LPS-induced Morphological Changes Research Articles

The triple combination DBDx alleviates cytokine storm and related lung injury

Cytokine storm is a life-threatening disorder, and therapeutic treatments are urgently needed. Here, we investigated the anti-cytokine storm efficacy of DBDx, a triple drug combination composed of dipyridamole, ubenimex and dexamethasone. Evaluated by lipopolysaccharide (LPS)-induced cytokine storm murine model, DBDx significantly improved survival rate and prolonged survival time of the model mice. Notably, the efficacy of DBDx was higher than that of dipyridamole, ubenimex and dexamethasone. Determined by ELISA, DBDx significantly reduced the LPS-stimulated serum levels of TNF-α, IL-6 and IL-1β in mice. Luminex assay showed that DBDx suppressed the serum levels of a wide variety of inflammatory cytokines and chemokines, which was more potent than dexamethasone alone. Otherwise, DBDx exerted similar inhibitory effects on cytokine profiles in bronchoalveolar lavage fluid. Histopathological observation showed that DBDx significantly reduced the LPS-induced thickening of alveolar septum, indicating its suppression of capillary congestion, edema and neutrophil infiltration in the lung. Ultra-structure analysis showed that DBDx suppressed the LPS-induced morphological changes of microvilli in type II pneumocytes. In vitro experiment showed that DBDx inhibited IL-6 and TNF-α secretion in THP-1 cells, and downregulated TLR4/NF-κB/HIF-1α signaling pathway. All of these results demonstrate that DBDx, a triple combination of clinical orally-administered drugs, can alleviate cytokine storm and related lung injury. DBDx is beneficial for treating cytokine storm disorders.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon β (IFNβ), tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-β-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

NF-κB-mediated anti-inflammatory effects of an organic light-emitting diode (OLED) device in lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models.

Inflammation is the body's physiological response to harmful agents. However, if not regulated properly, inflammation can become pathological. Macrophages are key players in the inflammatory process, and modulate the immune response. Due to the side effects of anti-inflammatory drugs, non-pharmaceutical therapies for inflammatory diseases must be developed. Photobiomodulation is a non-invasive therapeutic approach to treating certain pathological conditions using light energy. Light-emitting diodes (LEDs) are commonly used as light sources for photobiomodulation treatment, but their clinical applications are limited. Organic LEDs (OLEDs) are thin, lightweight and flexible, enabling consistent and even delivery of light energy to target areas; this makes OLED promising components for therapeutic devices. In the present study, we examined the effects of OLED treatment on inflammation in vitro using a lipopolysaccharide (LPS)-induced macrophage RAW264.7 cell model, and in vivo using a pinna skin mouse model. We found that LPS-induced morphological changes and inflammatory cytokine expression were significantly reduced in RAW264.7 cells subjected to OLED treatment compared to the LPS-induced controls. This work provides evidence for the anti-inflammatory effects of OLEDs, demonstrating their potential to be incorporated into medical devices in the future.

Ginsenoside Rg1 Mitigates Porcine Intestinal Tight Junction Disruptions Induced by LPS through the p38 MAPK/NLRP3 Inflammasome Pathway.

Inflammation leads to porcine tight junction disruption of small intestinal epithelial cells, resulting in intestinal dysfunction. Herein, we established lipopolysaccharide (LPS)-induced in-vivo and in-vitro inflammatory models. The results revealed that LPS induced tight junction disruption in IPEC-J2 cells by downregulating tight-junction-related protein zonula occludens-1 (ZO-1), occludin and claudin-1 expression, while ginsenoside Rg1 rescued such inhibition and abrogated the upregulated expression of phosphorylation p38 MAPK. The p38 MAPK inhibitor (SB203580) showed a similar effect with Rg1 and attenuated the LPS-induced inhibition of ZO-1, occludin and claudin-1 expression, which is consistent with the reduced expression of NLRP3 inflammasome and IL-1β. Furthermore, the specific inhibitors of NLRP3 and IL-1β result in increased expression of tight-junction-related protein, demonstrating that p38 MAPK signaling was associated with Rg1 suppression of tight junction disruption. Besides, LPS treatment decreased the expression of ZO-1, occludin and claudin-1 through p38 MAPK signaling, and caused abnormal morphological changes in murine ileum. Meanwhile, Rg1 attenuated the decreased expression of ZO-1, occludin and claudin-1 and partially alleviated LPS-induced morphological changes in murine ileum. In summary, these findings characterized a novel mechanism by which Rg1 alleviates LPS-induced intestinal tight junction disruption by inhibiting the p38 MAPK-mediated NLRP3 inflammasome pathway.

