IL-6促进小鼠小胶质细胞系BV2坏死性凋亡

目的 探究在神经炎性反应中小胶质细胞凋亡的调控机制。方法 脂多糖(lipopolysaccharide, LPS)诱导小鼠小胶质细胞系BV2,构建神经炎性反应细胞模型,使用白介素-6(IL-6)拮抗剂塞妥昔单抗(siltuximab)处理LPS诱导的BV2细胞。CCK-8法检测细胞增殖;流式细胞测量术检测细胞凋亡;ELISA检测IL-6与TNF-α含量;RT-qPCR检测BV2细胞M1极化标志物IL-1β、IFN-γ与M2极化标志物CD206、Arg-1表达;Western blot检测JAK-STAT3信号通路关键蛋白及坏死性凋亡相关蛋白(RIP1)和RIP3表达。结果 LPS诱导后BV2细胞的增殖活力下降,凋亡增加,炎性因子IL-6与TNF-α含量增加(P<0.01)。M1极化标志物IL-1β、IFN-γ表达增加(P<0.01),M2极化标志物CD206、Arg-1表达减少(P<0.01)。JAK-STAT3通路关键蛋白磷酸化增加(P<0.01),RIP1、RIP3蛋白表达增加(P<0.01)。IL-6拮抗剂siltuximab处理细胞后,JAK-ST...     

CpG寡聚脱氧核苷酸(CpG ODN)协同促进脂多糖诱导的小鼠巨噬细胞增殖与迁移

目的探讨CpG寡聚脱氧核苷酸(CpG ODN)对脂多糖(LPS)诱导的巨噬细胞增殖与迁移能力的影响及机制。方法使用1 mg/L LPS处理小鼠RAW264.7巨噬细胞建立体外炎症细胞模型,采用CCK-8法检测CpG ODN(500 nmol/L)对LPS诱导的巨噬细胞增殖的影响,采用Transwell~(TM)实验检测CpG ODN对细胞迁移能力的影响;采用Western blot法检测p38丝裂原激活蛋白激酶(MAPK)、 c-Jun氨基末端激酶(JNK)、胞外信号调节激酶(ERK)、核因子κBp65(NF-κB p65)的蛋白磷酸化水平,同时使用以上通路的抑制剂SB203580、 SP600125、 PD98059、 BAY11-7082,探讨CpG ODN发挥效应的机制;采用实时定量PCR检测CpG ODN对LPS诱导产生的单核细胞趋化蛋白1(MCP-1)、环加氧酶2(COX2)mRNA水平的影响。结果 CpG ODN协同促进LPS诱导的巨噬细胞增殖与迁移,并促进COX2、 MCP-1的转录,增强JNK、 ERK信号通路蛋白的磷酸化水平,并且JNK与ERK信号通路激酶抑制剂可有效降低CpG ODN的协同效应。结论 CpG ODN可通过JNK与ERK途径协同促进LPS诱导的巨噬细胞增殖与迁移并促进COX2、 MCP-1的转录。     

巨噬细胞程序性坏死的研究进展

程序性坏死是具有独特信号通路并受信号分子调控的有序坏死。巨噬细胞的程序性坏死和其他细胞一样,为胱天蛋白酶(caspase)非依赖性,由受体相互作用蛋白激酶1 (RIP1)和RIP3介导形成死亡诱导信号复合体,进而被诱导产生。巨噬细胞的程序性坏死在多种炎症性疾病中作用显著,阐明巨噬细胞程序性坏死的调控机制及其在炎症相关性疾病发病中的作用,对研究炎症相关性疾病的发病机制及治疗策略具有非常重要的作用。我们主要总结了巨噬细胞程序性坏死的信号通路及其在感染性疾病、动脉粥样硬化、肿瘤和自身免疫性疾病中的作用。     

巨噬细胞程序性坏死的研究进展北大核心CSCD

程序性坏死是具有独特信号通路并受信号分子调控的有序坏死。巨噬细胞的程序性坏死和其他细胞一样,为胱天蛋白酶(caspase)非依赖性,由受体相互作用蛋白激酶1 (RIP1)和RIP3介导形成死亡诱导信号复合体,进而被诱导产生。巨噬细胞的程序性坏死在多种炎症性疾病中作用显著,阐明巨噬细胞程序性坏死的调控机制及其在炎症相关性疾病发病中的作用,对研究炎症相关性疾病的发病机制及治疗策略具有非常重要的作用。我们主要总结了巨噬细胞程序性坏死的信号通路及其在感染性疾病、动脉粥样硬化、肿瘤和自身免疫性疾病中的作用。     

