LPS-exposed Cells Research Articles

DHA/AA alleviates LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3 to inhibit inflammasome complexes assembly

Pyroptosis is a novel type of programmed cell death associated with the pathogenesis of many inflammatory diseases. Docosahexaenoic acid (DHA) and Arachidonic acid (AA) is widely involved in inflammatory pathological processes. However, the effect and mechanism of DHA and AA on pyroptosis in Kupffer cells are poorly understood. The present study demonstrated that DHA and AA ameliorated lipopolysaccharide (LPS)-induced Kupffer cells pyroptosis by reversing the increased expression of NLRP3 inflammasome complex, GSDMD, IL-1β, IL-18, and PI-stained positive rate. Next, the study revealed that GPR120 silencing eliminated the anti-pyroptosis of DHA and AA in LPS-induced Kupffer cells, suggesting that DHA and AA exerted their effect through GPR120 signaling. Importantly, GPR120 endocytose and binds to NLRP3 under LPS stimulation. Furthermore, co-immunoprecipitation showed that DHA and AA promoted the interaction between GPR120 and NLRP3 in LPS-exposed Kupffer cells, thus inhibiting the self-assembly of NLRP3 inflammasome complex. Finally, the study verified that DHA and AA alleviated hepatic injury through inhibiting Kupffer cells pyroptosis in vivo. The findings indicated that DHA and AA alleviated LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3, it might become a potential therapeutic approach hepatic injury.

HEMA-induced oxidative stress inhibits NF-κB nuclear translocation and TNF release from LTA- and LPS-stimulated immunocompetent cells

Objective: The release of inflammatory cytokines from antigen-stimulated cells of the immune system is inhibited by resin monomers such as 2-hydroxyethyl methacrylate (HEMA). Although the formation of oxidative stress in cells exposed to HEMA is firmly established, the mechanism behind the inhibited cytokine secretion is only partly known. The present investigation presents evidence regarding the role of HEMA-induced oxidative stress in the secretion of the pro-inflammatory cytokine TNFα from cells exposed to the antigens LTA (lipoteichoic acid) or LPS (lipopolysaccharide) of cariogenic microorganisms using BSO (L-buthionine sulfoximine) or NAC (N-acetyl cysteine) to inhibit or stabilize the amounts of the antioxidant glutathione.Method: RAW264.7 mouse macrophages were treated with LTA, LPS or HEMA in the presence of BSO or NAC for 1h or 24h. Secretion of TNFα from cell cultures was analyzed by ELISA, and the formation of reactive oxygen (ROS) or nitrogen species (RNS) was determined by flow cytometry. Protein expression was detected by Western blotting.Results: The release of TNFα in both LTA- and LPS-exposed cells was decreased by HEMA, and this concentration-dependent inhibitory effect was amplified by BSO or NAC. LTA- and LPS-stimulated expression of the redox-sensitive transcription factor NF-αB (p65) in cell nuclei decreased in the presence of HEMA because the translocation of p65 from the cytosol was prevented by oxidative stress specifically increased by the monomer.Conclusions: A disturbance of the cellular redox balance, particularly induced by HEMA, is a crucial factor in the inhibition of LTA- and LPS-stimulated signalling pathways leading to TNFα secretion.

FBW7 protects against spinal cord injury by mitigating inflammation-associated neuronal apoptosis in mice

Spinal cord injury (SCI) leads to severe and long-lasting neurological disability. Presently, the lack of effective therapies for SCI is largely attributable to an incomplete understanding of its pathogenesis. F-box and WD repeat domain-containing protein 7 (FBW7, also known as FBXW7) is a type of E3 ubiquitin ligase complex, and plays essential roles in regulating different pathological and physiological processes. In this study, we attempted to explore the effects of FBW7 on SCI progression by the in vivo and in vitro experiments. SCI mice showed significantly reduced expression of FBW7 in spinal cord tissues. Promoting FBW7 expression via intrathecal injection of AAV9/FBW7 effectively improved locomotor function in SCI mice. Neuronal death in spinal cords of SCI mice was obviously ameliorated by FBW7 over-expression, along with greatly decreased expression of cleaved Caspase-3. In addition, microglial activation in spinal cord specimens was detected in SCI mice through increasing Iba-1 expression levels, which was, however, attenuated in SCI mice injected with AAV9/FBW7. Additionally, FBW7 over-expression dramatically restrained inflammatory response in spinal cord tissues of SCI mice, as evidenced by the down-regulated expression of tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) through blocking the activation of nuclear factor-κB (NF-κB) signaling. These anti-inflammatory effects of FBW7 were confirmed in LPS-stimulated mouse microglial BV2 cells. Finally, our in vitro studies showed that conditional medium (CM) collected from LPS-incubated BV2 cells markedly induced apoptosis in the isolated primary spinal neurons; However, this effect was overtly ameliorated by CM from LPS-exposed BV2 cells over-expressing FBW7. Thus, FBW7-regulated inflammation in microglial cells was involved in the amelioration of neuronal apoptosis during SCI development. Collectively, these findings illustrated that FBW7 expression was down-regulated in spinal cords of SCI mice, and promoting its expression could effectively mitigate SCI progression by repressing microglial inflammation and neuronal death.

