Suppressive effects of levobupivacaine on endotoxin-induced microglial activation

Background

We sought to elucidate the effects of levobupivacaine on modulating endotoxin-induced upregulation of inflammatory mediators and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways in activated microglia.

Materials and methods

Confluent murine microglia (BV-2) were treated with endotoxin (lipopolysaccharide, 50 ng/mL) or endotoxin plus levobupivacaine (5, 25, or 50 μM) and denoted as the LPS, LPS + L(5), LPS + L(25), and LPS + L(50) groups, respectively. Levobupivacaine was administered immediately after endotoxin. Control groups were run simultaneously.

Results

The concentrations of inflammatory mediators, including macrophage inflammatory protein-2 (P = 0.023 and 0.016), tumor necrosis factor-α (P = 0.025 and 0.020), interleukin (IL)-1β (P = 0.018 and 0.014), IL-6 (P = 0.029 and 0.023), nitric oxide (P = 0.025 and 0.026), and prostaglandin E₂ (P = 0.028 and 0.016) of the LPS + L(25) and LPS + L(50) groups were significantly lower than those of the LPS group. The concentrations of macrophage inflammatory protein-2 (P = 0.035), IL-1β (P = 0.024), nitric oxide (P = 0.031), and prostaglandin E₂ (P = 0.036) but not tumor necrosis factor-α and interleukin-6 of the LPS + L(5) group were also significantly lower than those of the LPS group. These data revealed that effects of endotoxin on upregulating inflammatory mediators were inhibited by levobupivacaine. Moreover, effects of endotoxin on activating NF-κB, including inhibitor-κB degradation, NF-κB nuclear translocation, and NF-κB–DNA binding, were also inhibited by levobupivacaine. Similarly, effects of endotoxin on activating MAPKs, including extracellular signal–regulated kinase, c-jun N-terminal kinase, and p38 MAPK, were also significantly inhibited by levobupivacaine.

Conclusions

Levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators and activation of NF-κB and MAPKs signaling pathways in activated microglia.     

Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells

Background

Tea brewed from the leaves of persimmon or Rosa agrestis have several medical functions including treating allergy, antiatopic dermatitis, and anti-inflammatory effects. The objective of this study was to investigate the molecular mechanisms of astragalin, a main flavonoid component isolated from these herbs, in modifying lipopolysaccharide (LPS)-induced signaling pathways in primary cultured mouse mammary epithelial cells (mMECs).

Materials and methods

The mMECs were treated with LPS in the absence or presence of different concentrations of astragalin. The expression of proinflammatory cytokines tumor necrosis factor α, and interleukin 6, as well as nitric oxide production were determined by enzyme-linked immunosorbent assay and Griess reaction, respectively. Cyclooxygenase-2, inducible nitric oxide synthase, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), inhibitor protein of NF-κB (IκBα), P38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were measured by Western blot.

Results

The results showed that astragalin suppressed the expression of tumor necrosis factor α, interleukin 6, and nitric oxide in a dose-dependent manner in mMECs. Western blot results showed that the expression of inducible nitric oxide synthase and cyclooxygenase-2 was inhibited by astragalin. Besides, astragalin efficiently decreased LPS-induced TLR4 expression, NF-κB activation, IκBα degradation, and the phosphorylation of p38, extracellular signal-regulated kinase in BMECs.

Conclusions

Our results indicated that astragalin exerts anti-inflammatory properties possibly via the inactivation of TLR4-mediated NF-κB and mitogen-activated protein kinases signaling pathways in LPS-stimulated mMECs. Thus, astragalin may be a potential therapeutic agent for bovine mastitis.     

Isotetrandrine protects against lipopolysaccharide-induced acute lung injury by suppression of mitogen-activated protein kinase and nuclear factor-kappa B

Background

Mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways are pleiotropic regulator of many genes involved in lipopolysaccharide (LPS)-induced acute lung injury (ALI). The present study aimed to reveal the protective effect of isotetrandrine (ITD), a small molecule inhibitor, on various aspects of LPS-induced inflammation in vitro and in vivo.

Methods

In vitro, RAW 264.7 cells were pretreated with different dose of ITD 1 h before treatment with 1 mg/L of LPS. In vivo, to induce ALI, male BALB/c mice were injected intranasally with LPS and treated with ITD (20 and 40 mg/kg) 1 h before LPS.

