检索结果-南通市图书馆

烙灸对兔激素性股骨头坏死组织中iKKβ、iκbα表达的影响

在线全文

学校读者我要写书评

暂无评论

宁夏医科大学学报 2024年第5期46卷 440-445,452页

作者：徐玉娟刘园园陶莹陈坤陈梦莹连佳伟朱宁宁夏医科大学银川750004 宁夏回族自治区宁东医院银川750411 宁夏医科大学总医院宁夏医科大学第一临床医学院银川750004

目的探讨烙灸治疗早期激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)的作用机制。方法将新西兰兔按随机数表法分为空白组、模型组、烙灸组、抑制剂组,每组6只,采用内毒素联合激素的方法制备SONFH模型。... 详细信息

目的探讨烙灸治疗早期激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)的作用机制。方法将新西兰兔按随机数表法分为空白组、模型组、烙灸组、抑制剂组,每组6只,采用内毒素联合激素的方法制备SONFH模型。烙灸组进行4周的烙灸治疗,抑制剂组给予iκbα磷酸化抑制剂bay11-7082(1 mg·kg-1)腹腔注射3周。干预前后分别观察实验兔一般状况变化;HE染色观察各组实验兔股骨头组织病理学变化;Western blot和RT-qPCR分别检测股骨头组织中iKKβ、磷酸化iκbα(p-iκbα)蛋白及mRNA的表达变化;ELiSA检测血清中核因子κb(nuclear factor kappa-b,NF-κb)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,iL-6)水平。结果与空白组比较,模型组实验兔逐渐出现精神萎靡、毛发暗淡等情况,股骨头组织骨小梁出现断裂、稀疏等改变,iKKβ、p-iκbα蛋白水平,iKKβmRNA水平及血清中NF-κb、TNF-α、iL-6水平均升高(P均iKKβ、p-iκbα蛋白水平,iKKβmRNA水平及血清中NF-κb、TNF-α、iL-6水平均降低(P均iκbα蛋白及mRNA表达均升高(P均b通路有关。

关键词：烙灸激素性股骨头坏死 TLR4/NF-κb通路 iKKβ iκbα

iKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and iκbα

在线全文

学校读者我要写书评

暂无评论

Acta Pharmaceutica Sinica b 2022年第2期12卷 651-664,F0004页

作者： Juan Liu Yuxin Zhuang Jianlin Wu Qiang Wu Meixian Liu Yue Zhao Zhongqiu Liu Caiyan Wang Linlin Lu Yingjiao Meng Kawai Lei Xiaojuan Li Qibiao Wu Elaine Lai-Han Leung Zhengyang Guo Liang Liu Ting Li State Key Laboratory of Quality Research in Chinese Medicine/Macao institute for Applied Research in Medicine and Health Macao University of Science and TechnologyMacao SAR 999078China international institute for Translational Chinese Medicine Guangzhou University of Chinese MedicineGuangzhou 510006China Guangdong Provincial Key Laboratory of New Drug Screening/Laboratory of Anti-inflammatory and immunomodulatory Pharmacology School of Pharmaceutical SciencesSouthern Medical UniversityGuangzhou 510515China Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory infectious Disease Macao University of Science and TechnologyMacao SAR 999078China Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People’s Republic of China Macao University of Science and TechnologyMacao SAR 999078China

inhibitor of nuclear factor kappa-b kinase subunit beta(iKKβ)is one of important kinases in inflammation to phosphorylate inhibitor of nuclear factor kappa-b(iκbα)and then activate nuclear factor kappa-b(NF-κb).inhibition of iKKβhas been a therapeutic strategy for inflammatory and autoimmune diseases.Here we report that iKKβis constitutively activated in healthy donors and healthy ikkβ^(C46A)(cysteine 46 mutated to alanine)knock-in mice although they possess intensive iKKβ-iκbα-NF-κb signaling activation.These indicate that iKKβactivation probably plays homeostatic role instead of causing inflammation.Compared to ikkβWTlittermates,lipopolysaccharides(LPS)could induce high mortality rate in ikkβ^(C46A) mice which is correlated to breaking the homeostasis by intensively activating p-iκbα-NF-κb signaling and inhibiting phosphorylation of 5’adenosine monophosphate-activated protein kinase(p-AMPK)expression.We then demonstrated that iKKβkinase domain(KD)phosphorylates AMPKa1 via interacting with residues Thr183,Ser184,and Thr388,while iKKβhelix-loop-helix motifs is essential to phosphorylate iκbαaccording to the previous reports.Kinase assay further demonstrated that iKKβsimultaneously catalyzes phosphorylation of AMPK and iκbαto mediate homeostasis.Accordingly,activation of AMPK rather than inhibition of iKKβcould substantially rescue LPS-induced mortality in ikkβ^(C46A) mice by rebuilding the homeostasis.We conclude that iKKβactivates AMPK to restrict inflammation and iKKβmediates homeostatic function in inflammation via competitively phosphorylating AMPK and iκbα.

