Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells

Cordyceps militaris, a caterpillar-grown traditional medicinal mushroom, produces an important bioactive compound, cordycepin (3'-deoxyadenosine). Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus and anti-infection activities. The molecular mechanisms of cordycepin on pharmacological and biochemical actions of macrophages in inflammation have not been clearly elucidated yet. In the present study, we tested the role of cordycepin on the anti-inflammation cascades in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. In LPS-activated macrophage, nitric oxide (NO) production was inhibited by butanol fraction of C. militaris and the major component of C. militaris butanol faction was identified as cordycepin by high performance liquid chromatography. To investigate the mechanism by which cordycepin inhibits NO production and inducible nitric oxide synthase (iNOS) expression, we examined the activation of Akt and MAP kinases in LPS-activated macrophage. Cordycepin markedly inhibited the phosphorylation of Akt and p38 in dose-dependent manners in LPS-activated macrophage. Moreover, cordycepin suppressed tumor necrosis factor (TNF-alpha) expression, IkappaB alpha phosphorylation, and translocation of nuclear factor-kappaB (NF-kappaB). The expressions of cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly decreased in RAW 264.7 cell by cordycepin. Taken together, these results suggest that cordycepin inhibits the production of NO production by down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB activation, Akt and p38 phosphorylation. Thus, cordycepin may provide a potential therapeutic approach for inflammation-associated disorders.     

PI3K信号通路在LPS诱导RAW264.7细胞表达sTREM-1中的作用

目的：探讨磷脂酰肌醇3-激酶(PI3K)信号通路在脂多糖(LPS)诱导RAW264.7细胞表达可溶性髓样细胞触发受体-1(sTREM-1)中的作用。方法培养小鼠巨噬细胞株RAW264.7,采用相同浓度的LPS在不同时间诱导RAW264.7细胞,应用Western blot法分别检测PI3K蛋白表达水平,RT-PCR法检测PI3K mRNA表达水平,酶联免疫吸附(ELISA)法检测细胞培养血清中sTREM-1表达水平。用不同浓度PI3K特异性抑制剂LY294002处理细胞,观察上述指标变化。结果 LPS可时间依赖性地诱导RAW264.7细胞PI3K蛋白、PI3K mRNA的表达;LY294002可浓度依赖性地抑制PI3K蛋白、PI3K mRNA的表达;LY294002阻断PI3K信号转导通路后,LPS对sTREM-1表达的诱导作用受到显著抑制,并且具有剂量依赖性。结论 LPS通过PI3K信号通路诱导RAW264.7细胞表达sTREM-1。     

bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways

Extensive research has focused on finding effective strategies to prevent or improve recovery from brain ischemia and reperfusion (I/R) injury. The basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some central nervous system (CNS) disorders, including ischemic injury. In this study, we demonstrate that bFGF administration can improve locomotor activity and inhibit the ER stress induced in the CA1 region of the hippocampus in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response proteins CHOP, XBP-1, ATF-6 and caspase-12 that are induced by H(2)O(2) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signaling pathways, PI3K/Akt and ERK1/2. Inhibition of the PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and U0126, respectively, partially reduce the protective effect of bFGF. Taken together, our results indicate that the neuroprotective role of bFGF involves the suppression of ER stress in the ischemic oxidative damage models and oxidative stress-induced PC12 cell injury, and these effects is underlying the activation of the PI3K/Akt and ERK1/2 signal pathway.     

Recent developments in anti-cancer agents targeting PI3K, Akt and mTORC1/2

Inappropriate PI3K signaling is one of the most frequent occurrences in human cancer and is critical for tumor progression. A variety of genetic mutations and amplifications have been described affecting key components of this pathway, with implications not only for tumorigenesis but also for resistance to targeted agents. Emerging preclinical research has significantly advanced our understanding of the PI3K pathway and its complex downstream signalling, interactions and crosstalk. This knowledge, combined with the limited clinical antitumor activity of mTOR complex 1 inhibitors, has led to the development of rationally designed drugs targeting key elements of this pathway, such as pure PI3K inhibitors (both pan-PI3K and isoform-specific), dual PI3K/ mTOR inhibitors, Akt inhibitors, and mTOR complexes 1 and 2 catalytic site inhibitors. This review will focus primarily on an analysis of newly developed inhibitors of this pathway that have entered clinical trials, and recently registered patents in this field.     

