Curcumin attenuates oxidative stress following downhill running-induced muscle damage.

Downhill running causes muscle damage, and induces oxidative stress and inflammatory reaction. Recently, it is shown that curcumin possesses anti-oxidant and anti-inflammatory potentials. Interestingly, curcumin reduces inflammatory cytokine concentrations in skeletal muscle after downhill running of mice. However, it is not known whether curcumin affects oxidative stress after downhill running-induced muscle damage. Therefore, the purpose of this study was to investigate the effects of curcumin on oxidative stress following downhill running induced-muscle damage. We also investigated whether curcumin affects macrophage infiltration via chemokines such as MCP-1 and CXCL14. Male C57BL/6 mice were divided into four groups; rest, rest plus curcumin, downhill running, or downhill running plus curcumin. Downhill running mice ran at 22 m/min, -15% grade on the treadmill for 150 min. Curcumin (3mg) was administered in oral administration immediately after downhill running. Hydrogen peroxide concentration and NADPH-oxidase mRNA expression in the downhill running mice were significantly higher than those in the rest mice, but these variables were significantly attenuated by curcumin administration in downhill running mice. In addition, mRNA expression levels of MCP-1, CXCL14 and F4/80 reflecting presence of macrophages in the downhill running mice were significantly higher than those in the rest mice. However, MCP-1 and F4/80 mRNA expression levels were significantly attenuated by curcumin administration in downhill running mice. Curcumin may attenuate oxidative stress following downhill running-induced muscle damage.

Alpha-tocopheryl succinate induces rapid and reversible phosphatidylserine externalization in histiocytic lymphoma through the caspase-independent pathway.

Phosphatidylserine (PS) externalization is a key feature of apoptotic cell death and plays an important role in clearance of apoptotic cells by phagocytes. PS externalization during apoptosis is generally an irreversible event mediated by caspase activation and is accompanied by other apoptotic events. We report here that an apoptosis inducer alpha-tocopheryl succinate (TOS) can induce PS externalization that is independent of apoptosis and reversible in the absence of fetal bovine serum (FBS) in histiocytic lymphoma U937 cells. In the presence of FBS, TOS induced PS externalization via a caspase-dependent mechanism accompanied by mitochondrial depolarization, cell shrinkage, increase of caspase-3 activity, and chromatin condensation. In contrast, in the absence of FBS, TOS induced the rapid PS externalization which was not accompanied by other apoptotic events. The PS externalization was reversible by removing TOS and was not involved in Ca(2+)-dependent scramblase activation and thiol oxidation of aminophospholipid translocase. A similar PS externalization was also induced by cholesteryl hemisuccinate (CS), the other succinate ester. These results suggested that the mechanism of TOS- and CS-induced PS externalization in the absence of FBS was different from it occurring during typical apoptosis.

Exhaustive exercise reduces tumor necrosis factor-alpha production in response to lipopolysaccharide in mice.

OBJECTIVE: Stressful exercise reduces the plasma pro-inflammatory cytokine concentration in response to lipopolysaccharide (LPS). The aim of this study was to clarify the mechanism of exhaustive exercise-induced suppression of the plasma tumor necrosis factor (TNF)-alpha concentration in response to LPS. METHODS: Male C3H/HeN mice (n = 66) were randomized to treadmill running to exhaustion (Ex) or a sedentary (Non-Ex) condition. Monocytes and splenic macrophages were collected from some animals, and other animals were injected with LPS (1 mg/kg) immediately after the exercise. The liver, lung and spleen tissues in the mice were removed 30 min after the LPS injection for determination of TNF-alpha mRNA expression. Blood and tissue samples were collected for determination of TNF-alpha and TNF receptors (TNFR) 1 h after the LPS injection. RESULTS: Although there was a significant suppression in LPS-induced plasma TNF-alpha in the Ex mice when compared to the Non-Ex mice (p < 0.01), soluble TNFR in plasma was not affected by the exercise. There was no change in cell-surface expression of Toll-like receptor 4 (TLR4) and in LPS-induced TNF-alpha mRNA expression and TNFR content in tissues between the Ex and Non-Ex groups. Interestingly, TNF-alpha contents in the liver, lung and spleen of the Ex mice were significantly lower than those of the Non-Ex group (p < 0.01, p < 0.01 and p < 0.05, respectively). CONCLUSION: These data suggest that exhaustive exercise-induced suppression of the plasma TNF-alpha concentration despite LPS stimulation might depend on translation of TNF-alpha in tissues.

