Gambogic acid protects from endotoxin shock by suppressing pro-inflammatory factors in vivo and in vitro

Objective

Gambogic acid (GBA) targeted Heat shock protein 90 (Hsp90) and prohibited TNF-α/NF-κB signaling pathway. It can be inferred that the anti-inflammatory activity of GBA results from inhibiting the cytokine production via NF-κB signaling pathway. We used the RAW264.7 cell line and the endotoxin shock mouse model to confirm the hypothesis that GBA protects mice from endotoxin shock by suppressing cytokine synthesis.

Method

RAW264.7 cells were cultured and the endotoxin shocked mice model was constructed. ELISA was employed to evaluate the change of cytokine secretion levels. The effects of GBA on the activation of NF-κB signaling pathway were also determined by western blot and immune-fluorescent analysis. Cell viability was determined by MTT assay, and the cell migration was tested by wound healing assay.

Result

Our results demonstrated that GBA significantly inhibited the LPS-induced release of pro-inflammatory factors both in cell lines and mice serum, thereby protecting mice from endotoxin shock. Furthermore, we observed that the reduction of inflammatory cytokines interleukin 1-beta, interleukin 6 and TNF-α resulted from the Hsp90’s client protein IKK degradation and the suppression of NF-κB pathway. Moreover, GBA suppressed the migration of LPS-induced RAW264.7 cells.

Conclusion

Our results indicate that GBA has a potential both as an antitumor and anti-inflammatory therapeutic agent.     

Anti-inflammatory effects of Hwang-Heuk-San,a traditional Korean herbal formulation,on lipopolysaccharide-stimulated murine macrophages

Background

Hwang-Heuk-San (HHS), a Korean traditional herbal formula comprising four medicinal herbs, has been used to treat patients with inflammation syndromes and digestive tract cancer for hundreds of years; however, its anti-inflammatory potential is poorly understood. The aim of the present study was to investigate the anti-inflammatory effects of HHS using a lipopolysaccharide (LPS)-activated RAW 264.7 macrophage model.

Methods

The inhibitory effects of HHS on LPS-induced nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production were examined using Griess reagent and enzyme-linked immunosorbent assay (ELISA) detection kits. The effects of HHS on the expression of inducible NO synthase (iNOS), IL-1β and TNF-α, their upstream signal proteins, including nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and activator protein (AP-1), were also investigated.

Results

A noncytotoxic concentration of HHS significantly reduced the production of NO, IL-1β and TNF-α in LPS-stimulated RAW 264.7 cells, which was correlated with reduced expression of iNOS, IL-1β and TNF-α at the mRNA and protein levels. HHS efficiently blocked the phosphorylation of MAPKs, especially that of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not that of the p38 MAPK. The reduced production of inflammatory molecules by HHS was followed by decreased activity of NF-κB and AP-1.

Conclusions

These results suggest that HHS may offer therapeutic potential for treating inflammatory diseases accompanied by macrophage activation.

     

Protective effect of gossypol on lipopolysaccharide-induced acute lung injury in mice

Objective

Gossypol has been reported to have anti-inflammatory properties. The purpose of this study was to evaluate the effect of gossypol on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice.

Methods

Male BALB/c mice were pretreated with gossypol 1 h before intranasal instillation of LPS. Then, 7 h after LPS administration, the myeloperoxidase in histology of lungs, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were determined. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 were detected by western blot.

Results

Gossypol markedly attenuated the LPS-induced histological alterations in the lung and inhibited the production of TNF-α, IL-1β and IL-6. Additionally, gossypol reduced the inflammatory cells in BALF, decreased the wet/dry ratio of lungs and inhibited the phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 caused by LPS.

Conclusion

The data suggest that anti-inflammatory effects of gossypol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPKs signaling pathways. Gossypol may be a promising potential therapeutic reagent for ALI treatment.     

Effects of exogenous annexin-1 on lipopolysaccharide-induced proliferation and reactive oxygen species production partially through modulation of CRAC channels but independent of NF-κB pathway

Objective

To investigate the effects of exogenous annexin-1 (ANXA1) on lipopolysaccharide(LPS)-induced proliferation, reactive oxygen species (ROS) production, and calcium signal transduction in RAW264.7 macrophages.

Methods

RAW264.7 macrophages were treated with or without LPS in the absence or presence of ANXA1. The proliferation effects were detected by Cell Counting Kit-8 assay. ROS were quantified by flow cytometry and fluorescence microscopy. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was analyzed by laser confocal scanning microscopy. IκBα degradation and NF-κB translocation were tested by Western blot.

