【作者】 张莉；

【导师】 张晓杰；

【作者基本信息】 山东中医药大学 ， 中医外科学， 2023， 博士

【摘要】 研究背景银屑病是以多基因遗传为基础,协同环境因素共同刺激诱导的慢性炎症性皮肤病,免疫紊乱促进了银屑病的发病过程。T细胞是银屑病皮损区主要的免疫反应细胞,活化的Th17细胞可以分泌白细胞介素(interleukin,IL)如IL-17A、IL-17F、IL-22等细胞因子。IL-17刺激角质形成细胞,促使其激活和增殖,激活的角质形成细胞又会分泌炎性细胞因子、趋化因子以及抗菌肽,进一步募集中性粒细胞等迁移到炎症部位,从而产生微脓肿表现。同时这些细胞因子还可以刺激角质形成细胞产生炎症因子及趋化因子等,在银屑病皮损中形成恶性循环,进一步加重银屑病的病症,促进银屑病的病程。因此,明确炎症相关因子在银屑病中的表达水平及其在发病机制中的作用在治疗银屑病中至关重要。表皮基底层角质形成细胞的有丝分裂速度较正常皮肤增加是银屑病特征性的病理生理表现之一,其可导致表皮增厚(棘层肥厚)、表皮嵴延长,角化过度和不完全的角质形成细胞分化及成熟异常,角质层细胞异常堆积,形成鳞屑。在银屑病发生和发展过程中,炎症相关因子刺激角质形成细胞活化,激活的角质形成细胞又可以分泌产生炎症因子及抗凋亡因子等,进一步促进角质形成细胞的增殖、炎症和抗凋亡作用,促进银屑病的进展。因此抑制角质形成细胞增殖或抗凋亡作用是治疗银屑病的有效途径。白芍是一味用药历史悠久的经典中药,现代药理研究表明,白芍具有保护心血管、保肝、镇痛、抗炎等药理作用。白芍总苷是来源于中药白芍干燥根的提取物,其主要萃取成分为芍药苷,是白芍总苷发挥作用的主要成分。白芍总苷在银屑病的治疗中具有良好疗效,但其在机体免疫调节和抗炎症反应中的药物机理仍不明确。微小RNA(microRNA,miRNA)是一种小的非编码RNA,在细胞内通过与靶基因mRNA3’UTR区结合,发挥RNA沉默作用,在基因表达转录后和翻译水平调控基因表达。研究表明许多中药通过调控miRNA的表达发挥治疗作用,而在白芍总苷治疗银屑病的研究中,关于miRNA调控的研究尚未见报道。第一章 白芍总苷治疗斑块型银屑病的临床观察目的观察白芍总苷治疗斑块型银屑病的临床疗效以及安全性。方法1.收集斑块型银屑病患者将其随机分为观察组与对照组。观察组口服白芍总苷联合卡泊三醇外用、窄谱中波紫外线(NB-UVB)照射进行治疗,对照组外用卡泊三醇、NB-UVB照射治疗。2.收集并分析两组患者信息,包括性别、年龄、病程。3.记录两组患者治疗前银屑病皮损面积及严重度指数(PASI)、皮肤病生活质量指数(DLQI)、记录治疗第4周末、第8周末时PASI及第8周末时DLQI。计算PASI改善率,PASI 50、PASI 75及PASI 90应答率,有效率,进行比较。4.记录治疗期间不良反应情况。5.应用SPSS26.0软件进行数据分析。数据计量资料用均值±标准差表示(x±s),计数资料用频数(%)表示,应用t检验,P<0.05为差异有统计学意义。结果1.观察组与对照组比较,两组在性别、年龄、病程、治疗前PASI及DLQI无统计学差异(P>0.05)。2.治疗4周后,观察组PASI与对照组比较显著降低,(P<0.05),观察组PASI 50、75 应答率高于对照组(PASI50:63.33%vs 16.67%、PASI75:3.33%vs0,P<0.01);观察组有效率优于对照组(63.33%vs 16.67%,P<0.01),两组比较有统计学差异。治疗8周后,观察组的PASI、DLQI与对照组比较明显降低(P<0.01),观察组PASI 50、75、90 应答率显著高于对照组(PASI 50:100.00%vs 86.67%、PASI 75:76.67%vs 33.33%、PASI 90:36.67%vs 3.33%,P<0.05);观察组的有效率优于对照组(100.00%vs 86.70%,P<0.05)。3.治疗期间,观察组与对照组均无严重不良反应,治疗结束后随访6个月,观察组PASI 90患者的复发率明显低于对照组(P<0.01)。结论白芍总苷治疗斑块型银屑病疗效确切,不良反应少,患者依从性高。第二章 白芍总苷通过调控角质形成细胞STAT3的表达和磷酸化抑制炎症和抗凋亡因子的表达目的探讨白芍总苷治疗银屑病的相关分子靶点及治疗机理。方法1.收集并分析临床银屑病病人的全基因转录组表达(RNA-Seq)测序结果和基因芯片分析(RNAArray assay)结果,分析与银屑病发病相关的主要信号通路与分子靶点。根据第一章中的研究,同时收集了入组银屑病患者血液,使用酶联免疫吸附测定(ELISA)技术,分析观察组和对照组患者治疗前、后血液中IL-17的含量变化。2.使用不同浓度的IL-17刺激人永生化角质形成细胞(HaCaT),在不同时间后采用CCK8法检测细胞活性的变化,并确定IL-17的最佳作用浓度及作用时间。在IL-17刺激的HaCaT细胞中加入不同浓度的白芍总苷处理后,检测细胞活性的变化,并确定白芍总苷的最佳浓度及最佳作用时间。3.根据RNA-seq和RNA Array assay分析的结果,使用实时荧光定量PCR(qRT-PCR)检测IL-17诱导的HaCaT细胞的主要炎症相关因子和抗凋亡因子的表达,并与加入白芍总苷进行干预后的表达结果进行对比。