Beta-naphthoflavone inhibits LPS-induced inflammation in BV-2 cells via AKT/Nrf-2/HO-1-NF-κB signaling axis

Numerous studies have shown that over-activation of microglia could cause neuroinflammation and release pro-inflammatory mediators, which could result in neurodegenerative diseases, like Parkinson’s disease, Alzheimer’s disease etc. Beta-naphthoflavone (BNF) has anti-oxidant and anti-inflammatory effects in borderline tissues, but BNF has not been reported the effect associated with neuroinflammation. Therefore, the purpose of this experiment is to inquiry the impact and mechanism of BNF on neuroinflammation. The results indicated that BNF significantly inhibited the production of pro-inflammatory mediators (inducible nitric-oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) andinterleukin-6 (IL-6)) in LPS-exposed BV-2 cells. Analysis of western blot results found that BNF accelerated the activation of AKT/Nrf-2/HO-1 signaling pathway and suppressed NF-κB pathway activation. Further study showed that BNF inhibited activation of NF-κB pathway via promoting HO-1, and SnPP IX (a HO-1 inhibitor) could inhibit anti-inflammatory function of BNF. We also found that BNF reduced the apoptosis rate of Human neuroblastoma cells (SHSY5Y) and mouse hippocampal neuron cell line (HT22) by inhibiting release of inflammatory mediators in LPS-exposed BV2 cells. In a word, our results suggested that BNF could inhibit inflammatory response via AKT/Nrf-2/HO-1-NF-κB signaling axis in BV-2 cells and exerts neuroprotective impact via inhibiting the activation of BV2 cells.

Inhibition of histone deacetylases is the major pathway mediated by astaxanthin to antagonize LPS-induced inflammatory responses in mammary epithelial cells.

Mastitis is amajor inflammatory response of the mammary gland due to various pathogenic invasions and is a serious disease that affects the production yield and health status of cows. Astaxanthin (AST), a xanthophyll carotenoid, is a secondary metabolite synthesized by microalgaeand yeasts that has been reported to suppress various inflammatory responses. However, the protective effect of AST on lipopolysaccharide (LPS)-induced mammary epithelial cells has not yet been reported. The present study results indicated that AST treatment markedly attenuated the oxidative stress markers and nitric oxide (NO) while improving the anti-oxidant enzymes in LPS exposed cells. On the other hand, LPS-exposed cells showed nuclear translocation of nuclear factor-κB (NF-κB) with the activation of inflammatory cytokines such as monocyte chemoattractantprotein-1, tumor necrosis factor-α, interferon-γ, and interleukin-6 (IL-6). In addition, mRNA expression analysis revealed that the histone deacetylase (HDAC) -1, -2, -3, -6, -7 and pentraxin 3 (PTX3) expressions were increased in the LPS group. Furthermore, the activity of HDAC was increased to 2-fold with a significant reduction in the histone acetyltransferase activity in cells exposed to LPS. However, AST was able to inhibit the nuclear translocation of NF-κB with attenuated HDAC activity. Intriguingly, HDAC inhibition studies demonstrated that the cytokines such as IL-4, IL-8, granulocyte-mcrophage colony stimulating factor, C-reactive protein, IL-17A, and IL-22 were significantly suppressedwhichwere upregulated in LPS treatment; while AST was found acting byimproving the anti-inflammatory cytokineIL-10, and thioredoxin reductase levels. Collectively, these findings provide novel insights into the role of HDACs in regulating cellular processes involved in the pathogenesis of LPS-induced mastitis as well as the potential use of AST as a therapeutic in treatment for controlling disease progression.

