NF-κB-SREBPs途径介导巨噬细胞胆固醇流出和炎症因子的产生
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摘要
动脉粥样硬化（atherosclerosis, As）是一种慢性炎症性病理过程，炎症刺激在动脉粥样硬化的起始、进展以及并发症的产生中起重要作用。核因子-kappa B(NF-κB)是炎症因子产生的关键转录因子。在动脉粥样硬化斑块中可检测到活化的NF-κB，特异性的沉默或抑制NF-κB通路可减轻动脉粥样硬化斑块面积，并减少其并发症。NF-κB促动脉粥样硬化的机制主要是由炎症因子介导，但近期的研究发现NF-κB可促进巨噬细胞脂质蓄积，这为我们认识NF-κB的促动脉粥样硬化机制提供了新的研究方向。
固醇调节元件结合蛋白（Sterol-regulatory element binding proteins, SREBPs）是体内胆固醇生物合成和摄取相关基因的关键调节因子。目前已确定的SREBPs异构体有3种：SREBP-1a、1c和2。SREBPs彼此之间存在相关性，但也有差异：SREBP-1a和SREBP-1c易于激活脂肪酸和三酰甘油合成过程中的基因，而SREBP-2易于激活LDL受体基因和多个胆固醇合成所必需的基因。在SREBPs的内含子区域，含有新发现的一类microRNA（smiRNAs），miR-33s。已知miRNAs是一类具有转录后调节活性的单链小分子RNA，已有研究表明miRNAs参与调控脂质代谢及炎症因子表达，介导As的形成。miR-33s的主要靶基因是三磷酸腺苷结合盒A1（ATP binding cassette transporterA1， ABCA1）。由于ABCA1是介导细胞脂质流出的主要膜转运体，抑制其表达后将导致细胞内蓄积大量的脂质，最终形成泡沫细胞。体内实验发现，抑制miR-33s表达，可促进ABCA1表达和胆固醇逆向转运（RCT），并明显减少斑块面积。因此，miR-33s已成为防治As性疾病的重要靶标。然而，体内对miRNAs的调控研究目前还处在起步阶段，miR-33s在体内的受控机制还远未阐明。
越来越多的证据显示固有免疫应答与多种疾病包括As密切相关。NOD样受体（NLRs）是细胞内感受微生物或非微生物信号的模式识别受体，NLRs能形成细胞内大的蛋白复合体，称为“炎性体”，包括NLRs、caspase1和ASC。炎性体的作用是形成一个活化caspase1的支架，促进pro-IL-1β和pro-IL-18分别转化为成熟的IL-1β和IL-18。IL-1β和IL-18与As的发展关系密切，在实验性的小鼠中发现，若缺乏IL-1β或IL-18，则明显限制As的发展。然而，炎性体在体内的调控机制还不甚清楚。
通过生物信息学分析，我们发现miR-33s宿主基因SREBPs的启动子区存在NF-κB反应元件（κBREs），提示NF-κB可能通过直接结合SREBPs的启动子，进而调控SREBPs和miR-33s的表达。为了探讨SREBPs和miR-33s在NF-κB促As中的可能作用及机制，因此，我们首先在第一部分中观察了NF-κB对巨噬细胞胆固醇流出及炎症因子表达的影响，然后，在第二部分中我们研究了NF-κB对SREBPs和miR-33s表达的作用及机制，并探讨了miR-33s在NF-κB抑制胆固醇流出中的作用，以及SREBPs在NF-κB促炎症因子释放和炎性体表达中的作用，接着，在第三部分中，我们在体内观察了NF-κB-SREBPs途径在As和炎症反应中的作用，最后，在第四部分中，我们观察了具有明显抗炎效应的白桦脂酸（betulinic acid，BA）在体内外对NF-κB-SREBPs途径的影响，并深入探讨了其作用机制。本研究为揭示NF-κB-SREBPs途径在As发生发展中的可能作用和机制提供新的研究思路。
第一部分NF-κB对巨噬细胞胆固醇流出及炎症因子表达的影响
目的：观察NF-κB对巨噬细胞胆固醇流出及相关转运体ABCA1的影响，并观察NF-κB对炎症因子表达的影响。
方法：培养THP-1细胞株，用160nmol/L的佛波酯(phorbol12-myristate13-acetate，PMA)刺激细胞24h，使其诱导分化为巨噬细胞。以100μg/ml的氧化型低密度脂蛋白（oxidized low density lipoprotein，oxLDL）分别孵育THP-1和RAW264.7巨噬细胞，使其转化为泡沫细胞。两种细胞分别分为3组：对照组，LPS组（10ng/ml，24h）和LPS+PDTC组（LPS10ng/ml，PDTC50μM，24h）。以液体闪烁计数法检测细胞内胆固醇的流出，荧光定量PCR检测ABCA1的mRNA表达，Western blotting免疫印迹法检测ABCA1的蛋白表达，酶联免疫吸附法（ELISA）法检测肿瘤坏死因子α（tumor necrosis factor α, TNF-α）、白细胞介素1β（interleukin-1β, IL-1β）、和IL-6的表达。
结果：用LPS处理THP-1和RAW264.7巨噬细胞源性泡沫细胞后，载脂蛋白A1（apolipoproteinA-I, apoA-I）介导的胆固醇流出明显减少；相应的，ABCA1mRNA和蛋白含量明显下降。使用NF-κB特异性的抑制剂PDTC处理巨噬细胞，可明显减少LPS所致的胆固醇流出下降，并减弱LPS所致的ABCA1表达下降。此外，PDTC处理可明显抑制LPS诱导的炎症因子TNF-α、IL-1β和IL-6的分泌。
结论：①NF-κB可抑制巨噬细胞胆固醇流出，下调ABCA1表达；②NF-κB可促进TNFα、IL-6和IL-1β等炎症因子产生。
第二部分SREBPs介导NF-κB调控胆固醇流出和炎症因子表达的机制
目的：探讨NF-κB调控巨噬细胞胆固醇流出和炎症因子表达的可能分子机制。
方法：培养THP-1细胞株，以PMA和ox-LDL建立THP-1巨噬细胞源性泡沫细胞模型。10ng/ml LPS处理3h后，加入放线菌素D（actinomycin D,Act D）（5μg/ml）终止转录，采用定量PCR检测ABCA1mRNA的表达，观察ABCA1mRNA稳定性变化。以LPS处理细胞，观察NF-κB对miR-33s及其宿主基因SREBPs表达影响的浓度效应和时间效应。PDTC处理，观察抑制NF-κB对miR-33s/SREBPs的表达变化。制备SREBPs启动子报告基因质粒，与表达载体p50(pRSV-NF-κB-1)和p65（pRSV-RelA）共同转染HEK-293T细胞，采用双荧光素酶报告基因分析NF-κB对SREBPs启动子的激活作用。染色体免疫共沉淀（ChIP）检测NF-κB是否可直接结合在SREBPs的启动子上。用miR-33a/b mimic（40nM）增加miR-33s的表达，用拮抗剂anti-miR-33a/b（60nM）降低miR-33s的表达，检测ABCA1表达以及细胞胆固醇的流出情况，判断miR-33s在其中的作用。用SREBPs siRNA处理巨噬细胞，研究SREBPs在炎症因子表达中的作用。
结果：NF-κB活化后，ABCA1mRNA的降解速度明显加快，NF-κB可增加细胞SREBP-2/miR-33a和SREBP-1a/miR-33b mRNA的表达，以及SREBPs蛋白水平。转染NF-κB后，SREBPs启动子荧光素酶报告基因值明显升高，而κBRE突变后，NF-κB对SREBPs启动子的作用消除。ChIP检测进一步证明了NF-κB可直接结合到SREBPs启动子上。LPS活化NF-κB后，再加入miR-33a/b mimic，ABCA1表达进一步下降，相反，加入anti-miR-33a/b后，NF-κB对ABCA1的抑制作用得到有效缓解。相应的，加入miR-33a/b mimic后，胆固醇流出明显下降，而加入anti-miR-33a/b后，胆固醇流出得以部分恢复。SREBP-2或SREBP-1抑制后，明显降低LPS诱导的IL-1β上调，但对TNF-α、IL-6和IL-18的表达无明显影响。炎性体检测发现，抑制SREBPs后，NLRP1表达下降，而NLRP3无明显影响。进一步使用siRNA抑制NLRP1的表达，发现NF-κB对IL-1β的诱导作用明显下降。
