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.

Isoflavone Attenuates the Nuclear Transcription Factor Kappa B (NF-<i>κ</i>B) Activation on MPP⁺-Induced Apoptosis of PC12 Cells

Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.

siRNA阻断NF-κB信号通路抑制胃癌SGC-7901细胞的增殖及侵袭

目的采用RNA干扰(RNA interference,RNAi)技术下调胃癌细胞株SGC-7901中NF-κB p65基因的表达,探讨其对肿瘤细胞增殖活性和侵袭能力的影响。方法利用阳离子脂质体LipofectamineTM2000将化学合成的人NF-κB p65的小干扰RNA(small interference RNA,siRNA)转染入胃癌细胞株SGC-7901中。采用RT-PCR法测定细胞内NF-κB p65mRNA的表达;酶联免疫吸附测定法(ELISA)检测NF-κB亚单位p65的DNA结合活性的改变;采用MTT法测定细胞增殖活性的变化;利用Transwell侵袭实验检测细胞体外侵袭能力的变化。结果与对照组比较,化学合成的人NF-κB p65siRNA能有效地抑制SGC-7901细胞中NF-κB p65mRNA的表达(P<0.05);RelAsiRNA组的p65亚单位与DNA结合活性明显低于对照组(P<0.05),并且RelA siRNA组中SGC-7901细胞的体外侵袭力减弱,增殖活性降低。结论特异的siRNA可以有效阻断NF-κB信号通路,影响人胃癌细胞的增殖活性和体外侵袭能力。

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。

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.

A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-κB were labled with DIG by terminal transferase After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film The results showed that nuclear proteins binded specifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-κB in PMA group was more than that in PMA+PDTC group It is suggested that detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories

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。

Nuclear Factor kappa B p65 Expression in Mouse Cochlea

Nuclear factor kappa B(NF-κB) is one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli,including inflammatory cytokines, phorbol esters, growth factors, and bacterial and viral products. The aim of this study is to demonstrate NF-κB expression in the mouse cochlea and its enhancement in response to lipopolysaccharides(LPS) and kanamycin(KA) treatment. Methods KA treatment consisted of subcutaneous KA injections at 700 mg/kg twice a day with an eight-hour interval between the two injections for 3 or 7 days. For animals in the LPS treatment group, a single dose of 0.3 mg LPS dissolved in 0.2 ml sterile saline were injected into both bullae through the tympanic membrane and kept there for 3 hours. Animals in the control group received subcutaneous saline injection for 7 days. Following immmunohistochemichal processing with rabbit polyclonal anti-NF-κB p65 antibodies, cryosections of the cochlea were examined for expression of NF-κB p65 in various structures in the cochlea. Results NF-κB p65 expression, identified by presence of brown reaction products characteristic of DAB immunohistochemistry, was visible in the spiral ligament, spiral prominence, tectorial membrane(TM), spiral ganglion and nerve fibers. Relatively weak NF-κB p65 expression was also visualized in the organ of Corti. Within the organ of Corti, the inner hair cells(IHC), outer hair cells(OHC), inner pillar cells(IP), outer pillar cells (OP), Deiter’s cells(DC), and Boettcher’s cells exhibited stronger staining than the inner sulcus cells, Hensen’s cells(HC) and Claudius’cells. No NF-κB p65 expression was seen in the nucleus of the IHC and OHC. NF-κB p65 expression was increased in animals exposed to LPS or KA, demonstrating significant differences in the staining between control animals and LPS/KA-treated animals. NF-κB p65 expression was not significantly different between LPS treated and KA treated animals or between 3 and 7 days in KA-treated animals. Conclusion LPS and KA exposure increases expression of NF-κB p65 in the mouse cochlea.

Lipopolysaccharide enhances the inhibition of NF-κB expression in NNK-mediated peritoneal macrophages

Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated primary mouse peritoneal macrophages in vitro. Methods： The activity of peritoneal rnacrophages treated with different concentrations of LPS was detected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK （100-500 μM）, with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry （ICC） and Western- blot, respectively. Results： The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages （P 〈 0.01）. Simultaneously, LPS （25 μg/mL） increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, especially when cotreated with LPS （25 μg/mL, P 〈 0.01 ）. Furthermore, NNK treatment （500 μM） with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB （P 〈 0.005）. Conclusion： LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.

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.

