Glycyrrhizin attenuates rat ischemic spinal cord injury by suppressing inflammatory cytokines and HMGB1

Aim:

To investigate the neuroprotective effect of glycyrrhizin (Gly) against the ischemic injury of rat spinal cord and the possible role of the nuclear protein high-mobility group box 1 (HMGB1) in the process.

Methods:

Male Sprague-Dawley rats were subjected to 45 min aortic occlusion to induce transient lumbar spinal cord ischemia. The motor functions of the animals were assessed according to the modified Tarlov scale. The animals were sacrificed 72 h after reperfusion and the lumbar spinal cord segment (L2–L4) was taken out for histopathological examination and Western blotting analysis. Serum inflammatory cytokine and HMGB1 levels were analyzed using ELISA.

Results:

Gly (6 mg/kg) administered intravenously 30 min before inducing the transient lumbar spinal cord ischemia significantly improved the hind-limb motor function scores, and reduced the number of apoptotic neurons, which was accompanied by reduced levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the plasma and injured spinal cord. Moreover, the serum HMGB1 level correlated well with the serum TNF-α, IL-1β and IL-6 levels during the time period of reperfusion.

Conclusion:

The results suggest that Gly can attenuate the transient spinal cord ischemic injury in rats via reducing inflammatory cytokines and inhibiting the release of HMGB1.     

Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms

Aim:

To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice.

Methods:

Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay.

Results:

Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury.

Conclusion:

Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.     

Cannabidiol attenuates delayed-type hypersensitivity reactions via suppressing T-cell and macrophage reactivity

Aim:

To investigate the effects cannabidiol (CBD) on delayed-type hypersensitivity (DTH) reactions and antigen-induced T-cell cytokine expression.

Methods:

DTH was induced by subcutaneous ovalbumin (OVA) challenge to the footpads of mice sensitized with OVA. Inflammatory reactions were measured by footpad swelling and histological analysis. Antigen-induced cytokine expression by OVA-primed splenocytes was measured using ELISA and RT-PCR.

Results:

CBD (1-10 mg/kg) administration, in a dose-dependent fashion, significantly attenuated inflammatory reactions associated with DTH in the footpads of mice sensitized and challenged with OVA. Histological examination revealed that CBD suppressed the infiltration of T cells and macrophages, and the expression of interferon (IFN)-γ and tumor necrosis factor-α, two pro-inflammatory cytokines implicated in DTH in the inflammatory site. In contrast, the expression of interleukin (IL)-10 in the footpads was enhanced by CBD administration. In addition, CBD at concentrations devoid of cytotoxic effects (1-4 μmol/L) attenuated OVA-induced IFN-γ production by OVA-primed splenocytes, whereas IL-4 was unaffected.

Conclusion:

CBD curbs DTH reactions via suppressing the infiltration and functional activity of T cells and macrophages in the inflammatory site, suggesting a therapeutic potential for CBD for the treatment of type IV hypersensitivity.     

High glucose stimulates TNFα and MCP-1 expression in rat microglia via ROS and NF-κB pathways

Aim:

To investigate whether high glucose stimulates the expression of inflammatory cytokines and the possible mechanisms involved.

Methods:

ELISA and real-time PCR were used to determine the expression of the inflammatory factors, and a chemiluminescence assay was used to measure the production of reactive oxygen species (ROS).

Results:

Compared to low glucose (10 mmol/L), treatment with high glucose (35 mmol/L) increased the secretion of tumor necrosis factor (TNF)α and monocyte chemotactic protein-1 (MCP-1), but not interleukin (IL)-1β and IL-6, in a time-dependent manner in primary cultured rat microglia. The mRNA expression of TNFα and MCP-1 also increased in response to high glucose. This upregulation was specific to high glucose because it was not observed in the osmotic control. High-glucose treatment stimulated the formation of ROS. Furthermore, treatment with the ROS scavenger NAC significantly reduced the high glucose-induced TNFα and MCP-1 secretion. In addition, the nuclear factor kappa B (NF-κB) inhibitors MG132 and PDTC completely blocked the high glucose-induced TNFα and MCP-1 secretion.

Conclusion:

We found that high glucose induces TNFα and MCP-1 secretion as well as mRNA expression in rat microglia in vitro, and this effect is mediated by the ROS and NF-κB pathways.     