H2S counteracts proinflammatory effects of LPS through modulation of multiple pathways in human cells.

Hydrogen sulfide donors reduce inflammatory signaling in vitro and in vivo. The biological effect mediated by H2S donors depends on the kinetics of the gas release from the donor molecule. However, the molecular mechanisms of H2S-induced immunomodulation were poorly addressed. Here, we studied the effect of two different hydrogen sulfide (H2S)-producing agents on the generation of the LPS-induced inflammatory mediators. Importantly, we investigated the transcriptomic changes that take place in human cells after the LPS challenge, combined with the pretreatment with a slow-releasing H2S donor-GYY4137. We investigated the effects of GYY4137 and sodium hydrosulfide on the release of proinflammatory molecules such as ROS, NO and TNF-α from LPS-treated human SH-SY5Y neuroblastoma and the THP-1 promonocytic cell lines. Transcriptomic and RT-qPCR studies using THP-1 cells were performed to monitor the effects of the GYY4137 on multiple signaling pathways, including various immune-related and proinflammatory genes after combined action of LPS and GYY4137. The GYY4137 and sodium hydrosulfide differed in the ability to reduce the production of the LPS-evoked proinflammatory mediators. The pre-treatment with GYY4137 resulted in a drastic down-regulation of many TNF-α effectors that are induced by LPS treatment in THP-1 cells. Furthermore, GYY4137 pretreatment of LPS-exposed cells ameliorates the LPS-mediated induction of multiple pro-inflammatory genes and decreases expression of immunoproteasome genes. Besides, in these experiments we detected the up-regulation of several important pathways that are inhibited by LPS. Based on the obtained results we believe that our transcriptomic analysis significantly contributes to the understanding of the molecular mechanisms of anti-inflammatory and cytoprotective activity of hydrogen sulfide donors, and highlights their potential against LPS challenges and other forms of inflammation.

Gracillin inhibits apoptosis and inflammation induced by lipopolysaccharide (LPS) to alleviate cardiac injury in mice via improving miR-29a

Sepsis induces critical myocardial dysfunction, resulting in an increased mortality. Gracillin (GRA) is a natural steroidal saponin, showing strong capacities of anti-inflammation, but its pharmacological effects on lipopolysaccharide (LPS)-induced acute cardiac injury still remain unclear. In this study, we attempted to explore if GRA was effective to attenuate cardiac injury in LPS-challenged mice and the underlying mechanisms. First, we found that GRA treatments markedly up-regulated the expression of miR-29a in cardiomyocytes. LPS-induced cytotoxicity in cardiomyocytes was significantly alleviated by GRA treatment, as evidenced by the improved cell viability and reduced lactate dehydrogenase (LDH) release. In addition, LPS-triggered apoptotic cell death was clearly ameliorated in cardiomyocytes co-treated with GRA. Notably, LPS-exposed cells showed significantly reduced expression of miR-29a, while being rescued by GRA treatment. In vivo, LPS apparently impaired cardiac function in mice, which was, however, alleviated by GRA administration. In addition, GRA markedly attenuated apoptosis in hearts of LPS-challenged mice by decreasing the expression of cleaved Caspase-3. LPS-triggered inflammatory response in cardiac tissues was also suppressed by GRA through blocking nuclear factor κB (NF-κB) signaling pathway. We also found that miR-29a expression was highly reduced in hearts of LPS-treated mice but was rescued by GRA pretreatment. Besides, miR-29a mimic alleviated LPS-induced apoptosis and inflammation in cardiomyocytes; however, LPS-caused effects were further accelerated by miR-29a. Of note, the protective effects of GRA on LPS-injured cardiac tissues were significantly abrogated by miR-29a suppression. In conclusion, our findings demonstrated that GRA exerted an effective role against LPS-induced acute cardiac injury through impeding apoptosis and inflammation regulated by miR-29a.

