Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

火针对带状疱疹后遗神经痛大鼠疼痛阈值及NF-κB表达的影响

目的观察火针对带状疱疹后遗神经痛大鼠机械性痛阈值、脊髓核转录因子-κB(nuclear factor-κB,NF-κB)表达的影响,探讨火针治疗带状疱疹后遗神经痛的可能机制。方法将30只雄性SD大鼠,随机分为3组,即空白对照组、模型组、火针组,每组大鼠均为10只。通过VZV接种建立带状疱疹后神经痛(postherpetic neuralgia,PHN)动物模型。于接种前1 d,接种后1 d、4 d、7 d、14d和21 d分别进行机械性痛阈(paw withdrawal threshold,PWT)测定。接种后第7天开始,火针组进行火针治疗,隔日治疗1次,7次1个疗程。第21天,取各组大鼠L4、5段脊髓,用免疫组化法测定脊髓NF-κBp65蛋白表达情况。结果与空白对照组比较,模型组从造模后7 d开始出现PWT值下降(P<0.01),造模后第14天、21天时仍处于较低水平(P<0.01)。与模型组比较,火针组第7天治疗后开始出现PWT值上升(P<0.01),第14天、21天时PWT值上升明显(P<0.01)。与空白对照组比较,模型组脊髓组织中NF-κBp65表达明显增多,差异有统计学意义(P<0.01),与模型组比较,火针组脊髓组织中NF-κBp65表达明显减低,差异有统计学意义(P<0.01)。结论火针可缓解PHN大鼠PWT值,抑制NF-κB的表达,可能是火针治疗PHN的机制之一。

NF-κB、Bcl-2与酒精性肝病

酒精性肝病(ALD)是由于长期过度饮酒而引发的一系列肝脏损害疾病。现研究表明肝细胞的凋亡对该病的发生、发展起着十分重要的作用。其中核因子κB(NF-κB)、B细胞淋巴瘤-2基因(Bcl-2)与ALD关系密切。ALD患者肝细胞内活化的NF-κB,通过刺激大量炎性细胞因子释放,引发肝组织炎症、纤维化、坏死和凋亡,同时通过调控凋亡蛋白酶(Caspase)、Bcl-2、死亡受体等基因来干预肝细胞凋亡;被激活的Bcl-2,除本身具有抗凋亡功能外,还与NF-κB以复合物的形式发挥抗凋亡作用,同时与同家族促凋亡蛋白Bax以二聚体的形式依比例对肝细胞凋亡发挥抑制或促进作用。肝细胞凋亡和抗凋亡的动态失衡将成为酒精性肝病发病的重要途径之一。

Effects of IκBα and its mutants on NF-κB and p53 signaling pathways

AIM： To study the effects of IκBα and its mutants （IκBαM, IκBα3N, IκBαM44C） on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed. METHODS： pECFP-IκBα, pECFP-IκBαM （amino acides 1-317, Ser32, 36A）, pECFP-IκBα243N （amino acides 1-243）, pECFP-IκBα244C （amino acides 24±317）, pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-Cl as a control. 30 h after the transfection, location patterns of NF-κB, p53 and IκBα（IκBαM, IκBα243N, IκBα224C） were observed by a laser scanning microscope （LSM510/ConfoCor2, Zeiss）. RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IκBα and its mutants on the transprition level of NF-κB, NF-κB downstream target gene TNF-α, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments β-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS： Cells that were transfected with p53- DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IκBα, CFP-IκBαM, and CFP-IκBα243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IκBα244C revealed a predominant nuclear localization. The rnRNA levels of p65, TNF-α, p53 and Bax in CFP-IκBα transfected cells did not change significantly, while in YFP-p65/CFP-IκBα co-transfected cells, IκBα decreased the transcription of p65 downstream gene TNF-α （2.24 ± 0.503） compared with the YFP-p65/ CFP-C1 co-transfected cells （5.08 ± 0.891） （P 〈 0.05）. Phosphorylation defective IκBα （IκBαM） decreased the transcription levels of all the four genes compared with the control （P 〈 0.05）. The N terminus of IκBα（IκBα243N） increased the transcription of NF-κB （1.84 ± 0.176） and TNF-α （1.51 ± 0.203） a little bit. However, the C terminus of IκBα（IκBα244C） increased the transcription of NF-κB, TNF-α, p53 and Bax significantly （8.29 ± 1.662, 14.16 ± 2.121, 10.2 ± 0.621, 3.72 ± 0.346） （P 〈 0.05）. The CCK-8 experiment also showed that IκBα244C and p53 synergistically mediate apoptosis. CONCLUSIONS： IκBα and its mutants （IκBαM, IκBα243N, IκBαM244C） have different effects on NF- KB and p53 signaling pathways, according to their different structures. IκBαbounds with NF-KB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways, p53 and IκBα244C may be co-factor in inducing apoptosis. The C terminal of IκBαnhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Promising Effects of Zerumbone on the Regulation of Tumor-promoting Cytokines Induced by TNF-α-activated Fibroblasts