关键词： iKKβ Homeostasis Kinase domain AMPK inflammation iκbα Anti-inflammatory drug Phosphorylation

Histone deacetylase 4 inhibits NF-κb activation by facilitating iκbα sumoylation

在线全文

学校读者我要写书评

暂无评论

Journal of Molecular Cell biology 2020年第12期12卷 933-945页

作者： Qi Yang Jielin Tang Chonghui Xu He Zhao Yuan Zhou Yanyi Wang Min Yang Xinwen Chen Jizheng Chen Department of Gastroenterology Guangzhou Women and Children's Medical CenterGuangzhou 510623China State Key Laboratory of Virology Wuhan Institute of VirologyCenter for Biosafety Mega-ScienceChinese Academy of SciencesWuhan 430071China University of Chinese Academy of Sciences Beijing 100049China Guangzhou institutes of biomedicine and Health Chinese Academy of SciencesGuangzhou 510530China

Protein modification by small ubiquitin-like modifier(SUMO)is an important regulatory mechanism for multiple cellular pro-cesses.Although the canonical pathway involving the ubiquitylation or phosphorylation of iκbα has been well characterized,little is known about the sumoylation of ikba in the control of NF-κb activity.Here,we find that histone deacetylase 4(HDAC4)negatively regulates tumor necrosis factor-alpha-or lipopolysaccharide-triggered NF-κb activation.HDAC4 belongs to the SUMO E3 ligase family and can directly sumoylate iκbα.The cytoplasm location of HDAC4 is essential for iκbαsumoylation.The Cys292 of HDAC4 is a key site for its SUMO E3 ligase activity.The sumoylation of ikbc prevents its polyubiquitination and degradation be-cause these two modifications occur both at the Lys21.Our findings reveal a previously undiscovered role for HDAC4 in the inflammatory response as a SUMO E3 ligase for iκbαsumoylation.Our work provides insight into mechanisms ensuring optimal mediation of the NF-κb pathway.

关键词： histone deacetylase 4 sumoylation iκbα NF-κb SUMO E3 ligase

Nuclear Factor-κb Signaling Pathway Constitutively Activated in Esophageal Squamous Cell Carcinoma Cell Lines and inhibition of Growth of Cells by Small interfering RNA

维普期刊数据库评论

在线全文

维普期刊数据库

学校读者我要写书评

暂无评论

Acta biochimica et biophysica Sinica 2006年第5期38卷 318-326页

作者： Fang TiAN Wei-Dong ZANG Wei-Hong HOU Hong-Tao LiU Le-Xun XUE Laboratory for Cell biology,Medical College,Zhengzhou University,Zhengzhou 450052,China Laboratory for Cell biology Medical College Zhengzhou UniversityZhengzhou 450052 China

Although constitutive nuclear factor(NF)-kb activation has been reported in many humantumors,the role of the NF-kb pathway in esophageal squamous cell carcinoma(ESCC)has not been known.in this study,NF-kb pathway in two ESCC cell lines was investigated using immunocytochemistry,Westernblot and reverse transciiption-polymerase chain reaction.The activation of NF-kb DNA binding was determinedby electrophoretic mobility-shift assay.RNA interference was used to specifically inhibit the expression ofp65.Growth of cells Was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.The results showed that p50,p65,ikbα,p-ikbα and ikb kinase β were expressed and mainly localized in thecytoplasm.Reverse transcription-polymerase chain reaction results showed the constitutive expressions ofp50,p65 and ikbα mRNA in the two ESCC cell lines.Furthermore,the nuclear extracts revealed that p50and p65 translocated to the nucleus had DNA-binding activity.Finally,small interfering RNA of p65 decreasedthe expression of p65,and the viability of ceils transfected with p65 small interfering RNA was significantlysuppressed at the same concentration of 5-fluorouracil(Pis study showed that there was the constitutively activated NF-kb signaling pathway in the ESCC celllines.RNA interference targeting at p65 increased the sensitivity of the ESCC cell lines to 5-fluorouracil,suggesting that NF-kb might be a good target for cancer treatment.