Transduced Tat-CIAPIN1 reduces the inflammatory response on LPS- and TPA-induced damages

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1), known as an anti-apoptotic and signal-transduction protein, plays a pivotal role in a variety of biological processes. However, the role of CIAPIN1 in inflammation is unclear. We investigated the protective effects of CIAPIN1 in lipopolysaccharide (LPS)-exposed Raw 264.7 cells and against inflammatory damage induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in a mouse model using cell-permeable Tat-CIAPIN1. Transduced Tat-CIAPIN1 significantly reduced ROS production and DNA fragmentation in LPS-exposed Raw 264.7 cells. Also, Tat-CIAPIN1 inhibited MAPKs and NF-κB activation, reduced the expression of Bax, and cleaved caspase-3, COX-2, iNOS, IL-6, and TNF-α in LPS-exposed cells. In a TPA-induced animal model, transduced Tat-CIAPIN1 drastically decreased inflammation damage and inhibited COX-2, iNOS, IL-6, and TNF-α expression. Therefore, these findings suggest that Tat-CIAPIN1 might lead to a new strategy for the treatment of inflammatory skin disorders.

Dexmedetomidine Inhibits Neuroinflammation by Altering Microglial M1/M2 Polarization Through MAPK/ERK Pathway.

Neuroinflammation is critical in the pathogenesis of neurological diseases. Microglial pro-inflammatory (M1) and anti-inflammatory (M2) status determines the outcome of neuroinflammation. Dexmedetomidine exerts anti-inflammatory effects in many neurological conditions. Whether dexmedetomidine functions via modulation of microglia M1/M2 polarization remains to be fully elucidated. In the present study, we investigated the anti-inflammatory effects of dexmedetomidine on the neuroinflammatory cell model and explored the potential mechanism. BV2 cells were stimulated with LPS to establish a neuroinflammatory model. The cell viability was determined with MTT assay. NO levels were assessed using a NO detection kit. The protein levels of IL-10, TNF-α, iNOS, CD206, ERK1/2, and pERK1/2 were quantified using Western blotting. LPS significantly increased pro-inflammatory factors TNF-α and NO, and M1 phenotypic marker iNOS, and decreased anti-inflammatory factor IL-10 and M2 phenotypic marker CD206 in BV2 cells. Furthermore, exposure of BV2 cells to LPS significantly raised pERK1/2 expression. Pretreatment with dexmedetomidine attenuated LPS-elicited changes in p-ERK, iNOS, TNF-α, NO, CD206 and IL-10 levels in BV2 cells. However, co-treatment with dexmedetomidine and LM22B-10, an agonist of ERK, reversed dexmedetomidine-elicited changes in p-ERK, iNOS, TNF-α, NO, CD206 and IL-10 levels in LPS-exposed BV2 cells. We, for the first time, showed that dexmedetomidine increases microglial M2 polarization by inhibiting phosphorylation of ERK1/2, by which it exerts anti-inflammatory effects in BV2 cells.

NLRP3 Inflammasome Activation by MicroRNA-495 Promoter Methylation May Contribute to the Progression of Acute Lung Injury

Acute lung injury (ALI) is a pulmonary disorder that causes acute respiratory failure, thus leading to relative high mortality worldwide. However, the molecular mechanisms of ALI remain largely unknown. MicroRNA (miRNA)-dependent control of gene expression at a post-transcriptional level has been recently reported. Herein, we identify a candidate miRNA, miR-495, that affects the progression of ALI. Alveolar macrophages (NR8383) were treated with 1 μg/mL lipopolysaccharide (LPS) to establish a cell-injury model. Combined with the data from western blot, methylation-specific PCR, methylated DNA immunoprecipitation, and chromatin immunoprecipitation assays, NLRP3 inflammasome activation and methylation-dependent repression of miR-495 were found in LPS-exposed NR8383 cells. Dual-luciferase reporter gene assay and miR-495 gain-of-function experiments confirmed that NLRP3 was a target of miR-495. Next, the expression of miR-495 and NLRP3 was overexpressed or silenced to assess their effects on NLRP3 inflammasome activation, alveolar macrophage inflammation, and pyroptosis in vitro. As demonstrated, overexpressed miR-495 alleviated alveolar macrophage inflammation and pyroptosis and inhibited NLRP3 inflammasome activation by negatively regulating the NLRP3 gene. Consistently, elevated miR-495 alleviated lung injury and reduced the neutrophil infiltration and inflammation in rat models of LPS-induced ALI. Taken together, the data in our study demonstrated that methylation of the miR-495 promoter could downregulate miR-495, whose elevation could attenuate the activation of the NLRP3 inflammasome to protect against ALI, which provides novel therapeutic targets for ALI treatment.

