Inflammatory mechanism of cerebral ischemia-reperfusion injury with treatment of stepharine in rats

BackgroundIncreasing evidence has shown that microglia-induced neuroinflammation is involved in the pathogenesis of ischemic stroke. Stepharine, one of the alkaloids extracted from Stephania japonica (Thunb.) Miers, exhibited strong inhibitory effect on microglial overactivation. However, it is not known whether it has the potential to prevent ischemic stroke.MethodsThe neuroprotective and anti-neuroinflammatory effects of stepharine were investigated in vivo and in vitro, using a rat model of middle cerebral artery occlusion (MCAO) and lipopolysaccharide (LPS)-stimulated BV-2 cells, respectively.ResultsIn vivo, stepharine (500 μg/kg) suppressed neurological deficits scores, brain water content and cerebral infarct volume induced by MCAO. Moreover, stepharine (500 μg/kg) inhibited NeuN⁺ cells loss and Iba-1⁺ cells increase in the MCAO ischemic cortex. In vitro, stepharine (10, 30 μM) substantially inhibited nitric oxide release as well as the mRNA and protein expression of pro-inflammatory mediators [inducible nitric oxide synthase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β] in LPS-activated BV-2 cells. LPS-induced increase of TLR4 expression, IκBα phosphorylation, and NF-κB p65 nuclear translocation was inhibited by stepharine (10, 30 μM). Molecular docking analysis showed that stepharine directly interacted with TLR4. SPR assay further confirmed that stepharine could bind to the TLR4/MD2 complex. Meanwhile, stepharine exhibited neuroprotective effects on SH-SY5Y cells cultured with LPS-treated conditioned medium.ConclusionOur study demonstrated for the first time that stepharine improved the outcomes in MCAO rats, reduced neuronal loss, and suppressed microglial overactivation via the inhibition of TLR4/NF-κB pathway. These results suggest that stepharine might be a potential therapeutic agent for the treatment of ischemic stroke.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Neuroprotection of Andrographolide Against Microglia-Mediated Inflammatory Injury and Oxidative Damage in PC12 Neurons

Andrographolide from leaves of Andrographis paniculata has been known to possess various bioactivities. In the present study, we aimed to explore the neuroprotection of andrographolide against inflammation-mediated injury and oxidative damage. In initial studies, our findings showed that pretreatment with andrographolide could effectively reduce neuronal cell death caused by LPS-induced conditioned supernatants. The further results indicated that this neuroprotective effect may be mainly due to the inhibition on the production of NO, TNF-α, IL-6, ROS, iNOS and enhancement of expression of anti-inflammatory marker CD206. Moreover, mechanism study revealed that the anti-inflammatory activity of andrographolide may be related to the suppression of nuclear translocation of NF-κB as well as the activation of Nrf2 and HO-1. Our study also showed that andrographolide could scavenge ROS and protect PC12 cells against H₂O₂- and 6-OHDA-mediated oxidative damage. In addition, several derivatives of andrographolide were prepared for evaluating the role of 3, 14, 19-hydroxy group on anti-inflammatory effect and cytoprotection of andrographolide. In conclusion, andrographolide protected neurons against inflammation-mediated injury via NF-κB inhibition and Nrf2/HO-1 activation and resisted oxidative damage via inhibiting ROS production. Our results will contribute to further exploration of the therapeutic potential of andrographolide in relation to neuroinflammation and neurodegenerative diseases.

     

Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis,oxidative stress,and inflammation in pulmonary endothelial cells

Acute respiratory distress syndrome (ARDS) is a heterogenous syndrome characterised by diffuse alveolar damage, with an increase in lung endothelial and epithelial permeability. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses antiapoptotic and antioxidative effects in distinct situations. In the present study, the protective effects and potential molecular mechanisms of LBP against lipopolysaccharide (LPS)-induced ARDS were investigated in the mice and in the human pulmonary microvascular endothelial cells (HPMECs). The data indicated that pretreatment with LBP significantly attenuated LPS-induced lung inflammation and pulmonary oedema in vivo. LBP significantly reversed LPS-induced decrease in cell viability, increase in apoptosis and oxidative stress via inhibiting caspase-3 activation and intracellular reactive oxygen species (ROS) production in vitro. Moreover, the scratch assay verified that LBP restored the dysfunction of endothelial cells (ECs) migration induced by LPS stimulation. Furthermore, LBP also significantly suppressed LPS-induced NF-κB activation, and subsequently reversed the release of cytochrome c. These results showed the antiapoptosis and antioxidant LBP could partially protect against LPS-induced ARDS through promoting the ECs survival and scavenging ROS via inhibition of NF-κB signalling pathway. Thus, LBP could be potentially used for ARDS against pulmonary inflammation and pulmonary oedema.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.     