Results

Exogenous ANXA1 inhibited LPS-induced proliferation and ROS production in a dose-dependent manner. LPS evoked [Ca²⁺]_i increase through CRAC channels, and ANXA1 suppressed LPS-induced [Ca²⁺]_i increase in a dose-dependent manner. The CRAC channels were associated with LPS-induced proliferation and ROS production. Exogenous ANXA1 had no effect on LPS-induced IκB degradation and NF-κB translocation.

Conclusions

ANXA1 inhibited LPS-induced proliferation and ROS production in RAW264.7 macrophages partially through modulation of CRAC channels but independent of the NF-κB pathway.     

Forsythin inhibits lipopolysaccharide-induced inflammation by suppressing JAK-STAT and p38 MAPK signalings and ROS production

Objective

Forsythin (FOR) is an active ingredient extracted from the fruit of the medicinal plant Forsythia suspensa (Thunb.) Vahl. Here, we investigated the effect of FOR on LPS-induced inflammatory response and the underlying molecular mechanisms in RAW264.7 macrophages.

Materials and methods

RAW264.7 cells were pre-treated with or without FOR and then stimulated with or without LPS. The productions of TNF-α, IL-1β, IL-6, PGE₂ and NO were determined by ELISA and nitrite analysis, respectively. The expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were measured by Western blotting and RT-PCR analysis. The activations of signaling molecules were detected by Western blotting using phosphorylation specific antibodies. Reactive oxygen species (ROS) production was determined by ROS assay.

Results

LPS-induced productions of IL-1β, IL-6, TNF-α, NO and PGE₂ were inhibited by FOR in a dose-dependent manner. FOR also suppressed the LPS-elevated expressions of iNOS and COX-2. Further investigations revealed that FOR significantly inhibited the LPS-induced activations of JAK-STATs and p38 MAPKs, but not of IKKα/β in LPS-stimulated RAW264.7 cells. Additionally, FOR interfered with both JAK-STATs and p38 MAPKs signaling pathways to modulate the expressions of IL-1β, IL-6, TNF-α, iNOS and COX-2. Furthermore, FOR reduced the LPS-induced ROS accumulation, validating that FOR serves as an antioxidant.

Conclusions

Our data suggested that FOR exerts anti-inflammatory action, at least in part, via suppressing LPS-induced activation of JAK-STATs and p38 MAPKs signalings and production of ROS in macrophage cells.     

Baicalein reduces lipopolysaccharide-induced inflammation via suppressing JAK/STATs activation and ROS production

Objective

To investigate the precise molecular mechanisms by which baicalein exerts beneficial biochemical activities in RAW264.7 macrophages treated with LPS.

Materials and methods

RAW264.7 cells were cultured in the absence or presence of baicalein together with or without LPS. iNOS and COX-2 expression were measured by western blot and RT-PCR analyses. TNF-α, IL-1β, and IL-6 were determined by using double-antibody sandwich ELISA. Phosphorylations of JAK1 and JAK2, and of STAT1 and STAT3 were detected by western blotting. Nuclear translocation of STAT1 and STAT3 was visualized by confocal microscopy. ROS production was detected by ROS assay.

Results

Baicalein significantly reduced the phosphorylation of STAT1 and STAT3 and the phosphorylation of JAK1 and JAK2, but without affecting MAPKs phosphorylation in LPS-stimulated RAW264.7 cells. Baicalein suppressed the nuclear translocation of STAT1 and STAT3 and inhibited production of iNOS upon LPS-stimulation, resulting in the inhibition of releases of NO and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, in a dose-dependent manner. In addition, we found that baicalein reduced the LPS-induced accumulation of ROS, confirming that baicalein serves as an antioxidant.

Conclusions

Our results suggested that suppressing JAK/STATs activation and interfering with ROS production might contribute to the anti-inflammatory action of baicalein in macrophages.     

The Yin and Yang actions of North American ginseng root in modulating the immune function of macrophages

Background

Immuno-modulatory effects of ginseng, including both immuno-stimulatory and immuno-suppressive effects, have been widely reported. This study aims to determine whether the paradoxical immuno-modulatory effect is related to unique phytochemical profiles of different North American (NA) ginseng, namely aqueous (AQ) and alcoholic (ALC) extracts.

Methods

AQ and ALC extracts were prepared and their immuno-bioactivity were studied in vitro in murine macrophages (Raw 264.7) through measuring the direct stimulatory production of pro-inflammatory mediator and cytokines as well as the suppression of lipopolysaccharide (LPS)-stimulatory response by the two extracts. Gel permeation chromatography was used to fractionate and isolate phytochemicals for characterization of ginseng extracts.