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO₂), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200 mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.     

Chlorogenic acid inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 cells through suppressing NF-κB and JNK/AP-1 activation

Chlorogenic acid (CGA) is a naturally occurring phenolic acid in human diet. Data obtained from in vivo and in vitro experiments demonstrate that CGA mostly presents anti-oxidant and anti-carcinogenic activities. Here we show that CGA also inhibits lipopolysaccharide (LPS)-induced inflammatory response[AU1] in RAW 264.7 cells. Our results indicated that CGA significantly decreased LPS-induced up-regulation of cyclooxygenase (COX-2) at protein and mRNA levels in RAW 264.7 cells and as a result it inhibited prostaglandin E₂ (PGE₂) release from LPS-treated RAW 264.7 cells. In the further experiments, LPS-induced activation of nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)-c-Jun-activator protein (AP-1) pathway were suppressed significantly by CGA. In addition, CGA did not affect phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38. In conclusion, CGA suppresses LPS-induced COX-2 expression via attenuating the activation of NF-κB and JNK/AP-1 signaling pathways suggesting that CGA, the polyphenol compound in our food, could exert anti-inflammatory effects through inhibiting PGE₂ production.     

小檗碱通过调控PI3K/Akt通路抑制喉癌细胞生长和转移

目的探讨小檗碱对喉癌Hep2细胞增殖、凋亡和侵袭的影响以及相关作用机制。方法培养Hep2细胞分为空白对照组和5、10、20μmol·L-1小檗碱组,CCK-8检测Hep2的增殖活性,流式细胞术检测细胞凋亡情况,划痕实验检测细胞迁移情况,Transwell法检测细胞侵袭能力,Western blot法检测凋亡蛋白酶激活因子1(Apaf1)、剪切后半胱天冬酶(cl-caspase)-9、cl-caspase-3蛋白、血管内皮生长因子(VEGF)、基质金属蛋白酶(MMP)-2、MMP-9、磷脂酰肌醇3激酶(PI3K)、磷酸化蛋白激酶B(p-Akt)和p-p65蛋白表达水平。结果与空白对照组相比,5、10、20μmol·L-1小檗碱组细胞增殖活性显著降低(P <0.01);凋亡细胞百分比显著增加(P <0.01),Apaf1、cl-caspase-9和cl-caspase-3蛋白水平显著上调(P <0.01);划痕愈合率显著降低(P <0.01),侵袭细胞数目显著减少(P <0.01);细胞中VEGF、MMP-2、MMP-9、PI3K、p-Akt、p-p65蛋白水平显著降低(P <0.01),且均呈浓度依赖性。结论小檗碱通过调控PI3K/Akt通路抑制喉癌细胞增殖、侵袭和迁移,诱导细胞凋亡。     

4,7-Dimethoxy-5-methyl-1,3-benzodioxole from Antrodia camphorata inhibits LPS-induced inflammation via suppression of NF-κB and induction HO-1 in RAW264.7 cells

Several benzenoid compounds have been isolated from Antrodia camphorata are known to have excellent anti-inflammatory activity. In this study, we investigated the anti-inflammatory potential of 4,7-dimethoxy-5-methyl-1,3-benzodioxole (DMB), one of the major benzenoid compounds isolated from the mycelia of A. camphorata. DMB significantly decreased the LPS-induced production of pro-inflammatory molecules, such as nitric oxide (NO), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW264.7 cells. In addition, DMB suppressed the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner. Moreover, DMB significantly suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB), and this inhibition was found to be associated with decreases in the phosphorylation and degradation of its inhibitor, inhibitory κB-α (IκB-α). Moreover, we found that DMB markedly inhibited the protein expression level of Toll-like receptor 4 (TLR4). Furthermore, treatment with DMB significantly increased hemoxygenase-1 (HO-1) expression in RAW264.7 cells, which is further confirmed by hemin, a HO-1 enhancer, significantly attenuated the LPS-induced pro-inflammatory molecules and iNOS and TLR4 protein levels. Taken together, the present study suggests that DMB may have therapeutic potential for the treatment of inflammatory diseases.     