4.使用生物信息技术及生物信息学数据库GTRD(gtrd(biouml.org))预测与IL-17 诱导的炎症和抗凋亡因子相关的转录因子。敲除转录因子基因后,使用 qRT-PCR、蛋白免疫印迹(Western Blot,WB)及免疫荧光染色技术检测IL-17刺激的HaCaT细胞中炎症相关因子和抗凋亡因子表达,并与白芍总苷处理细胞模型后的结果进行对比,验证相关转录因子的表达调控作用,分析白芍总苷的治疗机理。5.实验采用随机分组,并使用Graphpad-Prism 6软件,采用t检验对结果进行分析,其中qRT-PCR和WB结果以PBS处理的Control组为参照,以ACTIN为内参进行比较。结果1.全基因转录组表达测序结果和基因芯片结果分析显示,IL-17相关信号通路在银屑病的发展中起主导地位,并且其下游分子靶点IL-1 B、CXCL1/2/10、CCL20等参与了银屑病的病理过程。2.银屑病患者血液ELISA结果分析显示,患者血液中IL-17的含量显著高于正常人。白芍总苷治疗后(观察组),与对照组相比,IL-17的含量明显降低。3.细胞活性结果显示,使用IL-17刺激HaCaT细胞的最佳浓度为12.5 mmol/L,最佳处理时间为24 h。加入不同浓度白芍总苷处理后发现20 μ g/mL浓度效果最佳,处理时间为24 h。白芍总苷干预后,IL-17诱导的HaCaT细胞活性明显降低,炎症相关因子IL-1 B、CXCL1/2/10、CCL20 mRNA水平的表达明显降低。4.生物信息学预测发现信号转导及转录激活蛋白3(STAT3)是IL-1 B、CXCL1/2/10、CCL20潜在的转录调节因子,同时STAT3也是抗凋亡因子BCL-XL的转录因子。敲除STAT3后,炎症相关因子IL-1 B、CXCL1/2/10、CCL20的表达明显降低,同时细胞抗凋亡因子BCL-XL的表达显著降低。5.使用白芍总苷处理银屑病细胞模型后,STAT3 mRNA水平无明显改变,但蛋白水平的表达和磷酸化水平明显降低,同时细胞抗凋亡蛋白BCL-XL mRNA水平和蛋白水平的表达均明显降低。免疫荧光染色结果与WB结果一致。结论白芍总苷通过抑制STAT3蛋白水平的表达和磷酸化活性降低角质形成细胞炎症相关因子和抗凋亡因子的表达。第三章 白芍总苷通过mi R-125a/b降低STAT3的表达抑制角质形成细胞炎症及抗凋亡的研究目的探究白芍总苷通过抑制STAT3蛋白表达发挥治疗作用的分子机理。方法1.根据文献报道,使用qRT-PCR检测与银屑病治疗相关的miRNA的表达。2.使用miRNA数据库(TargetScanHuman8.0)分析STAT3与miRNA之间的联系。使用双荧光素酶报告实验确认miRNA与STAT3的靶向调控作用。3.过表达miRNA后检测STAT3及炎症相关因子IL-1 B、CXCL1/2/10、CCL20及抗凋亡因子BCL-XL的表达。4.实验采用随机分组,并使用Graphpad-Prism 6软件,采用t检验对结果进行分析。其中qRT-PCR和WB结果以PBS处理的Control组为参照,以ACTIN为内参进行比较。结果1.使用IL-17诱导HaCaT细胞,与PBS处理的HaCaT细胞对照组相比较:与银屑病相关的miRNA中,miR-31在IL-17诱导的HaCaT细胞中表达升高,上升2.55±0.46倍,(P<0.05),miR-146a表达升高,变化倍数为2.19±0.18倍,(P<0.05);其他相关miRNA如miR-203、miR-125a/b、miR-197、miR-520a与对照组相比无明显变化。当使用白芍总苷处理IL-17诱导的HaCaT细胞后,与IL-17诱导的HaCaT细胞相比:miR-31表达下调,变化倍数为0.51±0.11倍,(P<0.01);miR-146a表达降低,变化倍数为0.46±0.12倍,(P<0.05);miR-125a及miR-125b的表达水平明显升高,升高倍数为5.80±0.64倍和6.10±0.35倍,(P<0.001);miR-520a表达升高,升高倍数为 2.77±0.33 倍,(P<0.05)。miR-31、miR-146、miR-520a、miR-125a 及 miR-125b 的表达变化与白芍总苷的治疗存在相关性,其中miR-125a/b的变化倍数最为明显。2.TargetScan数据库分析显示STAT3是miR-125a/b可能的靶基因,双荧光素酶证实miR-125a/b对STAT3的表达具有直接调控作用。当过表达miR-125a/b后,STAT3蛋白水平的表达明显降低。3.过表达miR-125a/b后,IL-17诱导的HaCaT细胞中炎症相关因子CXCL1/2/10、CCL20及IL-1 B的表达明显降低,STAT3 mRNA表达水平无变化,但蛋白水平表达明显降低;同时,抗凋亡因子BCL-XL mRNA和蛋白水平的表达均明显降低。结论白芍总苷通过诱导miR-125a/b靶向抑制STAT3蛋白水平的表达,降低炎症相关因子CXCL1/2/10、CCL20、IL-1 B和抗凋亡因子BCL-XL的表达,发挥治疗作用。更多还原

【Abstract】 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.更多还原