结论：①SREBPs是NF-κB的直接靶基因；②NF-κB通过促进miR-33s表达调控ABCA1及胆固醇流出；③NF-κB通过促进SREBPs表达调控NLRP1炎性体及IL-1β产生。
第三部分NF-κB对apoE-/-小鼠动脉粥样硬化病变及体内炎症因子表达水平的影响
目的：以apoE基因敲除（apoE-KO）小鼠为研究对象，观察NF-κB对体内动脉粥样硬化病变、胆固醇逆向转运（RCT）、炎症及NF-κB-SREBPs途径相关分子的表达变化。
方法：8周龄雄性apoE-KO小鼠以普通饲料饲养，并随机分为三组：对照组、LPS组和LPS+PDTC组（每组15只）。其中，对照组每周腹腔注射与实验组同等剂量的生理盐水；LPS组每周腹腔注射LPS (2.5mg/kg body wt)1次；LPS+PDTC组每周腹腔注射LPS (2.5mg/kg body wt)和PDTC(50mg/kg body wt)1次。8周后处死动物，采用酶氧化法检测甘油三酯（TG）、总胆固醇（TC）等血脂水平；油红O染色观察主动脉和主动脉窦处脂质蓄积情况；免疫组织化学法检测巨噬细胞标志物CD68和NF-κB的表达；Masson氏染色法检测斑块处胶原纤维面积；ELISA法检测血清炎症细胞因子(TNF-α、IL-1β和MCP-1等)的表达。液体闪烁计数仪检测小鼠巨噬细胞来源的RCT效率。提取小鼠腹腔巨噬细胞，荧光定量PCR法和western-blot法检测SREBPs、miR-33、ABCA1、ABCG1、NLRPs和NF-κB的表达。
结果：与对照组比较，LPS组小鼠血浆TG、TC和LDL-C含量明显增高，而HDL-C则水平降低，加入PDTC后，HDL-C水平有明显回升。PDTC明显减少LPS所致的主动脉窦和主动脉上的As病变面积增加；增加粪便中的胆固醇流出，而肝脏和血液中的胆固醇流出没有明显改变；降低小鼠腹腔巨噬细胞中miR-33表达，促进ABCA1和ABCG1的表达。PDTC处理后，与LPS组比较，巨噬细胞浸润减少而胶原纤维增加；斑块处NF-κB的表达下降；血液中TNF-α、IL-1β和MCP-1等炎症因子表达水平降低；腹腔巨噬细胞中SREBPs和NLRPs的表达下降。
结论：①体内NF-κB活化可促进miR-33和SREBPs表达，抑制ABCA1和ABCG1的表达，抑制体内RCT并促进As；②体内NF-κB活化可增加炎症因子水平，并促进NLRPs表达。
第四部分白桦脂酸对ABCA1表达和炎症因子释放的影响及机制
目的：观察白桦脂酸对ABCA1表达和炎症因子释放的影响并探讨其机制。
方法：培养THP-1细胞株，以PMA和ox-LDL建立THP-1巨噬细胞源性泡沫细胞模型。以不同浓度（0,0.5,1,2μg/ml）BA处理24h或1μg/ml BA处理不同时间（0,12,24and48h），然后再加入LPS（10ng/ml）作用24h，观察BA预处理对LPS诱导的胆固醇流出和ABCA1表达抑制的影响。高效液相色谱检测细胞内胆固醇含量；荧光定量PCR检测BA处理对miRNA-33s及其宿主基因SREBPs表达的影响；用miR-33a/b mimic增加miR-33s的表达，用拮抗剂anti-miR-33a/b降低miR-33s的表达，检测ABCA1表达情况，判断miR-33s在BA促胆固醇流出中的作用；western-blot法检测NF-κB的核转录水平，使用NF-κB特异性的抑制剂PDTC (50μM)或Bay11-7082(5μM)处理细胞，再加入BA和LPS，荧光定量PCR检测miR-33s和ABCA1的mRNA表达，液体闪烁计数法检测细胞内胆固醇的流出，以明确NF-κB在BA促胆固醇流出中的作用；进一步检测细胞质中IκBα、磷酸化IκBα和细胞核中磷酸化NF-κB p65的表达，以及巨噬细胞炎症因子的分泌情况，以明确BA的抗炎作用及其机制。以apoE基因敲除小鼠为研究对象，观察BA对体内动脉粥样硬化病变和体内炎症反应的影响。
结果：BA预处理明显减弱LPS对巨噬细胞胆固醇流出和ABCA1表达的抑制，下调细胞内总胆固醇、胆固醇酯和游离胆固醇的含量。BA预处理有效抑制miR-33s及其宿主基因SREBPs表达。加入anti-miR-33a/b后，与单加LPS组比较，ABCA1的表达明显升高，而加入miR-33a/b mimic，ABCA1的表达则无明显改变；加入BA后，再加入anti-miR-33a/b，ABCA1的表达无明显变化，而加入miR-33a/b mimic，ABCA1的含量明显下降。BA处理可抑制NF-κB的核转录水平； PDTC或Bay11-7082可增强BA对miR-33s的抑制作用，同时增强BA对ABCA1表达及胆固醇流出的促进作用。BA处理后，细胞质中磷酸化IκBα的表达明显下降，细胞核中磷酸化NF-κB p65的表达，炎症因子分泌水平降低。LPS组小鼠较对照组小鼠血浆TG、TC和LDL-C含量明显增高，而HDL-C则水平降低，BA处理后，TG、TC和LDL-C含量下降，HDL-C水平明显回升。组LPS组比较，BA处理组主动脉窦和主动脉上的As病变面积减少；小鼠主动脉miR-33的表达下降；ABCA1表达水平升高；NF-κB的核转录水平下降；血液中炎症因子TNF-α、IL-6和IL-1β表达水平降低。
结论：①BA抗小鼠动脉粥样硬化的作用与其抗炎和降低血脂功能有关；②NF-κB-SREBPs途径参与了BA的抗动脉粥样硬化作用。
Atherosclerosis (As) is a chronic inflammatory pathological processes.Inflammation plays an important role in the initiation, progress, and complications ofatherosclerosis. Nuclear factor-kappa B (NF-κB) is one of the key transcriptionfactors in inflammatory cytokine production. Activated NF-κB has demonstrated inhuman atherosclerotic lesions. Specificity silence or inhibition of NF-κB pathwaycould reduce atherosclerotic plaque area and its complications. The mechanisms thatNF-κB promotes atherosclerosis were mainly mediated by inflammatory factors, butrecent studies found that NF-κB could promote macrophage lipid accumulation,which provide us a new research direction to recognize the proatherogenicmechanisms of NF-κB.