TNF-α诱导MLO-Y4细胞发生RIP3介导的程序性坏死

目的:探讨肿瘤坏死因子α(TNF-α)能否诱导小鼠长骨骨样细胞株MLO-Y4发生程序性坏死及其发生机制。方法:将MLO-Y4细胞分为正常对照(control)组、TNF-α处理组、TNF-α+necrostatin-1(Nec-1)处理组、TNF-α+Z-VAD处理组和TNF-α+受体相互作用蛋白3(RIP3)-siRNA组。用流式细胞术检测各组细胞凋亡或坏死率,透射电镜鉴定细胞形态学变化,用Western blot法测定RIP1、RIP3和cleaved caspase-3的蛋白水平,应用激光共聚焦显微镜观察RIP1和RIP3蛋白的共表达,应用荧光标记法检测各组细胞活性氧(ROS)水平。结果:TNF-α诱导MLO-Y4细胞24 h,凋亡和坏死率明显高于control组(P0.01)。与TNF-α组相比,Nec-1、Z-VAD和RIP3-siRNA均能降低细胞的凋亡或坏死率(P0.01)。在TNF-α组可见大量坏死样细胞,在Z-VAD组仍可见到坏死样细胞,而在Nec-1和RIP3-siRNA组未见到坏死样细胞。Western blot实验结果显示Nec-1可有效抑制RIP1蛋白表达,而Z-VAD对RIP1和RIP3蛋白表达无影响,RIP3-siRNA可有效降低RIP3蛋白表达(P0.01)。与TNF-α组比较,Nec-1可有效降低RIP1-RIP3蛋白共表达阳性细胞百分率(P0.01),而Z-VAD对其无影响。与control组相比,TNF-α组的ROS水平明显增高(P0.01);与TNF-α组相比,Nec-1、Z-VAD及RIP3-siRNA均能有效抑制ROS水平(P0.01)。结论:TNF-α能诱导MLO-Y4细胞发生RIP3介导的程序性坏死;ROS可能是MLO-Y4细胞程序性坏死的执行者。     

去乙酰化酶抑制剂VPA 对巨噬细胞极化过程的影响

目的:本研究通过分析去乙酰化酶抑制剂对巨噬细胞极化过程中组蛋白修饰的影响,以及对巨噬细胞极化过程的影响,分析去乙酰化酶抑制剂是否可以通过改变巨噬细胞的组蛋白修饰,进而影响巨噬细胞极化的过程,旨在为治疗自身免疫性疾病提供新的思路。方法:利用脂多糖(Lipopolysaccharide,LPS)和干扰素γ(Interferon-γ,IFN-γ)诱导J774.1巨噬细胞24 h,白细胞介素4(Interleukin-4,IL-4)诱导J774.1巨噬细胞24 h,并在诱导过程中加入2 mmol/L丙戊酸(Valproic acid,VPA),收集巨噬细胞,实时免疫荧光定量PCR和ELISA法检测巨噬细胞极化的特异性标记基因的表达,流式细胞术和细胞免疫荧光法检测组蛋白修饰情况。结果:J774.1细胞经LPS和IFN-γ诱导24 h极化为M1型巨噬细胞;经IL-4刺激24 h极化为M2型巨噬细胞,VPA处理后的M1型巨噬细胞组蛋白H3K9的乙酰化程度升高,标记基因白细胞介素6(Interleukin-6,IL-6)、诱导性一氧化氮合酶(Inducible nitric oxide synthase,i NOS)、CC趋化因子配体2[Chemokine(C-C motif)ligand 2,CCL2]的表达量降低,CD86的表达量升高;VPA处理后的M2型巨噬细胞组蛋白H3K9的乙酰化程度也升高,标记基因精氨酸酶(Arginase-1,Arg-1)、Fizz-1(Found in inflammatory zone-1,Fizz-1)、甘露糖受体(CD206)、Ym1的表达量升高。结论:巨噬细胞的极化状态和组蛋白的修饰具有一定的相关性,VPA在M1型巨噬细胞诱导体系中可以促使M1型巨噬细胞向M2型巨噬细胞转化,但是在M2型巨噬细胞诱导体系中,却抑制了M1型巨噬细胞的特异性基因的表达。     

