationship between the changed STAT3 and miRNA,and the dual luciferase reporter assay was used to confirm the targeted regulation of miRNA and STAT3.3.The expression of STAT3 was detected after miRNA overexpression.After overexpression of miRNA,the expression of inflammatory factor IL-1B,CXCL1/2/10,CCL20 and anti-apoptotic factor BCL-XL in the psoriasis cell model were detected.4.The experiment was divided into random groups,and the Graphpad-Prism 6 software was used to analyze the results by t test.The results of qRT-PCR and WB were compared in the Control group treated with PBS,and ACTIN was used as the internal reference.Result1.After treatment of HaCaT cells with IL-17,miRNAs related to psoriasis were detected by qRT-PCR.The results showed that miR-31 increased by 2.55±0.46 times in the psoriasis cell model,(P<0.05),miR-146a was up-regulated 2.19±0.18 times,(P<0.05),but other miRNAs such as miR-203,miR-125a/b,miR-197 and miR-520a were not changed significantly.After the cell model treated with total glucosides of paeony,the expression of miR-31 was down-regulated,and the change factor was 0.51±0.11 times,(P<0.01).The expression of miR-146a was down-regulated and the change factor was 0.46±0.12 times,(P<0.05).The expression of miR-520a increased,and the increase was 2.77±0.33 times,(P<0.05).However,the expression levels of miR-125a and miR-125b were significantly increased,the increasing folds were 5.80±0.64 times and 6.10±0.35 times,(P<0.001).It shows that the expression changes of miR-31,miR-146,miR-520a,miR-125a and miR-125b are correlated with the treatment of total glucosides of paeony,and the change fold of miR-125a/b is the most obvious.2.TargetScan database analysis showed that STAT3 was a possible target gene of miR125a/b,and double luciferase confirmed that miR-125a/b had a direct regulatory effect on the expression of STAT3.When miR-125a/b was overexpressed,the expression of STAT3 protein level was significantly reduced.3.After overexpression of miR-125a/b,the expression of the inflammatory factors CXCL1/2/10,CCL20 and IL-1B in the psoriasis cell model were significantly reduced,and the expression of the anti-apoptotic factor BCL-XL in both mRNA level and protein level were significantly reduced.ConclusionTotal glucosides of paeony plays a therapeutic role by promoting the expression of miR125a/b to regulate the expression of inflammatory related factors CXCL1/2/10,CCL20,IL1B and anti-apoptotic factor BCL-XL by inhibiting the expression of STAT3 in the psoriasis cell model. |
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