Inflammation plays an important role in the development of several cancers.Inflammatory cytokines,including tumor necrosis factor-α(TNF-α),are associated with the induction of inflammation.Chronic inflammation contributes to the progression of cancer through several mechanisms,including increased cytokine production and activation of transcription factors,such as nuclear factor-κB(NF-κB).Zerumbone(ZER),a component of subtropical ginger(Zingiber zerumbet Smith),seems to have anti-inflammatory,anti-cancer,and antioxidant activities.In this study,we aimed to explore the protective function and mechanisms of ZER against TNF-α-induced cancer-promoting cytokines.We found that the viability of stimulated human fibroblast cell lines was reduced after treatment with ZER(IC50=18µmol/L),compared to un-stimulated fibroblasts(IC50=40µmol/L).Besides,ZER inhibited mRNA expression and protein secretion of transforming growth factor-β(TGF-β),interleukin-33(IL-33),monocyte chemoattractant protein-1(MCP-1),and stromal cell-derived factor 1(SDF-1),which were produced by TNF-α-induced fibroblasts,as measured by quantitative real time-PCR(qRT-PCR)and ELISA assays.The mRNA expression levels of TGF-β,IL-33,SDF-1,and MCP-1 showed 8,5,2.5,and 4-fold reductions,respectively.Moreover,secretion of TGF-β,IL-33,SDF-1,and MCP-1 was reduced to 3.65±0.34 ng/mL,6.3±0.26,1703.6±295.2,and 5.02±0.18 pg/mL,respectively,compared to the untreated group.In addition,the conditioned media(CM)of TNF-α-stimulated fibroblasts increased the NF-κB expression in colorectal cancer cell lines(HCT-116 and Sw48),while in the vicinity of ZER,the expression of NF-κB was reversed.Considering the significant effects of ZER,this component can be used as an appropriate alternative herbal treatment for cancer-related chronic inflammation.

EYA4通过抑制NF-κB依赖的RAP1反式激活抑制肝细胞癌的生长和侵袭

背景与目的我们之前研究表明,眼缺失蛋白同源物4(eyes absent homolog 4,EYA4)是果蝇眼部发育相关的眼缺乏蛋白家族成员之一,在肝细胞癌(hepatocellular carcinoma,HCC)标本中常发生甲基化和沉默,并与患者生存期短密切相关。本研究旨在探讨EYA4在HCC中作为肿瘤抑制因子的作用机制。方法转染EYA4表达质粒(pEYA4)构建稳定表达EYA4的人HCC细胞系Huh-7和PLC/PRF/5(PLC)。通过BALB/c裸鼠皮下注射稳定转染细胞建立异种移植肿瘤。组织标本来自75例病理诊断为HCC的患者。采用实时定量聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)、蛋白质免疫印迹和免疫组织化学的方法检测EYA4在细胞系、异种移植物和临床标本中的表达;研究了稳定转染细胞系的细胞增殖、克隆形成、侵袭性和肿瘤形成。利用基因表达芯片筛选EYA4调节的基因。通过共转染EYA4和带Flag标签的RAS相关蛋白1A(RAS-related protein 1A,RAP1A)基因的表达质粒(pEYA4和Flag-RAP1A)、功能研究、染色质免疫共沉淀、免疫荧光染色和细胞泛素化分析等方法,研究了EYA4对核因子-κB(nuclear factor-κB,NF-κB)/RAS相关蛋白1(RAS-related protein 1,RAP1)信号通路的影响。结果恢复HCC细胞系中EYA4的表达可抑制细胞的增殖、抑制克隆形成、降低细胞的侵袭性和抑制异种移植肿瘤生长,在体外实验中Flag-RAP1A可逆转pEYA4的抑制作用。用肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)激活NF-κB通路可增加p65与RAP1A基因启动子的结合并上调RAP1蛋白的表达。用BAY 11-7085和p65 siRNA抑制NF-κB通路,成功阻断了TNF-α诱导的RAP1上调。EYA4拮抗了TNF-α诱导的NF-κB抑制因子α(inhibitor of NF-κBα,IκBα)的磷酸化和泛素化以及p65的核易位和反式激活,进而抑制了NF-κB的活性和RAP1的表达。用calyculin A阻断EYA4的丝氨酸/苏氨酸磷酸酶活性可显著消除其对NF-κB活性的抑制作用。此外,EYA4的表达与HCC临床标本中IκBα/RAP1活性呈负相关。结论我们的研究结果为明确EYA4是真正的肿瘤抑制因子提供了功能和机制的理论基础,该肿瘤抑制因子可抑制NF-κB/RAP1信号通路的异常激活,从而抑制HCC生长和侵袭。