Alkannin attenuates lipopolysaccharide-induced lung injury in mice via Rho/ROCK/NF-κB pathway.

We investigated the effects and associated mechanism of alkannin (AL) on lipopolysaccharide (LPS)-induced acute lung injury in a mouse model. Pretreatment with AL in vivo significantly reduced the lung wet/dry weight ratio and inhibited lung myeloperoxidase activity and malondialdehyde content, while increasing superoxide dismutase activity. Hematoxylin and eosin staining demonstrated that AL attenuated lung histopathological changes. In addition, AL-inhibited overproduction of proinflammatory cytokines in bronchoalveolar lavage fluid and lung tissues in LPS-injured mice and LPS-exposed A549 cells. Further analysis showed that AL-inhibited induction of the Rho/ROCK/NF-κB pathway via LPS-induced inflammation in mice and A549 cells. Fasudil, a selective ROCK inhibitor, showed similar effects. Overall, the findings indicate that AL suppresses the expression of messenger RNAs and proteins associated with Rho/ROCK/NF-κB signaling to effectively ameliorate lung injury.

Inhibition activity of Robusta coffee beans polyphenol extract on the production of TNF-α neutrophil cells

Polyphenols are one of the active substances in the Robusta coffee beans with various benefits for humans’ health including anti-inflammation. neutrophil cell (polymorphonuclear PMN) plays a significant role as the primary immune response against foreign agent. Inflammatory response is characterized by the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). Hence, the purpose of this study is to determine anti-inflammation capacity of Robusta coffee beans polyphenols extract on the TNF-α production in neutrophil cells. Neutrophil was derived from human peripheral venous blood by means of gradient density method. One hundred microliter of neutrophil (1,5x103 cell) was incubated with 3.13%, 6.25%, 12.5%, 25% polyphenol extract and exposed with 100 μl of Lipopolysaccharides (LPS) 25 mg/ml. The production of TNF-α were determined by ELISA. Data were analyzed by one way Anova. Polyphenols of Robusta coffee bean extract was shown to inhibit TNF-α production in LPS-exposed neutrophil cells compared to controls. Whereas, LPS-exposed neutrophil increased TNF-α production. The most effective concentration to inhibit TNF-α production was 12.5%. It was concluded that polyphenols of Robusta coffee beans has anti-inflammatory properties as indicated by its ability to decrease TNF-α levels.

TRAF1 meditates lipopolysaccharide-induced acute lung injury by up regulating JNK activation

Acute lung injury (ALI) is served as a severe life-threatening disease. However, the pathogenesis that contributes to ALI has not been fully understood. Tumor necrosis factor receptor-associated factor 1 (TRAF1) interacts with multiple regulators, performing its diverse role in biological functions. However, the effects of TRAF1 on ALI remain unknown. In this study, we attempted to explore the role of TRAF1 in ALI progression. The findings suggested that TRAF1-knockout (KO) markedly attenuated LPS-induced severe mortality rate in murine animals. LPS-elicited histological alterations in pulmonary tissues were significantly alleviated by TRAF1-deletion. Additionally, TRAF1 knockout effectively attenuated lung injury, as evidenced by the reduced lung wet/dry (W/D) weight ratio, as well as decreased bronchoalveolar lavage fluid (BALF) protein levels and neutrophil infiltration. Meanwhile, TRAF1 deletion markedly lessened inflammation, oxidative stress and apoptosis in BALF and/or lung tissues. The levels of pro-inflammatory cytokines stimulated by LPS were down-regulated by TRAF1 ablation, along with the inactivation of nuclear factor κB (NF-κB). LPS-promoted reactive oxygen species (ROS) generation was decreased in TRAF1-KO mice, partly through the improvement of anti-oxidants. Apoptosis was also inhibited by TRAF1 deletion in lung tissues of LPS-challenged mice through the suppression of cleaved Caspase-3. Moreover, TRAF1 knockout significantly decreased c-Jun N-terminal kinase (JNK) activation and its down-streaming signal of c-Jun in pulmonary samples of LPS-induced mice. Importantly, the in vitro study suggested that promoting JNK activation markedly abrogated TRAF1 knockdown-attenuated inflammation, ROS production and apoptosis in LPS-exposed A549 cells. Therefore, our experimental results provided evidence that TRAF1 suppression effectively protected LPS-induced ALI against inflammation, oxidative stress and apoptosis through the suppression of JNK activity.