LPS诱导胰腺癌细胞Panc-1表达B7-H1

目的: 研究胰腺癌细胞株Panc-1在应用脂多糖(LPS)刺激前后B7-H1表达的变化,并探讨其可能的分子机制。方法: LPS刺激以及丝裂原活化蛋白激酶(MAPKs)的特异性抑制剂处理Panc-1细胞前后,应用Western blotting检测MAPKs信号通路中p38丝裂原活化蛋白激酶(p38)、细胞外信号调节蛋白激酶(ERK)和c-Jun氨基端激酶(JNK)磷酸化水平的变化,real-time PCR和Western blotting检测B7-H1 mRNA和蛋白的表达变化。结果: LPS刺激后B7-H1的表达显著上调,p38、ERK和JNK的磷酸化水平也明显上调,加入MAPKs特异性的抑制剂后,LPS诱导的p38、ERK和JNK的磷酸化被抑制,并且在p38和ERK的抑制剂处理后,B7-H1的表达也明显被抑制,而在JNK的抑制剂处理后B7-H1的表达没有显著变化。结论: LPS可以诱导胰腺癌细胞株Panc-1表达B7-H1,并且p38和ERK的活化在LPS诱导的B7-H1的表达过程中起着重要作用。     

洛美利嗪调控小鼠巨噬细胞极化的机制研究

目的:探究洛美利嗪对小鼠巨噬细胞M1型和M2型极化的调控作用及分子机制。方法:采用RTqPCR及Western blot实验检测洛美利嗪对小鼠骨髓来源的巨噬细胞和Raw 264.7小鼠巨噬细胞M1和M2型极化的影响;Western blot实验检测洛美利嗪对调控巨噬细胞极化的丝裂原活化蛋白激酶(MAPK)、核因子κB(NF-κB)和信号转导及转录激活因子6(STAT6)信号通路的影响。结果:洛美利嗪能够显著抑制脂多糖(LPS)诱导的M1型巨噬细胞炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和IL-1β的产生(P0.01),且剂量依赖性地降低M1型极化标志物诱导型一氧化氮合酶(iNOS)的表达(P0.05),同时促进IL-4诱导的M2型巨噬细胞标志物几丁质酶3样蛋白3(CHI3L3/Ym-1)、Fizz-1(found in inflammatory zone-1)和精氨酸酶1(Arg-1)的mRNA和蛋白表达(P0.05)。洛美利嗪能够剂量依赖性地抑制MAPK信号通路中p38 MAPK、细胞外信号调节激酶1/2(ERK1/2)和c-Jun氨基末端激酶1/2(JNK1/2)的磷酸化以及NF-κB信号通路中NF-κB p65的磷酸化,并促进核因子κB抑制因子α(IκBα)的表达(P0.05),进而介导巨噬细胞极化。结论:洛美利嗪可以通过调控MAPK和NF-κB信号通路进而剂量依赖性地抑制小鼠巨噬细胞M1型极化,同时洛美利嗪还可以显著促进M2型极化。     

PPARγ配体4i通过抑制NF-κB和MAPK信号通路抑制小鼠巨噬细胞炎性细胞因子的产生

目的探讨新型过氧化物酶体增殖物激活受体γ(PPARγ)配体4i的体外抗炎作用及机制。方法取对数生长期RAW264. 7小鼠腹腔巨噬细胞,经100 ng/m L脂多糖(LPS)诱导,采用ELISA检测10μmol/L 4i对巨噬细胞肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)分泌的影响;采用Western blot法检测4i对核因子κBp65(NF-κBp65)、核因子κB抑制蛋白α(IκBα)、c-Jun氨基端激酶(JNK)、胞外信号调节激酶1/2(ERK1/2)、p38丝裂原激活蛋白激酶(p38MAPK)活性的影响,加入不可逆的PPARγ拮抗剂GW9662(5μmol/L)探讨PPARγ在NF-κB和MAPK相关蛋白表达中的作用;采用SYBYL 8. 1软件进行分子对接分析探讨4i与PPARγ蛋白的结合特性。结果 4i显著抑制TNF-α和IL-6的产生呈时间依赖性;不同程度抑制NF-κBp65、IκBα、JNK、ERK1/2和p38MAPK蛋白的磷酸化水平,且抑制作用被GW9662所逆转; 4i能与PPARγ受体较好地结合。结论4i通过激活PPARγ抑制NF-κB和MAPK信号通路相关蛋白的活化,抑制TNF-α、IL-6等的产生,发挥抗炎作用。     