阻断 NF -κB 信号途径防治急性肺损伤的研究进展

目的：探讨以核因子κB（NF-κB）为靶点防治急性肺损伤（ALI）的分子生物学机制。方法应用CNKI、Medline、ScienceDirect 等数据库，查阅近年来相关文献，总结NF-κB在ALI中的作用机制与干预手段。结果 NF-κB在ALI中的信号转导途径及作用机制逐渐被揭示，大量研究证实，通过干预NF-κB上游信号通路、NF-κB抑制蛋白（ IκB）、IκB激酶（ IKK）等途径，能在一定限度内阻断细胞因子和炎症介质的释放，缓解ALI的炎症反应。结论阻断靶细胞中NF-κB通路、特异性抑制目的蛋白和基因表达，或许能成为未来治疗ALI的研究方向；不恰当的药物干预会破坏机体的免疫平衡状态，虽然NF-κB阻断剂已进入临床试验阶段，但多数仅局限于动物实验和细胞水平，实现临床防治尚需进行大量实验研究。

dsODNs 靶向封闭肺泡巨噬细胞中 NF -κB炎症信号通路的实验研究

目的：在确定脂质体转染双链寡聚脱氧核苷酸（ dsODNs ）最佳转染浓度及时间的基础上，评估脂质体转染的双链寡聚脱氧核苷酸（ dsODNs/Lipofectamin2000）竞争性抑制对肺泡巨噬细胞中核因子κB（ NF-κB）/DNA结合活性的影响，验证dsODNs-decoy策略靶向封闭肺泡巨噬细胞中NF-κB信号通路的可行性。方法支气管肺泡灌洗分离提取家兔肺泡巨噬细胞（ AMs）后体外培养，以Lipofectamine2000为载体转染dsODNs后对巨噬细胞进行干预，测定巨噬细胞中dsODNs/Lipofectamine2000的转染活性、转染效率及转染后对AMs的毒性；检测dsODNs/Lipofectamine2000转染对炎症因子（ IL -1α、IL -6、TNF -α等） mRNA 的表达；观察 dsODNs/Lipofectamine2000转染后NF -κB p65亚基的核移位情况。结果转染肺泡巨噬细胞dsODNs/Lipofectamine2000的最适比例为1∶5，最佳时间为6 h，此时细胞毒性适中，转染效率、荧光强度及细胞状态最佳；与LPS组比较，dsODNs/Lipofectamine2000转染组各种炎症介质mRNA的表达均明显抑制（P＜0．01）；细胞核中NF-κB p65亚基的表达明显少于细胞浆中。结论 dsODNs/Lipofecetamine2000能在体外有效转染AMs并成功抑制AMs中NF-κB信号通路相关炎症靶基因的转录表达，证实了dsODNs-decoy策略影响炎症效应细胞的可行性。

Hydrogen sulfide protects against amyloid beta-peptide induced neuronal injury via attenuating inflammatory responses in a rat model

Neuroinflammation has been recognized to play a critical role in the pathogenesis of Alzheimer＇s disease （AD）, which is pathologically characterized by the accumulation of senile plaques containing activated microglia and amyloid β-peptides （Aβ）. In the present study, we examined the neuroprotective effects of hydrogen sulfide （H2S） on neuroinflammation in rats with Aβ1-40 hippocampal injection. We found that Aβ-induced rats exhibited a disorder of pyramidal cell layer arrangement, and a decrease of mean pyramidal cell number in the CA1 hippocampal region compared with those in sham operated rats. NaHS （a donor of H2S, 5.6 mg/kg/d, i.p.） treatment for 3 weeks rescued neuronal cell death significantly. Moreover, we found that H2S dramatically suppressed the release of TNF-α, IL-1β and IL-6 in the hippocampus. Consistently, both immunohistochemistry and Western blotting assays showed that H2S inhibited the upregulation of COX-2 and the activation of NF-κB in the hippocampus. In conclusion, our data indicate that H2S suppresses neuroinflammation via inhibition of the NF-κB activation pathway in the Aβ-induced rat model and has potential value for AD therapy.