The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells

AimExposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells.Main methodsRAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.Key findingsAfter cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15–60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production.SignificanceDMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.     

Synergistic anti-inflammatory effects of silibinin and thymol combination on LPS-induced RAW264.7 cells by inhibition of NF-κB and MAPK activation

BackgroundCombination drug therapy has become an effective strategy for inflammation control. The anti‑inflammatory capacities of silibinin and thymol have each been investigated on its own, but little is known about the synergistic anti-inflammatory effects of these two compounds.PurposeThis study aims to investigate the synergistic anti-inflammatory effects of silibinin and thymol when administered in combination to lipopolysaccharide (LPS)-induced RAW264.7 cells.MethodsRAW264.7 cells were pre-treated with silibinin and thymol individually or in combination for 2 h before LPS stimulation. Cell viability was detected by the MTT assay. Nitric oxide (NO) production was measured by Griess reagent. Reactive oxygen species (ROS) was evaluated by 2’,7’-dichlorofluorescein-diacetate. ELISA was used to detect tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Western blot was performed to analyse the protein expression of LPS-induced RAW264.7 cells.ResultsWe observed a synergistic anti-inflammatory effect of silibinin and thymol when administered in combination to LPS-induced RAW264.7 cells. Silibinin combined with thymol (40 μM and 120 μM respectively, with the molar ratio 1:3) had more potent effects on the inhibition of NO, TNF-α, and IL-6 than those exerted by individual administration of these compounds in LPS-induced RAW264.7 cells. The combination of silibinin and thymol (40 μM and 120 μM respectively, with the molar ratio 1:3) strongly inhibited ROS and cyclooxygenase-2 (COX-2). More importantly, the combination of silibinin and thymol (40 μM and 120 μM respectively, with the molar ratio 1:3) was also successful in inhibiting nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activities. Our results suggest that the synergistic anti-inflammatory effects of silibinin with thymol were associated with the inhibition of NF-κB and MAPK signalling pathways.ConclusionThe combination of silibinin and thymol (40 μM and 120 μM, respectively, with the molar ratio 1:3) could inhibit inflammation by suppressing NF-κB and MAPK signalling pathways in LPS-induced RAW264.7 cells.     

The protective effect of 5-O-methylvisammioside on LPS-induced depression in mice by inhibiting the over activation of BV-2 microglia through Nf-κB/IκB-α pathway

Background5-O-methylvisammioside (MeV), also known as 4′-O-β-D-glucosyl-5-O-methylvisamminol, is a conventional marker compound for quality control of roots of Saposhnikovia diviaricata (Radix Saposhnikoviae), which exhibits anti-inflammatory and neuroprotective activities.PurposeAccording to the activity of MeV, we speculated that MeV may have antidepressant effect on LPS induced depression, and further explored its mechanism.Study DesignFirst, to explore the effect and mechanism of MeV on LPS-induced depression in mice, and then to further explore the effect and mechanism of MeV on LPS-activated BV-2 microglia.MethodsBy the OFT, EPM, TST and FST behavioral tests, to explore the effect of MeV pretreatment on the behavior of LPS-induced depression mice. ELISA and Griess method were used to detect the changes of the serum TNF-α and IL-6 levels, the hippocampus SOD and MDA levels, and the NO, SOD, MDA, TNF-α and IL-6 levels in the culture medium of LPS-stimulated BV-2 microglia. Western blot was used to analyze the protein expression in the Nf-κB/IκB-α and BDNF/TrkB pathway in the hippocampus of mice and BV-2 microglia.ResultsMeV (4 mg/kg, i.p.) pretreatment significantly improves the activity and exploration ability of LPS-induced depression mice, and reduces the immobility time. MeV inhibited the production of pro-inflammatory cytokines in the serum of mice induced by LPS, such as IL-6 and TNF-α. MeV also increased the levels of SOD and reduces the expression of MDA in the hippocampus, thus promoting the alleviation of depressive symptoms in mice. Western blotting analysis showed that the antidepressant activity of MeV was related to the decrease of Nf-κB nuclear transport, the inhibition of IκB-α phosphorylation, and the increase of BDNF and TrkB expression. MeV (40 μM) significantly reduced the contents of NO, MDA, TNF-α and IL-6 in the culture medium of LPS-stimulated BV-2 microglia, and increased the content of SOD.ConclusionMeV can regulate the neurotrophic factors in the mouse brain, reduce the content of inflammatory factors by the Nf-κB/IκB-α pathway, improve oxidative stress, and inhibit the excessive activation of LPS-stimulated BV -2 microglia. It effectively reversed the depression-like behAavior induced by LPS in mice.     