Albumin resuscitation protects against traumatic/hemorrhagic shock-induced lung apoptosis in rats

Objective: To determine the effects of albumin administration on lung injury and apoptosis in traumatic/hemorrhagic shock (T/HS) rats. Methods: Studies were performed on an in vivo model of spontaneously breathing rats with induced T/HS; the rats were subjected to femur fracture, ischemia for 30 min, and reperfusion for 20 min with Ringer's lactate solution (RS) or 5% (w/v) albumin (ALB), and the left lower lobes of the lungs were resected. Results: Albumin administered during reperfusion markedly attenuated injury of the lung and decreased the concentration of lactic acid and the number of in situ TdT-mediated dUTP nick-end labelling (TUNEL)-positive cells. Moreover, immunohistochemistry performed 24 h after reperfusion revealed increases in the level of nuclear factor κB (NF-κB), and phosphorylated p38 mitogen-activated protein kinase (MAPK) in the albumin-untreated group was down-regulated by albumin treatment when compared with the sham rats. Conclusion: Resuscitation with albumin attenuates tissue injury and inhibits T/HS-induced apoptosis in the lung via the p38 MAPK signal transduction pathway that functions to stimulate the activation of NF-κB.

Influence of catgut implantation at acupoints on splenic lymphocyte nuclear factor (NF-κB p65) and correlated signaling molecules (β2AR) in rats with experimental colitis

Objective To investigate the mechanisms of catgut implantation at acupoints on ulcerative colitis. Methods Eighteen SD rats were randomly divided into a normal control group （NC）, a model group （MO） and a catgut implantation group （CI） with 6 rats in each group. Animals in group MO and group CI were treated by trinitro-benzene-sulfonic acid （TNBS） to establish model with colitis. No other treatment was given to the rats in group MO, but catgut was implanted at ＂Shàngjùxū＂ （上 巨虚 ST 37）, ＂Tiānshū＂ （天枢 ST 25） and ＂Dàchángshū＂ （大肠俞 BL 25） in the rats in group CI. The symptoms of diarrhea and bloody stool, and changes in histopathology were detected 15 days after the treatment. Expressions of splenic lymphocyte nuclear factor κB p65（NF-κB p65）and correlated signaling molecules（β2AR）were detected by the western blot method. Results Diarrhea and mucus bloody purulent stool were soon controlled, and mucous injures were obviously improved in group CI. The NF-κB p65 value of splenic lymphocytes was signifi cantly increased （P0.01） and expression of β2AR remarkably reduced in group MO （P0.01）, compared with group NC. But, the NF-κB p65 value was significantly decreased （P0.01） and expression of β2AR remarkably increased in group CI （P 0.01） , compared with group MO. Conclusion Catgut implantation at acupoints is obviously effective in treating experimental colitis. Modulation of NF-κB p65 and the correlated signaling molecules β2AR may be involved in the mechanisms.

蠲痹历节清方对改良痛风性关节模型大鼠滑膜的TLR4,NF-κB,PPARγ的影响

目的：观察蠲痹历节清方对改良痛风性关节炎大鼠滑膜组织中Toll样受体4（Toll-like receptors 4,TLR4）,核转录因子kappa B（nuclear factor-kappa B,NF-κB）,过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptorγ,PPARγ）的影响,探讨蠲痹历节清方治疗急性痛风性关节炎局部组织炎症的可能作用机制。方法：将42只SD雄性大鼠,按随机数字表法选取6只为正常组,30只为造模组,造模组采用改良痛风性关节炎造模后随机分为模型组,蠲痹历节清方高、中、低剂量组（4 400,2 200,1 100 mg·kg^-1）,依托考昔组（11 mg·kg^-1）,吡格列酮组（20 mg·kg^-1）,每组6只。正常组及模型组的大鼠以生理盐水灌胃20 mL·kg^-1。每组每只大鼠每日灌胃给药2次,连续2 d后处死大鼠,取受试右踝关节,分离出滑膜组织,分为两部分,一部分用于病理形态学观察,一部分用逆转录聚合酶链式反应（RT-PCR）检测TLR4,NF-κB p65,PPARγmRNA的表达水平,蛋白质免疫印迹法（Western blot）检测TLR4,NF-κB p65,PPARγ蛋白表达水平。结果：与正常组比较,模型组中TLR4和NF-κB mRNA和蛋白表达显著升高（P〈0.01）,PPARγmRNA和蛋白表达升高（P〈0.05）;与模型组比较,吡格列酮组及蠲痹历节清方高、中、低剂量组TLR4和NF-κB mRNA和蛋白的表达显著降低（P〈0.01）,PPARγmRNA和蛋白的表达显著升高（P〈0.05）。结论：蠲痹历节清方可能通过上调PPARγ表达,抑制TLR4,NF-κB表达从而抑制急性痛风性关节炎局部组织炎症。