The therapeutic mechanism of Shenyuan Gan in lipopolysaccharide-induced neuroinflammation in BV2 microglial cells

ObjectiveTo study the therapeutic effects of Shenyuan Gan (参远苷, SYG) on the inflammatory response in BV2 microglial cells induced by lipopolysaccharide (LPS). MethodsThe cytotoxicity of SYG to BV2 microglial cells was evaluated using a Cell Counting Kit-8 (CCK-8) assay, and the effect of SYG concentrations on LPS-induced BV2 microglial cells was studied. The morphological changes were observed using an optical microscope. The nitric oxide (NO) concentration in cell culture supernatant was determined using Griess reagent. The expression of cytokines and inflammatory mediators were also measured by an enzyme-linked immunosorbent assay (ELISA). Western blot analysis was used to determine the levels of inducible NO synthase (iNOS), nuclear factor-kappa B (NF-κB) p65, alpha inhibitor of NF-κB (IκB-α), phosphorylation-IκB-α (p-IκB-α), NOD-like receptor 3 (NLRP3), and caspase-1 expression. Moreover, the expression of iNOS, NLRP3, and ionized calcium binding adapter molecule 1 (Iba1) was also observed using immunofluorescent staining. ResultsSYG had a low cytotoxic effect on BV2 microglial cells and could significantly decr-ease LPS-induced morphological changes of BV2 microglial cells (P < 0.05). ELISA results showed that SYG significantly inhibited the LPS-induced increase in interleukin (IL)-1β and IL-6 in BV2 microglia cells (P < 0.05), and Western blot analysis showed that the phosphorylation levels of iNOS, NF-κB p65, and IκB-α as well as NLRP3 and caspase-1 expression were also significantly decreased, and IκB-α expression was increased after SYG treatment (P < 0.05, compared with the LPS-treated group). The immunofluorescence results were consistent with the Western blot results, and Iba1 staining indicated that the cell morphology tended to be resting. These results indicate that SYG has a certain inhibitory effect on LPS-induced inflammation in BV2 microglial cells. ConclusionSYG can inhibit LPS-induced release of inflammatory factors in BV2 microglial cells by affecting the phosphorylation levels of NF-κB p65 and IκB-α. SYG is a valuable candidate for treating neuroinflammation-related diseases.

Elizabethkingia anophelis, an emerging pathogen, inhibits RAW 264.7 macrophage function.

Elizabethkingia anophelis is a pathogen that can cause a life-threatening infection in immunocompromised patients. The first case of E. anophelis infection was reported in 2013; subsequently, an increase in its incidence has been reported globally. Additionally, a mortality rate of more than 30% was observed in the US outbreak of 2015. To date, the pathogenic mechanisms underlying E. anophelis infection, such as toxin production, remain unclear. Since tissue macrophages act as the first line of defense against pathogens, in the present study the interactions between E. anophelis and a macrophage-like cell line RAW 264.7 were examined. Although E. anophelis showed no cytotoxicity toward RAW 264.7 macrophages, the infection inhibited LPS-induced morphological changes and activation of differentiation markers for the polarization of RAW 264.7 macrophages toward an M1-like phenotype. However, when the cell contact was restricted using Transwell inserts or bacterial culture supernatants were used instead of live bacteria, no such inhibition was observed. Moreover, it was shown that E. anophelis evaded phagocytosis. Overall, the results suggest that E. anophelis infection inhibits the differentiation of RAW 264.7 macrophages to a pro-inflammatory phenotype in a contact-dependent manner.

Edaravone protects rat astrocytes from oxidative or neurotoxic inflammatory insults by restoring Akt/Bcl-2/Caspase-3 signaling axis

Astrocytes are the major glia cells in the central nervous system (CNS). Increasing evidence indicates that more than to be safe-guard and supporting cells for neurons, astrocytes play a broad spectrum of neuroprotective and pathological functions. Thus, they are compelling models to decipher mechanistic insights of glia cells to CNS insults and for the development of drugs. Edaravone is a free radical scavenger with the capacity to eliminate hydroxyl radicals and lipid peroxides. In this study, we examined the neuroprotective effects of edaravone in rat astrocytes challenged by hydrogen peroxide (H2O2) or bacterial lipopolysaccharides (LPS), respectively. We discovered that edaravone attenuated H2O2-induced oxidative stress by reactivating the Akt signaling axis and antagonistically restoring the expression of apoptosis associated regulators such as Bcl-2 and Caspase-3. Consistently, inhibition of Akt signaling by LY294002 attenuated the anti-oxidative activity of edaravone. In addition, edaravone mitigated LPS-induced morphological changes in astrocytes and alleviated the inflammatory activation and expression of TNF-α, IL-1β, IL-6 and NOS2. In summary, our data suggested that edavarone effectively protects astrocytes from oxidative stress or infectious insults, which may pave a new avenue for its application in preclinical research and human disease therapeutics.