NAT10 regulates the LPS-induced inflammatory response via the NOX2-ROS-NF-κB pathway in macrophages

Periodontitis is a chronic osteolytic inflammatory disease resulting from complex dynamic interactions among bacterial pathogens and the host immune response. Macrophages play a vital role in the pathogenesis of periodontitis by triggering periodontal inflammation and inducing periodontium destruction. N-Acetyltransferase 10 (NAT10) is an acetyltransferase that has been shown to catalyse N4-acetylcytidine (ac4C) mRNA modification and is related to cellular pathophysiological processes, including the inflammatory immune response. Nevertheless, whether NAT10 regulates the inflammatory response of macrophages in periodontitis remains unclear. In this study, the expression of NAT10 in macrophages was found to decrease during LPS-induced inflammation. NAT10 knockdown significantly reduced the generation of inflammatory factors, while NAT10 overexpression had the opposite effect. RNA sequencing revealed that the differentially expressed genes were enriched in the NF-κB signalling pathway and oxidative stress. Both the NF-κB inhibitor Bay11-7082 and the ROS scavenger N-acetyl-L-cysteine (NAC) could reverse the upregulation of inflammatory factors. NAC inhibited the phosphorylation of NF-κB, but Bay11-7082 had no effect on the production of ROS in NAT10-overexpressing cells, suggesting that NAT10 activated the LPS-induced NF-κB signalling pathway by regulating ROS generation. Furthermore, the expression and stability of Nox2 was promoted after NAT10 overexpression, indicating that Nox2 may be a potential target of NAT10. In vivo, the NAT10 inhibitor Remodelin reduced macrophage infiltration and bone resorption in ligature-induced periodontitis mice. In summary, these results showed that NAT10 accelerated LPS-induced inflammation via the NOX2-ROS-NF-κB pathway in macrophages and that its inhibitor Remodelin might be of potential therapeutic significance in periodontitis treatment.     

Saikosaponin-D induces the pyroptosis of lung cancer by increasing ROS and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway

Saikosaponin-D (SSD), an active ingredient in Bupleurum chinense, exerts anticancer effects in various cancers by inhibiting cancer proliferation and inducing apoptosis. However, whether SSD can induce other forms of cell death is unknown. The current study aims to demonstrate that SSD can induce pyroptosis in non-small-cell lung cancer. In this study, HCC827 and A549 non-small-cell lung cancer cells were treated with different concentrations of SSD for 1.5 h. HE and TUNEL staining were used to verify cell damage caused by SSD. Immunofluorescence and western blotting were performed to verify the effect of SSD on the NF-κB/NLRP3/caspase-1/gasdermin D (GSDMD) pathway. Changes in inflammatory factors were detected by ELISAs. Finally, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was introduced to verify that SSD induces pyroptosis through the ROS/NF-κB pathway. The results of the HE and TUNEL staining showed that SSD resulted in balloon-like swelling of NSCLC cells accompanied by increased DNA damage. Immunofluorescence and western blot assays confirmed that SSD treatment activated the NLRP3/caspase-1/GSDMD pathway, stimulated an increase in ROS levels and activated NF-κB in lung cancer cells. The ROS scavenger N-acetylcysteine significantly attenuated SSD-induced NF-κB/NLRP3/caspase-1/GSDMD pathway activation and inhibited the release of the inflammatory cytokines IL-1β and IL-18. In conclusion, SSD induced lung cancer cell pyroptosis by inducing ROS accumulation and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway. These experiments lay the foundation for the application of SSD in the treatment of non-small-cell lung cancer and regulation of the lung cancer immune microenvironment.     

Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-κB pathway in microglia cells

Microglial activation plays an important role in neuroinflammation, which contributes to neuronal damage, and inhibition of microglial activation may have therapeutic benefits that could alleviate the progression of neurodegeneration. Recent studies have indicated that the antimalarial agent artemisinin has the ability to inhibit NF-κB activation. In this study, the inhibitory effects of artemisinin on the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-stimulated primary microglia. Our results show that artemisinin significantly inhibited LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO). Artemisinin significantly decreased both the mRNA and the protein levels of these pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) and increased the protein levels of IκB-α, which forms a cytoplasmic inactive complex with the p65-p50 heterodimeric complex. Artemisinin treatment significantly inhibited basal and LPS-induced migration of BV-2 microglia. Electrophoretic mobility shift assays revealed increased NF-κB binding activity in LPS-stimulated primary microglia, and this increase could be prevented by artemisinin. The inhibitory effects of artemisinin on LPS-stimulated microglia were blocked after IκB-α was silenced with IκB-α siRNA. Our results suggest that artemisinin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The data provide direct evidence of the potential application of artemisinin for the treatment of neuroinflammatory diseases.     

