关键词： 绵羊   营养剥夺   骨骼肌   转录组测序分析   应激、免疫和凋亡   ATF3  

摘要： 营养剥夺可使动物机体发生一系列生理生化反应,包括应激、免疫和凋亡,其在骨骼肌中的具体机制尚不清楚。本试验旨在从动物和细胞水平,基于转录组测序和生物信息学分析鉴定营养剥夺条件下绵羊骨骼肌应激、免疫和凋亡相关的差异表达基因(DEGs),为揭示相关机制奠定基础。首先通过对禁食条件下绵羊骨骼肌转录组测序(RNA-Seq)获得的DEGs进行GO和KEGG等分析,筛选了应激、免疫和凋亡相关DEGs及其通路,并在动物和细胞水平利用荧光定量PCR(q RT-PCR)对部分DEGs进行了验证。然后重点分析并研究了应激相关转录因子ATF3,利用生物信息学分析了其分子特性及基因启动子区转录调控元件,并扩增了其编码序列,合成了linker肽(GS),构建了ATF3-(GS)-EGFP融合表达载体p ATF3-EGFP,转染绵羊成肌细胞后通过荧光观察和q RT-PCR检测了其表达效果。结果显示:(1)通过对营养剥夺条件下绵羊骨骼肌转录组结果分析,共筛选出107个应激、免疫和凋亡相关DEGs,其中15个基因上调,92个基因下调。应激、免疫和凋亡相关DEGs分别有45、60和66个,其中免疫和凋亡共同相关DEGs有32个,应激和凋亡共同相关DEGs有25个,应激和免疫共同相关DEGs有22个,应激、免疫和凋亡共同相关DEGs有15个。GO富集注释到45、47和64个应激、免疫和凋亡相关DEGs。KEGG通路注释到26和4个免疫、凋亡相关DEGs。蛋白互作得到3个分值最高的网络,相关性分析进一步表明,3个子网络中可能相互关联的DEGs之间多数呈现出表达水平的显著相关性。q RT-PCR结果与转录组测序结果趋势一致,证明了测序结果的可靠性。(2)成功建立了绵羊成肌细胞营养剥夺应激模型,并对上述分析的部分DEGs进行了q RT-PCR检测。结果显示,与2%HS(马血清)相比,20%和60%PBS组绵羊成肌细胞中ATF3、TNFRSF12A、MAFF、NR4A3、TRIB3、PPP1R15A、SOX4、ANGPTL4、NR1D1、TP63和IKBKG m RNA表达量显著升高;GADD45B、NR4A1、BCL2L1和STAT5B m RNA表达量显著降低。(3)生物信息学分析显示绵羊ATF3蛋白定位于细胞核,二级结构主要由α螺旋和无规则卷曲组成,三级结构与二级结构保持一致且可信度高。进化树显示绵羊ATF3与牛、山羊、猪的同源性较高。绵羊ATF3基因启动子区存在CREB,NFKB,AP1和GRE等转录因子结合元件。酶切和测序结果表明p ATF3-EGFP融合表达载体构建成功,转染绵羊成肌细胞后,荧光观察和q RT-PCR结果显示ATF3成功过表达,为进一步深入研究奠定了基础。综上所述,本试验基于RNA-Seq筛选和鉴定,获得营养剥夺条件下绵羊骨骼肌中应激、免疫和凋亡功能相关的DEGs共107个,其中许多DEGs在三种功能间具有交叠性,部分DEGs存在互作关系和表达相关性。通过对候选基因ATF3生物信息学分析了解了其结构和特征,并利用体外重组技术实现了其在绵羊成肌细胞中的过表达。

关键词： Induced pluripotent stem cells   nerve conduit   sciatic nerve   scaffold   regenerative medicine   neurospheres   neural stem/progenitor cells   cell viability   transcription factor   neurotrophic factor   MARROW STROMAL CELLS   NEUROTROPHIC FACTOR   SCHWANN-CELLS   EXPRESSION   MOUSE   ATF3   GENERATION  

摘要： BACKGROUND: We previously demonstrated that a bioabsorbable nerve conduit coated with mouse induced pluripotent stem cell (iPSC)-derived neurospheres accelerated peripheral nerve regeneration in mice. OBJECTIVE: We examined the fate and utility of iPSC-derived neurospheres transplanted with nerve conduits for the treatment of sciatic nerve gaps in mice. METHODS: Complete 5-mm defects were created in sciatic nerves and reconstructed using nerve conduits that were either uncoated or coated with mouse iPSC-derived neurospheres. The survival of the neurospheres on the nerve conduits was tracked using an in vivo imaging. The localization of the transplanted cells and regenerating axons was examined histologically. The gene expression levels in the nerve conduits were evaluated. RESULTS: The neurospheres survived for at least 14 days, peaking at 4-7 days after implantation. The grafted neurospheres remained as Schwann-like cells within the nerve conduits and migrated into the regenerated axons. The expression levels of ATF3, BDNF, and GDNF in the nerve conduit coated with neurospheres were upregulated. CONCLUSIONS: Mouse iPSC-derived neurospheres transplanted with nerve conduits for the treatment of sciatic nerve defects in mice migrated into regenerating axons, survived as Schwann-like cells, and promoted axonal growth with an elevation in the expression of nerve regeneration-associated trophic factors.