甘草酸通过JAK/STAT1通路抑制IFN-γ诱导HaCaT细胞CXCL10的产生

目的研究甘草酸(GL)对IFN-γ诱导HaCaT细胞CXC趋化因子配体10(CXCL10)表达的影响。方法体外培养HaCaT细胞,以10 ng/m Lγ干扰素(IFN-γ)刺激HaCaT细胞,分别用0. 5 mmol/L GL、15 nmol/L CYT387及二者联合处理细胞。采用噻唑蓝(MTT)法检测细胞活力,实时荧光定量PCR检测CXCL10 mRNA水平,ELISA检测CXCL10蛋白水平,Western blot法检测Janus激酶1(JAK1)、JAK2、信号转导子与转录激活子1(STAT1)的磷酸化水平。结果 GL抑制IFN-γ对HaCaT细胞的损伤,且对HaCaT细胞活力无明显影响。GL与JAK1/2抑制剂CYT387均可下调IFN-γ诱导HaCaT细胞的CXCL10表达;且与CYT387处理相比,GL及联合CYT387处理显著下调CXCL10 mRNA表达。GL下调JAK/STAT1信号通路中JAK1、JAK2以及STAT1蛋白磷酸化水平。结论推测GL可能通过阻断IFN-γ介导的JAK/STAT1信号活化,从而抑制IFN-γ诱导HaCaT细胞分泌CXCL10。     

Necrostatin-1 Protects Against <Emphasis Type="SmallCaps">d</Emphasis>-Galactosamine and Lipopolysaccharide-Induced Hepatic Injury by Preventing TLR4 and RAGE Signaling

Fulminant hepatic failure (FHF) is a life-threatening clinical syndrome results in massive inflammation and hepatocyte death. Necroptosis is a regulated form of necrotic cell death that is emerging as a crucial control point for inflammatory diseases. The kinases receptor interacting protein (RIP) 1 and RIP3 are known as key modulators of necroptosis. In this study, we investigated the impact of necroptosis in the pathogenesis of FHF and molecular mechanisms, particularly its linkage to damage-associated molecular pattern (DAMP)-mediated pattern recognition receptor (PRR) signaling pathways. Male C57BL/6 mice were given an intraperitoneal injection of necrostatin-1 (Nec-1, RIP1 inhibitor; 1.8 mg/kg; dissolved in 2% dimethyl sulfoxide in phosphate-buffered saline) 1 h before receiving d-galactosamine (GalN; 800 mg/kg)/lipopolysaccharide (LPS; 40 μg/kg). Hepatic RIP1, RIP3 protein expression, their phosphorylation, and RIP1/RIP3 complex formation upregulated in the GalN/LPS group were attenuated by Nec-1. Nec-1 markedly reduced the increases in mortality and serum alanine aminotransferase activity induced by GalN/LPS. Increased serum high mobility group box 1 (HMGB1) and interleukin (IL)-33 release, HMGB1-toll-like receptor 4 and HMGB1-receptor for advanced glycation end products (RAGE) interaction, and nuclear protein expressions of NF-κB and early growth response protein-1 (egr-1) were attenuated by Nec-1. Our finding suggests that necroptosis is responsible for GalN/LPS-induced liver injury through DAMP-activated PRR signaling.     

RSK3 mediates necroptosis by regulating phosphorylation of RIP3 in rat retinal ganglion cells

Receptor-interacting protein 3 (RIP3) plays an important role in the necroptosis signaling pathway. Our previous studies have shown that the RIP3/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis occurs in retinal ganglion cell line 5 (RGC-5) following oxygen-glucose deprivation (OGD). However, upstream regulatory pathways of RIP3 are yet to be uncovered. The purpose of the present study was to investigate the role of p90 ribosomal protein S6 kinase 3 (RSK3) in the phosphorylation of RIP3 in RGC-5 cell necroptosis following OGD. Our results showed that expression of RSK3, RIP3, and MLKL was upregulated in necroptosis of RGC-5 after OGD. A computer simulation based on our preliminary results indicated that RSK3 might interact with RIP3, which was subsequently confirmed by co-immunoprecipitation. Further, we found that the application of a specific RSK inhibitor, LJH685, or rsk3 small interfering RNA (siRNA), downregulated the phosphorylation of RIP3. However, the overexpression of rip3 did not affect the expression of RSK3, thereby indicating that RSK3 could be a possible upstream regulator of RIP3 phosphorylation in OGD-induced necroptosis of RGC-5 cells. Moreover, our in vivo results showed that pretreatment with LJH685 before acute high intraocular pressure episodes could reduce the necroptosis of retinal neurons and improve recovery of impaired visual function. Taken together, our findings suggested that RSK3 might work as an upstream regulator of RIP3 phosphorylation during RGC-5 necroptosis.     