Sterol regulatory element binding proteins (SREBPs) is a key regulator of the genesthat control cholesterol biosynthesis and uptake. There are three forms of SREBP inmammals: SREBP-1a,-1c and-2. Although they undergo similar proteolyticactivation and share some target genes, SREBP-1a and-1c mainly stimulate fatty acidsynthesis, whereas SREBP-2acts primarily on the cholesterol biosynthetic genes andLDL receptor (LDLR) gene. The intron regions of SREBPs contain a newlydiscovered class of microRNAs (miRNAs), miR-33s. MiRNAs is a kind of smallsingle-stranded RNA molecules that have post-transcriptional regulatory activity.Studies have shown that miRNAs involved in the regulation of lipid metabolism,expression of inflammatory cytokines and formation of As. The main target genes ofmiR-33s is ATP-binding cassette transporter A1(ABCA1). As ABCA1is the mainmembrane transporter that mediates the outflow of cellular lipids, inhibiting itsexpression will results in intracellular accumulation of a large number of lipids and ultimately the formation of foam cells. In vivo studies demonstrated that antagonismof miR-33in mice promotes ABCA1expression and reverse cholesterol transport，butpromotes the regression of atherosclerosis. Thus, miR-33s has become an importanttarget for prevention and treatment of As diseases. However, the in vivo regulation ofmiRNAs is still in the initial stage, the mechanisms that control the expression ofmiR-33s in the body is far from clear.
There is a growing body of evidence pointing to the closely relationship betweenthe innate immune response and a variety of diseases including As. NOD-likereceptors (NLRs) are pattern recognition receptors that feeling microbial andnon-microbial signal in cells. NLRs can form large protein complexes within the cells,referred to as the " inflammasome", including the NLRs, caspase1and the ASC. Themain role of inflammasome is to form an bracket to activate caspase1, which couldpromote pro-IL-1β and pro-IL-18transform into mature IL-1β and IL-18, respectively.IL-1β and IL-18correlate closely to the development of As, the absence of IL-1β orIL-18significantly restrict the development of the As in mice. However, themechanisms that regulate the inflammasome in vivo are still unclear.
Through bioinformatics analysis, we found the promoter region of SREBPs, thehost gene of miR-33s, have the NF-κB response element (κBREs), suggesting thatNF-κB may be direct binding to SREBPs promoter and regulating the expression ofSREBPs/miR-33s. Therefore, in this study, we firstly observed the effects of NF-κBactivation on macrophages cholesterol efflux and inflammatory factors production inthe first part. Then, in the second part we explored the effect and regulatorymechanisms of NF-κB on SREBPs. We also investigated the the roles of miR-33s inNF-κB repressing cellular cholesterol efflux and the roles of SREBPs in NF-κBpromoting inflammatory cytokine release and inflammasome expression. Next, in thethird part we observed the effects of NF-κB-SREBPs pathway in As and inflammationreaction in vivo. Finally, we observed the effects of betulinic acid (BA), which hadproved to have significant anti-inflammatory effects, on NF-κB-SREBPs pathways invitro and in vivo, and explored its possible mechanisms in the fourth part. Our studiesprovide new insights for revealing the potential roles and mechanisms of NF-κB-SREBPs pathway in the development of As.
Part I: Effect of NF-κB Activation on Cholesterol Efflux andInflammatory Cytokines Production in Macrophages
Aims: To investigate the effect of NF-κB activation on macrophages cholesterolefflux and the expression of related membrane transporter ABCA1, and also observethe effect of NF-κB on inflammatory factors production.
Methods: Human THP-1monocytes were treated with phorbol12-myristate13-acetate (PMA)(160nmol/liter) for24h to differentiate into macrophages. THP-1and RAW264.7macrophages were then replaced to the serum-free medium containingoxLDL (100μg/ml) to become fully differentiated macrophage foam cells beforetheir use in experiments. Both cells were divided into3groups: control group, LPSgroup (10ng/ml,24h) and LPS+PDTC group (LPS10ng/ml, PDTC50μM,24h), respectively. The cholesterol efflux was assessed by liquid scintillation counting.The protein and mRNA expression of ABCA1were examined by westernimmunoblotting assays and real-time quantitative PCR, respectively. Theconcentrations of tumor necrosis factor-α (TNF-α), IL-6and interleukin-1β (IL-1β) incell culture supernatants were measured by enzyme-linked immunosorbent assay(ELISA).
Results: Apolipoprotein A1(apoA-I)-mediated cholesterol efflux wassignificantly reduced after treatment with LPS in THP-1and RAW264.7macrophage-derived foam cells. Accordingly, NF-κB activation clearly decreased thelevels of ABCA1mRNA and protein. Application of the NF-κB specific inhibitorPDTC significantly suppressed the LPS-induced down-regulation of cholesterol effluxand the expression of ABCA1. In addition, PDTC obviously inhibited the secretion ofinflammatory cytokines TNF-α, IL-1β and IL-6induced by LPS.
Conclusion:①The activation of NF-κB inhibits cholesterol efflux and theexpression of ABCA1in macrophages.②NF-κB activation promotes the secretion of inflammatory cytokines, such as TNFα, IL-6and IL-1β etc.
Part II: Molecular Mechanism of SREBPs-Mediated NF-κBRegulating Cholesterol Efflux and Secretion of InflammatoryCytokines
Aims: To investigate the possible molecular mechanisms of NF-κB activation onmacrophages cholesterol efflux and secretion of inflammatory cytokines
Methods: Human THP-1monocytes were preincubated with PMA and ox-LDLto form foam cells. Cells were treated with LPS (10ng/ml) for3h and followed bytreatment of act D (5μg/ml) to stop the transcription. ABCA1mRNA expression wasexamined by real-time quantitative PCR to observe the changes of ABCA1mRNAstability. Cells was treated by LPS and observed the concentration-dependent ortime-dependent effect of NF-κB activation on miR-33s and their host genes SREBPs.Observed the expression changes of miR-33s/SREBPs after inhibition of NF-κBactivation by PDTC treatment. Co-transfection of SREBPs promoter reporterplasmid with expression vector p50(pRSV-NF-κB-1) and p65(pRSV-RelA) intoHEK-293T cells, cells were lysed and luciferase activities were measured by using thedual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) assays wasperformed to detect if NF-κB have a direct binding to the promoter regions of theSREBPs genes. Human THP-1cells were transfected with40nM miRIDIAN miRNAmimics (miR-33a/b) or with60nM miRIDIAN miRNA inhibitors (anti-miR-33a/b) toassess the effects of gain and loss of function of miR-33on ABCA1expression andcholesterol efflux. Treatment of macrophages with SREBPs siRNA to study the roleof SREBPs in the expression of inflammatory cytokines.
Results: NF-κB activation significantly promotes ABCA1mRNA degradation,indicating that the reduction in mRNA is caused by destabilization. NF-κB activationincreased the SREBP-2/miR-33a and SREBP-1a/miR-33b mRNA expression in aconcentration-dependent manner when compared with a negative control. Accordingly, NF-κB also increase the SREBPs protein levels. The Luciferase-SREBPs reporterswere expressed at markedly higher levels as compared with the luciferase vectorwithout insert. Both deletions and mutations of κBRE abolished the enhancing effectsof NF-κB compared to the wild-type control construct. Further, ChIP assays with anantibody to the p65subunit of NF-κB demonstrated a direct binding of thistranscription factor to the promoter regions of the SREBPs genes. ABCA1mRNA andprotein levels were both reduced in THP-1cells transfected with excess wild-typehuman miR-33a/b mimic oligonucleotides compared to the control group. By contrast,the effects of NF-κB on ABCA1were abolished by anti-miR-33a/b. Accordingly,introduction of miR-33mimic into macrophages resulted in further repression of the[3H]-cholesterol efflux compared to the control oligonucleotides, whereas miR-33antisense oligonucleotides treatment led to a marked increase in [3H]-cholesterolefflux. The increase in IL-1β release observed in WT macrophages was significantlyreduced with SREBPs knockdown, but the repression of either SREBP-1or SREBP-2had no obvious effects on TNF-α, IL-6and IL-18expression. Treatment with siRNAfor SREBPs significantly down-regulated NF-κB-induced NLRP1protein expressionin THP-1cells without detectable effect on NLRP3. In addition, the effect of NF-κBon IL-1β expression in NLRP1siRNA cells was obviously suppressed.