Ethyl linoleate from garlic attenuates lipopolysaccharide-induced pro-inflammatory cytokine production by inducing heme oxygenase-1 in RAW264.7 cells

In the present study, an essential fatty acid, ethyl linoleate (ELA), was isolated from the cloves of Allium sativum, and its structure was elucidated by NMR and GC-MS analyses. In vitro systems were used to evaluate the anti-inflammatory activity of ELA. Our results indicate that ELA down-regulates inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and thereby reduces nitric oxide (NO) and prostaglandin E₂ production in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Immunofluorescent microscopy and western blot analyses revealed that these effects were mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of phosphorylation of mitogen activated protein kinases. Furthermore, ELA exerted its anti-inflammatory activity by inducing heme oxygenase-1 (HO-1) expression, as determined by HO-1 small interfering (Si) RNA system. Si RNA-mediated knock-down of HO-1 abrogated the inhibitory effects of ELA on the production of NO, TNF-α, IL-1β, and IL-6 in LPS-induced macrophages. These findings indicate the potential therapeutic use of ELA as an anti-inflammatory agent.     

GYF-17, a chloride substituted 2-(2-phenethyl)-chromone,suppresses LPS-induced inflammatory mediator production in RAW264.7 cells by inhibiting STAT1/3 and ERK1/2 signaling pathways

GYF-17, a 2-(2-phenethyl)-chromone derivative, was isolated from agarwood and showed superior activity of inhibiting NO production of RAW264.7 cells induced by LPS in our preliminary pharmacodynamic screening. In order to develop novel therapeutic drug for acute and chronic inflammatory disorders, the anti-inflammatory activity and underlying mechanism of GYF-17 were investigated in LPS-induced RAW264.7 cells. The results showed that GYF-17 could reduce LPS-induced expression of iNOS and then result in the decrement of NO production. More meaningful, the expression and secretion of key pro-inflammatory factors, including TNF-α, IL-6 and IL-1β, were intensively inhibited by GYF-17. Furthermore, GYF-17 also down regulated the expression of COX2 and the production of PGE2 which plays important role in causing algesthesia during inflammatory response. In mechanism study, GYF-17 selectively suppressed phosphorylation of STAT1/3 and ERK1/2 during the activation of NF-κB, MAPK and STAT signaling pathways induced by LPS. Collectively, GYF-17 can intensively suppress the production of LPS-induced inflammatory mediators in RAW264.7 cells by inhibiting STAT1/3 and ERK1/2 signaling pathways and thereby shows great potential to be developed into therapeutic drug for inflammatory diseases.     

Cryptochlorogenic acid attenuates LPS-induced inflammatory response and oxidative stress via upregulation of the Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages

Phenolic acids are found in natural plants, such as caffeic acid, rosmarinic acid, and chlorogenic acid. They have long been used as pharmacological actives, owing to their anti-inflammatory and antioxidant activities. Cryptochlorogenic acid (CCGA) is a special isomer of chlorogenic acid; the pharmacological effects and related molecular mechanisms of CCGA have been poorly reported. In the present study, the antioxidant and anti-inflammatory effects of CCGA in RAW 264.7 macrophages and the underlying mechanisms were investigated. The results revealed that CCGA dose-dependently inhibited LPS-induced production of NO, TNF-α, and IL-6 and blocked iNOS, COX-2, TNF-α, and IL-6 expressions. CCGA also significantly increased the GSH/GSSG ratio and SOD activity and reduced the MDA level. Moreover, CCGA suppressed the nuclear translocation of NF-κB by hindering the phosphorylation of IκB kinase (IKK) and degrading IκB. It also downregulated the phosphorylation of MAPKs. Our results indicated that CCGA significantly inhibited NF-κB activation by controlling the expression of pro-inflammatory factors and promoting the nuclear transfer of Nrf2. In conclusion, CCGA could attenuate LPS-induced inflammatory symptoms by modulating NF-κB/MAPK signaling cascades and inhibit LPS-induced oxidative stress via Nrf2 nuclear translocation.     

