TLR3在人胎盘滋养层细胞系Bewo中的抗乙型肝炎病毒感染作用

目的研究乙型肝炎病毒(HBV)感染对人胎盘滋养层细胞系Bewo中TLR3mRNA表达的影响,观察人胎盘滋养层细胞系Bewo中TLR3抗HBV感染的生物学作用。方法培养人胎盘滋养层细胞Bewo,利用PolyI∶C刺激Bewo细胞TLR3mRNA的表达,同时设对照组,实时荧光聚合酶链反应(realtimeRT-PCR)检测细胞经PolyI∶C刺激后TLR3mRNA的表达变化;同时以高浓度HBVDNA阳性血清与两组Bewo细胞共培养,荧光定量PCR法检测细胞上清液中HBVDNA含量的变化。结果Bewo细胞经PolyI∶C刺激后TLR3mRNA水平高于对照组(t=7.90,P<0.01),差异有统计学意义;用HBVDNA阳性血清感染细胞时,培养上清中均可检出HBVDNA,PolyI∶C组上清中HBVDNA含量低于对照组(t=7.088,P<0.01),差异有统计学意义。结论PolyI∶C刺激可使Bewo细胞TLR3mRNA表达上调,与HBV共孵育后,培养上清中HBV病毒载量低于对照组,提示TLR3在人胎盘滋养层细胞系Bewo遭受HBV感染时发挥了一定的抗病毒作用。     

基于NF-κB/NLRP3/Caspase-1信号轴探究白藜芦醇对痛风性肾病模型大鼠肾脏的保护作用机制

目的 基于NF-κB/NLRP3/Caspase-1信号轴探究白藜芦醇对痛风性肾病模型大鼠的肾脏的保护作用机制。方法 将60只SD雄性大鼠随机分为对照组、模型组、秋水仙碱（阳性对照,0.03 mg · kg^-1）组和白藜芦醇高、中、低剂量（1 000、500、250 mg·kg^-1）组,连续7 d ig给药,给药过程中,除对照组外,其余各组使用氧嗪酸钾合并尿酸钠的方法制备大鼠痛风性肾病模型。ELISA法检测大鼠血清中白细胞介素（IL）-1β、IL-18、尿酸、肌酐（SCr）、尿素氮（BUN）水平,肾脏组织匀浆中肿瘤坏死因子-α（TNF-α）、单核细胞趋化蛋白-1（MCP-1）、环氧合酶-2（COX-2）水平;HE、Masson染色观察肾脏组织细胞形态变化;PAS染色检测大鼠肾组织中肾小球损伤情况,TUNEL观察肾脏组织细胞DNA损伤情况;实时荧光定量PCR （qRT-PCR）、免疫组化法检测肾脏组织中核因子-κB （NF-κB）、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、半胱氨酸蛋白酶-1（Caspase-1） mRNA以及蛋白的表达量,最后采用分子对接研究白藜芦醇与NF-κB、NLRP3、Caspase-1的结合情况。结果 与模型组比较,秋水仙碱组及白藜芦醇各给药组大鼠血清中IL-1β、IL-18、SCr、BUN及肾脏TNF-α、MCP-1、COX-2水平显著降低（P<0.01）,白藜芦醇高剂量组尿酸、中和高剂量组BUN显著降低（P<0.05）;白藜芦醇各剂量组不同程度降低肾组织中胶原纤维化面积、肾小球阳性率以及肾组织细胞TUNEL染色阳性率,减缓病理损伤情况,其中高剂量组作用最显著（P<0.05）;qRT-PCR、免疫组化结果表明,白藜芦醇各给药组均抑制肾脏组织细胞中NF-κB、NLRP3、Caspase-1mRNA和蛋白的表达,其中高剂量组作用最显著（P<0.05）;分子对接结果进一步表明,白藜芦醇与NF-κB、NLRP3、Caspase-1结合状态良好,即白藜芦醇对NF-κB、NLRP3、Caspase-1具有良好的靶向调控作用。结论 白藜芦醇对痛风性肾病模型大鼠的肾脏保护作用可能为抑制NF-κB信号通路,进而抑制NLRP3的激活从而阻断Caspase-1招募IL-1β、IL-18,降低其分泌,遏制肾脏细胞程序性死亡的初始阶段细胞焦亡的发生,从而逆转痛风性肾病大鼠肾组织的炎症损伤。     