Results

AQ extract up-regulated the production of nitric oxide (NO), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) while ALC extract did not. ALC extract but not AQ extract suppressed LPS-induced macrophage NO and TNF-α production. These immuno-stimulatory and suppressive effects were exhibited at similar extract concentrations. Moreover, the macrophage-stimulating activity of the AQ extract was inhibited in the presence of ALC extract. Fractionation of AQ extract revealed the presence of two major peaks at 230 nm with average molecular weights of 73,000 and 37,000 Da. The first fraction had similar elution volume as the crude polysaccharide (PS) fraction isolated from the AQ extract, and it was the only bioactive species. Parallel fractionation study of ALC extract yielded similar elution profiles; however, both sub-fractions were devoid of PS. Fraction I of the ALC extract suppressed LPS-induced NO production dose-dependently.

Conclusion

ALC extract of NA ginseng, which was devoid of PS, was immuno-inhibitory whereas the AQ extract, which contained PS, was immuno-stimulatory. These extract-related anti-inflammatory and pro-inflammatory effects may be considered as the Yin and Yang actions of ginseng.     

MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult

Background

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD) through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases.

Methods

For in vitro studies, mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanism by which FLZ, a squamosamide derivative, mediates anti-inflammatory and neuroprotective effects in both lipopolysaccharide-(LPS)- and 1-methyl-4-phenylpyridinium-(MPP⁺)-mediated models of PD. For in vivo studies, a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP-) induced PD mouse model was used.

Results

FLZ showed potent efficacy in protecting dopaminergic (DA) neurons against LPS-induced neurotoxicity, as shown in rat and mouse primary mesencephalic neuronal-glial cultures by DA uptake and tyrosine hydroxylase (TH) immunohistochemical results. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E₂ (PGE₂). Mechanistic studies revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase (PHOX), the key microglial superoxide-producing enzyme. A critical role for PHOX in FLZ-elicited neuroprotection was further supported by the findings that 1) FLZ's protective effect was reduced in cultures from PHOX^-/- mice, and 2) FLZ inhibited LPS-induced translocation of the cytosolic subunit of p47^PHOX to the membrane and thus inhibited the activation of PHOX. The neuroprotective effect of FLZ demonstrated in primary neuronal-glial cultures was further substantiated by an in vivo study, which showed that FLZ significantly protected against MPTP-induced DA neuronal loss, microglial activation and behavioral changes.

Conclusion

Taken together, our results clearly demonstrate that FLZ is effective in protecting against LPS- and MPTP-induced neurotoxicity, and the mechanism of this protection appears to be due, at least in part, to inhibition of PHOX activity and to prevention of microglial activation.     

Antagonism of the histamine H4 receptor reduces LPS-induced TNF production in vivo

Objective

Antagonism of the histamine H₄ receptor (H₄R) has been shown to be anti-inflammatory in a number of preclinical disease models, however the exact mechanisms behind this are still being uncovered. In vitro, the receptor interacts with TLR and impacts inflammatory mediator production from a number of different cell types. Here it is shown that this interaction also occurs in vivo.

Materials and methods

Wild-type and H₄R deficient BALB/c mice received an i.p. injection of LPS in PBS in conjunction with p.o. JNJ 7777120 or JNJ 28307474 (H₄R antagonists). Two hours later blood was collected and TNF was measured.

Results

Two different H₄R antagonists inhibited LPS-induced TNF production in mice and this production was also reduced in H₄R-deficient mice. The TNF mRNA analysis showed that the major source of the cytokine was the liver and not blood, and that the H₄R antagonist only reduced the expression levels in the liver. Depletion or inactivation of macrophages reduced the TNF levels and eliminated the H₄R sensitivity. Treatment with an H₄R antagonist also reduced LPS-induced liver injury and blocked LPS-enhanced lung inflammation in mice.

Conclusion

The data support an interaction between H₄R and TLR activation in vivo that can drive inflammatory responses.     

Effect of chlorogenic acid on LPS-induced proinflammatory signaling in hepatic stellate cells

Objective and design

This study was aimed at investigating the effect of chlorogenic acid (CGA) on lipopolysaccharide (LPS)-induced proinflammatory signaling in hepatic stellate cells (HSCs).

Methods

An immortalized rat HSC line was cultured in vitro and treated with LPS in the absence or presence of CGA. Reactive oxygen species (ROS) production in the HSCs was monitored by flow cytometer using DCFH-DA. The protein expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), and p-IκB-α were determined by Western blot. The mRNA expression levels of TLR4, MyD88, monocyte chemotactic protein 1(MCP-1), and interleukin 6 (IL-6) were detected by RT-PCR. The levels of MCP-1 and IL-6 in the culture supernatant of HSCs were measured by ELISA.