Effects of Jian Pi Qing Chang Hua Shi decoction on mucosal injuries in a 2,4,6-trinitrobenzene sulphonic acid-induced inflammatory bowel disease rat model

ContextJian Pi Qing Chang Hua Shi decoction (JPQCHSD) has been considered as an effective remedy for the treatment of inflammatory bowel disease (IBD) in Chinese traditional medicine.ObjectiveWe evaluated the efficacy of JPQCHSD on 2-4-6-trinitrobenzene sulphonic acid (TNBS)-induced IBD rats and the responsible mechanisms.Materials and methodsExcept the rats of the control group (50% ethanol), Sprague–Dawley rats (180 ± 20 g) induced by TNBS (150 mg/kg in 50% ethanol), received water extract of JPQCHSD daily at 0, 9.5, 19, or 38 g/kg for 12 days. The rats were sacrificed, and their colons were removed to evaluate the disease activity index. Malondialdehyde (MDA), superoxide dismutase (SOD), myeloperoxidase (MPO), immunoglobulin A (IgA), tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and nuclear factor-κB were evaluated.ResultsJPQCHSD extract significantly reduced the disease activity index of TNBS-induced colitis with a median effective dose (ED₅₀) of 26.93 g/kg. MPO and MDA were significantly reduced in the 19 and 38 g/kg groups (ED₅₀ values 37.38 and 53.2 g/kg, respectively). The ED₅₀ values for the increased SOD and IgA were 48.98 and 56.3 g/kg. ED₅₀ values for inhibition of TNF-α, IL-1β, and IL-6 were 32.66, 75.72, and 162.06 g/kg, respectively.DiscussionJPQCHSD promoted mucosal healing in IBD rats via its anti-inflammation, immune regulation, and antioxidation properties.ConclusionsJPQCHSD has healing function on IBD. Further clinical trials are needed to demonstrate its efficacy and tolerance to IBD.     

Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-κB-dependent pathways

BACKGROUND AND PURPOSE Angiotensin-(1-7) [Ang-(1-7)] has anti-inflammatory effects in models of cardiovascular disease and arthritis, but its effects in asthma are unknown. We investigated whether Ang-(1-7) has anti-inflammatory actions in a murine model of asthma. EXPERIMENTAL APPROACH The effects of Ang-(1-7) alone or in combination with the MAS1 receptor antagonist, A779, were evaluated over a 4 day period in an ovalbumin-challenged mouse model of allergic asthma. On day 5, bronchoalveolar lavage was performed, and lungs were sectioned and assessed histologically for quantification of goblet cells, perivascular and peribronchial inflammation and fibrosis. Biochemical analysis of the pro-inflammatory ERK1/2 and IκB-α was assessed. In addition, the effect of Ang-(1-7) on proliferation of human peripheral blood mononuclear cells (HPBMC) was investigated. KEY RESULTS Ang-(1-7) attenuated ovalbumin-induced increases in total cell counts, eosinophils, lymphocytes and neutrophils. Ang-(1-7) also decreased the ovalbumin-induced perivascular and peribronchial inflammation, fibrosis and goblet cell hyper/metaplasia. Additionally, Ang-(1-7) reduced the ovalbumin-induced increase in the phosphorylation of ERK1/2 and IκB-α. These effects of Ang-(1-7) were reversed by the MAS1 receptor antagonist A779. Furthermore, Ang-(1-7) inhibited phytohaemagglutinin (PHA)-induced HPBMC proliferation. CONCLUSION AND IMPLICATIONS Ang-(1-7), via its MAS1 receptor, acts as an anti-inflammatory pathway in allergic asthma, implying that activation of the MAS1 receptor may represent a novel approach to asthma therapy.     

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action

Background and purpose

JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved.

Experimental approach

JZL184 and/or the CB₁ receptor antagonist, AM251 or the CB₂receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.

Key results

JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE₂ and PGD₂ were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.

Conclusion and implications

Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8     

Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E(2) production in human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE

Betulinic acid (BA) is a naturally occurring triterpenoid widely distributed throughout the plant kingdom. We previously reported that BA inhibits lipopolysaccharide (LPS)-induced interleukin-6 production through modulation of nuclear factor κB (NF-κB) in human peripheral blood mononuclear cells (hPBMCs). This study attempted to identify other mechanisms through which BA modulates LPS signalling in mononuclear cells. The effects of BA on signalling pathways downstream were focused on in this study.

EXPERIMENTAL APPROACH

We determined the ability of BA to interfere with p38 and extracellular regulated kinase (ERK) phosphorylation as well as Akt phosphorylation and nuclear factor-κB activation using LPS-activated hPBMCs as an in vitro model. LPS-induced endotoxin shock in mice was the in vivo model employed.