Effect of Apremilast on LPS-induced immunomodulation and inflammation via activation of Nrf2/HO-1 pathways in rat lungs

Lipopolysaccharides (LPS), the lipid component of gram-negative bacterial cell wall, is recognized as the key factor in acute lung inflammation and is found to exhibit severe immunologic reactions. Phosphodiesterase-4 (PDE-4) inhibitor: “apremilast (AP)” is an immune suppressant and anti-inflammatory drug which introduced to treat psoriatic arthritis. The contemporary experiment designed to study the protective influences of AP against LPS induced lung injury in rodents. Twenty-four (24) male experimental Wistar rats selected, acclimatized, and administered with normal saline, LPS, or AP + LPS respectively from 1 to 4 groups. The lung tissues were evaluated for biochemical parameters (MPO), Enzyme Linked Immunosorbent Assay (ELISA), flowcytometry assay, gene expressions, proteins expression and histopathological examination. AP ameliorates the lung injuries by attenuating immunomodulation and inflammation. LPS exposure upregulated IL-6, TNF-α, and MPO while downregulating IL-4 which were restored in AP pretreated rats. The changes in immunomodulation markers by LPS were reduced by AP treatment. Furthermore, results from the qPCR analysis represented an upregulation in IL-1β, MPO, TNF-α, and p38 whereas downregulated in IL-10 and p53 gene expressions in disease control animals while AP pretreated rats exhibited significant reversal in these expressions. Western blot analysis suggested an upregulation of MCP-1, and NOS-2, whereas HO-1, and Nrf-2 expression were suppressed in LPS exposed animals, while pretreatment with AP showed down regulation in the expression MCP-1, NOS-2, and upregulation of HO-1, and Nrf-2 expression of the mentioned intracellular proteins. Histological studies further affirmed the toxic influences of LPS on the pulmonary tissues. It is concluded that, LPS exposure causes pulmonary toxicities via up regulation of oxidative stress, inflammatory cytokines and stimulation of IL-1β, MPO, TNF-α, p38, MCP-1, and NOS-2 while downregulation of IL-4, IL-10, p53, HO-1, and Nrf-2 at different expression level. Pretreatment with AP controlled the toxic influences of LPS by modulating these signaling pathways.     

七氟烷通过P~(70S6K)/Nrf2信号通路诱导神经元HO-1mRNA表达

目的探讨七氟烷(Sevoflurane)诱导神经元血红素氧合酶-1(HO-1)基因表达的信号转导通路。方法将培养7d的新生大鼠海马神经元随机分为4组:正常培养组(C组)、2%七氟烷组(S1组)、4%七氟烷组(S2组)和4%七氟烷+Rapamycin组(R组)。C组神经元按正常培养方法培养。S1组和S2组神经元分别给予2%或4%七氟烷处理60min后继续培养24h。R组在神经元给予4%七氟烷处理同时在培养液中加入Rapamycin使其终浓度为10nmol.L-1后同S2组处理。收集神经元进行HO-1mRNA和P70S6K、Nrf2、AP-1和HO-1蛋白表达的检测。结果S1组P70S6K和Nrf2蛋白表达增加(vsC组,P<0.01),HO-1mRNA和HO-1蛋白表达增加(vsC组,P<0.01),AP-1蛋白表达变化不明显(vsC组,P>0.05)。S2组P70S6K和Nrf2蛋白表达增加(vsS1组,P<0.05),HO-1mRNA和HO-1蛋白表达增加(vsS1组,P<0.05),AP-1蛋白表达变化不明显(vsS1组,P>0.05)。R组P70S6K和Nrf2蛋白表达减少(vsS2组,P<0.01),HO-1mRNA和HO-1蛋白表达减少(vsS2组,P<0.01),AP-1蛋白表达变化不明显(vsS2组,P>0.05)。结论Sevoflurane通过P70S6K/Nrf2信号通路诱导神经元HO-1mRNA表达。     