Exhaustive exercise reduces TNF-α and IFN-α production in response to R-848 via toll-like receptor 7 in mice.

Stressful exercise results in temporary immune depression. However, the impact of exercise on the immune responses via toll-like receptor (TLR) 7, which recognizes the common viral genomic feature, single-stranded RNA, remains unclear. To clarify the effect of stressful exercise on immune function in response to viral infection, we measured the changes in the plasma concentration of tumor necrosis factor (TNF)-α and interferon (IFN)-α, which are induced downstream from the TLR-ligand interaction, in exhaustive-exercised mice immediately after treatment with the imidazoquinoline R-848, which can bind to and activate TLR7. Both exhaustive-exercised (EX) and non-exercised (N-EX) male C3H/HeN mice were injected with R-848 (5 mg kg(-1)), and blood samples were collected. In addition, RAW264 cells, which are mouse macrophage cells, were cultured 30 min after epinephrine (10 µM) or norepinephrine (10 µM) treatments, and were then stimulated with R-848 (10 µg ml(-1)). In addition, the effect of propranolol (10 mg kg(-1)) as blockade of ß-adrenergic receptors on R-848-induced TNF-α and IFN-α production in the exercised mice was examined. Both the TNF-α and IFN-α concentrations in the plasma of EX were significantly lower than those in the plasma of N-EX after R-848 injection (P < 0.05 and P < 0.01, respectively), although the R-848 treatment increased the plasma TNF-α and IFN-α concentrations in both groups (P < 0.01, respectively). The R-848-induced TNF-α production in RAW264 cells was significantly inhibited by epinephrine and norepinephrine pre-treatment, although IFN-α was not detected. The propranolol treatment completely inhibited exercise-induced TNF-α and IFN-α suppression in response to R-848 in the mice. These data suggest that EX induces a reduction in TNF-α and IFN-α production in response to R-848, and that these phenomena might be regulated by an exercise-induced elevation of the systemic catecholamines.

High dose of lipopolysaccharide pre-treatment prevents OVA-induced anaphylactic decreases in rectal temperature in the immunized mice.

It remains unclear whether lipopolysaccharide (LPS) pre-treatment, which prevents Th2-type responses via Toll-like receptor 4 (TLR4), inhibits anaphylaxis. To determine the dose-dependent effects of LPS pre-treatment on anaphylactic decreases in rectal temperature caused by ovalbumin (OVA) re-exposure in immunized mice, C3H/HeN mice were divided into vehicle/OVA (0 mg/kg LPS), L-LPS/OVA (0.5 mg/kg LPS), M-LPS/OVA (1.0 mg/kg LPS) and H-LPS/OVA (3.0 mg/kg LPS) groups. After receiving these treatments, the mice were systemically immunized with OVA. Negative control mice were not immunized with OVA (N-OVA). After measuring the serum levels of OVA-specific IgE and IgG1 antibodies, the mice were examined for changes in their rectal temperature and plasma histamine concentration after OVA re-exposure. The allergen-specific IgE and IgG1 concentrations in sera from L-LPS/OVA, M-LPS/OVA and H-LPS/OVA mice were significantly lower than those in sera from vehicle/OVA mice despite OVA immunization. However, the antibody levels in all OVA-immunized mice, with the exception of the IgG1 levels in H-LPS/OVA mice, were significantly higher than those in N-OVA mice. Interestingly, H-LPS/OVA mice were the only group that did not exhibit a decrease in rectal temperature, since the rectal temperatures in vehicle/OVA, L-LPS/OVA and M-LPS/OVA mice were significantly decreased by OVA re-exposure. Furthermore, the decrease in rectal temperature after OVA re-exposure in L-LPS/OVA mice, which did not exhibit an increase in the plasma histamine concentration, was significantly prevented by treatment with a platelet-activating factor (PAF) receptor antagonist alone. Taken together, the present results indicate that high-dose LPS pre-treatment may prevent anaphylaxis in OVA-immunized mice, and that this mechanism may depend on inhibition of the IgG-PAF pathway rather than the IgE-histamine pathway.