The tobacco smoke component, acrolein, suppresses innate macrophage responses by direct alkylation of c-Jun N-terminal kinase

The respiratory innate immune system is often compromised by tobacco smoke exposure, and previous studies have indicated that acrolein, a reactive electrophile in tobacco smoke, may contribute to the immunosuppressive effects of smoking. Exposure of mice to acrolein at concentrations similar to those in cigarette smoke (5 ppm, 4 h) significantly suppressed alveolar macrophage responses to bacterial LPS, indicated by reduced induction of nitric oxide synthase 2, TNF-α, and IL-12p40. Mechanistic studies with bone marrow-derived macrophages or MH-S macrophages demonstrated that acrolein (1-30 μM) attenuated these LPS-mediated innate responses in association with depletion of cellular glutathione, although glutathione depletion itself was not fully responsible for these immunosuppressive effects. Inhibitory actions of acrolein were most prominent after acute exposure (<2 h), indicating the involvement of direct and reversible interactions of acrolein with critical signaling pathways. Among the key signaling pathways involved in innate macrophage responses, acrolein marginally affected LPS-mediated activation of nuclear factor (NF)-κB, and significantly suppressed phosphorylation of c-Jun N-terminal kinase (JNK) and activation of c-Jun. Using biotin hydrazide labeling, NF-κB RelA and p50, as well as JNK2, a critical mediator of innate macrophage responses, were revealed as direct targets for alkylation by acrolein. Mass spectrometry analysis of acrolein-modified recombinant JNK2 indicated adduction to Cys(41) and Cys(177), putative important sites involved in mitogen-activated protein kinase (MAPK) kinase (MEK) binding and JNK2 phosphorylation. Our findings indicate that direct alkylation of JNK2 by electrophiles, such as acrolein, may be a prominent and hitherto unrecognized mechanism in their immunosuppressive effects, and may be a major factor in smoking-induced effects on the immune system.     

Mycophenolic acid enhanced lipopolysaccharide-induced interleukin-18 release in THP-1 cells via activation of the NLRP3 inflammasome

Abstract

Background: Interleukin (IL)-18 is a pro-inflammatory cytokine that has important functions in host defense. The maturation and secretion of IL-18 has been shown to be regulated by the NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome. Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), in association with lipopolysaccharide (LPS), is able to promote the secretion of IL-18, but the mechanism remains unknown. This study aims to explore the mechanism by which MPA synergizes with LPS to induced IL-18 release.

Methods: THP-1 cells were stimulated with LPS and MPA and treated with or without the inhibitors of caspase-1, Ac-YVAD-cmk or KCl; IL-18 in the supernatants was measured by ELISA. The intracellular protein levels of NF-κB p-p65, pro-IL-18, NLRP3, and cleaved caspase-1(p20) were measured by Western blot.

Results: We found that MPA alone failed to induce IL-18, whereas MPA enhanced LPS-mediated IL-18 release. MPA did not affect the intracellular protein levels of NF-κB p-p65 or pro-IL-18 but activated the NLRP3 inflammasome. Ac-YVAD-cmk or increasing extracellular K⁺ blocked the activation of caspase-1 and attenuated the release of IL-18.

Conclusions: Taken together, MPA synergized with LPS to induce the release of IL-18 via activating the NLRP3 inflammasome and increasing the degradation of pro-IL-18, rather than by enhancing the production of pro-IL-18.     

Toll-like receptor 4 mediates cross-talk between peroxisome proliferator-activated receptor gamma and nuclear factor-kappaB in macrophages

The peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in macrophages and plays an important role in suppressing the inflammatory response. Lipopolysaccharides (LPS), which activate Toll-like receptor 4 (TLR4), reduced PPARgamma expression and function in peritoneal macrophages and macrophage cell lines. Moreover, pretreatment with the synthetic PPARgamma ligand, rosiglitazone did not prevent LPS-mediated downregulation of PPARgamma. Inhibition of PPARgamma expression was not blocked by cycloheximide, indicating that de novo protein synthesis is not required for LPS-mediated suppression of PPARgamma. Destabilization of PPARgamma messenger RNA (mRNA) was not observed in LPS-stimulated macrophages, suggesting that LPS regulates the synthesis of PPARgamma mRNA. LPS had no effect on PPARgamma expression in macrophages from TLR4 knockout mice, whereas LPS inhibited PPARgamma expression in cells that had been reconstituted to express functional TLR4. Targeting the TLR4 pathway with inhibitors of MEK1/2, p38, JNK and AP-1 had no effect on PPARgamma downregulation by LPS. However, inhibitors that target NEMO, IkappaB and NF-kappaB abolished LPS-mediated downregulation of PPARgamma in LPS-stimulated macrophages. Our data indicate that activation of TLR4 inhibits PPARgamma mRNA synthesis by an NF-kappaB-dependent mechanism. Low-density genomic profiling of macrophage-specific PPARgamma knockout cells indicated that PPARgamma suppresses inflammation under basal conditions, and that loss of PPARgamma expression is sufficient to induce a proinflammatory state. Our data reveal a regulatory feedback loop in which PPARgamma represses NF-kappaB-mediated inflammatory signalling in unstimulated macrophages; however, upon activation of TLR4, NF-kappaB drives down PPARgamma expression and thereby obviates any potential anti-inflammatory effects of PPARgamma in LPS-stimulated macrophages.     

LPS resistance in monocytic cells caused by reverse signaling through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway

The transmembrane form of tumor necrosis factor (mTNF), expressed on activated monocytes (MO) and macrophages (MPhi), is able to induce apoptosis in human endothelial cells (EC). Apoptosis is mediated by two distinct mechanisms: direct cell contact and a yet-unidentified soluble protein, death factor X. In addition, mTNF acts as a receptor that transduces a "reverse signal" into MO/MPhi when bound to the TNF receptor on EC. Reverse signaling by mTNF confers resistance to bacterial lipopolysaccharide (LPS). Stimulation of reverse signaling by mTNF blocks the ability of MO/MPhi to produce death factor X and proinflammatory cytokines. We have investigated which signaling pathways are used by mTNF acting as receptor. Reverse signaling triggers two independent pathways that can be distinguished by protein kinase C (PKC) inhibitors. The suppression of LPS-induced death factor X is dependent on PKC, whereas the suppression of LPS-mediated cytokine release is not. LPS and reverse signaling stimulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. It is interesting that the activation of reverse signaling by mTNF renders MO/MPhi refractory to a subsequent activation of the MAPK/ERK pathway by LPS. Thus, reverse signaling achieves LPS resistance in monocytic cells through interference with key signal-transduction pathways.     

Inhibition of caspase 3 abrogates lipopolysaccharide-induced nitric oxide production by preventing activation of NF-kappaB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-kappaB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-kappaB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-kappaB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.     

Manipulation of necroptosis by Porphyromonas gingivalis in periodontitis development

To eliminate invading pathogens and keep homeostasis, host employs multiple approaches such as the non-inflammation associated-apoptosis, inflammation associated-necroptosis and pyroptosis, etc. Necroptosis is known as a highly pro-inflammatory form of cell death due to the release of massive damage-associated molecular patterns (DAMPs). For the first time, we reported that Porphyromonas gingivalis induced cellular necroptosis through receptor-interacting protein 1 (RIP1)/RIP3/mixed lineage kinase domain-like (MLKL) signaling pathway in monocytes. Necroptosis in THP-1 cells was induced by MLKL phosphorylation in vitro. P. gingivalis treated-THP-1 cells exhibited lower cell death rate with pretreatment of inhibitors RIP1 and MLKL, accompanied with attenuated TNF-α and IL-6 expressions. Moreover, the necroptosis risk was also reduced via gene silencing by RIP3 or MLKL in the P. gingivalis treated-THP-1 cell lines. We further explored P. gingivalis-induced necroptosis in animal models in vivo. Firstly, C57BL/6 mice were injected with P. gingivalis in the subcutaneous chamber model. Animals pretreated with MLKL inhibitor exhibited significantly enhanced P. gingivalis clearance; in addition, levels of TNF-α and IL-6 were notably decreased by 60% via MLKL inhibition. Secondly, P. gingivalis-induced periodontitis was utilized to investigate necroptosis related-periodontopathogensis. Positive staining of phosphorylated MLKL in mice periodontitis biopsies was detected to a higher degree, while larger amount of alveolar bone loss was observed in MLKL (−) group comparing to those in the MLKL (+) group. These findings may suggest that P. gingivalis play essential roles in necroptosis process during periodontitis, and our research may shed light on the further work on the related periodontopathogenesis investigation.