Conclusion:①SREBPs are direct NF-κB target genes.②NF-κB down-regulates ABCA1level and cholesterol efflux via promoting miR-33expression.③SREBPs increase NF-κB-induced IL-1β expression in macrophages throughactivation of NLRP1.
Part III: Effects of NF-κB on Atherosclerotic Lesions andinflammatory cytokines expression in apoE-/-Mice
Aims: To observe the effect of NF-κB activation on atherosclerotic lesion, RCT,inflammatory response and the changes of molecules expression related to NF-κB-SREBPs pathway in apoE-KO mice.
Methods: Six-week old male apoE-/-mice were obtained from LaboratoryAnimal Center of Peking University, China. All mice were fed a chow diet. At8weeks of age, apoE-/-mice were randomly divided into several groups (n=15pergroup). The LPS group was challenged intraperitoneally (i.p.) with LPS (2.5mg/kgbody wt) in200μL of PBS once every week. Mice in the PDTC group were injectedi.p. with PDTC (50mg/kg body wt)1h before LPS challenge. The control group wasreceived abdominal injections of PBS of the same volume at the same time. At week16, the mice were sacrificed, blood was obtained, and tissues were collected forfurther analysis. Triglyceride (TG), total cholesterol (TC), and HDL-C weredetermined by commercially enzymatic methods. Lipid accumulation in aorta andaortic sinus were evaluated by Oil Red O stain. The expression of CD68, a marker formacrophage, and NF-κB p65in lesions were tested by immuno-histochemistry.Masson’s staining for collagen. Plasma inflammatory cytokines (TNF-α、IL-1β andMCP-1etc.) expression was detected by ELISA. RCT efficiency of murinemacrophage cell was detected by liquid scintillation counting. The protein and mRNAexpression of SREBPs、miR-33、ABCA1、ABCG1、NLRPs and NF-κB in mouseperitoneal macrophages (MPMs) were examined by western immunoblotting assaysand real-time quantitative PCR, respectively.
Results: Plasma TG, TC and LDL-C levels were increased in LPS groupcompared with the control group, and the HDL-C levels were decreased. ApoE-/-miceinjected with LPS and PDTC significantly reduced lesion size in comparison to theLPS group. Treatment of LPS-challenged apoE-/-mice with PDTC significantlyreduced cholesterol transport from macrophages to feces in the apoE-/-mice. Incontrast, both LPS and PDTC had no significant effect on cholesterol tracer recoveryin the liver and plasma. Treatment with PDTC significantly promoted the expressionof both ABCA1and ABCG1and reduced the expression of miR-33a in mouseperitoneal macrophages. Moreover, there was a reduction in CD68+macrophagescontent and increase in total lesional collagen content in PDTC group compared withLPS group. The NF-κB expression was decreased when treated with PDTC, andinhibition of NF-κB activity could reduce the inflammatory cytokines release. Analysis of the expression of SREBPs and NLRPs revealed a significant increased inLPS-challenged apoE-/-mice peritoneal macrophages, and this effect could be partlyreversed by application of the NF-κB specific inhibitor PDTC.
Conclusion:①NF-κB activation in vivo can increase the xpression of miR-33and SREBPs, reduce the expression of ABCA1/ABCG1, inhibit the RCT in vivo andpromote the development of mice atherosclerotic lesions.②NF-κB activation canincrease the level of inflammatory cytokines in vivo and promote NLRPs expression.
Part Ⅳ: Effect and Mechanism of Betulinic Acid on ABCA1Expression and Inflammatory Cytokines Production
Aims: To investigate the effect and molecular mechanism of BA on ABCA1expression and inflammatory cytokines production in vitro and in vivo.
Methods: Human THP-1monocytes were preincubated with PMA and ox-LDLto form foam cells. Cells was pre-treated with BA (0,0.5,1,2μg/ml) for24hr or withBA (1μg/ml) for0,12,24and48hr, respectively, and then exposed them to LPS. Toobserved the effects of BA on cholesterol efflux and the expression of ABCA1inLPS-treated macrophages. HPLC was performed to determine cellular totalcholesterol, free cholesterol and cholesterol ester. Real-time quantitative PCR wasperformed the effect of BA on the expression of miR-33s and their host genesSREBPs. Cells were transfected with miR-33a/b mimics or with anti-miR-33a/b toassess the effects of miR-33in BA promoting ABCA1expression and cholesterolefflux. The protein expression of nuclear NF-κB was examined by westernimmunoblotting assays. THP-1macrophage-derived foam cells were pretreated withPDTC (50μM) or Bay11-7082(5μM) for24h, and cells were then incubated withLPS for another24h with or without pretreatment of BA. Expression of miR-33smRNA was confirmed by RT-PCR. Cellular cholesterol efflux was analyzed by liquidscintillation counting assays to access the effect of NF-κB in BA promotingcholesterol efflux. Protein levels of IκBα, p-IκBα and phosphorylation of p65were measured and analyzed. Furthermore, we examined the effect of BA onLPS-stimulated inflammatory cytokine production in macrophages to clarify theanti-inflammatory effect and mechanism of BA. In addition, we observed the effect ofBA on atherosclerotic lesion and inflammatory response in apoE-KO mice. Plasma TG,TC and LDL-C levels were increased in LPS group compared with the control group.Both TG and TC levels in animals treated with BA were decreased. Analysis of theplasma lipoproteins showed an increase in HDL cholesterol and a decrease in LDLcholesterol levels in group of BA treatment. LPS-injected apoE-/-mice treatedadditionally with BA have a significantly reduced lesion size. BA significantlyreduced the expression of miR-33and promoted the expression of ABCA1ascompared with those treated by LPS alone. BA also suppressed the activation ofNF-κB and decreased the plasma pro-inflammatory cytokines levels
Results: Pre-treated with BA clearly decreased the inhibition of cholesterolefflux and ABCA1mRNA and protein expression induced by LPS. Cellularcholesterol content decreased when cells were treated with BA. LPS significantlyincreased the miR-33s and SREBPs expression in THP-1macrophage-derived foamcells, whereas treatment with BA down-regulated the expression of miR-33s andSREBPs compared with those treated by LPS only. The effects of LPS on ABCA1were reversed by anti-miR-33a/b. Whereas ABCA1levels was not changedsignificantly in THP-1cells transfected with excess wild-type human miR-33a/bmimic oligonucleotides compared to the LPS group. In addition, the effect of BA onABCA1expression was reversed by transfected with miR-33a/b mimic. With the BAtreatment, the promoting effects of LPS at nuclear NF-κB p65protein levels wereeffectively abrogated. miR-33s expression in cells treated by the combination ofNF-κB specific inhibitor (PDTC or Bay11-7082) was significantly decreased, at thesame time, PDTC or Bay11-7082efficiently promote ABCA1expression and cellularcholesterol efflux induced by BA. BA treated cells showed significant decrease in thephospho-protein expression of IκBα and NF-κB p65. BA significantly inhibitedLPS-stimulated secretion of TNFα, IL-6and IL-1β.