Phosphatidylinositol 3-kinase-mediated HO-1/CO represses Fis1 levels and alleviates lipopolysaccharide-induced oxidative injury in alveolar macrophages.

Sepsis-related acute respiratory distress syndrome is characterized by marked oxidative stress and mitochondrial dysfunction lacking of specific therapy. Heme oxygenase (HO)-1 followed by endogenous carbon monoxide (CO) exerted a cytoprotective effect against multi-organ damage during sepsis. Additionally, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which serves as an upstream regulator of HO-1, was associated with inflammation and oxidative stress. Therefore, the purpose of the present study was to investigate whether the PI3K/Akt pathway was involved in the effects of HO-1/CO on the expression of mitochondrial fission 1 protein (Fis1). In the present study, CO releasing molecule-2 (CORM2), as the exogenous source of CO, plus LY294002, as a specific PI3K inhibitor, were pre-incubated in lipopolysaccharide (LPS)-simulated rat NR8383 alveolar macrophages. The results demonstrated that CORM2 improved cell viability, inhibited tumor necrosis factor-α levels, malondialdehyde contents, while elevating interleukin-10 levels and superoxide dismutase activities. In addition, pretreatment with CORM2 suppressed the fragmentation of mitochondria, upregulated the expressions of phosphorylated-Akt and HO-1 but downregulated the levels of Fis1 mRNA and protein in LPS-exposed cells. However, pretreatment with LY294002 significantly inhibited the phosphorylation of Akt, decreased HO-1 levels, aggravated mitochondrial fragmentation, increased Fis1 mRNA and protein levels, and reversed the above protective effects of CORM2. Collectively, the results of the present study indicated that the PI3K/Akt pathway mediated the cytoprotective effects of HO-1/CO on the transcription and translational levels of Fis1, and alleviated LPS-induced oxidative injury in alveolar macrophages.

Benefits of VCE-003.2, a cannabigerol quinone derivative, against inflammation-driven neuronal deterioration in experimental Parkinson\u2019s disease: possible involvement of different binding sites at the PPAR\u03b3 receptor

BackgroundNeuroprotection with cannabinoids in Parkinson’s disease (PD) has been afforded predominantly with antioxidant or anti-inflammatory cannabinoids. In the present study, we investigated the anti-inflammatory and neuroprotective properties of VCE-003.2, a quinone derivative of the non-psychotrophic phytocannabinoid cannabigerol (CBG), which may derive its activity at the peroxisome proliferator-activated receptor-γ (PPARγ). The compound is also an antioxidant.MethodsWe evaluated VCE-003.2 in an in vivo [mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS)] model of PD, as well as in in vitro (LPS-exposed BV2 cells and M-213 cells treated with conditioned media generated from LPS-exposed BV2 cells) cellular models. The type of interaction of VCE-003.2 at the PPARγ receptor was furtherly investigated in bone marrow-derived human mesenchymal stem cells (MSCs) and sustained with transcriptional assays and in silico docking studies.ResultsVCE-003.2 has no activity at the cannabinoid receptors, a fact that we confirmed in this study using competition studies. The administration of VCE-003.2 to LPS-lesioned mice attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons and, in particular, the intense microgliosis provoked by LPS in the substantia nigra, measured by Iba-1/Cd68 immunostaining. The analysis by qPCR of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the striatum showed they were markedly elevated by the LPS lesion and strongly reduced by the treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with the PPARγ inhibitor T0070907. We then moved to some in vitro approaches, first to confirm the anti-inflammatory profile of VCE-003.2 in cultured BV2 cells exposed to LPS. VCE-003.2 was able to attenuate the synthesis and release of TNF-α and IL-1β, as well as the induction of iNOS and cyclooxygenase-2 (COX-2) elicited by LPS in these cells. However, we found such effects were not reversed by GW9662, another classic PPARγ antagonist. Next, we investigated the neuroprotective effects of VCE-003.2 in cultured M-213 neuronal cells exposed to conditioned media generated from LPS-exposed cultured BV2 cells. VCE-003.2 reduced M-213 cell death, but again, such effects were not reversed by T0070907. Using docking analysis, we detected that VCE-003.2 binds both the canonical and the alternative binding sites in the PPARγ ligand-binding pocket (LBP). Functional assays further showed that T0070907 almost abolished PPARγ transcriptional activity induced by rosiglitazone (RGZ), but it did not affect the activity of VCE-003.2 in a Gal4-Luc system. However, T0070907 inhibited the effects of RGZ and VCE-003.2 on the expression of PPARγ-dependent genes upregulated in MSCs.ConclusionsWe have demonstrated that VCE-003.2 is neuroprotective against inflammation-driven neuronal damage in an in vivo model of PD and in in vitro cellular models of neuroinflammation. Such effects might involve PPARγ receptors, although in silico and in vitro experiments strongly suggest that VCE-003.2 targets PPARγ by acting through two binding sites at the LBP, one that is sensitive to T0070907 (canonical binding site) and other that is not affected by this PPARγ antagonist (alternative binding site).