Salmonella administration induces a reduction of wheel-running activity via a TLR5-, but not a TLR4, dependent pathway in mice.

In general, systemic bacterial infections induce sickness behavior. In mice, lipopolysaccharide (LPS), a component of gram-negative bacteria, strongly reduces physical activity via toll-like receptor (TLR) 4. However, gram-negative bacteria, such as Salmonella, also express flagella containing flagellin (FG) which binds to TLR5 and induces pro-inflammatory cytokine production. It is unclear whether FG induces sickness behavior. To determine whether Salmonella administration regulates the reduction of voluntary physical activity in mice, male C3H/HeN (wild type) and C3H/HeJ (tlr4 gene mutated) mice were administered living Salmonella (live) and examined for wheel-running activity. The production of TNF-alpha in RAW 264 cells was measured by the ELISA assay under both live and heat-killed (HK) Salmonella conditions in vitro. Wheel-running activity in both C3H/HeJ and C3H/HeN mice after i.p. injection of live Salmonella (1 x 10(6) CFU/kg) was significantly lower than that in vehicle groups (p < 0.01, respectively), although wheel-running activity in C3H/HeJ mice was not reduced after i.p. injection of HK Salmonella (1 x 10(6) CFU/kg). Furthermore, TNF-alpha production from RAW 264 cells with HK Salmonella treatment at the early phase was higher than that with live Salmonella treatment. Interestingly, gentamicin-treated (GMT) Salmonella, (which have bacterial flagella removed), did not induce reduction of wheel-running activity, although injection of the flagella-rich supernatant of GMT Salmonella significantly reduced it (p < 0.01). Indeed, FG treatment also induced reduction of wheel-running activity in mice (p < 0.01). Our findings suggest that the Salmonella-induced reduction of voluntary physical activity might be regulated by FG via TLR5, but not LPS via TLR4 in mice.

The reduction of voluntary physical activity after poly I:C injection is independent of the effect of poly I:C-induced interferon-beta in mice.

One characteristic of sickness behavior in mice is demonstrated by a reduction in voluntary wheel-running activity during infection. Among synthetic double-stranded (ds) RNAs, polyriboinosinic: polyribocytidylic acid (poly I:C) activates to produce interferon (IFN) -beta, which plays an important role in anti-viral activity and host-defense. However, how voluntary wheel-running activity is regulated during poly I:C infection is unknown. To determine whether poly I:C-induced IFN-beta production is responsible for reduced spontaneous physical activity, we measured poly I:C-induced changes in voluntary wheel-running activity in mice. In this experiment, the mice were injected with poly I:C (0-5 mg/kg i.v.) and/or anti-IFN-beta neutralizing antibody (1.5x10(5) U/kg i.v.). We also observed the direct effect of injection of recombinant IFN-beta (rIFN-beta: 5.0x10(4) and 2.5x10(5) U/kg) on wheel-running behavior. Poly I:C treatment dose-dependently reduced wheel-running activity, and induced an increase in plasma IFN-beta in mice. However the activity was not attenuated by the neutralizing antibody specific to IFN-beta treatment. Additionally, the wheel-running activity in rIFN-beta treated mice was maintained, although they showed a higher IFN-gamma inducible protein (IP)-10 concentration in plasma compared with that of the vehicle group. Our results suggest that the transient reduction in physical activity after poly I:C injection is induced dose dependently, but that the mediator might not be poly I:C-induced IFN-beta.