Conclusion:①Anti-inflammation and inhibition of plasma lipids are involvedin the mechanisms of BA treatment induces atherosclerosis regression in vivo.②NF-κB-SREBPs patherway involved in the reduction of atherosclerosis mediated byBA.
固醇调节元件结合蛋白（Sterol-regulatory element binding proteins, SREBPs）是体内胆固醇生物合成和摄取相关基因的关键调节因子。目前已确定的SREBPs异构体有3种：SREBP-1a、1c和2。SREBPs彼此之间存在相关性，但也有差异：SREBP-1a和SREBP-1c易于激活脂肪酸和三酰甘油合成过程中的基因，而SREBP-2易于激活LDL受体基因和多个胆固醇合成所必需的基因。在SREBPs的内含子区域，含有新发现的一类microRNA（smiRNAs），miR-33s。已知miRNAs是一类具有转录后调节活性的单链小分子RNA，已有研究表明miRNAs参与调控脂质代谢及炎症因子表达，介导As的形成。miR-33s的主要靶基因是三磷酸腺苷结合盒A1（ATP binding cassette transporterA1， ABCA1）。由于ABCA1是介导细胞脂质流出的主要膜转运体，抑制其表达后将导致细胞内蓄积大量的脂质，最终形成泡沫细胞。体内实验发现，抑制miR-33s表达，可促进ABCA1表达和胆固醇逆向转运（RCT），并明显减少斑块面积。因此，miR-33s已成为防治As性疾病的重要靶标。然而，体内对miRNAs的调控研究目前还处在起步阶段，miR-33s在体内的受控机制还远未阐明。
越来越多的证据显示固有免疫应答与多种疾病包括As密切相关。NOD样受体（NLRs）是细胞内感受微生物或非微生物信号的模式识别受体，NLRs能形成细胞内大的蛋白复合体，称为“炎性体”，包括NLRs、caspase1和ASC。炎性体的作用是形成一个活化caspase1的支架，促进pro-IL-1β和pro-IL-18分别转化为成熟的IL-1β和IL-18。IL-1β和IL-18与As的发展关系密切，在实验性的小鼠中发现，若缺乏IL-1β或IL-18，则明显限制As的发展。然而，炎性体在体内的调控机制还不甚清楚。
通过生物信息学分析，我们发现miR-33s宿主基因SREBPs的启动子区存在NF-κB反应元件（κBREs），提示NF-κB可能通过直接结合SREBPs的启动子，进而调控SREBPs和miR-33s的表达。为了探讨SREBPs和miR-33s在NF-κB促As中的可能作用及机制，因此，我们首先在第一部分中观察了NF-κB对巨噬细胞胆固醇流出及炎症因子表达的影响，然后，在第二部分中我们研究了NF-κB对SREBPs和miR-33s表达的作用及机制，并探讨了miR-33s在NF-κB抑制胆固醇流出中的作用，以及SREBPs在NF-κB促炎症因子释放和炎性体表达中的作用，接着，在第三部分中，我们在体内观察了NF-κB-SREBPs途径在As和炎症反应中的作用，最后，在第四部分中，我们观察了具有明显抗炎效应的白桦脂酸（betulinic acid，BA）在体内外对NF-κB-SREBPs途径的影响，并深入探讨了其作用机制。本研究为揭示NF-κB-SREBPs途径在As发生发展中的可能作用和机制提供新的研究思路。
第一部分NF-κB对巨噬细胞胆固醇流出及炎症因子表达的影响
目的：观察NF-κB对巨噬细胞胆固醇流出及相关转运体ABCA1的影响，并观察NF-κB对炎症因子表达的影响。
方法：培养THP-1细胞株，用160nmol/L的佛波酯(phorbol12-myristate13-acetate，PMA)刺激细胞24h，使其诱导分化为巨噬细胞。以100μg/ml的氧化型低密度脂蛋白（oxidized low density lipoprotein，oxLDL）分别孵育THP-1和RAW264.7巨噬细胞，使其转化为泡沫细胞。两种细胞分别分为3组：对照组，LPS组（10ng/ml，24h）和LPS+PDTC组（LPS10ng/ml，PDTC50μM，24h）。以液体闪烁计数法检测细胞内胆固醇的流出，荧光定量PCR检测ABCA1的mRNA表达，Western blotting免疫印迹法检测ABCA1的蛋白表达，酶联免疫吸附法（ELISA）法检测肿瘤坏死因子α（tumor necrosis factor α, TNF-α）、白细胞介素1β（interleukin-1β, IL-1β）、和IL-6的表达。
结果：用LPS处理THP-1和RAW264.7巨噬细胞源性泡沫细胞后，载脂蛋白A1（apolipoproteinA-I, apoA-I）介导的胆固醇流出明显减少；相应的，ABCA1mRNA和蛋白含量明显下降。使用NF-κB特异性的抑制剂PDTC处理巨噬细胞，可明显减少LPS所致的胆固醇流出下降，并减弱LPS所致的ABCA1表达下降。此外，PDTC处理可明显抑制LPS诱导的炎症因子TNF-α、IL-1β和IL-6的分泌。
结论：①NF-κB可抑制巨噬细胞胆固醇流出，下调ABCA1表达；②NF-κB可促进TNFα、IL-6和IL-1β等炎症因子产生。
第二部分SREBPs介导NF-κB调控胆固醇流出和炎症因子表达的机制
目的：探讨NF-κB调控巨噬细胞胆固醇流出和炎症因子表达的可能分子机制。
方法：培养THP-1细胞株，以PMA和ox-LDL建立THP-1巨噬细胞源性泡沫细胞模型。10ng/ml LPS处理3h后，加入放线菌素D（actinomycin D,Act D）（5μg/ml）终止转录，采用定量PCR检测ABCA1mRNA的表达，观察ABCA1mRNA稳定性变化。以LPS处理细胞，观察NF-κB对miR-33s及其宿主基因SREBPs表达影响的浓度效应和时间效应。PDTC处理，观察抑制NF-κB对miR-33s/SREBPs的表达变化。制备SREBPs启动子报告基因质粒，与表达载体p50(pRSV-NF-κB-1)和p65（pRSV-RelA）共同转染HEK-293T细胞，采用双荧光素酶报告基因分析NF-κB对SREBPs启动子的激活作用。染色体免疫共沉淀（ChIP）检测NF-κB是否可直接结合在SREBPs的启动子上。用miR-33a/b mimic（40nM）增加miR-33s的表达，用拮抗剂anti-miR-33a/b（60nM）降低miR-33s的表达，检测ABCA1表达以及细胞胆固醇的流出情况，判断miR-33s在其中的作用。用SREBPs siRNA处理巨噬细胞，研究SREBPs在炎症因子表达中的作用。
结果：NF-κB活化后，ABCA1mRNA的降解速度明显加快，NF-κB可增加细胞SREBP-2/miR-33a和SREBP-1a/miR-33b mRNA的表达，以及SREBPs蛋白水平。转染NF-κB后，SREBPs启动子荧光素酶报告基因值明显升高，而κBRE突变后，NF-κB对SREBPs启动子的作用消除。ChIP检测进一步证明了NF-κB可直接结合到SREBPs启动子上。LPS活化NF-κB后，再加入miR-33a/b mimic，ABCA1表达进一步下降，相反，加入anti-miR-33a/b后，NF-κB对ABCA1的抑制作用得到有效缓解。相应的，加入miR-33a/b mimic后，胆固醇流出明显下降，而加入anti-miR-33a/b后，胆固醇流出得以部分恢复。SREBP-2或SREBP-1抑制后，明显降低LPS诱导的IL-1β上调，但对TNF-α、IL-6和IL-18的表达无明显影响。炎性体检测发现，抑制SREBPs后，NLRP1表达下降，而NLRP3无明显影响。进一步使用siRNA抑制NLRP1的表达，发现NF-κB对IL-1β的诱导作用明显下降。