Paricalcitol attenuates lipopolysaccharide-induced inflammation and apoptosis in proximal tubular cells through the prostaglandin E2 receptor EP4.

BackgroundVitamin D is considered to exert a protective effect on various renal diseases but its underlying molecular mechanism remains poorly understood. This study aimed to determine whether paricalcitol attenuates inflammation and apoptosis during lipopolysaccharide (LPS)-induced renal proximal tubular cell injury through the prostaglandin E2 (PGE2) receptor EP4.MethodsHuman renal tubular epithelial (HK-2) cells were pretreated with paricalcitol (2 ng/mL) for 1 hour and exposed to LPS (1 μg/mL). The effects of paricalcitol pretreatment in relation to an EP4 blockade using AH-23848 or EP4 small interfering RNA (siRNA) were investigated.ResultsThe expression of cyclooxygenase-2, PGE2, and EP4 were significantly increased in LPS-exposed HK-2 cells treated with paricalcitol compared with cells exposed to LPS only. Paricalcitol prevented cell death induced by LPS exposure, and the cotreatment of AH-23848 or EP4 siRNA offset these cell-protective effects. The phosphorylation and nuclear translocation of p65 nuclear factor-kappaB (NF-κB) were decreased and the phosphorylation of Akt was increased in LPS-exposed cells with paricalcitol treatment. AH-23848 or EP4 siRNA inhibited the suppressive effects of paricalcitol on p65 NF-κB nuclear translocation and the activation of Akt. The production of proinflammatory cytokines and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were attenuated by paricalcitol in LPS exposed HK-2 cells. The cotreatment with an EP4 antagonist abolished these anti-inflammatory and antiapoptotic effects.ConclusionEP4 plays a pivotal role in anti-inflammatory and antiapoptotic effects through Akt and NF-κB signaling after paricalcitol pretreatment in LPS-induced renal proximal tubule cell injury.

The protective effects of epigallocatechin gallate on lipopolysa ccharide-induced hepatotoxicity: an in vitro study on Hep3B cells.

In the present study, our aim was to investigate the possible protective effects of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS)-induced hepatotoxicity by using Hep3B human hepatoma cells. Specifically, the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of LPS-associated cytotoxicity. Consequently, the hepatocellular carcinoma cell line Hep3B was chosen as a model for investigation of LPS toxicity and the effect of EGCG on LPS-exposed cells. The Hep3B human hepatoma cells were used for this study. The cytotoxic effects of chemicals (EGCG and LPS), AST and ALT levels, SOD and CAT activities, GSH-Px level and TNF-alpha and IL-6 levels were detected by using different biochemical and molecular methods. LPS and EGCG were applied to cells at various times and doses. The highest treatment dose of EGCG (400 µM) led to a dramatic decrease in SOD level and increase in CAT and GSH levels. Additionally, the highest dose of EGCG also led to a dramatic increase in TNF-alpha and IL-6 levels. On the other hand, effective doses of EGCG (200 and 100 µM) normalized all related parameters levels. LPS caused hepatotoxicity, but interestingly, a high dose of EGCG was found to be a cytotoxic agent in this study. However, other two doses of EGCG led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels. Further studies should examine the effect of EGCG on secondary cellular signaling pathways.