Toll样受体4在褐藻多糖硫酸酯干预治疗大鼠肾间质纤维化中的作用

目的:探讨Toll样受体4(TLR4)在大鼠肾间质纤维化(RIF)中的表达情况及褐藻多糖硫酸酯(FPS)干预治疗对其调节作用。方法:将54只大鼠随机分为假手术组、模型组和药物组,每组18只,模型组和药物组制作单侧输尿管梗阻(UUO)模型,分别于术后2、4、6周处死大鼠,观察大鼠血肌酐(Scr)、尿素氮(BUN)及肾组织病理改变,并应用免疫组织化学方法检测肾组织TLR4及核因子(NF)-κB的表达。结果:假手术组大鼠肾组织TLR4及NF-κB无表达或仅有轻度表达;与假手术组相比,模型组肾组织表现为纤维化病理改变,同时TLR4及NF-κB强阳性表达(P<0.05),并随病程延长及纤维化的进展表达呈上升趋势;与模型组相比,药物组TLR4及NF-κB的表达减少(P<0.05),有效降低了Scr、BUN,减轻了肾间质纤维化程度。结论:褐藻多糖硫酸酯可能通过调节TLR4的表达而减缓肾纤维化病程进展。     

Inhibition of toll-like receptor-4, nuclear factor-kappaB and mitogen-activated protein kinase by lignocaine may involve voltage-sensitive sodium channels

We have shown previously that lignocaine inhibits the upregulation of inducible nitric oxide synthase (iNOS), a crucial factor that initiates the systemic inflammatory response during sepsis, possibly through voltage-sensitive sodium channels (VSSC). Toll-like receptor-4 (TLR-4), nuclear factor (NF)-kappaB and mitogen activated protein kinases (MAPKs) participate in the upstream regulation of iNOS expression induced by endotoxin. In the present study, we investigated the effects of lignocaine in the regulation of the expression of these enzymes. The role of VSSC in the effects of lignocaine was also investigated. Confluent murine macrophages (RAW264.7 cells) were randomized to receive lipopolysaccharide (LPS; 100 ng/mL), LPS + lignocaine (50 micromol/L), LPS + tetrodotoxin (TTX; 1 micromol/L; a VSSC inhibitor), LPS + lignocaine + veratridine (Ver; 50 micromol/L; a VSSC activator) or LPS + TTX + Ver. After reacting with LPS for 0, 15, 30, 45 and 60 min, cell cultures were harvested and enzyme expression was evaluated. We found that LPS significantly increased the concentrations of TLR-4, NF-kappaB and MAPKs, including extracellular regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAPK, in activated macrophages. Lignocaine and TTX significantly attenuated the effects of LPS on TLR-4, NF-kappaB, ERK and p38 MAPK expression, but not on JNK. Veratridine mitigated the effects of lignocaine and TTX. These data demonstrate that lignocaine has significant inhibitory effects on the activation of TLR-4, NF-kappaB and MAPKs in activated macrophages. Moreover, these effects involve VSSC.     