结论：①SREBPs是NF-κB的直接靶基因；②NF-κB通过促进miR-33s表达调控ABCA1及胆固醇流出；③NF-κB通过促进SREBPs表达调控NLRP1炎性体及IL-1β产生。
第三部分NF-κB对apoE-/-小鼠动脉粥样硬化病变及体内炎症因子表达水平的影响
目的：以apoE基因敲除（apoE-KO）小鼠为研究对象，观察NF-κB对体内动脉粥样硬化病变、胆固醇逆向转运（RCT）、炎症及NF-κB-SREBPs途径相关分子的表达变化。
方法：8周龄雄性apoE-KO小鼠以普通饲料饲养，并随机分为三组：对照组、LPS组和LPS+PDTC组（每组15只）。其中，对照组每周腹腔注射与实验组同等剂量的生理盐水；LPS组每周腹腔注射LPS (2.5mg/kg body wt)1次；LPS+PDTC组每周腹腔注射LPS (2.5mg/kg body wt)和PDTC(50mg/kg body wt)1次。8周后处死动物，采用酶氧化法检测甘油三酯（TG）、总胆固醇（TC）等血脂水平；油红O染色观察主动脉和主动脉窦处脂质蓄积情况；免疫组织化学法检测巨噬细胞标志物CD68和NF-κB的表达；Masson氏染色法检测斑块处胶原纤维面积；ELISA法检测血清炎症细胞因子(TNF-α、IL-1β和MCP-1等)的表达。液体闪烁计数仪检测小鼠巨噬细胞来源的RCT效率。提取小鼠腹腔巨噬细胞，荧光定量PCR法和western-blot法检测SREBPs、miR-33、ABCA1、ABCG1、NLRPs和NF-κB的表达。
结果：与对照组比较，LPS组小鼠血浆TG、TC和LDL-C含量明显增高，而HDL-C则水平降低，加入PDTC后，HDL-C水平有明显回升。PDTC明显减少LPS所致的主动脉窦和主动脉上的As病变面积增加；增加粪便中的胆固醇流出，而肝脏和血液中的胆固醇流出没有明显改变；降低小鼠腹腔巨噬细胞中miR-33表达，促进ABCA1和ABCG1的表达。PDTC处理后，与LPS组比较，巨噬细胞浸润减少而胶原纤维增加；斑块处NF-κB的表达下降；血液中TNF-α、IL-1β和MCP-1等炎症因子表达水平降低；腹腔巨噬细胞中SREBPs和NLRPs的表达下降。
结论：①体内NF-κB活化可促进miR-33和SREBPs表达，抑制ABCA1和ABCG1的表达，抑制体内RCT并促进As；②体内NF-κB活化可增加炎症因子水平，并促进NLRPs表达。
第四部分白桦脂酸对ABCA1表达和炎症因子释放的影响及机制
目的：观察白桦脂酸对ABCA1表达和炎症因子释放的影响并探讨其机制。
方法：培养THP-1细胞株，以PMA和ox-LDL建立THP-1巨噬细胞源性泡沫细胞模型。以不同浓度（0,0.5,1,2μg/ml）BA处理24h或1μg/ml BA处理不同时间（0,12,24and48h），然后再加入LPS（10ng/ml）作用24h，观察BA预处理对LPS诱导的胆固醇流出和ABCA1表达抑制的影响。高效液相色谱检测细胞内胆固醇含量；荧光定量PCR检测BA处理对miRNA-33s及其宿主基因SREBPs表达的影响；用miR-33a/b mimic增加miR-33s的表达，用拮抗剂anti-miR-33a/b降低miR-33s的表达，检测ABCA1表达情况，判断miR-33s在BA促胆固醇流出中的作用；western-blot法检测NF-κB的核转录水平，使用NF-κB特异性的抑制剂PDTC (50μM)或Bay11-7082(5μM)处理细胞，再加入BA和LPS，荧光定量PCR检测miR-33s和ABCA1的mRNA表达，液体闪烁计数法检测细胞内胆固醇的流出，以明确NF-κB在BA促胆固醇流出中的作用；进一步检测细胞质中IκBα、磷酸化IκBα和细胞核中磷酸化NF-κB p65的表达，以及巨噬细胞炎症因子的分泌情况，以明确BA的抗炎作用及其机制。以apoE基因敲除小鼠为研究对象，观察BA对体内动脉粥样硬化病变和体内炎症反应的影响。
结果：BA预处理明显减弱LPS对巨噬细胞胆固醇流出和ABCA1表达的抑制，下调细胞内总胆固醇、胆固醇酯和游离胆固醇的含量。BA预处理有效抑制miR-33s及其宿主基因SREBPs表达。加入anti-miR-33a/b后，与单加LPS组比较，ABCA1的表达明显升高，而加入miR-33a/b mimic，ABCA1的表达则无明显改变；加入BA后，再加入anti-miR-33a/b，ABCA1的表达无明显变化，而加入miR-33a/b mimic，ABCA1的含量明显下降。BA处理可抑制NF-κB的核转录水平； PDTC或Bay11-7082可增强BA对miR-33s的抑制作用，同时增强BA对ABCA1表达及胆固醇流出的促进作用。BA处理后，细胞质中磷酸化IκBα的表达明显下降，细胞核中磷酸化NF-κB p65的表达，炎症因子分泌水平降低。LPS组小鼠较对照组小鼠血浆TG、TC和LDL-C含量明显增高，而HDL-C则水平降低，BA处理后，TG、TC和LDL-C含量下降，HDL-C水平明显回升。组LPS组比较，BA处理组主动脉窦和主动脉上的As病变面积减少；小鼠主动脉miR-33的表达下降；ABCA1表达水平升高；NF-κB的核转录水平下降；血液中炎症因子TNF-α、IL-6和IL-1β表达水平降低。
结论：①BA抗小鼠动脉粥样硬化的作用与其抗炎和降低血脂功能有关；②NF-κB-SREBPs途径参与了BA的抗动脉粥样硬化作用。
Atherosclerosis (As) is a chronic inflammatory pathological processes.Inflammation plays an important role in the initiation, progress, and complications ofatherosclerosis. Nuclear factor-kappa B (NF-κB) is one of the key transcriptionfactors in inflammatory cytokine production. Activated NF-κB has demonstrated inhuman atherosclerotic lesions. Specificity silence or inhibition of NF-κB pathwaycould reduce atherosclerotic plaque area and its complications. The mechanisms thatNF-κB promotes atherosclerosis were mainly mediated by inflammatory factors, butrecent studies found that NF-κB could promote macrophage lipid accumulation,which provide us a new research direction to recognize the proatherogenicmechanisms of NF-κB.