ResolvinD1 reduces apoptosis and inflammation in primary human alveolar epithelial type 2 cells

Lung epithelial apoptosis and inflammatory responses are important pathological processes in many pulmonary disorders. ResolvinD1 (RvD1), generated in inflammatory resolution processes, reduces inflammatory responses in animal models of lung diseases. The aim of this study was to investigate whether RvD1 attenuates apoptosis and proinflammatory responses in primary human alveolar epithelial type 2 cells (AEC2 cells) that are exposed to lipopolysaccharide (LPS) in vitro. We examined the percentage of apoptotic AEC2 cells by flow cytometry. The expression levels of cytokines and chemokines were determined by ELISA and microarray. The expression levels of molecular signaling modulators were evaluated by western blot. LPS-stimulated AEC2 cells pretreated with RvD1 exhibited a statistically significant reduction in apoptosis. The pretreatment of LPS-stimulated cells with RvD1 stimulated the phosphorylation of AKT and prevented the cleavage of caspase-3, the upregulation of Bax, and the downregulation of Bcl-2. The antiapoptotic effects of RvD1 were abrogated upon pretreatment with a PI3K inhibitor. In addition, RvD1 reduced the release of cytokines and chemokines, and inhibited the degradation and phosphorylation of IκB-α in LPS-stimulated AEC2 cells. RvD1 reduces apoptosis of LPS-exposed AEC2 cells by inducing the phosphorylation of AKT and attenuates the inflammatory response by suppressing the degradation and phosphorylation of IκB-α.

LPS stimulates IgM production invivo without help from non-B cells.

Gram-negative bacterial LPS induce murine B-cell activation and innate (polyclonal) Ab production. Mouse B cells express the LPS signaling receptor (TLR4), yet how LPS activates B-cell responses invivo is not known. Can LPS directly stimulate B cells to induce innate Ab production? Is activation of non-B cells also required? To address these questions, we transfused LPS-responsive (Tlr4(+/+)) or non-responsive (Tlr4(-/-)) B cells into LPS-responsive or non-responsive mice. Increased expression of the early activation markers CD69 and CD86 could be induced on transfused Tlr4(-/-) B cells by injecting LPS subcutaneously into Tlr4(+/+) mice, demonstrating indirect activation of B cells by TLR4-responsive non-B cells invivo, but the Tlr4(-) (/) (-) B cells did not increase serum IgM levels. In contrast, when Tlr4(-/-) recipients were transfused with Tlr4(+/+) B cells, LPS induced large amounts of serum IgM and LPS could also enhance specific Ab production to a protein that was co-injected with it (adjuvant response). Thus, LPS-exposed non-B cells mediated increased surface expression of early B-cell activation markers, but this response did not predict innate Ab responses or LPS adjuvanticity invivo Direct stimulation of B cells by LPS via TLR4 was necessary and sufficient to induce B cells to produce Ab invivo.

Effects of Lipopolysaccharide on Human First Trimester Villous Cytotrophoblast Cell Function In Vitro.

It has been shown that adverse obstetrical outcomes such as pre-eclampsia and intrauterine growth retardation correlate with maternal infection. In this study, we investigated mechanisms involved in infection-associated abnormalities in cytotrophoblast function. Primary human first trimester cytotrophoblast cells were isolated and treated with lipopolysaccharide (LPS). Levels of the cytokines and chemokines were measured and cytotrophoblast invasion was investigated. In addition, first trimester decidual macrophages were isolated and treated with the conditioned medium from LPS-treated cytotrophoblast cells, and macrophage migration was assessed. Coculturing decidual macrophages with cytotrophoblast cells was conducted to investigate macrophage costimulatory molecule and receptor expression and intracellular cytokine production. We found that LPS exposure increased cytotrophoblast production of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, and chemokines IL-8, macrophage inflammatory protein (MIP)-1alpha, and CXCL12 in a dose-dependent manner. In addition, LPS decreased cytotrophoblast invasion, and its effect was Toll-like receptor 4 (TLR4)-dependent and partly TNF-alpha-dependent. Conditioned medium from LPS-stimulated cytotrophoblast cells increased decidual macrophage migration and this effect was partly TLR4-dependent. Furthermore, coculturing decidual macrophages with LPS-exposed cytotrophoblast cells up-regulated macrophage CD80 and CD86 expression and intracellular TNF-alpha and IL-12p40 production, while down-regulating macrophage CD206 and CD209 expression and intracellular IL-10 secretion. LPS-stimulated macrophages also inhibited cytotrophoblast invasion. In conclusion, our results indicate that LPS increases the production of a subset of proinflammatory cytokines and chemokines by human first trimester cytotrophoblast cells, decreases cytotrophoblast invasion, and alters the cross talk between cytotrophoblast cells and decidual macrophages.