关键词： 通关藤注射液   紫杉醇   乳腺癌   ATF3   迁移侵袭  

摘要： 乳腺癌是女性发病率最高的恶性肿瘤。紫杉醇(Paclitaxel,PTX)是乳腺癌的一线化疗药物之一,可通过促进微管聚合并阻止其解聚而发挥抗肿瘤作用,但研究发现PTX可通过调控ATF3的表达引起乳腺癌的转移,影响PTX对乳腺癌治疗的有效性。ATF3作为转录激活因子参与乳腺癌的发生发展过程,但其在乳腺癌中的作用机制尚未完全阐明。通关藤注射液(Tong-Guan-Teng Injection,TGT)是临床常用的抗肝癌、肺癌等的辅助中成药,并可配合放化疗的辅助治疗。但TGT是否可增强PTX抗乳腺癌的增殖和转移作用,且作用机制是否与ATF3有关仍有待阐明。目的:研究ATF3介导的TGT增强PTX抗乳腺癌的作用,阐明TGT通过调控ATF3的表达增强PTX抗乳腺癌机制,为乳腺癌新的治疗靶点及中西医联合治疗方案提供给理论依据。方法:体外培养人乳腺癌MDA-MB-231细胞。TGT与PTX单用或联合作用于乳腺癌MDA-MB-231细胞,采用q RT-PCR和Western blot法检测ATF3 m RNA和蛋白的表达水平。Si RNA干扰ATF3的表达后,采用TGT和PTX单独或联合干预,q RT-PCR和Western blot检测ATF3及相关细胞因子的表达,划痕实验和Transwell实验检测细胞迁移和侵袭能力,Hoechst活细胞染色和流式细胞术检测细胞凋亡。采用MDA-MB-231细胞尾静脉注射的方法构建乳腺癌裸鼠肺转移模型,评价TGT和PTX两药联用对体内肿瘤转移的作用,免疫组化和Western blot法检测ATF3蛋白表达,HE染色观察转移组织中的细胞病理情况,ELISA检测血清中相关因子的表达。体外构建巨噬细胞和乳腺癌细胞共培养体系,干扰ATF3的表达,研究ATF3介导TGT联合PTX对巨噬细胞迁移和极化表型的影响,阐明TGT通过调控ATF3增强PTX抑制乳腺癌增殖和转移的机制。结果:1.通关藤注射液联合紫杉醇可影响ATF3的表达qRT-PCR和Western blot实验结果显示,PTX单用组可诱导ATF3 m RNA的高表达,而TGT联合PTX可降低ATF3 m RNA的表达;同样,与PTX单用组相比,TGT联合PTX组ATF3蛋白表达显著下调。流式细胞仪检测结果显示,TGT联合PTX与单药组相比,可显著抑制MDA-MB-231细胞凋亡;划痕和Transwell实验显示,TGT联合PTX与单药组相比,可显著抑制乳腺癌细胞的迁移和侵袭。结果表明,通关藤注射液增强紫杉醇抗乳腺癌作用可能与ATF3有关。2.通关藤注射液通过调控ATF3的表达增强紫杉醇体外抗乳腺癌的作用在ATF3-Si RNA干扰的MDA-MB-231细胞中,PTX可显著上调ATF3蛋白的表达,而TGT可以抵消PTX引起的ATF3蛋白表达的上调;TGT可显著抑制MDA-MB-231细胞的增殖和转移、诱导细胞凋亡,且TGT联合PTX组较PTX单药组作用更为显著。3.通关藤注射液增强紫杉醇在裸鼠体内的抗乳腺癌作用在尾静脉注射MDA-MB-231细胞的裸鼠乳腺癌肺转移模型中,与PTX单用或TGT单用相比,TGT与PTX联用对MDA-MB-231细胞尾静脉注射的肿瘤转移有更显著的抑制效果,且TGT高剂量组与PTX联用效果最佳。对肺组织病理形态观察的HE染色结果显示,TGT高剂量组与PTX联用后,大量肿瘤细胞坏死、细胞核皱缩且细胞形态发生改变;免疫组织化学检测结果显示,TGT联合PTX组ATF3的表达降低,且高剂量联合组效果更加显著;ELISA检测结果显示,裸鼠血清中细胞因子IL-1β和IL-6的表达显著降低。4.通关藤注射液通过ATF3影响巨噬细胞的极化作用结果显示,TGT可显著增强PTX对巨噬细胞的极化作用,提高了M1表型标志物CD86的表达,同时降低了M2表型标记物CD206的表达。与PTX单用组相比,TGT与PTX联用组乳腺癌细胞划痕愈合能力减弱;与乳腺癌细胞单培养组相比,共培养组细胞划痕愈合能力减弱;与共培养+PTX单用组比较,共培养+PTX+TGT组细胞在竖直方向迁移水平下降;Si ATF3干扰ATF3的表达后,与乳腺癌细胞单培养组相比,共培养+Si ATF3组细胞迁移水平显著下降。综上,TGT可通过ATF3促进巨噬细胞向M1型极化,增强PTX抗乳腺癌迁移的作用。结论:通关藤注射液通过调控ATF3的表达,增强紫杉醇的抗乳腺癌增殖和转移的作用,且作用机制可能与影响巨噬细胞极化有关。该研究将为中药与化疗药物联合治疗乳腺癌提供新靶点,同时也为乳腺癌的中西医结合治疗方案提供理论依据。