Sterol regulatory element binding proteins (SREBPs) is a key regulator of the genesthat control cholesterol biosynthesis and uptake. There are three forms of SREBP inmammals: SREBP-1a,-1c and-2. Although they undergo similar proteolyticactivation and share some target genes, SREBP-1a and-1c mainly stimulate fatty acidsynthesis, whereas SREBP-2acts primarily on the cholesterol biosynthetic genes andLDL receptor (LDLR) gene. The intron regions of SREBPs contain a newlydiscovered class of microRNAs (miRNAs), miR-33s. MiRNAs is a kind of smallsingle-stranded RNA molecules that have post-transcriptional regulatory activity.Studies have shown that miRNAs involved in the regulation of lipid metabolism,expression of inflammatory cytokines and formation of As. The main target genes ofmiR-33s is ATP-binding cassette transporter A1(ABCA1). As ABCA1is the mainmembrane transporter that mediates the outflow of cellular lipids, inhibiting itsexpression will results in intracellular accumulation of a large number of lipids and ultimately the formation of foam cells. In vivo studies demonstrated that antagonismof miR-33in mice promotes ABCA1expression and reverse cholesterol transport，butpromotes the regression of atherosclerosis. Thus, miR-33s has become an importanttarget for prevention and treatment of As diseases. However, the in vivo regulation ofmiRNAs is still in the initial stage, the mechanisms that control the expression ofmiR-33s in the body is far from clear.
There is a growing body of evidence pointing to the closely relationship betweenthe innate immune response and a variety of diseases including As. NOD-likereceptors (NLRs) are pattern recognition receptors that feeling microbial andnon-microbial signal in cells. NLRs can form large protein complexes within the cells,referred to as the " inflammasome", including the NLRs, caspase1and the ASC. Themain role of inflammasome is to form an bracket to activate caspase1, which couldpromote pro-IL-1β and pro-IL-18transform into mature IL-1β and IL-18, respectively.IL-1β and IL-18correlate closely to the development of As, the absence of IL-1β orIL-18significantly restrict the development of the As in mice. However, themechanisms that regulate the inflammasome in vivo are still unclear.
Through bioinformatics analysis, we found the promoter region of SREBPs, thehost gene of miR-33s, have the NF-κB response element (κBREs), suggesting thatNF-κB may be direct binding to SREBPs promoter and regulating the expression ofSREBPs/miR-33s. Therefore, in this study, we firstly observed the effects of NF-κBactivation on macrophages cholesterol efflux and inflammatory factors production inthe first part. Then, in the second part we explored the effect and regulatorymechanisms of NF-κB on SREBPs. We also investigated the the roles of miR-33s inNF-κB repressing cellular cholesterol efflux and the roles of SREBPs in NF-κBpromoting inflammatory cytokine release and inflammasome expression. Next, in thethird part we observed the effects of NF-κB-SREBPs pathway in As and inflammationreaction in vivo. Finally, we observed the effects of betulinic acid (BA), which hadproved to have significant anti-inflammatory effects, on NF-κB-SREBPs pathways invitro and in vivo, and explored its possible mechanisms in the fourth part. Our studiesprovide new insights for revealing the potential roles and mechanisms of NF-κB-SREBPs pathway in the development of As.
Part I: Effect of NF-κB Activation on Cholesterol Efflux andInflammatory Cytokines Production in Macrophages
Aims: To investigate the effect of NF-κB activation on macrophages cholesterolefflux and the expression of related membrane transporter ABCA1, and also observethe effect of NF-κB on inflammatory factors production.
Methods: Human THP-1monocytes were treated with phorbol12-myristate13-acetate (PMA)(160nmol/liter) for24h to differentiate into macrophages. THP-1and RAW264.7macrophages were then replaced to the serum-free medium containingoxLDL (100μg/ml) to become fully differentiated macrophage foam cells beforetheir use in experiments. Both cells were divided into3groups: control group, LPSgroup (10ng/ml,24h) and LPS+PDTC group (LPS10ng/ml, PDTC50μM,24h), respectively. The cholesterol efflux was assessed by liquid scintillation counting.The protein and mRNA expression of ABCA1were examined by westernimmunoblotting assays and real-time quantitative PCR, respectively. Theconcentrations of tumor necrosis factor-α (TNF-α), IL-6and interleukin-1β (IL-1β) incell culture supernatants were measured by enzyme-linked immunosorbent assay(ELISA).
Results: Apolipoprotein A1(apoA-I)-mediated cholesterol efflux wassignificantly reduced after treatment with LPS in THP-1and RAW264.7macrophage-derived foam cells. Accordingly, NF-κB activation clearly decreased thelevels of ABCA1mRNA and protein. Application of the NF-κB specific inhibitorPDTC significantly suppressed the LPS-induced down-regulation of cholesterol effluxand the expression of ABCA1. In addition, PDTC obviously inhibited the secretion ofinflammatory cytokines TNF-α, IL-1β and IL-6induced by LPS.
Conclusion:①The activation of NF-κB inhibits cholesterol efflux and theexpression of ABCA1in macrophages.②NF-κB activation promotes the secretion of inflammatory cytokines, such as TNFα, IL-6and IL-1β etc.
Part II: Molecular Mechanism of SREBPs-Mediated NF-κBRegulating Cholesterol Efflux and Secretion of InflammatoryCytokines
Aims: To investigate the possible molecular mechanisms of NF-κB activation onmacrophages cholesterol efflux and secretion of inflammatory cytokines
Methods: Human THP-1monocytes were preincubated with PMA and ox-LDLto form foam cells. Cells were treated with LPS (10ng/ml) for3h and followed bytreatment of act D (5μg/ml) to stop the transcription. ABCA1mRNA expression wasexamined by real-time quantitative PCR to observe the changes of ABCA1mRNAstability. Cells was treated by LPS and observed the concentration-dependent ortime-dependent effect of NF-κB activation on miR-33s and their host genes SREBPs.Observed the expression changes of miR-33s/SREBPs after inhibition of NF-κBactivation by PDTC treatment. Co-transfection of SREBPs promoter reporterplasmid with expression vector p50(pRSV-NF-κB-1) and p65(pRSV-RelA) intoHEK-293T cells, cells were lysed and luciferase activities were measured by using thedual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) assays wasperformed to detect if NF-κB have a direct binding to the promoter regions of theSREBPs genes. Human THP-1cells were transfected with40nM miRIDIAN miRNAmimics (miR-33a/b) or with60nM miRIDIAN miRNA inhibitors (anti-miR-33a/b) toassess the effects of gain and loss of function of miR-33on ABCA1expression andcholesterol efflux. Treatment of macrophages with SREBPs siRNA to study the roleof SREBPs in the expression of inflammatory cytokines.
Results: NF-κB activation significantly promotes ABCA1mRNA degradation,indicating that the reduction in mRNA is caused by destabilization. NF-κB activationincreased the SREBP-2/miR-33a and SREBP-1a/miR-33b mRNA expression in aconcentration-dependent manner when compared with a negative control. Accordingly, NF-κB also increase the SREBPs protein levels. The Luciferase-SREBPs reporterswere expressed at markedly higher levels as compared with the luciferase vectorwithout insert. Both deletions and mutations of κBRE abolished the enhancing effectsof NF-κB compared to the wild-type control construct. Further, ChIP assays with anantibody to the p65subunit of NF-κB demonstrated a direct binding of thistranscription factor to the promoter regions of the SREBPs genes. ABCA1mRNA andprotein levels were both reduced in THP-1cells transfected with excess wild-typehuman miR-33a/b mimic oligonucleotides compared to the control group. By contrast,the effects of NF-κB on ABCA1were abolished by anti-miR-33a/b. Accordingly,introduction of miR-33mimic into macrophages resulted in further repression of the[3H]-cholesterol efflux compared to the control oligonucleotides, whereas miR-33antisense oligonucleotides treatment led to a marked increase in [3H]-cholesterolefflux. The increase in IL-1β release observed in WT macrophages was significantlyreduced with SREBPs knockdown, but the repression of either SREBP-1or SREBP-2had no obvious effects on TNF-α, IL-6and IL-18expression. Treatment with siRNAfor SREBPs significantly down-regulated NF-κB-induced NLRP1protein expressionin THP-1cells without detectable effect on NLRP3. In addition, the effect of NF-κBon IL-1β expression in NLRP1siRNA cells was obviously suppressed.