Results

In vitro, the cytokine levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in supernatant were reduced by ITD. Meanwhile, in vivo, pulmonary inflammatory cell infiltration, myeloperoxidase activity, total cells, neutrophils, macrophages, along with the levels of tumor necrosis factor-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid were dose-dependently attenuated by ITD. Furthermore, our data showed that ITD significantly inhibited the activation of MAPK and NF-κB, which are induced by LPS in ALI model.

Conclusions

These results suggested that ITD dose-dependently suppressed the severity of LPS-induced ALI by inactivation of MAPK and NF-κB, which may involve the inhibition of tissue oxidative injury and pulmonary inflammatory process.     

Angelicin regulates LPS-induced inflammation via inhibiting MAPK/NF-κB pathways

Background

Angelicin is a furocoumarin found in Psoralea corylifolia L. fruit. The purpose of this study was to investigate the protective ability of angelicin against inflammation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-induced in vivo acute lung injury model.

Materials and methods

The concentrations of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 in the culture supernatants of RAW 264.7 cells were determined 24 h after LPS administration. ALI was induced by intratracheal instillation of LPS. Six hours after LPS inhalation, bronchoalveolar lavage fluid and lung tissue samples were obtained for enzyme-linked immunosorbent assay, histologic, and Western blotting analyses.

Results

The results showed that pretreatment with angelicin markedly downregulated TNF-α and IL-6 levels in vitro and in vivo, and significantly decreased the amount of inflammatory cells, lung wet-to-dry weight ratio, and myeloperoxidase activity in LPS-induced ALI mice. Furthermore, Western blotting analysis results demonstrated that angelicin blocked the phosphorylation of IκBα, NF-κBp65, p38 MAPK, and JNK in LPS-induced ALI.

Conclusions

These results suggest that angelicin was potentially advantageous to prevent inflammatory diseases by inhibiting NF-κB and MAPK pathways. Our data indicated that angelicin might be a potential new agent for prevention of inflammatory reactions and diseases in the clinic.     

Small Interfering RNA Targeting Nuclear Factor Kappa B to Prevent Vein Graft Stenosis in Rat Models

Background

Intimal hyperplasia plays an important role in vein graft stenosis. Inflammatory injury, especially nuclear factor kappaB (NF-κB) gene activation, is highly involved in stenosis progression. We examined whether neointimal hyperplasia and vein graft stenosis could be inhibited by silencing the NF-κB gene with small interference RNA (siRNA).

Methods

Sixty adult male Sprague-Dawley rats were randomly divided into a normal vein group, a vein graft group, a scrambled siRNA group, and an NF-κB siRNA group. We performed reverse interpositional grafting of the autologous external jugular vein to the abdominal aorta. Vein grafts were treated with liposome and gel complexes containing NF-κB siRNA or scrambled siRNA. The levels of monocyte chemoattractant protein -1, tumor necrosis factor-α, and NF-κB p65 in vessel tissues were evaluated after surgery for content of proliferating cell nuclear antigen (PCNA) and vascular wall thickness.

Results

NF-κB siRNA treated vein graft showed less neointimal formation and fewer positive PCNA cells (P < .05). In addition there were lower levels of, NF-κB p65 protein and of inflammatory mediators (P < .05) compared with the vein graft group.

Conclusion

Our study suggested that siRNA transfection suppressed NF-κB expression, reduced inflammatory factors, lessened neointimal proliferation, and suppressed PCNA.     

Lidocaine attenuates lipopolysaccharide-induced inflammatory responses in microglia

Background

Lidocaine has been used as a local anesthetic with anti-inflammatory properties, but its effects on neuroinflammation have not been well defined. In the present study, we investigated the prophylactic effects of lidocaine on lipopolysaccharide (LPS)-activated microglia and explored the underlying mechanisms.

Materials and methods

Microglial cells were incubated with or without 1 μg/mL LPS in the presence or absence of lidocaine, a p38 mitogen–activated protein kinase (p38 MAPK) inhibitor (SB203580), a nuclear factor-kappa B (NF-κB) inhibitor (pyrrolidine dithiocarbamate), or small interfering RNA. The protein and expression levels of inflammatory mediators, such as monocyte chemotactic protein 1, nitric oxide, prostaglandin E₂, interleukin 1β, and tumor necrosis factor α were measured using enzyme-linked immunosorbent assays and real-time polymerase chain reaction. The effect of lidocaine on NF-κB and p38 MAPK activation was evaluated using enzyme-linked immunosorbent assays, Western blot analysis, and electrophoretic mobility shift assay.

