Apigenin exhibits anti-inflammatory effects in LPS-stimulated BV2 microglia through activating GSK3β/Nrf2 signaling pathway

Abstract

Objective: Apigenin is a natural flavonoid compound extracted from Matricaria chamomilla. We evaluated the anti-inflammatory effects of apigenin in this study using the Lipopolysaccharide (LPS)-stimulated BV2 microglia.

Methods: BV2 cells were treated with apigenin for 1?h and then treated with LPS. The inflammatory cytokine productions were tested by qRT-PCR and ELISA. The expression of GSK3β, Nrf2, and NF-κB signaling pathways were measured by western blot analysis.

Results: Apigenin significantly attenuated LPS-induced TNF-α, IL-1β, and IL-6 production. Apigenin suppressed LPS-induced NF-κB activation. Furthermore, GSK3β, Nrf2, and HO-1 were concentration-dependently increased by apigenin. The suppression of apigenin on LPS-induced inflammatory response and NF-κB activation were prevented when Nrf2 was knocked out or by GSK3β inhibitor.

Conclusions: Collectively, apigenin suppressed LPS-induced microglia activation via activating GSK3β/Nrf2 signaling pathway.     

脂多糖促进大鼠肝星状细胞系HSC-T6细胞自噬

目的研究脂多糖(LPS)对大鼠肝星状细胞系HSC-T6自噬的影响及NF-κB通路在其中的作用。方法 1)常规培养的HSC-T6细胞以不同质量浓度(0、0.01、0.1、1和10 mg/L)的LPS分别处理不同时间(0、3、6、12、18和24 h)后,Western blot检测细胞内微管相关蛋白轻链Ⅱ(LC3Ⅱ)及Beclin1含量;2)常规培养的HSC-T6细胞随机分为对照组、LPS组、PDTC组、LPS+PDTC组、PMA组和LPS+PMA组,各组经相应处理后,Western blot检测各组LC3Ⅱ及Beclin1含量;免疫荧光法观察NF-κB P65细胞内分布情况;比色法检测各组细胞培养上清羟脯氨酸(Hyp)含量;ELISA检测细胞培养上清层黏蛋白(LN)及透明质酸(HA)含量。结果经LPS处理后HSC-T6细胞中LC3Ⅱ及Beclin1含量增加且在LPS质量浓度为0.1 mg/L作用6 h达到峰值,同时细胞培养上清中肝纤维化指标Hyp、LN和HA含量明显增加(P0.05);经PDTC预处理后,LPS刺激的HSC-T6中LC3Ⅱ及Beclin1含量增加,上清中Hyp、LN和HA含量明显降低(P0.05);而经PMA预处理则LC3Ⅱ、Beclin1含量降低而Hyp、LN和HA含量增加(P0.05)。结论 LPS可促进HSC-T6细胞自噬及NF-κB通路的活化,NF-κB的活化可能抑制HSC-T6细胞自噬。     

黄芪总皂苷对脂多糖诱导的BV2小胶质细胞的抗炎机制

目的:探讨黄芪总皂苷(TAS)对脂多糖(LPS)诱导的BV2小胶质细胞炎症损伤的抗炎作用机制.方法:用CCK-8法筛选出对细胞活力无抑制的药物浓度;用浓度为1 mg/L的LPS刺激BV2细胞24 h,建立细胞炎症模型;实验分为正常组、LPS组、高剂量(75 mg/L)TAS组和低剂量(50 mg/L)TAS组;应用流式...     

p21WAF1 modulates NF-kappaB signaling and induces anti-apoptotic protein Bcl-2 in Tax-expressing rat fibroblast

Of the cell cycle-associated genes regulated by human T-cell leukemia virus type-1 (HTLV-1) Tax, cyclin-dependent kinase (CDK) inhibitor p21WAF1 is upregulated in HTLV-1-infected cells. Previously, we reported that p21WAF1 stimulated Tax-dependent NF-kappaB activation which influences a variety of cellular processes, including proliferation, differentiation, and apoptosis. In HTLV-1-infected cells, Tax is primarily involved in the constitutive activation of NF-kappaB signaling. Here, we demonstrate that p21WAF1 affects Tax-dependent NF-kappaB signaling by inducing p100/52, an NF-kappaB-related protein. W4, a Tax-transformed rat fibroblast cell line, exhibits the constitutive activation of NF-kappaB signaling, potentially mediated by overexpression of RelB. Ectopic expression of p21WAF1 in W4 cells, which lack endogenous expression due to methylation of the p21WAF1 promoter, induces the expression of p100/52. Bcl-2 expression was also upregulated by ectopic p21WAF1 in this cell line, suggesting that p21WAF1 plays an important role in the regulation of apoptosis by modulating NF-kappaB signaling in Tax-expressing rat fibroblasts. We also address the expression of NF-kappaB-related proteins in HTLV-1-infected cells.     

