【作者】 王璐；

【导师】 王俊平；

【作者基本信息】 山西医科大学 ， 内科学（专业学位）， 2024， 博士

【摘要】 溃疡性结肠炎(ulcerative colitis,UC)是一种发生在结直肠黏膜的慢性特发性炎症性疾病,以腹痛、腹泻、血便为主要临床表现,病程迁延不愈,反复发作,进行性加重。其发病机制至今未明,公认的致病机制包括遗传,环境因素,异常的免疫反应,肠道屏障功能损伤,氧化应激和肠道菌群失调。目前UC的治疗药物主要包括氨基水杨酸类,皮质类固醇类,免疫抑制剂和生物制剂。然而,尚缺乏特效治疗药物且上述药物存在副作用多,部分患者出现原发性治疗不耐受和继发性耐药现象。中药治疗与UC症状类似的病症已有几千年的历史,其疗效确切,副作用小。但是,中医诊治体系与西医显著不同,导致中药治疗UC的理论依据及其科学性受到现代医学质疑,也导致中药在世界范围内的推广受阻。从中药单体的角度研究中药的治疗机制能够将复杂的问题具体化,已成为促进中药现代化的途径之一。和厚朴酚(honokiol,HKL)是中药厚朴的主要生物活性成分之一,具有广谱抗炎、抗菌、抗氧化、解痉、抗肿瘤、抗血栓、抗抑郁、抗焦虑作用及其他活性作用。本课题将从细胞、动物水平验证HKL对UC的治疗作用,并深入探讨其作用机制。目的:1.观察HKL对UC模型小鼠的疾病活动指数(disease activity index,DAI)、结肠长度、组织病理学评分这些反映疾病严重程度的指标的改善作用。2.测定HKL治疗组小鼠的结肠组织黏液屏障标志物表达量、紧密连接蛋白表达量、炎性因子表达量,探讨HKL对实验性UC的改善可能涉及的通路。3.体外观察HKL对RAW264.7小鼠巨噬细胞产生的炎症介质诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、环氧酶2(cyclooxygenase-2,COX2),炎症因子白细胞介素1β(interleukin 1β,IL1β)、白细胞介素6(interleukin 6,IL6)、肿瘤坏死因子α(tumor necrosis factorα,TNFα)表达的影响,对小鼠结肠上皮细胞(mouse colon epithelial cells,MCEC)的紧密连接蛋白(zonula occludens protein 1,ZO1)、Occludin、Claudin1的表达,细胞单层跨上皮电阻值(transepithelial electric resistance,TEER)的影响,从细胞水平探讨HKL对实验性UC所涉及的治疗作用。4.在细胞水平探索HKL的抑炎、抗氧化和屏障增强作用可能涉及的通路。方法:1.6-8周龄C57BL/6小鼠40只,随机分为对照组、单药组、模型组、治疗组四组,每组10只,实验周期12天,对照组全周期正常饮食;单药组全周期予HKL(40mg/kg)溶于橄榄油每日口服灌胃(10ul/g);模型组第1-5天正常饮食,第6-12天更换3%葡聚糖硫酸钠(dextran sulfate sodium,DSS)特殊饮水同时全周期橄榄油每日口服灌胃(10ul/g);治疗组第6-12天更换3%DSS特殊饮水同时全周期HKL(40mg/kg)溶于橄榄油每日口服灌胃(10ul/g)。每日记录小鼠的体重变化,粪便性状和便血情况,观察小鼠DAI。实验周期结束时颈脱臼法处死40只小鼠,分离小鼠回肠末端至肛门口结肠段,测量结肠长度。苏木素和伊红(hematoxylin and eosin,HE)染色结肠组织切片,依据炎症、隐窝损伤、溃疡和水肿情况进行组织病理学评分。实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,q RT-PCR)法测定组织IL-1β、IL-6、TNFα、i NOS、COX2、ZO1、Occludin、Claudin1 m RNA水平。免疫印迹(western blot,WB)测定组织ZO1、Occludin、Claudin1、p-p65、p65、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)蛋白表达水平。免疫组化观察结肠组织切片黏糖蛋白2(mucin-2,MUC2)、黏糖蛋白3(mucin-3,MUC3)、三叶肽因子3(trefoil factor 3,TFF3)表达情况。2.体外培养小鼠RAW264.7巨噬细胞、MCEC,采用不同浓度梯度的HKL作用上述细胞24小时,通过CCK-8测试细胞活性,筛选适宜的药物作用浓度。在RAW264.7细胞,通过qRT-PCR法测定IL-1β、IL-6、TNFα、iNOS、COX2 mRNA水平,细胞免疫荧光测定核因子-κB(nuclear factor-κB,NF-κB)p65、核因子红细胞2相关因子2(nuclear factor erythroid 2-related factor 2,NRF2)信号通路核转位情况,WB测定ZO1、Occludin、Claudin1蛋白表达水平;在MCEC,通过q RT-PCR法测定ZO1、Occludin m RNA水平,细胞免疫荧光测定ZO1、Occludin、Claudin1蛋白表达水平,WB测定HKL作用不同时间点ZO1、Occludin、Claudin1、磷酸化腺苷5’-单磷酸-活化蛋白激酶(phosphorylated adenosine 5′-monophosphate-activated protein kinase,p AMPK)、腺苷5’-单磷酸-活化蛋白激酶(adenosine 5′-monophosphate-activated protein kinase,AMPK)、去乙酰化酶3(sirtuin 3,SIRT3)、NRF2、血红素加氧酶1(Heme Oxygenase 1,HO1)蛋白表达水平,测定MCEC单层TEER。结果:1.HKL改善了DSS诱导的实验性UC小鼠的DAI,结肠长度缩短和组织病理学评分。2.与DSS模型组相比,HKL治疗组小鼠的IL-1β、IL-6、TNFα、iNOS、COX2m RNA水平显著降低。3.与DSS模型组相比,HKL治疗组小鼠的ZO1、occludin、Claudin1 m RNA表达水平和蛋白表达水平显著增加,MUC2、MUC3、TFF3表达水平也有所恢复。4.HKL抑制脂多糖(Lipopolysaccharide,LPS)刺激的RAW264.7小鼠巨噬细胞炎症细胞因子IL-1β、IL-6、TNFα基因表达,iNOS、COX2炎症介质蛋白表达。5.