Results

Lidocaine (≥2 μg/mL) significantly inhibited the release and expression of nitric oxide, monocyte chemotactic protein 1, prostaglandin E₂, interleukin 1β, and tumor necrosis factor α in LPS-activated microglia. Treatment with lidocaine also significantly inhibited the phosphorylation of p38 MAPK and the nuclear translocation of NF-κB p50/p65, increased the protein levels of inhibitor kappa B-α. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by SB203580, pyrrolidine dithiocarbamate, and small interfering RNA.

Conclusions

Prophylactic treatment with lidocaine inhibits LPS-induced release of inflammatory mediators from microglia, and these effects may be mediated by blockade of p38 MAPK and NF-κB signaling pathways.     

Levobupivacaine inhibits lipopolysaccharide-induced high mobility group box 1 release in vitro and in vivo

Background

The aim of the study was to investigate whether levobupivacaine (LB) suppressed lipopolysaccharide (LPS)-induced high mobility group box 1 (HMGB1) release in vitro and in vivo, and to determin its molecular mechanisms of action.

Materials and methods

RAW264.7 cells were treated with LPS and LB for 24 h. Levels of HMGB1, nuclear factor-kappa B (NF-κB) and phosphorylated p38 mitogen-activated protein kinase (MAPK) were measured by Enzyme-linked immunosorbent assay and Western blotting; the levels of HMGB1 messenger RNA were measured by real-time polymerase chain reaction. In addition, cecal ligation and puncture–induced septic C57BL/6 received LB infusion, and the levels of HMGB1 and functional parameters of multiple organs determined using several detection kits.

Results

LB inhibited HMGB1 release in vitro and in vivo. Furthermore, LB inhibited the translocation of NF-κB and phosphorylation of p38 MAPK in vitro. Mice treated with LB infusion improved survival in mice and significantly reduced cecal ligation and puncture–induced dysfunction of organs.

Conclusions

LB suppresses LPS-induced HMGB1 release in vitro and in vivo by partially inhibiting NF-κB/p38 MAPK pathways. LB can rescue mice from sepsis and protect against organ dysfunction in septic mice.     

Potential protective effect of nuclear factor-κB decoy oligodeoxynucleotides on endotoxin-induced liver injury

Purpose

We sought to study the protective effects of nuclear factor–κB decoy oligodeoxynucleotides (ODNs) on endotoxin-induced liver injury in a rat model.

Methods

Sixty Sprague-Dawley rats were randomly divided into a control (n = 20), a lipopolysaccharide (LPS) (n = 20), and an NF-κB decoy ODN group (n = 20). Liver and blood serum samples were collected at 24 hours after the operation. NF-κB binding activity was detected by an electrophoretic mobility shift assay, liver histopathology, by light microscopy; and cell apoptosis, by a terminal-deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. The serum of liver enzyme (aspartate transaminase [AST]) levels were measured using an automated biochemical analyzer and tumor necrosis factor (TNF)–α and interleukin (IL)-6 by enzyme-linked immunosorbent assays.

Results

NF-κB was dramatically activated after endotoxin-induced liver injury. Many hepatocytes underwent degeneration and necrosis in the LPS group. The expressions of AST, TNF-α, and IL-6 were significantly increased compared with the control group (P = .0005), However, NF-κB decoy ODNs altered these undesirable changes. On the other hand, IL-6 expression was not significantly decreased by the NF-κB decoy versus the LPS group (P = .0745).

Conclusions

NF-κB decoy strategy inhibited the binding activity of NF-κB, thus suppressing production of downstream cytokines which play crucial roles in protection from endotoxin-induced injury.     

Resveratrol ameliorates subacute intestinal ischemia-reperfusion injury

Background

Resveratrol has been shown to attenuate reactive oxygen species formation and protect against ischemia-reperfusion (I/R) injury. However, the effects of resveratrol against subacute intestinal I/R injury are not clearly elucidated. Therefore, this study was designed to investigate the effects and possible protective mechanisms of resveratrol on subacute intestinal I/R injury in mice.

Methods

BALB/c mice were subjected to 1 h ischemia by occluding the superior mesenteric artery and 24 h reperfusion. Histologic injury; myeloperoxidase, superoxide dismutase, and glutathione peroxidase activity; malondialdehyde level; inducible nitric oxide synthase (iNOS), Ac-NF-κBp65, and sirtuin 1 (SIRT1) expression; NF-κB translocation; and nitric oxide (NO) production were examined in treated with or without resveratrol in the absence or presence of pharmacologic inhibitors.