Results

CGA had no effect on expression of TLR4 and MyD88. However, the treatment of CGA can inhibit LPS-induced production of ROS in HSCs. Meanwhile, CGA can inhibit LPS-induced nuclear translocation of NF-κB and IκB-α phosphorylation in HSCs, as well as NAC (a ROS scavenger). The mRNA expression and the levels of MCP-1 and IL-6 in the culture supernatant of the HSCs in this study were elevated by LPS stimulation and inhibited by CGA treatment, as well as NAC and PDTC (a NF-κB inhibitor).

Conclusion

Our results indicate that CGA can efficiently inhibit LPS-induced proinflammatory responses in HSCs and the anti-inflammatory effect may be due to the inhibition of LPS/ROS/NF-κB signaling pathway.     

Omega-6 docosapentaenoic acid-derived resolvins and 17-hydroxydocosahexaenoic acid modulate macrophage function and alleviate experimental colitis

Objective

Enzymatically oxygenated lipid products derived from omega-3 and omega-6 fatty acids play an important role in inflammation dampening. This study examined the anti-inflammatory effects of n-6 docosapentaenoic acid-derived (17S)-hydroxy-docosapentaenoic acid (17-HDPAn-6) and (10,17S)-dihydroxy-docosapentaenoic acid (10,17-HDPAn-6) as well as n-3 docosahexaenoic acid-derived 17(R/S)-hydroxy-docosahexaenoic acid (17-HDHA).

Materials and methods

The effects of 17-HDPAn-6, 10,17-HDPAn-6 or 17-HDHA on activity and M1/M2 polarization of murine macrophage cell line RAW 264.7 were examined by phagocytosis assay and real-time PCR. To assess anti-inflammatory effects in vivo, dextran sodium sulfate (DSS) colitis was induced in mice treated with 17-HDPAn-6, 10,17-HDPAn-6, 17-HDHA or NaCl.

Results

Our results show that 17-HDPAn-6, 10,17-HDPAn-6 and 17-HDHA increase phagocytosis in macrophages in vitro and promote polarization towards the anti-inflammatory M2 phenotype with decreased gene expression of TNF-α and inducible Nitric oxide synthase and increased expression of the chemokine IL-1 receptor antagonist and the Scavenger receptor Type A. Intraperitoneal treatment with 17-HDPAn-6, 10,17-HDPAn-6, or 17-HDHA alleviated DSS-colitis and significantly improved body weight loss, colon epithelial damage, and macrophage infiltration.

Conclusion

These results suggest that DPAn-6-derived 17-HDPAn-6 and 10,17-HDPAn-6 as well as the DHA-derived 17-HDHA have inflammation-dampening and resolution-promoting effects that could be used to treat inflammatory conditions such as inflammatory bowel disease.     

Effect of salicin on gut inflammation and on selected groups of gut microbiota in dextran sodium sulfate induced mouse model of colitis

Objective and design

This study was undertaken to evaluate the anti-inflammatory effect of the pure compound salicin on dextran sulfate sodium (DSS)-induced colitis in a mouse model and to quantify the major gut bacteria during the treatment.

Material or subjects

Experimental colitis was induced in Swiss albino mice by dissolving 2 % DSS in their drinking water for 7 days. Five mice were used in each group.

Treatment

Salicin (100 and 200 mg per body weight) was administered daily through oral gavage for 7 days.

Methods

Disease activity index (DAI), colon length, myeloperoxidase (MPO) assay, pro-inflammatory cytokine expression, histological changes and absolute number of gut microbiota were measured after treatment. Student’s t test was applied for statistical analysis.

Results

Salicin significantly attenuated DSS-induced DAI scores, shortening of colon length and tissue MPO activity. Salicin administration also effectively and dose-dependently prevented pro-inflammatory cytokine expression in DSS-induced colitis mice. Histological examination indicated that salicin suppressed edema, mucosal damage and the loss of crypts induced by DSS. Oral administration of salicin in DSS-treated mice prevented loss of gut microbiota during the short period of treatment.

Conclusions

Salicin has an anti-inflammatory effect, and it may have therapeutic value in ameliorating inflammation during colitis.     

The involvement of the HO-1 pathway in the anti-inflammatory action of a sulfated polysaccharide isolated from the red seaweed Gracilaria birdiae

Objectives

The aim of this study was to investigate the involvement of the hemoxigenase-1 (HO-1) pathway in the anti-inflammatory action of a sulfated polysaccharide from the red seaweed Gracilaria birdiae (SP-Gb).