将LPS刺激RAW264.7细胞12小时后取上清液作用于MCEC细胞12小时,设定为模型组,HKL治疗组较模型组紧密连接蛋白ZO1、occludin的基因表达水平,ZO1、occludin、Claudin1的蛋白表达水平显著增加,MCEC单层TEER治疗组较模型组有所恢复。将40μmol HKL分别作用于MCEC 3小时、6小时、12小时,紧密连接蛋白ZO1、occludin、Claudin1的最大表达量发生在不同的时间点。6.在动物实验中,与DSS模型组相比较,HKL减少了治疗组结肠组织的NF-κB p65磷酸化水平、增加了PPARγ表达水平。7.在细胞实验中,与LPS模型组相比较,HKL减少了治疗组RAW264.7细胞的NF-κB p65磷酸化水平、恢复了PPARγ表达水平。免疫荧光显示HKL恢复了LPS对NRF2核转位的抑制作用,抑制了LPS对NF-κB p65核转位的促进作用。将40μmol HKL分别作用于MCEC 3小时、6小时、12小时,对AMPK磷酸化、SIRT3、NRF2和HO1表达水平在不同的时间点均有促进作用。结论:1.HKL对实验性UC小鼠的疾病具有改善作用。2.HKL可以减轻实验性UC小鼠的肠道炎症,改善肠黏膜屏障功能,抑制氧化应激。3.HKL的治疗作用可能涉及PPARγ/NF-κB p65、SIRT3/AMPK和NRF 2/HO1信号通路。更多还原

【Abstract】 Ulcerative colitis(UC)is a chronic idiopathic inflammatory disease that occurs in the colorectal mucosa.The main clinical manifestations are abdominal pain,diarrhea,and bloody stool.The course of the disease is prolonged,recurrent,and progressively aggravated.Its pathogenesis is still unclear,but it is widely recognized that the pathogenic mechanisms include genetic,environmental origin,abnormal immunoreaction,intestinal barrier dysfunction,oxidative stress,and intestinal flora imbalance.At present,the treatment of UC generally includes aminosalicylic acids,corticosteroids,immunosuppressive agents,and biological agents.However,there is still a lack of specific therapeutic drugs and the above drugs have many side effects,some patients have primary treatment intolerance and secondary drug resistance.Traditional Chinese medicine(TCM)has been used to treat diseases similar to UC symptoms for thousands of years,with definite efficacy and few side effects.However,the diagnosis and treatment system of traditional Chinese medicine is significantly different from that of Western medicine,which leads to the theoretical basis and scientific nature of TCM in the treatment of UC being questioned by modern medicine,and also leads to the blockage of the promotion of TCM worldwide.Studying the therapeutic mechanism of TCM from the perspective of TCM monomer can concretize complex problems and has become one of the ways to promote the modernization of TCM.Honokiol(HKL)is one of the main bioactive components of Magnolia officinalis,which has a broad spectrum of anti-inflammatory,antibacterial,anti-oxidation,spasmolysis,anti-tumor,anti-thrombotic,antidepressant,anti-anxiety effects and other activities.This study will verify the therapeutic effect of HKL on UC at the cell and animal levels and explore its mechanism in depth.Objective:1.To observe the effects of HKL on disease activity index(DAI),colon length,and histopathological score of UC model mice.2.The expression levels of mucus barrier markers,tight junction proteins,and inflammatory factors in the colon tissue of mice in the HKL treatment group were determined to explore the feasible pathways involved in the improvement of experimental UC by HKL.3.Inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX2)were