关键词： NF-κb RNA interference esophageal squamous cell carcinoma iκbα iκbα kinase

Triptolide-induced Apoptosis by inactivating Nuclear Factor-kappa b Apoptotic Pathway in Multiple Myeloma in vitro

在线全文

同方期刊数据库

学校读者我要写书评

暂无评论

Journal of Huazhong University of Science and Technology(Medical Sciences) 2011年第4期31卷 446-451页

作者：曾蓉曾令兰陈燕赵菲李睿文璐张纯 institute of Hematology Union HospitalTongji Medical CollegeHuazhong University of Science and Technology

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMi-8226 cells in vitro,as well as the roles of nuclear factor-kappa b（NF-κb） and iκbα was investigated.The effect of tritptolide on the growth of RPMi-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/Pi double staining assay.The expression of NF-κb and iκbα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κb apoptotic pathway in human multiple myeloma RPMi-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMi-8226 cells.besides,we observed the inhibition of NF-κb /p65 in the nuclear fraction was correlated with the increase in the protein expression of iκbα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κb/p65 and iκbα.

关键词： triptolide human multiple myeloma apoptosis NF-κb/p65 iκbα

SUMOylation and deacetylation affect NF-κb p65 activity induced by high glucose in human lens epithelial cells

在线全文

学校读者我要写书评

暂无评论

international Journal of Ophthalmology(English edition) 2019年第9期12卷 1371-1379页

作者： Xiao Han Xiao-Xuan Dong Ming-Yu Shi Li Feng Xin-Ling Wang Jin-Song Zhang Qi-Chang Yan Department of Ophthalmology the Fourth Affiliated Hospital of China Medical UniversityKey Laboratory of Lens Research of Liaoning ProvinceEye Hospital of China Medical UniversityShenyang110005Liaoning ProvinceChina Department of Ophthalmology the Fourth People’s Hospital of ShenyangShenyang110031Liaoning ProvinceChina

AiM: To explore the effects of iκbα SUMOylation and NF-κb p65 deacetylation on NF-κb p65 activity induced by high glucose in cultured human lens epithelial cells(HLECs).METHODS: HLECs(SRA01/04) were cultured with 5.5, 25, and 50 mmol/L glucose media for 24 h, and with 50 mmol/L glucose media for 0, 12, and 24 h respectively. SUMO1 and SiRT1 expressions were detected by reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot(Wb). iκbα and NF-κb p65 expressions were detected by Wb. With NAC, DTT, MG132 or Resveratrol(RSV) treatment, SUMO1 and SiRT1 expressions were detected by Wb. Protein expression localizations were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO1 or SiRT1 overexpression, as well as MG132 and RSV, on the nuclear expression and activity of iκbα and NF-κb p65 were analyzed by immunoblot and dual luciferase reporter gene assay.RESULTS: SUMO1 and SiRT1 expressions were influenced by high glucose in mRNA and protein levels, which could be blocked by NAC or DTT. SUMO1 was down-regulated by using MG132, and SiRT1 was up-regulated under RSV treatment. iκbα nuclear expression was attenuated and NF-κb p65 was opposite under high glucose, while iκbα and NF-κb p65 location was transferred to the nucleus. SUMO1 or SiRT1 overexpression and MG132 or RSV treatment affected the nuclear expression and activity of iκbα and NF-κb p65 under high glucose condition.CONCLUSiON: iκbα SUMOylation and NF-κb p65 deacetylation affect NF-κb p65 activity in cultured HLECs under high glucose, and presumably play a significant role in controlling diabetic cataract.