对羟基苯甲酸通过抑制NF-κB/caspase-1信号通路抗类风湿性关节炎的研究

目的 探讨天然酚酸类化合物对羟基苯甲酸（p-hydroxybenzoic acid,HA）对完全弗氏佐剂（complete Freund’s adjuvant,CFA）诱导的佐剂性关节炎（adjuvant arthritis,AA）模型的治疗作用,并对HA的作用机制进行初步分析。方法 除正常组外,向大鼠足跖皮内注射CFA 0.1 mL诱导AA大鼠模型。将AA模型大鼠随机分为模型组,HA低、中、高剂量组（2.5,5,10 mg·kg^-1）和阳性药物组（吲哚美辛,5 mg·kg^-1）,灌胃给予药物进行治疗干预,记录每组大鼠足肿胀体积、足肿胀厚度并进行关节肿胀评分;通过ELISA和qPCR检测不同剂量HA对炎症因子（TNF-α、IL-1β、IL-6）表达的影响;采用X-射线和HE染色观察足爪组织形态学和病理学变化;Western blotting检测caspase-1和NF-κB的表达。结果 HA能减轻AA大鼠关节肿胀（P<0.05）;从蛋白质和mRNA水平显著抑制炎症因子（TNF-α、IL-1β、IL-6）的产生（P<0.05）,并显著降低caspase-1和NF-κB蛋白表达水平;X-射线显示HA能减轻大鼠踝关节损伤,HE染色结果显示HA能显著抑制炎症细胞的浸润和软骨表层破坏等情况（P<0.05）,且这些结果均呈剂量依赖性。结论 HA可能通过抑制NF-κB/caspase-1信号通路缓解关节炎症状。     

Comparison of suppressive effects of demethoxycurcumin and bisdemethoxycurcumin on expressions of inflammatory mediators <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

Demethoxycurcumin and bisdemethoxycurcumin are the main active ingredients isolated from Curcumae Longae Radix. Recent studies demonstrated that both compounds exhibit antioxidative and anti-inflammatory effects as well as effects on cancer cell lines. In this study, we compared the activities of demethoxycurcumin and bisdemethoxycurcumin, and both compounds were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), cycloxygenase-2 (COX-2) and nuclear factor-kappaB (NF-κB) activity in a RAW 264.7 macrophage cell line. The evaluation:results suggested that the anti-inflammatory properties of demethoxycurcumin and bisdemethoxycurcumin were attributed to the inhibition of iNOS and COX-2 expression, as initiated by the inhibition of NF-κB activity. Additionally, both of them significantly inhibited carrageenan-induced paw edema in mice. Taken together, all of the results showed that the suppressive effect of demethoxycurcumin was stronger than that of bisdemethoxycurcumin, indicating that the methoxy group had enhanced demethoxycurcumin’s anti-inflammation effects.     

Cancer Cell Growth Inhibitory Effect of Bee Venom via Increase of Death Receptor 3 Expression and Inactivation of NF-kappa B in NSCLC Cells

Our previous findings have demonstrated that bee venom (BV) has anti-cancer activity in several cancer cells. However, the effects of BV on lung cancer cell growth have not been reported. Cell viability was determined with trypan blue uptake, soft agar formation as well as DAPI and TUNEL assay. Cell death related protein expression was determined with Western blotting. An EMSA was used for nuclear factor kappaB (NF-κB) activity assay. BV (1–5 μg/mL) inhibited growth of lung cancer cells by induction of apoptosis in a dose dependent manner in lung cancer cell lines A549 and NCI-H460. Consistent with apoptotic cell death, expression of DR3 and DR6 was significantly increased. However, deletion of DRs by small interfering RNA significantly reversed BV induced cell growth inhibitory effects. Expression of pro-apoptotic proteins (caspase-3 and Bax) was concomitantly increased, but the NF-κB activity and expression of Bcl-2 were inhibited. A combination treatment of tumor necrosis factor (TNF)-like weak inducer of apoptosis, TNF-related apoptosis-inducing ligand, docetaxel and cisplatin, with BV synergistically inhibited both A549 and NCI-H460 lung cancer cell growth with further down regulation of NF-κB activity. These results show that BV induces apoptotic cell death in lung cancer cells through the enhancement of DR3 expression and inhibition of NF-κB pathway.     