关键词： 铁死亡   ATF3   内质网应激   NOX4   胶质瘤  

摘要： 研究背景及目的:铁死亡(Ferroptosis)是一种新提出的程序性细胞死亡方式,这种死亡形式与神经退行性疾病、癌症、缺血再灌注损伤、脑外伤和脑出血等多种病理过程密切相关。铁死亡在形态学方面的特征是线粒体膜密度浓缩,体积变小。在生化方面的特点是由于铁的吸收、储存和输出不平衡导致的细胞内铁离子的积累。细胞内铁离子水平主要受转铁蛋白受体调节,转铁蛋白受体负责将细胞外的铁-转铁蛋白复合物通过clathrin介导的内吞作用运送到细胞内,细胞内的铁以铁蛋白的形式进行储存,而铁转运蛋白则负责铁离子向胞外输出。异常增多的铁离子能够使膜磷脂中的多不饱和脂肪酸过氧化,因而破坏细胞膜的完整性。这主要通过两种途径实现,一是作为非血红素含铁脂氧合酶的辅助因子参与酶促反应,另一种则是与过氧化氢发生芬顿反应,生成具有强过氧化能力的羟自由基。诱导铁死亡可以有效地清除前列腺癌、结直肠癌、肝癌甚至对顺铂耐药的肺癌细胞和胶质瘤干细胞等各种类型的恶性肿瘤细胞。因此,铁死亡诱导剂作为胶质瘤的治疗药物具有相当的潜力。转录激活因子3(ATF3)是属于ATF/CREB家族的具有亮氨酸拉链结构转录因子,在人胶质瘤中过度表达,并且其表达水平与肿瘤的恶性程度呈正相关性。ATF3在调控癌细胞死亡中起着双重作用。一方面,它能够抑制肝癌细胞的肿瘤发生和进展,抑制胆管癌细胞的上皮间质化转变,限制了人结直肠癌细胞的侵袭和迁移。另一方面,它可以通过改善AKT的磷酸化来保护乳腺癌细胞免受放疗的影响。ATF3在铁死亡过程中以及在细胞凋亡和坏死中均被上调。有研究发现,ATF3通过抑制x CT的表达,促进了纤维肉瘤细胞和视网膜色素上皮细胞铁死亡的发生。然而,ATF3在调控胶质瘤细胞铁死亡过程中的作用机制尚不明确。马钱子碱是从马钱子种子中提取的弱碱性吲哚生物碱,通常用于缓解关节炎和创伤性疼痛。最近的研究显示,马钱子碱对结肠腺癌、肝癌、多发性骨髓瘤、乳腺癌和胶质瘤等多种癌症具有强效的抗肿瘤活性。其抗肿瘤作用与诱导细胞凋亡和细胞周期阻滞、JNK激活、抑制血管生成和上皮间质化转变、抑制肿瘤细胞迁移和转移相关。然而,铁死亡和ATF3是否都参与马钱子碱诱导的胶质瘤细胞死亡仍不清除。因此,本研究旨在探讨铁死亡和ATF3在马钱子碱诱导的胶质瘤细胞死亡中的作用。