关键词： SUMOylation deacetylation NF-κb p65 iκbα diabetic cataract high glucose lens epithelial cells

鮸鱼microRNAs调控iκbα基因的分子机制研究

维普期刊数据库评论

在线全文

维普期刊数据库

学校读者我要写书评

暂无评论

鮸鱼microRNAs调控IκBα基因的分子机制研究

作者：杨鲤源上海海洋大学

学位级别：硕士

随着人们对水产品需求的日益上涨,单纯的捕捞和传统小规模养殖已经不能满足日常所需,因此水产品养殖的产业化,规模化成为必然。但随着养殖密度上升,一旦出现大规模的感染,将引起巨大的经济损失。鱼类抵御病害主要依靠先天免疫,因此,通... 详细信息

随着人们对水产品需求的日益上涨,单纯的捕捞和传统小规模养殖已经不能满足日常所需,因此水产品养殖的产业化,规模化成为必然。但随着养殖密度上升,一旦出现大规模的感染,将引起巨大的经济损失。鱼类抵御病害主要依靠先天免疫,因此,通过研究鮸鱼的免疫机制,以探索增强鮸鱼抵抗病原微生物的能力,从而减少病原微生物对养殖产业造成的经济损失。硬骨鱼与哺乳动物类似,先天免疫系统是宿主抵御病原体的第一道防线,它与适应性免疫系统合作,帮助宿主在不利的环境中生存。核因子转录因子κb(nuclear factor kappa-b,NF-κb)信号通路的激活,被认为是炎症发生过程中的重要过程。而NF-κb抑制剂蛋白α(inhibitorαof NF-κb,iκbα),可以抑制NF-κb信号通路,阻止NF-κb与DNA结合,在先天性免疫调控中,扮演十分重要的角色。microRNAs(miRNAs)是一种小的非编码RNA,被证明是一种负调控因子,可以调节许多生理生化过程,例如细胞的生长、增殖和机体的免疫反应等。在本次研究中,以miRNAs为起点,探究在鮸鱼中是否存在一种或多种miRNAs靶向iκbα,调控NF-κb信号通路,调节先天性免疫反应。研究结论如下:1.利用miRanda、Targe Scan软件预测miR-144和miR-122存在相同的潜在靶基因iκbα。为了探究miR-144和miR-122是否靶向iκbα,构建野生型iκbα的3’UTR双荧光素酶报告基因质粒和突变miR-144和miR-122与iκbα的3’UTR互补位点的双荧光素酶报告基因质粒。将这些质粒与miR-144和miR-122模拟物共转染EPC(epithelioma papulosum cyprini)细胞。检测荧光素酶活性,结果表明miR-122和miR-144作为负调控因子,靶向iκbα。2.已经确认miR-122和miR-144靶向iκbα,为探究iκbα基因是否在蛋白水平和mRNA水平均受到这两种miRNAs的调控。转染miR-122和miR-144的模拟物(模拟体和前体质粒)到鮸鱼肠细胞(Miichthys miiuy intestine cells,MiC)中,利用蛋白质印迹法(western blot)实验,验证了miR-122和miR-144模拟物抑制了iκbα蛋白表达,且实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)实验结果验证了miR-122和miR-144也可以从mRNA水平抑制iκbα基因的表达。同时,将miR-122和miR-144的抑制体转入MiC细胞中,得到的结果与模拟物相反。证实miR-122和miR-144作为负调控因子抑制iκbα基因的表达。3.在本次研究中发现,过表达p65对NF-κb、白细胞介素-8(interleukin-8,iL-8)和白细胞介素-1β(interleukin-1β,iL-1β)信号通路具有激活的功能,而过表达iκbα则对这个激活起抑制作用。4.基于iκbα对信号通路的抑制,在本研究中将荧光素酶报告基因质粒、iκbα质粒、p65质粒与miR-144和miR-122模拟体、前体、抑制体共转染。结果表明miR-122和miR-144靶向抑制iκbα表达,促进p65介导的NF-κb等信号通路的激活。综上所述,实验结果表明miR-122和miR-144为负调控因子,靶向iκbα的3’UTR,抑制了mRNA和蛋白质水平的表达,从而促进了p65对NF-κb信号通路的激活。本研究为miRNAs在硬骨鱼中免疫信号通路调控提供了理论基础。miRNAs作为一种负调节因子,通常情况下都是对通路的激活起抑制作用,而本文的miRNAs靶向iκbα,因靶基因所具有的功能,miRNAs对该通路起促进作用,促进了机体的先天性免疫反应,可能利于帮助宿主抵御病原的入侵,为提高免疫提出新的理论依据。本文将miRNAs作为该论文中的一环,丰富了在硬骨鱼中非编码RNA的免疫调控网络。