Molecular mechanism of the anti-inflammatory activity of a natural diarylnonanoid, malabaricone C

The spice-derived phenolic, malabaricone C (mal C), has recently been shown to accelerate healing of the indomethacin-induced gastric ulceration in mice. In this study, we explored its anti-inflammatory activity and investigated the underlying mechanism of the action. Mal C suppressed the microvascular permeability and the levels of tumor necrosis factor-α, interleukin-1β, and nitric oxide in the lipopolysaccharide (LPS)-administered mice. At a dose of 10 mg/kg, it showed anti-inflammatory activity comparable to that of omeprazole (5 mg/kg) and dexamethasone (50 mg/kg). It also reduced the expression and activities of inducible nitric oxide synthase, cyclooxygenase-2, as well as the pro- vs anti-inflammatory cytokine ratio in the LPS-treated RAW macrophages. Mal C was found to inhibit LPS-induced NF-kB activation in RAW 264.7 cells by blocking the MyD88-dependent pathway. Mal C suppressed NF-κB activation and iNOS promoter activity, which correlated with its inhibitory effect on IκB phosphorylation and degradation, and NF-κB nuclear translocation, in the LPS-stimulated macrophages. It also inhibited LPS-induced phosphorylation of p38 and JNK, which are also upstream activators of NF-κB, without affecting Akt phosphorylation. Mal C also effectively blocked the PKR-mediated activation of NF-κB. These findings indicate that mal C exerts an anti-inflammatory effect through NF-κB-responsive inflammatory gene expressions by inhibiting the p38 and JNK-dependent canonical NF-κB pathway as well as the PKR pathway, and is a potential therapeutic agent against acute inflammation.     

Forsythiaside A Exhibits Anti-inflammatory Effects in LPS-Stimulated BV2 Microglia Cells Through Activation of Nrf2/HO-1 Signaling Pathway

Inflammation and oxidative stress have been reported to play critical roles in the pathogenesis of neurodegenerative disease. Forsythiaside A, a phenylethanoside product isolated from air-dried fruits of Forsythia suspensa, has been reported to have anti-inflammatory and antioxidant effects. In this study, the anti-inflammatory effects of forsythiaside A on LPS-stimulated BV2 microglia cells and primary microglia cells were investigated. The production of inflammatory mediators TNF-α, IL-1β, NO and PGE₂ were detected in this study. NF-κB, nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) expression were detected by western blot analysis. Our results showed that forsythiaside A significantly inhibited LPS-induced inflammatory mediators TNF-α, IL-1β, NO and PGE₂ production. LPS-induced NF-κB activation was suppressed by forsythiaside A. Furthermore, forsythiaside A was found to up-regulate the expression of Nrf2 and HO-1. In conclusion, this study demonstrates that forsythiaside A inhibits LPS-induced inflammatory responses in BV2 microglia cells and primary microglia cells through inhibition of NF-κB activation and activation of Nrf2/HO-1 signaling pathway.     

Protective effect of 1,2,4-benzenetriol on LPS-induced NO production by BV2 microglial cells

Summary Hydroxyhydroquinone or 1,2,4-benzenetriol (BT) detected in the beverages has a structure that coincides with the water-soluble form of a sesame lignan, sesamol. We previously showed that sesame antioxidants had neuroprotective abilities due to their antioxidant properties and/or inducible nitric oxide synthase (iNOS) inhibition. However, studies show that BT can induce DNA damage through the generation of reactive oxygen species (ROS). Therefore, we were interested to investigate the neuroprotective effect of BT in vitro and in vivo. The results showed that instead of enhancing free radical generation, BT dose-dependently (10–100 μM) attenuated nitrite production, iNOS mRNA and protein expression in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. BT significantly reduced LPS-induced NF-κB and p38 MAPK activation. It also significantly reduced the generation of ROS in H₂O₂-induced BV-2 cells and in H₂O₂-cellfree conditions. The neuroprotective effect of BT was further demonstrated in the focal cerebral ischemia model of Sprague–Dawley rat. Taken together, the inhibition of LPS-induced nitrite production might be due to the suppression of NF-κB, p38 MAPK signal pathway and the ROS scavenging effect. These effects might help to protect neurons from the ischemic injury.     

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A₂ (cPLA₂) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA₂ and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA₂ expression in RAW 264.7 cells.