研究方法:1、MTT法检测马钱子碱对人胶质瘤各细胞系细胞的增殖能力抑制程度;LDH释放试验检测马钱子碱诱导的胶质瘤细胞死亡程度以及各抑制剂对马钱子碱诱导细胞死亡的影响;2、对细胞内游离二价铁离子浓度测定,采用细胞亚铁离子检测试剂盒,对不同浓度马钱子碱在给定作用时间条件下诱导人胶质瘤细胞内二价游离铁离子浓度的变化情况;检测各抑制剂对马钱子碱导致的人胶质瘤细胞亚铁离子浓度变化的影响。3、Western Blot检测马钱子碱对胶质瘤细胞作用不同时间导致的相应蛋白表达情况,以及基因沉默ATF3及NOX4后马钱子碱对胶质瘤细胞作用样品中相关蛋白含量变化的影响。4、通过MDA含量测定,研究马钱子碱诱导人胶质瘤细胞脂质氧化程度及DFO、FAC、Fer-1、Lip-1、4-PBA、GSH等抑制剂对马钱子碱诱导的人胶质瘤胶质瘤脂质氧化程度的影响。5、通过H2O2浓度测量,检测马钱子碱诱导人胶质瘤细胞活性氧自由基产生情况,以及DFO等抑制剂对马钱子碱诱导人胶质瘤细胞活性氧产生的影响。6、通过NOX氧化酶检测马钱子碱诱导胶质瘤氧化应激应对机制的影响;以及DFO等抑制剂对马钱子碱诱导人胶质瘤抗氧化应激的活性的影响。7、通过siRNA沉默ATF3、NOX4研究其对马钱子碱诱导胶质瘤细胞铁死亡过程的影响。8、通过裸鼠荷瘤实验接种小鼠胶质瘤转移瘤模型,腹腔注射马钱子碱,观察马钱子碱对胶质瘤体内模型的作用;瘤体组织检测相关亚铁离子浓度、氧化应激相关指标和相应蛋白表达情况。结果:1、马钱子碱能够抑制人胶质瘤细胞的增殖能力并且诱导细胞铁死亡。2、马钱子碱能够提高人胶质瘤细胞内亚铁离子浓度和脂质过氧化程度。3、ATF3参与马钱子碱诱导胶质瘤细胞铁死亡过程4、ATF3参与并促进马钱子碱诱导胶质瘤细胞内过氧化氢过度累积。5、ATF3通过激活NOX4和抑制xCT表达促进过氧化氢生成。6、马钱子碱诱导胶质瘤细胞内质网应激并建立内质网应激-过氧化氢累积正反馈环。7、荷裸鼠显示马钱子碱能够抑制体内胶质瘤细胞增殖,并伴有肿瘤组织内亚铁离子,MDA,H2O2水平均提高、内质网应激以及半胱氨酸和GSH消耗。结论:1、马钱子碱在体内和体外均能抑制胶质瘤增殖并诱导胶质瘤细胞死亡。2、马钱子碱诱导胶质瘤细胞发生死亡过程中伴随了细胞内铁离子浓度的增加和脂质过氧化程度上升。3、马钱子碱通过ATF3调控胶质瘤细胞铁死亡。4、马钱子碱通过激活胶质瘤细胞ATF3调控细胞内亚铁离子浓度和过氧化氢含量。5、马钱子碱通过增加过氧化氢水