Up-regulation of BDNF in Astrocytes by TNF-α: A Case for the Neuroprotective Role of Cytokine

Tumor necrosis factor-alpha (TNF-α) is widely known to be involved in physiological and pathophysiological processes of the brain where this proinflammatory cytokine is implicated with regulation of inflammatory and survival components. We report that TNF-α up-regulates exon-IV-bdnf mRNA and brain-derived neurotrophic factor (BDNF) protein in primary astrocytes. The BDNF protein was detectable both in cellular lysate and in the extracellular medium. Activation of NF-κB by TNF-α and inhibition of TNF-α-induced BDNF expression by Δp65 (a dominant-negative mutant) and NEMO-binding domain peptide (an inhibitor of NF-κB) suggests that TNF-α induces BDNF expression through the activation of NF-κB. Similarly, TNF-α induced the activation of C/EBPβ and the expression of BDNF was sensitive to overexpression of ΔC/EBPβ (a dominant-negative mutant) and ETO (an inhibitor of C/EBPβ). Among three MAP kinases, TNF-α-induced BDNF up-regulation was sensitive only to inhibitors of ERK MAP kinase. However, the ERK MAP kinase pathway was coupled to activation of C/EBPβ but not NF-κB. Taken together, this study identifies a novel property of TNF-α in inducing the expression of BDNF via NF-κB and C/EBPβ in astrocytes that may be responsible for neurotrophic activity of the cytokine.     

香雪抗病毒口服液体外抗甲型流感病毒及炎症因子的作用机制研究

目的 探讨香雪抗病毒口服液对甲型流感病毒的活性及其在炎症免疫应答中的潜在作用机制。方法 采用噻唑蓝（MTT）染色法检测香雪抗病毒口服液对MDCK细胞和A549细胞的细胞毒性作用。采用细胞病变抑制法（CPE）及空斑减少实验对香雪抗病毒口服液进行抗病毒活性检测。采用免疫荧光法检测NP蛋白。实时荧光定量PCR测定炎症细胞因子的表达情况。采用蛋白印迹实验检测香雪抗病毒口服液对炎症相关信号通路的蛋白表达的影响。结果 香雪抗病毒口服液对甲型流感病毒A/PR/8/34(H1N1)和A/Aichi/2/68(H3N2)均有一定的抗病毒抑制作用,半数细胞毒性浓度（TC50）分别为42.12、79.36 mg/mL,半数抑制浓度（IC50）分别为8.665、5.260 mg/mL,并可减少H1N1病毒空斑形成。香雪抗病毒口服液能呈剂量相关性地降低H1N1诱导的A549细胞炎症因子肿瘤坏死因子-α(TNF-α)、视黄酸（维甲酸）诱导基因蛋白I(RIGI)、趋化因子（CC基序）配体5(CCL5)、巨噬细胞炎症蛋白-1β(MIP-1β)、干扰素诱导蛋白-10(IP-10)、白细胞介素-10(IL-10)、λ干扰...     

Inhibitory effects of clotrimazole on TNF-α-induced adhesion molecule expression and angiogenesis

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-α-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-α-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-α-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-κB activation, as CLT inhibited TNF-α-induced ROS generation as well as NF-κB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-α-induced monocyte adhesion was also mimicked by the specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-α have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-α-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-α-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-α-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.     

Suppression of phorbol ester-induced NF-kappaB activation by capsaicin in cultured human promyelocytic leukemia cells

Capsaicin, a major pungent constituent of red pepper (Capsicum annuum L.) possesses a vast variety of pharmacologic and physiologic activities. Despite its irritant properties, the compound exerts anti-inflammatory and anti-nociceptive effects. Previous studies from this laboratory revealed that capsaicin, when topically applied onto dorsal skin of female ICR mice, strongly attenuated activation of NF-kappaB and AP-1 induced by the typical tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), which may account for its anti-tumor promoting activity in mouse skin. In the present work, we have found that capsaicin suppresses TPA-stimulated activation of NF-kappaB through inhibition of IkappaB alpha degradation and blockade of subsequent nuclear translocation of p65 in human promyelocytic leukemia HL-60 cells. Methylation of the phenolic hydroxyl group of capsaicin abolished its inhibitory effect on NF-kappaB DNA binding. Likewise, TPA-induced activation of AP-1 was mitigated by capsaicin treatment.     