Lipopolysaccharide-induced monocyte chemotactic protein-1 is enhanced by suppression of nitric oxide production, which depends on poor CD14 expression on the surface of skeletal muscle.

It is known that lipopolysaccharide (LPS)-induced monocyte chemotactic protein (MCP)-1 secretion from tissues recruits monocytes from the circulation, but the mechanism of the LPS-induced MCP-1 production in skeletal muscle is largely unexplained. To clarify the effect of LPS on MCP-1 production in skeletal muscle cells, C2C12 cells from a mouse skeletal muscle cell line, and RAW 264.7 cells from a mouse macrophage cell line, were used to assess production of LPS-induced MCP-1, nitric oxide (NO) and interferon (IFN)-beta. In addition, we evaluated inducible NO synthases (iNOS) mRNA expression using RT-PCR, and cell surface expression of CD14 and toll-like receptor (TLR) 4 using flow cytometry. In C2C12 cells, LPS stimulation increased MCP-1 production (p < 0.01), but combined treatment with LPS and NO inducer, diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (NONOate), significantly inhibited its production (p < 0.01). LPS stimulation neither induced production of NO nor of IFN-beta, which is an NO inducer. Recombinant IFN-beta stimulation, on the other hand, enhanced LPS-induced NO production (p < 0.01). Interestingly, we found that surface expression of CD14, which regulates IFN-beta production, in C2C12 cells was much lower than that in RAW 264.7 cells, although TLR4 expression on C2C12 cells was similar to that on RAW 264.7 cells. These data suggest that the reduced NO production in response to LPS may depend on low expression of CD14 on the cell surface of skeletal muscle, and that it may enhance LPS-induced MCP-1 production. Together, these functions of skeletal muscle could decrease the risk of bacterial infection by recruitment of monocytes.

Beta-adrenergic receptor blockade attenuates the exercise-induced suppression of TNF-alpha in response to lipopolysaccharide in rats.

Stressful exercise has been found to reduce the proinflammatory cytokine response to lipopolysaccharide (LPS). In this study, we aimed to determine whether receptor antagonists for corticosterone or catecholamines would increase the LPS-induced tumor necrosis factor-alpha (TNF-alpha) response after exhaustive exercise. Female F344 rats were randomly assigned to one of five groups: control (vehicle), RU-486 (glucocorticoid receptor antagonist, 30 mg/kg)-, propranolol [nonselective beta-adrenergic receptor (AR) blockade, 30 mg/kg]-, atenolol (beta(1)-AR blockade, 30 mg/kg)-, or ICI 118551 (beta(2)-AR blockade, 30 mg/kg)-treated groups. Each antagonist was given intraperitoneally 30 min prior to exercise or control period. Exercised rats ran until exhaustion on a treadmill at gradually increasing speeds, from 10 to 36 m/min at 15% grade. Immediately postexercise or control period all rats were injected with LPS (1 mg/kg, i.v.). Plasma TNF-alpha was reduced by prior exercise to approximately 10% of that of sedentary controls (p < 0.01). Plasma TNF-alpha concentration in exercised RU-486-treated rats was significantly different than that of nonexercised rats (19.2%, p < 0.01) and not different from exercised rats. However, pretreatment of rats with the nonselective beta-AR blocker propranolol almost completely reversed the exercise-induced suppression of plasma TNF-alpha in response to LPS. beta(1)-AR pretreatment almost completely attenuated the exercise-induced suppression of LPS-induced plasma TNF-alpha while beta(2)-antagonism had a partial effect. These results indicate that exercise-induced catecholamines, acting through beta-ARs (especially the beta(1)-AR), are responsible for the exercise-induced suppression of plasma TNF-alpha after LPS administration.