detected in RAW264.7 macrophages treated with HKL in vitro.The effect of inflammatory factors interleukin 1β(IL1β),interleukin 6(IL6),and tumor necrosis factor α(TNFα)expression,The expression of zonula occludens protein 1(ZO1),Occludin,and Claudin1 in mouse colon epithelial cells(MCEC)was detected by Western blot.The transepithelial electric resistance(TEER)value of the cell monolayer was determined to explore the therapeutic effect of HKL on experimental UC at the cellular level.4.To explore the possible pathways involved in the anti-inflammatory,anti-oxidation,and barrier enhancement effects of HKL at the cellular level.Methods:1.Forty C57BL/6 mice aged 6-8 weeks were randomly divided into four groups: control group,single drug group,model group,and treatment group,with 10 mice in each group.The experimental period was 12 days.The single drug group was given HKL(40mg/kg)dissolved in olive oil by oral gavage(10ul/g)daily for the whole cycle;The mice in the model group were fed with a normal diet from day 1 to day 5 and were given 3% dextran sulfate sodium(DSS)drinking water and olive oil by oral gavage(10ul/g)from day 6 to day12.The treatment group was given 3%DSS in drinking water and HKL(40mg/kg)dissolved in olive oil by oral gavage every day(10ul/g)from day 6 to day 12.The changes in body weight,fecal characteristics,and hematochezia were recorded daily,and DAI was observed.At the end of the experimental period,40 mice were sacrificed by the cervical dislocation method.The colon segment from the terminal ileum to the anal orifice of the mice was isolated,and the colon length was measured.Hematoxylin and eosin(HE)staining was used to evaluate the histopathological scores of inflammation,crypt injury,ulceration,and edema.The m RNA levels of IL-1β,IL-6,TNFα,iNOS,COX2,ZO1,Occludin,and Claudin1 were measured by real-time quantitative polymerase chain reaction(qRT-PCR).Western blot(WB)was used to detect ZO1,Occludin,Claudin1,p-p65,p65,and peroxisome proliferatoractivated receptor γ(PPARγ)protein expression.Immunohistochemistry was used to observe the expression of mucin-2(MUC2),mucin-3(MUC3),and trefoil factor 3(TFF3)in colon tissue sections.2.Mouse RAW264.7 macrophages and MCEC were cultured in vitro,and the above cells were treated with different concentration gradients of HKL for 24 hours.The cell activity was tested by CCK-8 to screen the appropriate drug concentration.In RAW264.7cells,the m RNA levels of IL-1β,IL-6,TNFα,i NOS,and COX2 were measured by qRTPCR.The nuclear translocation of nuclear factor-κB(NF-κB)p65 and nuclear factor erythroid 2-related factor 2(NRF2)signaling pathway was determined by cell immunofluorescence.The protein expression levels of ZO1,Occludin,and Claudin1 were determined by WB.In MCEC,the Mrna levels of ZO1 and