E3泛素连接酶RNF121对脂多糖诱导的巨噬细胞表达促炎性细胞因子的影响

目的探讨E3泛素连接酶RNF121对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞表达促炎性细胞因子的调控作用。方法 LPS刺激小鼠腹腔巨噬细胞,Western blot检测RNF121的表达。用RNA干扰(si-RNF121)降低RNF121的表达,小鼠腹腔巨噬细胞经LPS刺激后,实时荧光定量PCR法检测TNF-α和IL-6的mRNA水平,ELISA检测细胞培养上清中TNF-α和IL-6的浓度,Western blot检测小鼠腹腔巨噬细胞中P65及磷酸化P65(p-P65)的表达水平,利用双荧光素酶报告基因法检测NF-κB的活性。结果 LPS诱导小鼠腹腔巨噬细胞后,RNF121的蛋白水平显著降低(P0.05),蛋白酶抑制剂MG132能够显著增加RNF121的蛋白水平。si-RNF121降低RNF121的表达后,给予LPS刺激小鼠腹腔巨噬细胞,IL-6和TNF-α的表达水平均显著下降(P0.05),转录因子P65的磷酸化(p-P65)水平及NF-κB的活性均显著降低(P0.05)。结论 E3泛素连接酶RNF121通过调控NF-κB的活性从而影响小鼠巨噬细胞促炎性细胞因子TNF-α及IL-6的表达。     

Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.     

Neurotrophic factors increase tumor necrosis factor-alpha-induced nuclear translocation of NF-kappaB in rat PC12 cells

Neurotrophic factors regulate neuronal survival and differentiation and control neurite outgrowth by binding to tyrosine kinase receptors, the Trks, and a tumor necrosis factor (TNF) receptor-like molecule, p75 neurotrophin receptor. A proinflammatory cytokine, TNF, also affects survival and apoptotic death in neuronal cells. However, it is still unclear whether neurotrophic factors and TNF co-operate the intracellular signaling. Using green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1), we examined here the effects of TNF-alpha and neurotrophic factors on the nuclear translocation of NF-kappaB in PC12 cells. TNF-alpha induced gradually the translocation of GFP-NF-kappaB1 from the cytoplasm to the nucleus within 60 min. Pretreatment of lactacystin which is a proteasome-specific inhibitor suppressed significantly the nuclear translocation of GFP-NF-kappaB1 after TNF-alpha stimulation. In addition, we found that co-stimulation of TNF-alpha and neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) increased greatly the nuclear translocation of GFP-NF-kappaB1 whereas neither NGF nor BDNF itself induced the translocation. These results suggested that there is a close correlation between the signaling pathways via TNF receptors and neurotrophin receptors for the NF-kappaB activation, and that NGF and BDNF enhance TNF-alpha-induced nuclear translocation of NF-kappaB.     

Repeated administration of mirtazapine inhibits development of hyperalgesia/allodynia and activation of NF-kappaB in a rat model of neuropathic pain

Antidepressants have been widely used to treat neuropathic pain for many years. However, the mechanisms of their analgesic actions are little known and remain controvertible. Recent studies indicate that cytokines in central nervous system (CNS) play a critical role in the pathological states of pain. The present study was designed to explore the effects and most appropriate dosage of mirtazapine in treating neuropathic pain and its possible neuroimmune mechanisms. L5 spinal nerve transection was done to produce hyperalgesia in rats. Mirtazapine (10, 20 and 30 mg/kg, respectively) was orally administered daily for 14 days, beginning from the 5th day after nerve transection. Mechanical and thermal hyperalgesia was measured using Von-Frey filament and Hargreaves tests before and after the surgery. Rats were then sacrificed on days 3, 7, 14, 21 post-administration. The inflammatory cytokines production such as TNFalpha, IL-1beta, IL-10 and nuclear factor kappa B (NF-kappaB) activity in brain was quantified using enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assay (EMSA). We found that mirtazapine (20 and 30 mg/kg) can markedly attenuate mechanical and thermal hyperalgesia produced by nerve transection, most significantly on the 14th day. The elevated TNFalpha, IL-1beta and NF-kappaB in brain were accordingly reduced, while the expression of increased IL-10 were even stimulated after repeated mirtazapine administration. Our data could conclude that mirtazapine suppressed neuropathic pain partially through inhibiting cerebral proinflammatory cytokines production and NF-kappaB activation in CNS.     