Ethanol Extract of Lilium Bulbs Plays an Anti-Inflammatory Role by Targeting the IKK[Formula: see text]/[Formula: see text]-Mediated NF-[Formula: see text]B Pathway in Macrophages.

Lilium bulbs have long been used as Chinese traditional medicines to alleviate the symptoms of various human inflammatory diseases. However, mechanisms of Lilium bulb-mediated anti-inflammatory activity and the bioactive components in Lilium bulbs remain unknown. In the present study, the anti-inflammatory activity of Lilium bulbs and the underlying mechanism of action were investigated in macrophages using Lilium bulb ethanol extracts (Lb-EE). In a dose-dependent manner, Lb-EE inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and bone marrow-derived macrophages (BMDMs) without causing significant cytotoxicity. Lb-EE also down-regulated mRNA expression of inflammatory genes in LPS-stimulated RAW264.7 cells, which included inducuble nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]). Furthermore, Lb-EE markedly restored LPS-induced morphological changes in RAW264.7 cells to a normal morphology. HPLC analysis identified quercetin, luteolin, and kaempferol as bioactive components contained in Lb-EE. Mechanistic studies in LPS-stimulated RAW264.7 cells revealed that Lb-EE suppressed MyD88- and TRIF-induced NF-[Formula: see text]B transcriptional activation and the nuclear translocation of NF-[Formula: see text]B transcription factors. Moreover, Lb-EE inhibited IKK[Formula: see text]/[Formula: see text]-induced activation of the NF-[Formula: see text]B signaling pathway and IKK inhibition significantly reduced NO production in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that Lb-EE plays an anti-inflammatory role by targeting IKK[Formula: see text]/[Formula: see text]-mediated activation of the NF-[Formula: see text]B signaling pathway during macrophage-mediated inflammatory responses.

P38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1.

Inflammation and autophagy are two critical cellular processes. The relationship between these two processes is complex and includes the suppression of inflammation by autophagy. However, the signaling mechanisms that relieve this autophagy-mediated inhibition of inflammation to permit a beneficial inflammatory response remain unknown. We find that LPS triggers p38α mitogen-activated protein kinase (MAPK)-dependent phosphorylation of ULK1 in microglial cells. This phosphorylation inhibited ULK1 kinase activity, preventing it from binding to the downstream effector ATG13, and reduced autophagy in microglia. Consistently, p38α MAPK activity is required for LPS-induced morphological changes and the production of IL-1β by primary microglia in vitro and in the brain, which correlates with the p38α MAPK-dependent inhibition of autophagy. Furthermore, inhibition of ULK1 alone was sufficient to promote an inflammatory response in the absence of any overt inflammatory stimulation. Thus, our study reveals a molecular mechanism that enables the initial TLR4-triggered signaling pathway to inhibit autophagy and optimize inflammatory responses, providing new understanding into the mechanistic basis of the neuroinflammatory process.

Neuroprotective erythropoietin attenuates microglial activation, including morphological changes, phagocytosis, and cytokine production

Erythropoietin (EPO), a hematopoietic hormonal cytokine induced in response to hypoxia, has neuroprotective effects. EPO receptor (EPOR) is expressed in microglia, resident immune cells in the brain. However, the effect of EPO on microglial activation is not clear. In the present study, we demonstrated that the EPOR is highly expressed in microglia, rather than in neurons or astrocytes, in in vitro experiments. Therefore, we investigated whether EPO could attenuate lipopolysaccharide (LPS)-mediated activation of microglia in vitro. The BV-2 microglial cell line was treated with LPS in the absence or presence of EPO. In the presence of EPO, microglial expression of LPS-induced inflammatory cytokine genes was significantly decreased. In addition, EPO suppressed the LPS-induced phagocytic activity of BV-2 cells towards fluorescent beads, as well as induction of inducible nitric oxide synthase. In in vivo experiments, EPO significantly decreased the LPS-induced expression of inflammatory cytokine genes in mouse brains. Furthermore, morphological analysis of cortical microglia in the brains of mice stimulated with LPS revealed that combined treatment with EPO alleviated LPS-induced morphological changes in the microglia. These data indicate that EPO attenuates microglial activation, including morphological changes in vivo, phagocytosis in vitro, and the production of inflammatory cytokines in vivo and in vitro. Further investigation of EPO modulation of LPS-induced microglial activation may contribute to the development of novel neuroprotective therapies.