KEY RESULTS

BA inhibited LPS-induced COX-2 protein expression and prostaglandin E₂ production and also attenuated LPS-induced ERK and Akt phosphorylation, but not p38 in hPBMCs. BA abolished LPS-induced IκBα phosphorylation and thus normalized the levels of IκBα in cytosol. BA also inhibited LPS-induced reactive oxygen species formation and lactate dehydrogenase release. Interestingly, BA improved the life span of mice in endotoxin shock and also inhibited PGE₂ production and myeloperoxidase activity in vivo.

CONCLUSIONS AND IMPLICATIONS

BA modulates LPS-induced COX-2 expression in hPBMCs by inhibiting ERK and Akt pathways as well as by modulating IκBα phosphorylation. At the same time, no cell toxicity was observed. The effect of the drug was confirmed through in vivo experiments. The study gives an insight into the molecular mechanisms of BA.     

A nitric oxide/Ca(2+)/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose:

Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Experimental approach:

In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation.

Key results:

Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca²⁺ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca²⁺]_i.

Conclusions and implications:

These data identified the NO/Ca²⁺/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.     

Kynurenic acid attenuates multiorgan dysfunction in rats after heatstroke

Aim:

To assess whether systemic delivery of kynurenic acid improves the outcomes of heatstroke in rats.

Methods:

Anesthetized rats were divided into 2 major groups and given vehicle solution (isotonic saline 0.3 mL/kg rat weight) or kynurenic acid (30–100 mg in 0.3 mL saline/kg) 4 h before the start of thermal experiments. They were exposed to an ambient temperature of 43 °C for 68 min to induce heatstroke. Another group of rats were exposed to room temperature (26 °C) and used as normothermic controls. Their core temperatures, mean arterial pressures, serum levels of systemic inflammatory response molecules, hypothalamic values of apoptotic cells and neuronal damage scores, and spleen, liver, kidney and lung values of apoptotic cells were determined.

Results:

The survival time values during heatstroke for vehicle-treated rats were decreased from the control values of 475–485 min to new values of 83–95 min. Treatment with KYNA (30–100 mg/kg, iv) 4 h before the start of heat stress significantly and dose-dependently decreased the survival time to new values of 152–356 min (P<0.05). Vehicle-treated heatstroke rats displayed hypotension, hypothalamic neuronal degeneration and apoptosis, increased serum levels of tunor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and interleukin-10 (IL-10), and spleen, liver, kidney, and lung apoptosis. KYNA preconditioning protected against hypotension but not hyperthermia and attenuated hypothalamic neuronal degeneration and apoptosis during heatstroke. KYNA preconditioning attenuated spleen, kidney, liver, and lung apoptosis and up-regulated serum IL-10 levels but down-regulated serum TNF-α and ICAM-1 levels during heatstroke.

Conclusion:

Our results suggest that systemic delivery of kynurenic acid may attenuate multiorgan dysfunction in rats after heatstroke.     

Inhibition of glycogen synthase kinase-3beta attenuates the development of carrageenan-induced lung injury in mice

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) is a ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and has recently been implicated in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3beta inhibition in a model of acute inflammation. Here, we have investigated the effects of TDZD-8, a potent and selective GSK-3beta inhibitor, in a mouse model of carrageenan-induced pleurisy. EXPERIMENTAL APPROACH: Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: accumulation of fluid containing a large number of neutrophils (PMNs) in the pleural cavity, infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx), prostaglandin E2 (PGE2), tumour necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM-1 and P-selectin, iNOS, COX-2 as well as nitrotyrosine as determined by immunohistochemical analysis of lung tissues. KEY RESULTS: Administration of TDZD-8 (1, 3 or 10 mg kg(-1), i.p.), 30 min prior to injection of carrageenan, caused a dose-dependent reduction in all the parameters of inflammation measured. CONCLUSIONS AND IMPLICATIONS: Thus, based on these findings we propose that inhibitors of the activity of GSK-3beta, such as TDZD-8, may be useful in the treatment of various inflammatory diseases.     

Cryptotanshinone inhibits LPS-induced proinflammatory mediators via TLR4 and TAK1 signaling pathway

Cryptotanshinone (CTN), one of the major constituents of tanshinones, was investigated for anti-inflammatory activity in the murine macrophage cell line RAW 264.7. CTN inhibited the production of nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. Since CTN was considered as inhibiting LPS-triggered phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation, we consequently evaluated the expression of toll-like receptor 4 (TLR4) and CD14, as well as phosphorylation of TGF-β-activated kinase 1 (TAK1). CTN reduced the expression of CD14 and TLR4, and suppressed LPS-induced phosphorylation of TAK1. Furthermore, CTN significantly increased the survival rate against LPS challenge in D-galactosamine-sensitized mice, which was in line with in vitro results. These results suggested that CD14/TLR4 and TAK1 might be the potential molecular targets for addressing the protective effects of CTN on LPS-induced inflammatory effects in macrophages.     