Modulating effects of Astragalus polysaccharide on immune disorders via gut microbiota and the TLR4/NF-κB pathway in rats with syndrome of dampness stagnancy due to spleen deficiency

The syndrome of dampness stagnancy due to spleen deficiency(DSSD)is relatively common globally.Although the pathogenesis of DSSD remains unclear,evidence has suggested that the gut microbiota might play a significant role.Radix Astragali,used as both medicine and food,exerts the effects of tonifying spleen and qi.Astragalus polysaccharide(APS)comprises a macromolecule substance extracted from the dried root of Radix Astragali,which has many pharmacological functions.However,whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown.Here,we used DSSD rats induced by high-fat and low-protein(HFLP)diet plus exhaustive swimming,and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes,decreased the levels of interleukin-1β(IL-1β),IL-6,and endotoxin,and suppressed the Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)pathway.Moreover,a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size(LEfSe).APS increased the diversity of the gut microbiota and changed its composition,such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella,and increasing that of Parasutterella,Parabacteroides,Clostridium XIVb,Oscillibacter,Butyricicoccus,and Dorea.APS also elevated the contents of short-chain fatty acids(SCFAs).Furthermore,the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes.In general,our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota,especially for some bacteria involving immune and inflammatory response and SCFA production,as well as the TLR4/NF-κB pathway.This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD.

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation

Porcine epidemic diarrhea virus（PEDV） is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon（IFN） signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid（N） protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid（poly（I：C）） in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly（I：C）-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB（NF-κB） nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

Therapeutic Effect of Crocin on Diabetic Retinopathy in Rats Based on TLR4/My D88/NF-κB Pathway

Objective:To study the therapeutic effect of crocin on diabetic retinopathy(DR)in rats based on toll-like receptor 4(TLR4)/myeloid differentiation factor 88(My D88)/nuclear factor-κB(NF-κB)pathway.Methods:Thirty SPF SD rats were used in the experiment,which were randomly divided into DR group,control group and crocin group,with 10 rats in each group.The DR rat model was established by feeding the rats in both the DR group and crocin group with a high glucose and high fat diet,along with intraperitoneal injection(IP)of streptozotocin.Crocin IP was administered to the rats in the crocin group,whereas the rats in the DR group and control group received an equivalent dosage of saline IP for 12 weeks.A comparison was made among the three groups regarding retinal thickness,vascular permeability,expression of TLR4/My D88/NF-κB pathway protein,levels of inflammatory factors,and levels of Bcl-2,Bax,and Bcl-2/Bax.Results:The DR group and crocins group exhibited a lower retinal thickness compared to the control group,while the crocins group displayed a higher thickness than the DR group.The DR group and crocins group had higher retinal vascular permeability than the control group,and the crocins group had lower retinal vascular permeability than the DR group(P<0.05).TLR4,My D88,and P-NF-κB relative expressions were higher in the DR and crocin groups than in the control group,whereas TLR4,My D88,and P-NF-κB relative expressions were lower in the crocin group than in the DR group(P<0.05).The DR group and crocin group exhibited elevated levels of inflammatory cytokines compared to the control group,while the crocin group displayed decreased levels in comparison to the DR group(P<0.05).The DR group and crocin group exhibited lower levels of Bcl-2 and Bcl-2/Bax compared to the control group,whereas the control group displayed higher levels of Bax.The crocin group exhibited elevated levels of Bcl-2 and Bcl-2/Bax compared to the DR group,whereas the DR group displayed diminished levels of Bax(P<0.05).Conclusion:Crocin has the potential to enhance the retinal thickness and vascular permeability of DR rats,and the inhibition of the TLR4/My D88/NF-κB pathway by crocin could play a crucial role in impeding the advancement of DR.