关键词： 高尿酸肾病   纤维化   组蛋白修饰   ATF3   增强子  

摘要： 目的: 肾纤维化是高尿酸肾病(HN)的主要病理改变,也是慢性肾脏病(CKD)逐步发展为终末期肾病(ESRD)的最终途径。组蛋白修饰是参与机体生命活动的关键表观调控机制。研究发现组蛋白乙酰化修饰可调控基因元件增强子的活性状态,并在急性肾损伤、肾癌等多种肾脏疾病的进展中发挥重要作用,但高尿酸肾病中组蛋白乙酰化修饰的情况及作用是未知的。转录激活因子3(ATF3)在多种脏器纤维化效应细胞中高表达,同时ATF3可与组蛋白乙酰化修饰酶结合以调控基因表达,然而在高尿酸肾病中ATF3是否存在基于调控组蛋白乙酰化修饰参与肾脏纤维化过程的机制尚无人研究。本研究以表观遗传学中组蛋白修饰为切入点,首先探索高尿酸肾病中组蛋白修饰的动态变化及其功能,再进一步明确ATF3是否调控组蛋白乙酰化修饰影响增强子活性状态从而影响高尿酸肾病纤维化过程。 方法: 本研究将C57BL/6小鼠及ATF3基因敲除小鼠随机分配为对照组和模型组,通过腺嘌呤和氧嗪酸钾混合物诱导建立高尿酸肾病小鼠模型,21天后处死收取血、肾脏组织。检测血肌酐、尿素氮、血尿酸水平,肾脏组织PAS、Masson染色观察病理变化;利用RT-q PCR明确ATF3表达情况,WB检测ATF3基因敲除前后造成的各组小鼠肾脏ATF3、肾纤维化相关蛋白变化。各组小鼠肾脏组织进行RNA-Seq获取基因表达谱变化,GO、KEGG对差异基因进行功能富集分析,筛选肾纤维化相关基因;基于H3K27ac、H3K4me3为抗体的Ch IP-seq鉴定增强子、启动子功能状态;利用软件Diffbind对增强子组间差异进行比较分析,明确疾病特异增强子数量及种类;GREAT匹配疾病特异增强子及差异表达基因相应调控关系;利用软件HOMER在野生型小鼠增强子区域挖掘上游参与调控增强子活性状态的关键转录因子。C57BL/6小鼠肾组织ATF3为抗体的Ch IP-seq获得HN中ATF3在基因组上与增强子共定位区域,明确ATF3对增强子的调控作用。 结果: 口服灌胃腺嘌呤和氧嗪酸钾混合物21天,野生型造模组小鼠血尿酸升高,血清肾功指标(SCr、BUN)明显升高,Masson染色显示肾脏出现纤维化,提示高尿酸肾病小鼠模型建立成功。H3K27ac的Ch IP-seq对比分析发现HN后基因组整体乙酰化水平存在显著组间差异,有大量因组蛋白乙酰化变化而发生活性改变的增强子,其中共有14996个高尿酸肾病特有的增强子。造模前后基因转录水平同步发生显著变化,差异表达的基因中上调基因功能主要与炎症反应、肾纤维化相关,而下调的基因则主要富集于各类物质代谢通路。进一步整合组蛋白修饰及转录组数据后发现活化的增强子与上调基因在染色质组上共定位,下调基因则无上述规律,同时就整体表观变化与转录水平关系而言,增强子的活化程度与差异基因的表达量呈正相关,且大多数基因与疾病特异增强子之间存在对应的调控关系。基于Motif富集分析结果,结合转录因子自身转录水平及其在H3K27ac区域富集强度的差异,发现ATF3在变化的增强子区域富集,且自身表达差异显著。ATF3 KO小鼠造模后血肌酐、尿素氮、血尿酸水平降低,PAS和Masson染色显示肾脏损伤和纤维化程度得到改善,肾纤维化标志蛋白Collagen I、Fibronectin表达下降。敲除ATF3的小鼠造模后肾组织增强子大量失活,伴随大量基因表达水平的显著下降,其中下调的差异表达基因主要富集在与纤维化密切相关的TLR、MAPK、IL-17等炎症反应信号通路,在纤维化基因Heyl的上游发现ATF3与疾病特异增强子的直接结合。 结论: 转录因子ATF3在高尿酸肾病肾脏纤维化中扮演重要角色,其参与组蛋白乙酰化修饰的变化,改变疾病特异增强子的功能状态和数量,进而调控肾脏纤维化相关信号通路上的基因表达,发挥对肾脏纤维化的促进作用。

关键词： DNA-binding proteins   Liver fibrosis  

摘要： The excessive accumulation of extracellular matrix (ECM) is a key feature of liver fibrosis and the activated hepatic stellate cells (HSCs) are the major producer of ECM proteins. However, the precise mechanisms and target molecules that are involved in liver fibrosis remain unclear. In this study, we reported that activating transcription factor 3 (ATF3) was over-expressed in mice and human fibrotic livers, in activated HSCs and injured hepatocytes (HCs). Both in vivo and in vitro study have revealed that silencing ATF3 reduced the expression of pro-fibrotic genes and inhibited the activation of HSCs, thus alleviating the extent of liver fibrosis, indicating a potential protective role of ATF3 knockdown. However, ATF3 was not involved in either the apoptosis or proliferation of HCs. In addition, our data illustrated that increased nuclear localization of ATF3 promoted the transcription of fibrogenic genes and lnc-SCARNA10, which functioned as a novel positive regulator of TGF-beta signaling in liver fibrogenesis by recruiting SMAD3 to the promoter of these genes. Interestingly, further study also demonstrated that lnc-SCARNA10 promoted the expression of ATF3 in a TGF-beta /SMAD3-dependent manner, revealing a TGF-beta /ATF3/lnc-SCARNA10 axis that contributed to liver fibrosis by activating HSCs. Taken together, our data provide a molecular mechanism implicating induced ATF3 in liver fibrosis, suggesting that ATF3 may represent a useful target in the development of therapeutic strategies for liver fibrosis.