Protection against lipopolysaccharide-induced sepsis and inhibition of interleukin-1beta and prostaglandin E2 synthesis by silymarin

Silymarin is known to have hepatoprotective and anticarcinogenic effects. Recently, anti-inflammatory effect of silymarin is attracting an increasing attention, but the mechanism of this effect is not fully understood. Here, we report that silymarin protected mice against lipopolysaccharide (LPS)-induced sepsis. In this model of sepsis, silymarin improved the rate of survival of LPS-treated mice from 6 to 38%. To further investigate the mechanism responsible for anti-septic effect of silymarin, we examined the inhibitory effect of silymarin on interleukin-1beta (IL-1beta) and prostaglandin E2 (PGE2) production in macrophages. Silymarin dose-dependently suppressed the LPS-induced production of IL-1beta and PGE2 in isolated mouse peritoneal macrophages and RAW 264.7 cells. Consistent with these results, the mRNA expression of IL-1beta and cyclooxygenase-2 was also completely blocked by silymarin in LPS-stimulated RAW 264.7 cells. Moreover, the LPS-induced DNA binding activity of nuclear factor-kappaB/Rel was also inhibited by silymarin in RAW 264.7 cells. Taken together, these results demonstrate that silymarin has a protective effect against endotoxin-induced sepsis, and suggest that this is mediated, at least in part, by the inhibitory effect of silymarin on the production of IL-1beta and PGE2.     

Curcumin inhibits LPS-induced inflammation in rat vascular smooth muscle cells in vitro via ROS-relative TLR4-MAPK/NF-κB pathways

Aim:

To investigate whether curcumin (Cur) suppressed lipopolysaccharide (LPS)-induced inflammation in vascular smooth muscle cells (VSMCs) of rats, and to determine its molecular mechanisms.

Methods:

Primary rat VSMCs were treated with LPS (1 μg/L) and Cur (5, 10, or 30 μmol/L) for 24 h. The levels of MCP-1, TNF-α, and iNOS were measured using ELISA and real-time RT-PCR. NO level was analyzed with the Griess reaction. Western-blotting was used to detect the activation of TLR4, MAPKs, IκBα, NF-κB p65, and the p47^phox subunit of NADPH oxidase in the cells.

Results:

Treatment of VSMCs with LPS dramatically increased expression of inflammatory cytokines MCP-1 and TNF-α, expression of TLR4 and iNOS, and NO production. LPS also significantly increased phosphorylation of IκBα, nuclear translocation of NF-κB (p65) and phosphorylation of MAPKs in VSMCs. Furthermore, LPS significantly increased production of intracellular ROS, and decreased expression of p47^phox subunit of NADPH oxidase. Pretreatment with Cur concentration-dependently attenuated all the aberrant changes in LPS-treated VSMCs. The LPS-induced overexpression of MCP-1 and TNF-α, and NO production were attenuated by pretreatment with the ERK inhibitor PD98059, the p38 MAPK inhibitor SB203580, the NF-κB inhibitor PDTC or anti-TLR4 antibody, but not with the JNK inhibitor SP600125.

Conclusion:

Cur suppresses LPS-induced overexpression of inflammatory mediators in VSMCs in vitro via inhibiting the TLR4-MAPK/NF-κB pathways, partly due to block of NADPH-mediated intracellular ROS production.     

Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway

Sulforaphane (SFN) is a natural isothiocyanate that is present in cruciferous vegetables such as broccoli and cabbage. Previous studies have shown that SFN is effective in preventing carcinogenesis induced by carcinogens in rodents, which is related in part to its potent anti-inflammation properties. In the present study, we compared the anti-inflammatory effect of SFN on LPS-stimulated inflammation in primary peritoneal macrophages derived from Nrf2 (+/+) and Nrf2 (−/−) mice. Pretreatment of SFN in Nrf2 (+/+) primary peritoneal macrophages potently inhibited LPS-stimulated mRNA expression, protein expression and production of TNF-α, IL-1β, COX-2 and iNOS. HO-1 expression was significantly augmented in LPS-stimulated Nrf2 (+/+) primary peritoneal macrophages by SFN. Interestingly, the anti-inflammatory effect was attenuated in Nrf2 (−/−) primary peritoneal macrophages. We concluded that SFN exerts its anti-inflammatory activity mainly via activation of Nrf2 in mouse peritoneal macrophages.