猪NF-κB p65/p50亚基的原核表达及其对猪繁殖与呼吸综合征病毒增殖的影响

为了研究经原核表达的猪细胞核转录因子NF-κB p65/p50融合蛋白对猪繁殖与呼吸综合病毒(PRRSV)体外增殖影响,本文采用RT-PCR技术扩增出编码成熟p50区和p65开放阅读框(ORF),将其克隆至pET21a(+)中,转化至Rosetta感受态细胞,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,采用聚丙烯酰胺凝胶电泳(SDSPAGE)和蛋白免疫印迹法(Western-blot)对表达产物进行鉴定;纯化复性后添加到DMEM细胞维持液中,观察p65/p50对Marc145细胞毒性,确定最佳使用浓度;探讨最佳浓度的p65/p50对PRRSV体外感染增殖活力的影响,通过实时荧光定量PCR方法研究PRRSV感染时相,绘制PRRSV增殖曲线。成功构建了大量以包涵体形式表达Mr均约为70kD的p65/p50融合蛋白的p65/p50-pET21a(+)原核表达载体;Western-blot显示p50和p65重组蛋白可分别与兔抗人p50多克隆血清、兔抗人p65纯化抗体反应。确定p65/p50最佳添加浓度为0.4μg/mL。通过实时荧光定量PCR技术研究PRRSV感染时相,结果表明NF-κB p65/p50可促进PRRSV感染Marc-145CPE出现及病毒早期增殖;但CPE出现后,病毒增殖受到抑制,病毒滴度水平显著降低(P<0.05)。该结果为进一步研究PRRS的发病机理及治疗策略提供了新思路。

Catalpol ameliorates LPS-induced endometritis by inhibiting inflammation and TLR4/NF-κB signaling

Catalpol is the main active ingredient of an extract from Radix rehmanniae,which in a previous study showed a protective effect against various types of tissue injury.However,a protective effect of catalpol on uterine inflammation has not been reported.In this study,to investigate the protective mechanism of catalpol on lipopolysaccharide(LPS)-induced bovine endometrial epithelial cells(bEECs)and mouse endometritis,in vitro and in vivo inflammation models were established.The Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qRT-PCR),western blot(WB),and immunofluorescence techniques.The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factorα(TNF-α),interleukin(IL)-1β,and IL-6,and chemokines such as C-X-C motif chemokine ligand 8(CXCL8)and CXCL5,both in bEECs and in uterine tissue.From the experimental results of WB,qRT-PCR,and immunofluorescence,the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group.The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase(MPO)activity.The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.

Ds-echinoside A,a new triterpene glycoside derived from sea cucumber,exhibits antimetastatic activity via the inhibition of NF-κB-dependent MMP-9 and VEGF expressions

Ds-echinoside A (DSEA),a non-sulfated triterpene glycoside,was isolated from the sea cucumber Pearsonothuria graeffei.In vitro and in vivo investigations were conducted on the effects of DSEA on tumor cell adhesion,migration,invasion,and angiogenesis.In this study,we found that DSEA inhibited the proliferation of human hepatocellular liver carcinoma cells Hep G2,with a half-maximal inhibitory concentration (IC50) of 2.65 μmol/L,and suppressed Hep G2 cell adhesion,migration,and invasion in a dose-dependent manner.DSEA also reduced tube formation of human endothelial cells ECV-304 on matrigel in vitro and attenuated neovascularization in the chick embryo chorioallantoic membrane (CAM) assay in vivo.Immunocytochemical analysis revealed that DSEA significantly decreased the expression of matrix metalloproteinase-9 (MMP-9),which plays an important role in the degradation of basement membrane in tumor metastasis and angiogenesis.DSEA also increased the protein expression level of tissue inhibitor of metalloproteinase-1 (TIMP-1),an important regulator of MMP-9 activation.From the results of Western blotting,the expressions of nuclear factor-kappa B (NF-κB) and vascular endothelial growth factor (VEGF) were found to be remarkably reduced by DSEA.These findings suggest that DSEA exhibits a significant antimetastatic activity through the specific inhibition of NF-κB-dependent MMP-9 and VEGF expressions.