基于TLR4/NF-κB/NLRP3信号通路探讨脉络舒通丸保护小鼠脑缺血再灌注损伤的作用机制

目的:探讨脉络舒通丸对小鼠脑缺血再灌注损伤的保护作用及机制。方法:60只雄性ICR小鼠按体质量随机分为假手术对照组、模型对照组、尼莫地平0.024g/kg组和脉络舒通丸1.8、3.6、7.2 g/kg组,每组10只。各组灌胃给予相应药物或水预处理7 d,采用线栓法制备小鼠大脑中动脉阻塞(MCAO)模型。再灌注24 h后评估各组小鼠神经功能评分、脑含水量及脑梗死体积占比;HE染色观察缺血核心区脑组织病理学改变;ELISA法检测脑组织匀浆中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6含量;免疫印迹法分析小鼠缺血脑组织与半暗带组织中TLR4、p-NF-κB p65、NLRP3、ASC、cleaved Caspase-1、cleaved IL-1β蛋白表达。结果:与假手术对照组相比,模型对照组小鼠神经功能评分、脑含水量、脑梗死体积占比明显升高(P<0.05或P<0.01),HE染色显示脑组织产生损伤性病理改变,脑组织TNF-α、IL-1β及IL-6的含量均显著升高(P<0.01),缺血脑组织与半暗带组织中TLR4、p-NF-κB p65、NLRP3、ASC、cleaved Caspase-1、cleaved IL-1β蛋白表达均显著上调(P<0.01);与模型对照组相比,脉络舒通丸3.6、7.2 g/kg组神经功能评分、脑含水量、脑梗死体积占比明显减少(P<0.05或P<0.01);脉络舒通丸各组小鼠脑组织病理学均有显著改善,脑组织TNF-α、IL-1β及IL-6的含量明显降低(P<0.05或P<0.01);脉络舒通丸7.2 g/kg组小鼠缺血脑组织与半暗带组织中TLR4、p-NF-κB p65、NLRP3、ASC、cleaved Caspase-1、cleaved IL-1β蛋白表达显著下调(P<0.01)。结论:脉络舒通丸可通过调控TLR4/NF-κB/NLRP3信号通路,抑制神经炎症反应,从而达到对小鼠脑缺血再灌注损伤的保护作用。

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.

African Swine Fever Virus MGF360-12L Inhibits Type Ⅰ Interferon Production by Blocking the Interaction of Importin α and NF-κB Signaling Pathway

African swine fever(ASF)is an infectious transboundary disease of domestic pigs and wild boar and spreading throughout Eurasia.There is no vaccine and treatment available.Complex immune escape strategies of African swine fever virus(ASFV)are crucial factors affecting immune prevention and vaccine development.MGF360 genes have been implicated in the modulation of the IFN-Ⅰresponse.The molecular mechanisms contributing to innate immunity are poorly understood.In this study,we demonstrated that ASFV MGF360-12 L(MGF360 families 12 L protein)significantly inhibited the mRNA transcription and promoter activity of IFN-βand NF-κB,accompanied by decreases of IRF3,STING,TBK1,ISG54,ISG56 and AP-1 m RNA transcription.Also,MGF360-12 L might suppress the nuclear localization of p50 and p65 mediated by classical nuclear localization signal(NLS).Additionally,MGF360-12 L could interact with KPNA2,KPNA3,and KPNA4,which interrupted the interaction between p65 and KPNA2,KPNA3,KPNA4.We further found that MGF360-12 L could interfere with the NF-κB nuclear translocation by competitively inhibiting the interaction between NF-κB and nuclear transport proteins.These findings suggested that MGF360-12 L could inhibit the IFN-Ⅰproduction by blocking the interaction of importinαand NF-κB signaling pathway,which might reveal a novel strategy for ASFV to escape the host innate immune response.

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.