关键词： vascular calcification   vascular smooth muscle cells   miRNA   miR-27a-3p   activating transcription factor 3  

摘要： Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor of morbidity and mortality associated with these diseases. Herein, we report that reduction of microRNA-27a-3p (miR-27a-3p) causes an increase in activating transcription factor 3 (ATF3), a novel osteogenic transcription factor, in vascular smooth muscle cells. Both microRNA (miRNA) and mRNA microarrays were performed with rat vascular smooth muscle cells, and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatics analysis. Inorganic phosphate significantly reduced the expression of miR-27a-3p in A10 cells. The transcript level was also reduced in vitamin D3-administered mouse aortas. miR-27a-3p mimic reduced calcium deposition, whereas miR-27a-3p inhibitor increased it. The Atf3 mRNA level was upregulated in a cellular vascular calcification model, and miR-27a-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-27a-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-27a-3p may contribute to the development of vascular calcification by de-repression of ATF3.

关键词： Cancer   immunotherapy   immune  

摘要： With the support by the National Natural Science Foundation of China,the research team led by *** Xiang (陈翔)and *** Hong (刘洪)at the Xiangya Hospital,Central South University,uncovered the role of adenosine signaling pathway in cancer immunotherapy,which was published in Cancer Cell (2020,37:324—339).

关键词： Panax notoginseng saponins   myocardial infarction   ventricular remodeling inflammation  

摘要： Objective:To explore whether Panax notoginseng saponins(PNS)exhibits heart protective effect in myocardial infarction(MI)rats and to identify the potential signaling pathways ***:MI rats induced by ligating the left anterior descending(LAD)coronary artery were assigned to sham coronary artery ligation or coronary artery *** 36 Sprague-Dawley rats were randomly divided into sham group(distilled water,n=9),MI group(distilled water,n=9),PNS group(PNS,40 mg/kg daily,n=9)and fosinopril group(FIP,1.2 mg/kg daily,n=9)according to a random number *** left ventricular morphology and function were conducted by *** alterations were evaluated by the stainings of HE and *** serum levels of C reactive protein(CRP),tumor necrosis factorα(TNF-α),growth differentiation factor-15(GDF-15)and the ratio of metalloproteinase-9(MMP-9)and tissue inhibitor of MMP-9(TIMP-1)were determined by *** levels of activating transcription factor 3(ATF3),mitogen-activated protein kinase kinase 3(MAP2 K3),p38 mitogen-activated protein kinase(p38 MAPK),phosphorylation of p38 MAPK(p-p38 MAPK),transforming growth factor-β(TGF-β1),collagenⅠ,nuclear factor kappa B p65(NFκB p65),phosphorylation of NFκB p65(p-NFκB p65),and phosphorylation of inhibitory kappa Bα(p-IκBα)in hearts were measured by Western blot and immunohistochemical staining,***:PNS improved cardiac function and fibrosis in MI rats(P<0.05).The serum levels of CRP,TNF-α,GDF-15 and the ratio of MMP9/TIMP1 were reversed by PNS in MI *** expressions of TGF-β1,collagenⅠ,MAP2 K3,p38 MAPK,p-p38 MAPK,NFκB p65,p-NFκB p65,and p-IκBαwere down-regulated,while ATF3 increased with the treatment of PNS(P<0.05).Conclusions:PNS may improve cardiac function and fibrosis in MI rats via regulating ATF3/MAP2 K3/p38 MAPK and NFκB signaling *** results suggest the potential of PNS in preventing the development of ventricular remodeling in MI rats.