Cyclooxygenase-2 is over-expressed in Chinese esophageal squamous cell carcinoma, and correlated with NF-kappaB: an immunohistochemical study

BACKGROUND: Cyclooxygenase-2 (COX-2), the inducible isoform of the key enzyme in the synthesis of prostaglandins, has been found to be over-expressed in several human cancers. The aim of the present study was to investigate immunohistochemical expression of COX-2 in esophageal squamous cell carcinoma (SCC) and relationship with clinicopathological parameters and NF-kappaB. METHODS: Expression of COX-2 and NF-kappaB was investigated in 69 cases of esophageal SCC by immunohistochemistry and the correlation of COX-2 expression with clinicopathological features and NF-kappaB staining was examined. RESULTS: Thirty-one esophageal SCC (31/69, 44.9%) had positive expression of COX-2. COX-2 was expressed significantly higher in well-differentiated tumors (16/23, 69.6%) than that in moderate (13/34, 38.2%) and poor (2/12, 16.7%) differentiation (P = 0.034). COX-2 expression was increasingly progressive with the advance of the clinical stages significantly (P = 0.045). The correlation between COX-2 (47/99, 47.5%) and NF-kappaB/p50 (54/99, 54.5%) immunostaining was statistically significant (P = 0.030). CONCLUSIONS: COX-2 is over-expressed in esophageal SCC, especially in a well differentiation, correlated with tumor progression, and possibly regulated by NF-kappaB.     

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

SB203580 enhances interleukin-1 receptor antagonist gene expression in IFN-gamma-stimulated BV2 microglial cells through a composite nuclear factor-kappaB/PU.1 binding site

Interleukin-1 receptor antagonist (IL-1ra) is a naturally occurring antagonist of IL-1alpha and IL-1beta binding to the IL-1 receptors and alleviates various inflammatory reactions. Therefore, the upregulation of IL-1ra expression is important for preventing and/or treating inflammatory diseases including many neurodegenerative diseases. This study found that SB203580, which is generally known as a p38 MAP kinase inhibitor and an anti-inflammatory agent, increased the level of IL-1ra expression in IFN-gamma-stimulated BV2 microglial cells. This effect is believed to occur through the inhibition of protein kinase B (PKB), independently of the p38 MAP kinase pathways. Further mechanistic studies using an IL-1ra promoter revealed that a composite NF-kappaB/PU.1 binding site plays an important role in this SB203580-mediated upregulation of IL-1ra. Considering that IFN-gamma is a major stimulator of the innate and adaptive immune responses, the upregulation of anti-inflammatory IL-1ra expression by SB203580 in the IFN-gamma-stimulated microglia might provide a new therapeutic modality for various inflammatory diseases of the central nervous system.     

肌肽对糖尿病肾病大鼠氧化应激及NF-κB信号通路的影响

目的　探讨肌肽对糖尿病肾病（DN）大鼠肾组织的保护作用及其对氧化应激、NF-κB信号通路的影响。 方法　60只SPF级8周龄雄性SD大鼠，随机选取12只为对照组，其余予以高糖高脂饮食+链脲佐菌素腹腔注射建立糖尿病模型。注射链脲佐菌素3 d后，将符合糖尿病标准大鼠随机分为模型组、肌肽（100、300、900 mg/kg）组。肌肽各组分别灌胃100、300、900 mg/kg肌肽，每日1次。8周后，检测空腹血糖（FBG）、血清肌酐（Scr）、尿素氮（BUN）、24 h尿微量白蛋白（mAlb）。PAS染色法观察大鼠肾形态学变化；试剂盒检测肾组织的超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-Px）含量；免疫组织化学及Western blot检测肾组织P-NF-κB P65蛋白的表达。 结果　 DN大鼠建模成功。与模型组相比，肌肽组肾组织病理损伤明显减轻。肌肽各组大鼠mAlb、FBG、BUN水平下降，呈量-效依赖性关系（P<0.05），而SOD、GSH、GSH-Px的含量逐级升高，同时MDA和P-NF-κB P65含量减少。 结论　肌肽对DN模型大鼠肾组织具有保护作用，其机制可能与抑制氧化应激和NF-κB信号通路异常激活有关。     

牛珀至宝微丸对内毒素休克肺组织核转录因子-κB表达的调控

目的观察牛珀至宝微丸对内毒素休克肺损伤时核转录因子-κB表达的影响。方法静脉注射内毒素(LPS)1.5mg/kg、腹腔注射D-氨基半乳糖(D-GalN)100mg/kg造成内毒素休克模型,用牛珀至宝微丸作预治疗处理,免疫组化方法检测NF-κB在肺组织内的表达。结果LPS组阳性表达部位在细胞核,牛珀至宝微丸组表达部位主要在细胞质。牛珀至宝微丸降低NF-κB表达,肺损伤减轻。结论证实牛珀至宝微丸能降低内毒素休克时肺组织NF-κB表达,改变其表达部位,提示牛珀至宝微丸对内毒素休克造成的肺损伤的保护作用可能是通过调控肺组织NF-κB而产生的。