Suppression of hepatitis B virus-derived human hepatocellular carcinoma by NF-κB-inducing kinase-specific siRNA using liver-targeting liposomes

Hepatitis B virus triggers an increase of NF-κB inducing kinase (NIK)-dependent NF-κB activation, followed by the promotion of hepatocellular carcinoma (HCC). Here, we examined the inhibitory effect of NIK-specific siRNA on NF-κB signaling and HCC. The results of this study indicated that these siRNAs suppressed HBV-derived HCC by regulating NIK activation. To exert a protective effect from degradation enzyme, cationic liposomes were contrived and modified to contain β-sitosterol glucoside to target the asialoglycoprotein receptors in liver cancer cells. The cationic dimyristoyl diacyltrimethylammonium propane liposomes were prepared by a reverse-phase evaporation method with slight modification. β-Sitosterol glucoside was added to the lipid mixture at the beginning of the liposome preparation for the purpose of liver targeting. These liposomes assisted the delivery of the siRNA to specific cells and protected it from various lyases, followed by the ultimate suppression of HCC.     

Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON’s considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.     

CD200–CD200R Regulation of Microglia Activation in the Pathogenesis of Parkinson’s Disease

The role of CD200–CD200R signaling in immune regulation of the central nervous system has become a popular field of research in recent years. Many studies have shown that there is a close correlation between CD200–CD200R, microglia activation, and Parkinson’s disease (PD). This review discusses the above relationship, highlighting (1) the gene mapping and molecular structure of CD200 and CD200R, (2) the distribution and expression of CD200 and CD200R in the nervous system, (3) the effect of CD200–CD200R signaling on microglia activation, and (4) the role of microglia activation in the pathogenesis and progression of PD. Finally, we discuss the status of current studies on the regulation of microglia activation in PD and strongly suggest that it is very promising to regulate microglia activation in PD via targeting CD200–CD200R signaling pathways.     

胃复春胶囊通过NF-κB信号通路诱导胃癌细胞凋亡并抑制胃癌细胞转移作用

目的 研究胃复春胶囊对人胃癌细胞株MKN-49P的作用,为胃复春胶囊在抗消化道肿瘤方面的应用提供新的依据。方法 将SD大鼠随机分为2组,即对照组、胃复春胶囊组。胃复春胶囊组取胶囊内容物,以生理盐水混悬,按300 g/（kg·d）剂量ig给药,对照组大鼠ig 2 mL生理盐水,共5 d。末次给药后2 h,在无菌条件下,ip 10%水合氯醛（350 mg/kg）麻醉,腹主动脉取血,分离血清,经灭活处理后,置于—20℃冰箱保存备用。用不同质量浓度（0、2.5、5、10、20、30、50、80、100 μg/mL）的胃复春胶囊含药血清处理MKN-49P细胞,采用MTT法检测细胞活力。加入各质量浓度（25、50、100 μg/mL）胃复春胶囊含药血清,流式细胞术Annexin V/PI双染色检测细胞凋亡情况,Hoechst染色法检测细胞形态,细胞划痕实验测定细胞的迁移能力,Western blotting法检测信号传导与转录激活因子（STAT）蛋白、核转录因子-κB（NF-κB）信号通路及与细胞凋亡相关的蛋白表达情况。结果 胃复春胶囊含药血清能够剂量和时间相关地降低MKN-49P细胞的活力,并可诱导MKN-49P细胞凋亡。Hoechst染色显示细胞膜通透性增强;细胞划痕实验结果显示,与对照组（0 μg/mL）相比,不同浓度胃复春胶囊含药血清（25、50、100 μg/mL）可浓度相关性地抑制MKN-49P细胞的迁移。MKN-49P细胞经过25、50、100 μg/mL胃复春胶囊含药血清分别处理24 h后,MKN-49P细胞中STAT3的磷酸化减弱,NF-κB信号通路相关的蛋白表达量下调,抗凋亡蛋白表达量下降而促凋亡蛋白表达量增加。结论 胃复春胶囊含药血清能抑制胃癌细胞的增殖、诱导细胞凋亡,并显著降低细胞迁移能力,其作用机制推测与NF-κB信号通路有关。