关键词：鮸鱼 iκbα miR-144 miR-122 先天免疫反应

TMEPAi与Nedd4协同调控NF-κb信号途径机制与功能研究

同方学位论文库评论

在线全文

同方学位论文库

学校读者我要写书评

暂无评论

TMEPAI与Nedd4协同调控NF-κB信号途径机制与功能研究

作者：张亚新天津科技大学

学位级别：硕士

NF-κb信号在多种癌症中异常激活,并且通过调控细胞增殖,细胞侵袭和炎症反应等相关基因的表达参与肿瘤的发生发展。前列腺跨膜蛋白TMEPAi在多种肿瘤组织中高表达,与肿瘤的发生发展密切相关。前期探究发现TMEPAi能够与iκbα在体外相互作... 详细信息

NF-κb信号在多种癌症中异常激活,并且通过调控细胞增殖,细胞侵袭和炎症反应等相关基因的表达参与肿瘤的发生发展。前列腺跨膜蛋白TMEPAi在多种肿瘤组织中高表达,与肿瘤的发生发展密切相关。前期探究发现TMEPAi能够与iκbα在体外相互作用,另有研究报道TMEPAi能够与E3泛素连接酶Nedd4相互作用。本论文将进一步解析TMEPAi促进肿瘤发生发展的机制,深入研究TMEPAi、Nedd4与iκbα相互作用以及三者协同调控NF-κb信号的作用与功能。通过CRiSPR-Cas9技术成功构建敲除TMEPAi,Nedd4的He La细胞系。采用免疫共沉淀证明TMEPAi,Nedd4和iκbα三者在细胞中存在相互作用,可以形成三元复合体;并且TMEPAi作为衔接蛋白介导Nedd4与iκbα在体内相互作用。采用体内泛素化实验证明敲除TMEPAi、Nedd4抑制iκbα的泛素化,同时过表达TMEPAi与Nedd4能够协同增强iκbα的泛素化,而表达TMEPAi-2YA突变体则不能,说明TMEPAi与Nedd4协同促进iκbα泛素化降解依赖于TMEPAi与Nedd4的相互作用。通过荧光素酶报告基因实验、Western blot和免疫荧光技术以及q RT-PCR实验探究TMEPAi与Nedd4对NF-κb信号的协同激活作用,实验证明TMEPAi与Nedd4能够协同促进NF-κb信号报告基因p NF-κb-Luc活性、p65核易位以及NF-κb下游靶基因(包括Cyclin D1、c-Myc、iCAM-1、MMP9、i NOS、COX-2、TNF-α)的表达。通过MTT以及Ki-67实验证明TMEPAi和Nedd4能够协同增强He La细胞活力,促进细胞增殖,并且TMEPAi和Nedd4的促细胞生长活性依赖于NF-κb信号的激活。体内裸鼠荷瘤实验发现敲除TMEPAi、Nedd4能够抑制荷瘤裸鼠肿瘤的生长,其中对照组的平均肿瘤重量为0.49g,而敲除TMEPAi、Nedd4组的平均肿瘤重量仅为0.07g、0.18g。并且在肿瘤组织中敲除TMEPAi、Nedd4也能够抑制iκbα的降解。综上所述,TMEPAi、Nedd4和iκbα在细胞内可以形成三元复合物,TMEPAi通过募集Nedd4泛素化降解iκbα,促进p65入核,激活NF-κb信号,促进NF-κb下游靶基因的表达,进而促进肿瘤的发生发展。本论文研究揭示了TMEPAi通过激活NF-κb信号促进肿瘤发生发展的新机制,为以TMEPAi为靶点治疗肿瘤奠定了理论基础。

关键词： TMEPAi Nedd4 iκbα NF-κb信号途径

痛风致病基因PLAA在单核细胞内的功能及其调控机制研究

同方学位论文库评论

在线全文

同方学位论文库

学校读者我要写书评

暂无评论

痛风致病基因PLAA在单核细胞内的功能及其调控机制研究

作者：冉紫荆青岛大学

学位级别：硕士

目的:遗传因素是痛风发病的关键因素,而尿酸单钠晶体(MSU)诱发痛风炎症反应的遗传背景仍处于未知阶段。以原发性痛风家系为对象,基于全基因组测序分析,课题组发现了一个全新的痛风致病基因PLAA,但其中的致病机制仍不清楚。PLAA基因编码... 详细信息