关键词： 弥漫性大B细胞淋巴瘤   激活转录因子3   p53   预后   增殖   多柔比星  

摘要： 目的探讨激活转录因子3（Activating Transcription Factor 3,ATF3）与弥漫性大B细胞淋巴瘤（Diffuse Large B-cell Lymphoma,DLBCL）患者临床病理学特征及预后之间的关系;探讨ATF3对DLBCL细胞增殖能力的影响以及不同ATF3表达水平对DLBCL细胞药物敏感性的影响,及其可能的分子机制。方法应用免疫组织化学染色方法检测119例DLBCL患者肿瘤组织中ATF3及突变型p53（Mutant Type Tumor Protein 53,mutp53）蛋白的表达水平;建立稳定高表达ATF3及低表达ATF3的活化B细胞样（Activated B-cell-like,ABC）DLBCL TMD8细胞株和生发中心B细胞样（Germinal center B-cell-like,GCB）DLBCL BJAB细胞株,应用RT-PCR及Western Blot方法鉴定ATF3表达水平;CCK8细胞增殖实验检测不同ATF3表达水平对DLBCL细胞体外增殖能力的影响,RT-PCR检测48小时体外培养后不同ATF3表达水平的DLBCL细胞中BCL2、P21、RELA等基因m RNA的表达情况;多柔比星（Doxorubicin,DOX）化疗药物敏感实验研究不同ATF3表达水平下DLBCL细胞对DOX的耐药性。结果119例DLBCL患者中ATF3阳性表达38例（38/119,31.93%）,阴性表达81例（81/119,68.07%）,mutp53阳性表达43例（43/119,36.13%）,阴性表达76例（76/119,63.87%）,ATF3蛋白表达与p53蛋白表达水平之间具有显著统计学意义（P＜0.05）,ATF3蛋白表达水平与患者临床分期之间的相关性具有显著统计学意义（P＜0.05）,与性别、年龄、IPI评分、Hans分型之间无显著相关性（P＞0.05）,生存分析显示ATF3蛋白阳性表达者较ATF3蛋白阴性表达者的总体生存时间显著缩短（P＜0.05）;细胞增殖实验显示BJAB细胞组体外增殖活性显著高于TMD8组细胞,与对照组相比,ATF3表达水平上调后TMD8和BJAB细胞体外增殖活性均显著增高,ATF3表达水平下调后TMD8和BJAB细胞体外增殖活性均显著降低,各组差异均具有统计学意义（P＜0.05）;在各组细胞进行48小时体外培养后,RT-PCR分析显示ATF3表达上调后,TMD8细胞组中BCL2、RELA、HER2、P21表达水平升高,BJAB细胞组中P21、BCL2、HER2、RELA、PDCD5、CASP3、BIRC5表达水平升高;ATF3表达下调后,TMD8细胞组中HER2、CDKN2A、CCND-1表达升高,BJAB细胞组中CASP3、HER2表达升高;加入DOX药物后,下调TMD8和BJAB细胞内ATF3的表达水平后,两组肿瘤细胞存活数量均较对照组减少,上调ATF3表达水平后,两组肿瘤细胞存活数量均多于对照组,TMD8组细胞存活情况优于BJAB组。结论DLBCL中ATF3表达水平与p53蛋白表达呈正相关;ATF3的表达水平可作为DLBCL患者的预后指标;上调ATF3表达可增加DLBCL细胞体外增殖活性及对DOX的耐药性,并可能通过调节BCL2、RELA、HER2等基因参与调控DLBCL肿瘤细胞体外增殖及耐药的过程。

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： 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： EXPRESSION   TARGET   AXIS   RNA  

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： ACTIVATING TRANSCRIPTION FACTOR-3   OSTEOBLASTIC DIFFERENTIATION   UP-REGULATION   IN-VITRO   CALCIFICATION   MICRORNA   EXPRESSION   PROLIFERATION   CARDIOMYOCYTES   PATHWAY  

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： 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： 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： anti-inflammation   honeyberry   inflammatory response   Lonicera caerulea   macrophage   NF-KAPPA-B   HEME OXYGENASE-1   SIGNALING PATHWAYS   IN-VITRO   BLUE HONEYSUCKLE   FERULIC ACID   INFLAMMATION   EXPRESSION   MAPK   ANTIOXIDANT  

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