Results

Resveratrol significantly ameliorated subacute intestinal I/R injury accompanied with the decrease of NO production as well as iNOS expression. In addition, resveratrol obviously upregulated the expression of SIRT1 and inhibited the activity of NF-κB. After application of iNOS inhibitor S-methylisothiourea and NF-κB inhibitor pyrrolidine dithiocarbamate, the protective effect of resveratrol was significantly augmented by attenuating iNOS and NO production, indicating that resveratrol exerted its protective effect on intestinal I/R injury via NF-κB-mediated iNOS pathway. Furthermore, the protective effect of resveratrol was correlated with SIRT1, because application of SIRT1 inhibitor nicotinamide strikingly weakened the protective effect of resveratrol.

Conclusions

Taken together, our findings showed that resveratrol protects intestinal subacute I/R injury via the SIRT1-NF-κB pathway in an iNOS-NO-dependent manner. Therefore, resveratrol has a potential clinical prospect for further development of anti-injury therapy.     

Effects of sleep deprivation on pain-related factors in the temporomandibular joint

Background

The objective of this study was to investigate the effects of experimental sleep deprivation (SD) on the temporomandibular joint (TMJ) in rats by examining pain-related factors and to determine the possible involvement of estrogen and NF (nuclear factor) κB signaling in the TMJ synovial membrane.

Methods

The influence of SD, conducted in rats using the modified multiple platform method, was estimated by observing behavioral manifestations and examining changes in serum hormone levels. The morphologic changes of synovial tissue were observed with light microscopy and the serum levels of estrogen were measured by radioimmunoassay. Activation of NF-κB in the synovial membrane was examined using an immunofluorescence technique, and the expression levels of interleukin (IL) 1β, IL-6, tumor necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase were measured with real-time polymerase chain reaction.

Results

The SD group showed evidence of elevated anxiety and stress, and increased plasma levels of estradiol compared with the control group. The activity of NF-κB was significantly enhanced and translocation of NF-κB p65 was evident in the synovial membrane after SD. The expression of pain-related factors IL-1β, IL-6, cyclooxygenase-2, tumor necrosis factor α, and inducible nitric oxide synthase in the synovial membrane significantly increased after SD.

Conclusions

These results indicate that SD increases serum levels of estrogen and induces alterations in pain-related factors in the TMJ. The NF-κB pathway has been associated with the regulation of these inflammatory cytokines and plays an important role in temporomandibular disorders.     

Inhibitory effects of rosiglitazone on lipopolysaccharide-induced inflammation in a murine model and HK-2 cells

Background: Inflammation may play an important role in the pathogenesis of kidney disease. Agonists of the peroxisome proliferator-activated receptor-γ (PPAR-γ), such as rosiglitazone, have been recently demonstrated to regulate inflammation by modulating the production of inflammatory mediators. The purpose of this study was to examine the effects of rosiglitazone on lipopolysaccharide (LPS)-induced kidney inflammation and to explore the mechanism of its renoprotection. Methods: Mice were treated with LPS with or without pretreatment with rosiglitazone. Blood urea nitrogen (BUN), creatinine levels, the urinary albumin-to-creatinine ratio, macrophage infiltration, monocyte chemoattractant protein-1 (MCP-1) expression, PPAR-γ expression, and NF-κB and PPAR-γ activity were investigated. HK-2 cells were maintained under defined in vitro conditions, treated with either rosiglitazone and/or the PPAR-γ antagonist GW9662, and then stimulated with LPS. MCP-1, IL-8, IL-6, NF-κB activity and PPAR-γ expression were investigated. Results: Compared to the LPS only group, pretreatment with rosiglitazone in vivo significantly attenuated the BUN levels macrophage infiltration, MCP-1 overexpression and NF-κB activity (p < 0.05). Rosiglitazone also restored PPAR-γ expression and protein activity, which were reduced significantly in the LPS only group (p < 0.05). Furthermore, in the LPS-stimulated HK-2 cells, rosiglitazone downregulated MCP-1, IL-8 and IL-6 expression as well as NF-κB activation and increased PPAR-γ expression (p < 0.05). These effects were diminished by GW9662. Conclusion: These results showed that pretreatment with rosiglitazone could attenuate kidney inflammation through the activation of PPAR-γ, suppression of MCP-1 overproduction and NF-κB activation. Rosiglitazone had a protective effect via a PPAR-γ-dependent pathway in LPS-treated HK-2 cells.