目的:遗传因素是痛风发病的关键因素,而尿酸单钠晶体(MSU)诱发痛风炎症反应的遗传背景仍处于未知阶段。以原发性痛风家系为对象,基于全基因组测序分析,课题组发现了一个全新的痛风致病基因PLAA,但其中的致病机制仍不清楚。PLAA基因编码磷脂酶A2激活蛋白,在单核/巨噬细胞中高表达,且先前报道称与NF-κb信号通路的激活有关。本研究主要以尿酸盐晶体处理的人巨噬细胞和PLAA单核细胞特异性敲除小鼠为实验对象,研究PLAA基因在痛风炎症中的作用及其分子机制。方法:1.构建PLAA敲低和过表达的THP-1细胞模型。1.1.MSU晶体处理THP-1来源巨噬细胞,应用RT-PCR及Western blot检测PLAA敲减和过表达效率,iL-1β的转录水平及Pro-iL-1β的蛋白水平;Western blot及ELiSA检测iL-1β的分泌,观察PLAA对关键炎症因子iL-1β的影响;1.2.Western blot检测iκbα总蛋白及磷酸化水平,NF-kb磷酸化水平探究PLAA对MSU诱导的NF-κb活化的影响;1.3.过表达PLAA,通过RT-PCR检测iKKα/β及其上游信号通路的转录水平;1.4.过表达PLAA,通过RT-PCR及Western blot检测NLRP3炎症小体组分的变化;1.5.敲低/过表达PLAA后,通过RT-PCR检测iκbα的转录水平;通过免疫共沉淀探究PLAA与VCP,iκbα,NLRP3的内源性相互作用,初步探究PLAA对NF-κb的调控机制。2.构建PLAA单核细胞特异性敲除小鼠模型。2.1.分离Plaa 小鼠和对照小鼠骨髓单核细胞,L929细胞上清培养,将其诱导为巨噬细胞,通过Western blot检测PLAA敲除效率;LPS预刺激后给予MSU晶体处理,RT-PCR及Western blot检测PLAA敲除效率,pro-iL-1β的蛋白水平以及iL-1β的转录及分泌情况;2.2.在Plaa 及对照小鼠骨髓巨噬细胞中通过Western blot检测NF-κb通路和NLRP3炎症小体中关键蛋白的表达以验证PLAA调控炎症的机制;2.3.通过将MSU混悬液注射于小鼠足垫建立小鼠痛风性关节炎模型,测量注射后0,4,8,12,24,48,72h的足垫厚度及小鼠机械痛阈以探究生理状态下PLAA缺失对痛风炎症的影响;2.4.MSU混悬液注射于小鼠足垫,在注射8小时后收集两组小鼠足垫软组织,通过Western blot检测iκbα,NLRP3蛋白水平。结果:1.在THP-1细胞系中,RT-PCR及Western blot结果显示MSU诱导的iL-1β表达随PLAA敲低而下调;过表达PLAA逆转了iL-1β的表达。此外,相比空载病毒细胞系,PLAA过表达细胞系中MSU诱导后的iκbα及P65的磷酸化水平均明显增加,而iκbα总蛋白水平降低,同时,在敲低PLAA后NF-κb活化相关蛋白出现相反趋势。进一步发现过表达PLAA后NLRP3的mRNA及蛋白水平均升高,ASC,pro-Caspase-1蛋白水平没有显著变化,但培养上清中Caspase-1成熟体分泌明显升高。2.在体外培养的小鼠巨噬细胞中给予MSU刺激后,发现Plaa缺陷使得iL-1βmRNA,Pro-iL-1β蛋白水平以及细胞上清中iL-1β明显降低,NF-κb激活减弱,伴随着iκbα总蛋白水平增加及NLRP3蛋白的降低。3.小鼠急性痛风性关节炎模型结果显示,足垫肿胀均在8h达到高峰,Plaa 小鼠足垫肿胀程度及痛觉始终低于对照小鼠。Western blot结果显示Plaa 小鼠关节炎足垫组织中iκbα总蛋白表达水平高于对照小鼠而NLRP3蛋白的表达则低于对照小鼠。4.在THP-1细胞中过表达PLAA后发现iKK激酶及其上游MyD88,TRAF6的转录水平升高,但iκbα在敲减或过表达PLAA后转录水平均无差异。内源免疫共沉淀实验观察到PLAA与VCP、iκbα存在结合。结论:PLAA缺陷可以减弱小鼠痛风模型中的炎症反应。在细胞水平,PLAA可以增强NF-κb的活化导致NLRP3和Pro-iL-1β的表达增加,继而促进NLRP3炎症小体激活,最终导致iL-1β分泌增加进而调控痛风炎症。

关键词：痛风性关节炎致病基因 PLAA iκbα NLRP3