Homocysteine induces cell cycle G1 arrest in endothelial cells through the PI3K/Akt/FOXO signaling pathway

OBJECTIVE: High levels of homocysteine (Hcy) induce a sustained injury on arterial endothelial cells, which accelerates the development of thrombosis and atherosclerosis. Hcy specifically inhibits the growth of endothelial cells. The present study investigated the signaling pathways underlying this cell-cycle effect. METHODS: Human umbilical venous endothelial cells were treated with Hcy, and/or LY294002, okadaic acid, peroxovanadate (PV), antisense Akt, phosphorylation of Akt and FKHRL1 proteins. p27(kip1) protein levels were measured with Western blotting, and Akt kinase activity and cell cycle were measured with immunoprecipitation and flow cytometry, respectively. RESULTS: We demonstrate that Hcy induces dephosphorylation of Akt and FKHRL1 and upregulates the cyclin-dependent kinase inhibitors p27(kip1) in a time- and dose-dependent manner. Phosphatidylinositol-3 kinase (PI3K) activator PV and phosphatase 2A inhibitor okadaic acid could reverse it, which suggests it was dependent on PI3K activity. Moreover, Hcy induces cell cycle G1 phase arrest prevented by pretreatment with PV and okadaic acid. Transfection with specific antisense oligonucleotides to Akt further proves the observations. CONCLUSIONS: The studies implied that a novel signaling pathway, PI3K/Akt/FOXO, might play an important role in mediating cell cycle G1 arrest in endothelial cells.     

Pristimerin inhibits neuronal inflammation and protects cognitive function in mice with sepsis-induced brain injuries by regulating PI3K/Akt signalling

ContextSepsis is a systemic inflammatory disease; pristimerin exhibits strong antibacterial, anti-inflammatory and antioxidant properties.ObjectivesWe explored whether pristimerin protected against cognitive dysfunction and neuroinflammation in C57BL/6 J mice with sepsis-induced brain injuries.Materials and methodsSepsis was induced by intraperitoneal administration of 2 mg/kg lipopolysaccharide (LPS). C57BL/6 J mice were separated into four groups (n = 10 per group): positive control, negative control, pristimerin 10 mg/kg and pristimerin 100 mg/kg. Pristimerin was administered orally for 28 days prior to LPS administration and for six days thereafter. Behavioural changes were assessed one day after LPS administration using the Morris water maze and via neurological dysfunction scoring. Molecular pathogenesis was explored by measurement of malondialdehyde, superoxide dismutase, reactive oxygen species and inflammatory cytokine levels in mouse brains. Neuronal apoptosis was evaluated using the TUNEL assay. The levels of p-Akt/Akt, p-PI3K/PI3K, mTOR, Bax, Bcl-2 and caspase-3 proteins were determined via Western blotting.ResultsPristimerin improved cognitive function and reduces the neurological score to 1.15 ± 0.03. Pristimerin significantly reduced all cytokine levels: TNF-α by 18 ± 0.6 pg/mg, IL-1β by 43 ± 1.3 pg/mg and IL-6 by 34 ± 1.12 pg/mg. There was significant (p < 0.01) improvement in PI3K/Akt signalling and histopathological changes in the brain tissue of sepsis induced brain injured rats.ConclusionsPristimerin ameliorated neuronal injury by regulating PI3K/Akt signalling in mice with sepsis-induced brain injuries. Pristimerin may merit further development for clinical applications.