Conclusion:①SREBPs are direct NF-κB target genes.②NF-κB down-regulates ABCA1level and cholesterol efflux via promoting miR-33expression.③SREBPs increase NF-κB-induced IL-1β expression in macrophages throughactivation of NLRP1.
Part III: Effects of NF-κB on Atherosclerotic Lesions andinflammatory cytokines expression in apoE-/-Mice
Aims: To observe the effect of NF-κB activation on atherosclerotic lesion, RCT,inflammatory response and the changes of molecules expression related to NF-κB-SREBPs pathway in apoE-KO mice.
Methods: Six-week old male apoE-/-mice were obtained from LaboratoryAnimal Center of Peking University, China. All mice were fed a chow diet. At8weeks of age, apoE-/-mice were randomly divided into several groups (n=15pergroup). The LPS group was challenged intraperitoneally (i.p.) with LPS (2.5mg/kgbody wt) in200μL of PBS once every week. Mice in the PDTC group were injectedi.p. with PDTC (50mg/kg body wt)1h before LPS challenge. The control group wasreceived abdominal injections of PBS of the same volume at the same time. At week16, the mice were sacrificed, blood was obtained, and tissues were collected forfurther analysis. Triglyceride (TG), total cholesterol (TC), and HDL-C weredetermined by commercially enzymatic methods. Lipid accumulation in aorta andaortic sinus were evaluated by Oil Red O stain. The expression of CD68, a marker formacrophage, and NF-κB p65in lesions were tested by immuno-histochemistry.Masson’s staining for collagen. Plasma inflammatory cytokines (TNF-α、IL-1β andMCP-1etc.) expression was detected by ELISA. RCT efficiency of murinemacrophage cell was detected by liquid scintillation counting. The protein and mRNAexpression of SREBPs、miR-33、ABCA1、ABCG1、NLRPs and NF-κB in mouseperitoneal macrophages (MPMs) were examined by western immunoblotting assaysand real-time quantitative PCR, respectively.
Results: Plasma TG, TC and LDL-C levels were increased in LPS groupcompared with the control group, and the HDL-C levels were decreased. ApoE-/-miceinjected with LPS and PDTC significantly reduced lesion size in comparison to theLPS group. Treatment of LPS-challenged apoE-/-mice with PDTC significantlyreduced cholesterol transport from macrophages to feces in the apoE-/-mice. Incontrast, both LPS and PDTC had no significant effect on cholesterol tracer recoveryin the liver and plasma. Treatment with PDTC significantly promoted the expressionof both ABCA1and ABCG1and reduced the expression of miR-33a in mouseperitoneal macrophages. Moreover, there was a reduction in CD68+macrophagescontent and increase in total lesional collagen content in PDTC group compared withLPS group. The NF-κB expression was decreased when treated with PDTC, andinhibition of NF-κB activity could reduce the inflammatory cytokines release. Analysis of the expression of SREBPs and NLRPs revealed a significant increased inLPS-challenged apoE-/-mice peritoneal macrophages, and this effect could be partlyreversed by application of the NF-κB specific inhibitor PDTC.
Conclusion:①NF-κB activation in vivo can increase the xpression of miR-33and SREBPs, reduce the expression of ABCA1/ABCG1, inhibit the RCT in vivo andpromote the development of mice atherosclerotic lesions.②NF-κB activation canincrease the level of inflammatory cytokines in vivo and promote NLRPs expression.
Part Ⅳ: Effect and Mechanism of Betulinic Acid on ABCA1Expression and Inflammatory Cytokines Production
Aims: To investigate the effect and molecular mechanism of BA on ABCA1expression and inflammatory cytokines production in vitro and in vivo.
Methods: Human THP-1monocytes were preincubated with PMA and ox-LDLto form foam cells. Cells was pre-treated with BA (0,0.5,1,2μg/ml) for24hr or withBA (1μg/ml) for0,12,24and48hr, respectively, and then exposed them to LPS. Toobserved the effects of BA on cholesterol efflux and the expression of ABCA1inLPS-treated macrophages. HPLC was performed to determine cellular totalcholesterol, free cholesterol and cholesterol ester. Real-time quantitative PCR wasperformed the effect of BA on the expression of miR-33s and their host genesSREBPs. Cells were transfected with miR-33a/b mimics or with anti-miR-33a/b toassess the effects of miR-33in BA promoting ABCA1expression and cholesterolefflux. The protein expression of nuclear NF-κB was examined by westernimmunoblotting assays. THP-1macrophage-derived foam cells were pretreated withPDTC (50μM) or Bay11-7082(5μM) for24h, and cells were then incubated withLPS for another24h with or without pretreatment of BA. Expression of miR-33smRNA was confirmed by RT-PCR. Cellular cholesterol efflux was analyzed by liquidscintillation counting assays to access the effect of NF-κB in BA promotingcholesterol efflux. Protein levels of IκBα, p-IκBα and phosphorylation of p65were measured and analyzed. Furthermore, we examined the effect of BA onLPS-stimulated inflammatory cytokine production in macrophages to clarify theanti-inflammatory effect and mechanism of BA. In addition, we observed the effect ofBA on atherosclerotic lesion and inflammatory response in apoE-KO mice. Plasma TG,TC and LDL-C levels were increased in LPS group compared with the control group.Both TG and TC levels in animals treated with BA were decreased. Analysis of theplasma lipoproteins showed an increase in HDL cholesterol and a decrease in LDLcholesterol levels in group of BA treatment. LPS-injected apoE-/-mice treatedadditionally with BA have a significantly reduced lesion size. BA significantlyreduced the expression of miR-33and promoted the expression of ABCA1ascompared with those treated by LPS alone. BA also suppressed the activation ofNF-κB and decreased the plasma pro-inflammatory cytokines levels
Results: Pre-treated with BA clearly decreased the inhibition of cholesterolefflux and ABCA1mRNA and protein expression induced by LPS. Cellularcholesterol content decreased when cells were treated with BA. LPS significantlyincreased the miR-33s and SREBPs expression in THP-1macrophage-derived foamcells, whereas treatment with BA down-regulated the expression of miR-33s andSREBPs compared with those treated by LPS only. The effects of LPS on ABCA1were reversed by anti-miR-33a/b. Whereas ABCA1levels was not changedsignificantly in THP-1cells transfected with excess wild-type human miR-33a/bmimic oligonucleotides compared to the LPS group. In addition, the effect of BA onABCA1expression was reversed by transfected with miR-33a/b mimic. With the BAtreatment, the promoting effects of LPS at nuclear NF-κB p65protein levels wereeffectively abrogated. miR-33s expression in cells treated by the combination ofNF-κB specific inhibitor (PDTC or Bay11-7082) was significantly decreased, at thesame time, PDTC or Bay11-7082efficiently promote ABCA1expression and cellularcholesterol efflux induced by BA. BA treated cells showed significant decrease in thephospho-protein expression of IκBα and NF-κB p65. BA significantly inhibitedLPS-stimulated secretion of TNFα, IL-6and IL-1β.
Conclusion:①Anti-inflammation and inhibition of plasma lipids are involvedin the mechanisms of BA treatment induces atherosclerosis regression in vivo.②NF-κB-SREBPs patherway involved in the reduction of atherosclerosis mediated byBA.
引文
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