Occludin were determined by qRT-PCR,and the protein expression levels of ZO1,Occludin,and Claudin1 were determined by cellular immunofluorescence.ZO1,Occludin,Claudin1,phosphorylated adenosine 5’-monophosphate-activated protein kinase(p AMPK),adenosine 5’-monophosphate-activated protein kinase(AMPK),deacetylase 3(SIRT3),NRF2,and Heme Oxygenase 1(HO1)protein expression levels were detected by WB at different time points after HKL treatment.The TEER of the MCEC monolayer was measured.Results:1.HKL improved DAI,colon length shortening,and histopathological score in DSSinduced experimental UC mice.2.Compared with the DSS model group,the m RNA levels of IL-1β,IL-6,TNFα,iNOS,and COX2 in the HKL treatment group were significantly decreased.3.Compared with the DSS model group,the m RNA and protein expression levels of ZO1,Occluding,and Claudin1 in the HKL treatment group were significantly increased,and the expression levels of MUC2,MUC3,and TFF3 were also restored.4.HKL inhibited the m RNA expression of inflammatory cytokines IL-1β,IL-6,TNFαand the protein expression of Inos and COX2 in lipopolysaccharide(LPS)stimulated RAW264.7 mouse macrophages.5.LPS stimulated RAW264.7 cells for 12 hours and then the supernatant was taken to act on MCEC for 12 hours,which was set as the model group.Compared with the model group,the gene expression levels of tight junction proteins ZO1 and Occluding and the protein expression levels of ZO1,Occluding,and Claudin1 in the HKL treatment group were significantly increased.The TEER in the treatment group recovered somewhat compared with the model group of MCEC.When 40μmol HKL was applied to MCEC for 3,6,and 12 hours,the maximum expression of tight junction proteins ZO1,Occluding,and Claudin1 occurred at different time points.6.In the animal experiment,HKL reduced the phosphorylation level of NF-κB p65 and increased the expression level of PPARγ in the colon tissues of the treatment group compared with the DSS model group.7.In the cell experiment,compared with the LPS model group,HKL reduced the phosphorylation level of NF-κB p65 and restored the expression level of PPARγ in RAW264.7 cells in the treatment group.Immunofluorescence showed that HKL restored the inhibitory effect of LPS on NRF2 nuclear translocation and inhibited the promoting effect of LPS on NF-κB p65 nuclear translocation.Treatment of MCEC with 40μmol HKL for 3 h,6 h,and 12 h promoted AMPK phosphorylation and SIRT3,NRF2,and HO1 expression levels at different time points.Conclusion:1.HKL could improve the symptoms in experimental UC mice.2.HKL can alleviate intestinal inflammation,enhance intestinal mucosal barrier function,and inhibit oxidative stress in experimental UC mice.3.The therapeutic effect of HKL may involve PPARγ/ NF-κB p65,SIRT3/ AMPK,and NRF 2/ HO1 signaling pathways.更多还原