关键词： Breast cancer   ATF3   miRNA   lncRNA   circRNA  

摘要： We previously reported that activating transcription factor 3 (ATF3), an adaptive response gene, plays a dichotomous role in regulating several molecular processes during breast cancer progression. ATF3 promoted the expression of runt-related transcription factor 2 (Runx2, a metastatic gene) and activated matrix metalloproteinase 13 (MMP13, an invasive gene), thereby fostering proliferation and bone-metastasis of the breast cancer cells. Targeting ATF3 may mitigate the metastatic spread of breast cancer and improve the patient's lifespan. Non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are the new-era regimens that are currently utilized for diagnosis and treatment of a variety of malignancies including cancer. mir-3674 putatively targets ATF3, but its expression was significantly increased in human breast cancer cells (MDA-MB231), compared to normal human mammary epithelial cells (MCF-10A). Our in silico analysis identified a few lncRNAs and circRNAs showing their putative binding sites for miR-3674. Thus, mir-3674, despite its abundance in the MDA-MB231 cells, could not effectively target ATF3, which could be due to the sponging mechanism of lncRNAs and circRNAs towards mir-3674. More extensive in vitro and in vivo studies are required to validate this and expand the diagnostic and therapeutic perspectives of breast cancer.

关键词： ATF3   Atherosclerosis   CREB   Indoxyl sulfate   Neointimal formation   Uremia  

摘要： Background and aims: Uremic patients are characterized by an increased risk of atherosclerotic cardiovascular diseases. Vascular smooth muscle cell (VSMC) proliferation contributes to neointimal formation, a main pathological feature in atherosclerosis. Activation of CREB/ATF3 signaling is pivotal in VSMC proliferation, yet its role in uremic atherosclerosis is unknown. This study aimed to explore whether CREB/ATF3 signaling is involved in the molecular mechanism underlying neointimal formation in uremia. Methods and results: Treatment of VSMCs with uremic toxin (indoxyl sulfate [IS]) activated cAMP/CREB/ATF3/cyclin D signaling, which was reflected by increased VSMC proliferation. Blocking cAMP/PKA/CREB/ATF3 signaling attenuated the promoting effect of IS on cyclin D1 expression and VSMC proliferation. Loss-of-function and time-dependent experiments showed that ATF3 lies downstream of the CREB signaling. Mutational analysis of cyclin D1 promoter along with chromatin immunoprecipitation assays showed that CREB/ATF3 signaling participated in IS-induced cyclin D transcription. In vivo, phosphorylated CREB (an active form of CREB) and ATF3 were prominently upregulated in the neointima of experimental uremic rats, the atherosclerotic plaques of uremic ApoE(-/-) mice, and the iliac arteries of uremic patients. Notably, the use of lentivirus to knock down ATF3 in the neointima of balloon-injured arteries could suppress the effect of uremia in vivo, including neointimal formation and cyclin D expression. Conclusions: In this study, we demonstrated that CREB/ATF3-related signaling may be involved in IS-induced VSMC proliferation and the pathogenesis of neointimal formation during uremia.

关键词： anti-inflammation   honeyberry   inflammatory response   Lonicera caerulea   macrophage  

摘要： Honeyberry (Lonicera caerulea) has long been used as a traditional medicine in China, Japan and northern Russia. Functional studies of honeyberry have mainly focused on the fruits, which have been reported to exert various pharmacological activities, including anti-inflammatory activity, with limited or no studies on the other parts of the plant, such as the leaves and branches. In the present study, the anti-inflammatory effects of extracts of the leaves (HBL), branches (HBB) and fruit (HBF) of honeyberry plant were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. HBL and HBB significantly inhibited the production of pro-inflammatory mediators in LPS-stimulated RAW264.7 cells, and the inhibitory effects of HBL and HBB were stronger than those of HBF. HBL and HBB blocked the nuclear accumulation of p65 independently of I kappa B-alpha. HBL did not inhibit the phosphorylation of ERK1/2 or p38;however, HBB effectively inhibited the phosphorylation of p38 but not ERK1/2. HBL and HBB increased the expression of heme oxygenase-1 (HO-1) protein by inducing the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) through the activation of the reactive oxygen species (ROS)/p38 pathway;the reduction in inducible nitric oxide synthase (iNOS) and interleukin-1 beta (IL-1 beta) expression by HBL and HBB was inhibited by HO-1 knockdown. In addition, HBL and HBB increased the expression of activating transcription factor-3 (ATF3), and the reduction in iNOS and IL-1 beta expression by HBL and HBB was inhibited by ATF3 knockdown. Collectively, HBL and HBB inhibited LPS-induced nuclear factor-kappa B activation by blocking the nuclear accumulation of p65, increasing HO-1 expression through activation of the ROS/p38/Nrf2 pathway, and increasing ATF3 expression. Furthermore, HBB inhibited LPS-induced p38 phosphorylation. These findings suggest that HBL and HBB may have great potential as natural products for the development of anti-inflammatory dr