Analysis of cardiovascular disease-related NF-κB-regulated genes and microRNAs in TNFα-treated primary mouse vascular endothelial cells

Activated nuclear factor-κB(NF-κB)plays an important role in the development of cardiovascular disease(CVD)through its regulated genes and microRNAs(miRNAs).However,the gene regulation profile remains unclear.In this study,primary mouse vascular endothelial cells(pMVECs)were employed to detect CVD-related NF-κB-regulated genes and miRNAs.Genechip assay identified 77 NF-κB-regulated genes,including 45 upregulated and 32 downregulated genes,in tumor necrosis factorα(TNFα)-treated pMVECs.Ten of these genes were also found to be regulated by NF-κB in TNFα-treated He La cells.Quantitative real-time PCR(q RT-PCR)assay confirmed the upregulation of Egr1,Tnf,and Btg2 by NF-κB in the TNFα-treated p MVECs.The functional annotation revealed that many NF-κB-regulated genes identified in pMVECs were clustered into classical NF-κB-involved biological processes.Genechip assay also identified 26 NF-κB-regulated miRNAs,of which 21 were upregulated and 5 downregulated,in the TNFα-treated pMVECs.Further analysis showed that nine of the identified genes are regulated by seven of these mi RNAs.Finally,among the identified NF-κB-regulated genes and miRNAs,5 genes and 12 miRNAs were associated with CVD by miRWalk and genetic association database analysis.Taken together,these findings show an intricate gene regulation network raised by NF-κB in TNFα-treated p MVECs.The network provides new insights for understanding the molecular mechanism underlying the progression of CVD.

EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-κB-dependent RAP1 transactivation

Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.

Effect of Palrnatine on lipopolysaccharide-induced acute lung injury by inhibiting activation of the Akt/NF-κB pathway

Inflammation plays an important role in the development of acute lung injury(ALI).Severe pulmonary inflammation can cause acute respiratory distress syndrome(ARDS)or even death.Expression of proinflammatory interleukin-1β(IL-1β)and inducible nitric oxide synthase(iNOS)in the process of pulmonary inflammation will further exacerbate the severity of ALI.The purpose of this study was to explore the effect of Palrnatine(Pa)on lipopolysaccharide(LPS)-induced mouse ALI and its underlying mechanism.Pa,a natural product,has a wide range of pharmacological activities with the potential to protect against lung injury.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo.Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus.We also used molecular modeling to further clarify the mechanism of action.The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1β,and significantly reduce the protein level of the proinflammatory protease iNOS,in both in vivo and in vitro models induced by LPS.Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B(Akt)/nuclear factor-κB(NF-κB)signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells.Through molecular dynamics simulation,we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt.These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

Inflammatory Mechanism of Total Flavonoids of Chrysanthemum and Medicated Serum on Castrated Dry Eye Animal and Cell Models

Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.

Therapeutic Effect and Mechanism of New Maixian Powder on DSS-induced UC Rats

[Objectives] To study the therapeutic effect and mechanism of New Maixian Powder on ulcerative colitis( UC) rats through observing its regulatory effect on the protein kinase R-like endoplasmic reticulum kinase( PERK)/eukaryotic translation initiation factor-2α( e IF-2α)/nuclear transcription factor-kappa B( NF-κB) signaling pathway. [Methods]First,60 SD rats were randomly divided into normal group,model group,mesalazine group,and New Maixian Powder low,medium and high dose groups,10 rats each group. Then,dextran sulfate sodium( DSS) was used to induce UC rats. The mesalazine group was given 0. 42 g/( kg·d) of mesalazine sustained-release granule suspension,New Maixian Powder low,medium and high dose groups were given 1. 5,3,and 6 g/( kg·d) of New Maixian Powder suspension,respectively,and other groups were given an equal volume of physiological saline,continuous intragastric administration for 14 d. Next,the disease activity index( DAI) of UC rats was evaluated; the expression of NF-κB in serum was measured by enzyme-linked immunosorbent assay( ELISA); the expression of PERK and e IF-2α protein and m RNA in colon tissue was detected by Western blot and real-time quantitative polymerase chain reaction( RT q-PCR). [Results] Compared with the normal group,the DAI score and serum NF-κB level in the model group were significantly higher( P < 0. 05),and PERK and e IF-2α protein and m RNA levels in the colon tissue were increased( P < 0. 05); compared with the model group,the DAI score decreased and serum NF-κB level declined in the New Maixian Powder group,and the expression of PERK and e IF-2α protein and m RNA in New Maixian Powder medium dose and high dose groups declined( P < 0. 05). [Conclusions]New Maixian Powder has good therapeutic effect on UC rats,and its mechanism may be connected with the inhibition of the activation of PERK/e IF-2α/NF-κB signaling pathway.