Methods

SP-Gb (5, 10 and 20?mg/kg) was administered to Wistar rats in a peritonitis model using carrageenan or a paw edema model using carrageenan or dextran. To analyze the involvement of HO-1 in the anti-inflammatory activity of SP-Gb, the animals were pretreated subcutaneously with a specific HO-1 inhibitor (ZnPP IX). To evaluate the systemic effects, SP-Gb (10?mg/kg) was administered to mice intraperitoneally before waiting for 48?h or for 14?days.

Results

SP-Gb (10?mg/kg) caused an anti-inflammatory effect that was evidenced by a decrease in leukocytes in the peritoneal cavity. SP-Gb also reduced the paw edema induced by carrageenan and inhibited the paw edema induced by dextran in the first half-hour. After being inhibited by ZnPP IX, the anti-inflammatory effect of SP-Gb on carrageenan-induced rat paw edema was not observed. SP-Gb did not cause mortality or significant changes in the biochemical, hematological and histopathological parameters.

Conclusion

SP-Gb may be used as a tool for further investigations into the inflammatory processes associated with the hemoxigenase-1 pathway.     

Concentration dependent anti-inflammatory effects thrombin on polyphosphate-mediated inflammatory responses in vitro and in vivo

Aim and objective

Recent results indicate that polyphosphate (polyP) released by human endothelial cells can function as a pro-inflammatory mediator, and it has been reported that low thrombin concentrations mediate anti-inflammatory activities. This study was undertaken to investigate whether low thrombin concentrations can modulate polyP-mediated inflammatory responses in human umbilical vein endothelial cells (HUVECs) and in mice.

Methods

Concentration dependent anti-inflammatory effects of thrombin such as barrier protection, inhibition of cell adhesion molecule expression and inhibition of monocytes adhesion and migration toward human endothelial cells against polyP-mediated pro-inflammatory activities were tested in vitro and in vivo. The concentration-dependent effects of thrombin on polyP-induced nuclear factor (NF)-κB activation and the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were also tested.

Results

We found that at low concentrations (25–75 pM), thrombin inhibits polyP-mediated barrier disruption, the expressions of cell adhesion molecules, and leukocyte to HUVEC adhesion/migration. Interestingly, polyP-induced NF-κB activation and the production of TNF-α and IL-6 were inhibited by low thrombin concentrations in HUVECs. These anti-inflammatory functions of thrombin were confirmed in polyP-injected mice.

Conclusion

These results suggest that thrombin at 25–75 pM may have therapeutic potential for various systemic inflammatory diseases.     

Nuciferine alleviates LPS-induced mastitis in mice via suppressing the TLR4-NF-κB signaling pathway

Background

Nuciferine, a major bioactive component from the lotus leaf, has been reported to have notable anti-inflammatory activities such as renal inflammation and acute lung injury in previous studies. Mastitis is one of the most prevalent diseases in the dairy cattle, which causes large economic losses for the dairy industry. However, the effects of nuciferine on lipopolysaccharide (LPS)-induced mastitis have not been reported.

Methods and results

Here, we investigated the anti-inflammatory effects of nuciferine on LPS-induced mastitis in mice and illuminated its potential mechanism on the TLR4-mediated signaling pathway in mouse mammary epithelial cells (mMECs). Histopathological changes and myeloperoxidase (MPO) activity assay showed that nuciferine treatment significantly alleviated the LPS-induced injury of mammary gland flocculus, inflammatory cells infiltration. qPCR and ELISA assays indicated that nuciferine dose-dependently reduced the levels of TNF-α and IL-1β, which indicated that nuciferine might have therapeutic effects on mastitis. Furthermore, nuciferine treatment significantly decreased the expression of TLR4 in a dose-dependent manner. Besides, nuciferine was also found to suppress LPS-induced NF-κB activation.

Conclusion

These findings indicate that nuciferine potently ameliorates LPS-induced mastitis by inhibition of the TLR4-NF-κB signaling pathway.

     

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

Objective

Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E₂ (PGE₂) are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE₂ by HGFs were examined.

Methods

HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE₂ levels were evaluated by ELISA.

Results

H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE₂ production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE₂ production. Up to 10 ??g/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE₂ production, but they were significantly increased at 100 ??g/ml. Similarly, 0.01 ??g/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE₂ production, but they were significantly increased at concentrations of 0.1 and 1 ??g/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE₂ production.

Conclusion

These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE₂ production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 ??g/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.