Trans-cinnamaldehyde improves memory impairment by blocking microglial activation through the destabilization of iNOS mRNA in mice challenged with lipopolysaccharide

Microglia activation and neuroinflammation are critically involved in pathogenesis of neurodegenerative disorders. Patients with neurodegenerative disorders often suffer memory impairment and currently there is no effective treatment for inflammation-led memory impairment. Trans-cinnamaldehyde (TCA) isolated from medicinal herb Cinnamomum cassia has been shown to exhibit anti-inflammatory capability. However, the potential of TCA to be used to improve memory impairment under neuroinflammation has not been explored. Primary microglia stimulated by lipopolysaccharide (LPS) were used to evaluate the potential anti-neuroinflammatory effects of TCA by examining the production of nitric oxide (NO), expression of inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines, and activation of MAPKs. A mouse model of LPS-induced memory impairment was established to assess the neuroprotective effects of TCA against memory deficit and synaptic plasticity inhibition by both behavioral tests and electrophysiological recordings. TCA pretreatment decreased LPS-induced morphological changes, NO production and IL-1β release in primary microglia. Decreased NO production was due to the accelerated degradation of iNOS mRNA in LPS-stimulated microglia through TCA's inhibitory effect on MEK1/2-ERK1/2 signaling pathway. TCA was able to reduce the levels of iNOS and phosphorylated ERK1/2 in hippocampus of mice challenged with LPS. Most importantly, TCA significantly lessened memory deficit and improved synaptic plasticity in LPS-challenged mice. This study demonstrates that TCA suppressed microglial activation by destabilizing iNOS mRNA, which leads to improved memory impairment in mice suffering neuroinflammation.

Silymarin Inhibits Morphological Changes in LPS-Stimulated Macrophages by Blocking NF-κB Pathway.

The present study showed that silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), inhibited lipopolysaccharide (LPS)-induced morphological changes in the mouse RAW264.7 macrophage cell line. We also showed that silymarin inhibited the nuclear translocation and transactivation activities of nuclear factor-kappa B (NF-κB), which is important for macrophage activation-associated changes in cell morphology and gene expression of inflammatory cytokines. BAY-11-7085, an NF-κB inhibitor, abrogated LPS-induced morphological changes and NO production, similar to silymarin. Treatment of RAW264.7 cells with silymarin also inhibited LPS-stimulated activation of mitogen-activated protein kinases (MAPKs). Collectively, these experiments demonstrated that silymarin inhibited LPS-induced morphological changes in the RAW264.7 mouse macrophage cell line. Our findings indicated that the most likely mechanism underlying this biological effect involved inhibition of the MAPK pathway and NF-κB activity. Inhibition of these activities by silymarin is a potentially useful strategy for the treatment of inflammation because of the critical roles played by MAPK and NF-κB in mediating inflammatory responses in macrophages.

A novel anti‑cancer effect of resveratrol: reversal of epithelial‑mesenchymal transition in prostate cancer cells.

Carcinoma progression is associated with the loss of epithelial features and the acquisition of a mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT). Resveratrol, a natural polyphenolic compound found in grapes, berries and peanuts, has a wide range of pharmacological properties, including anti-tumor metastasis properties. The underlying mechanism through which resveratrol inhibits metastasis of prostate cancer (PCa) is not yet fully understood; however, it is thought to be associated with the disruption of EMT. In the present study, lipopolysaccharide (LPS) was used to trigger EMT in PC-3 and LNCaP PCa cell lines, and the cell lines were subsequently treated with resveratrol. The results demonstrated that exposure of PC-3 and LNCaP cells to LPS resulted in morphological alterations characteristic of EMT, as well as an increase in the expression of the mesenchymal marker vimentin and a decrease in the expression of E-cadherin. In addition, LPS exposure resulted in an increase in cell motility, along with an upregulation of the transcription factor glioma-associated oncogene homolog 1 (Gli1). However, treatment with resveratrol inhibited LPS-induced morphological changes, decreased the expression of LPS-induced markers of EMT and inhibited the expression of Gli1, resulting in the inhibition of in vitro cell motility and invasiveness. These results provide a novel perspective for the anti-invasion mechanism of resveratrol, suggesting that the effect is in part due to its ability to inhibit the EMT process through the Hedgehog signaling pathway.

GBE50 Attenuates Inflammatory Response by Inhibiting the p38 MAPK and NF- κ B Pathways in LPS-Stimulated Microglial Cells.

Overactivated microglia contribute to a variety of pathological conditions in the central nervous system. The major goal of the present study is to evaluate the potential suppressing effects of a new type of Ginko biloba extract, GBE50, on activated microglia which causes proinflammatory responses and to explore the underlying molecular mechanisms. Murine BV2 microglia cells, with or without pretreatmentof GBE50 at various concentrations, were activated by incubation with lipopolysaccharide (LPS). A series of biochemical and microscopic assays were performed to measure cell viability, cell morphology, release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and signal transduction via the p38 MAPK and nuclear factor-kappa B (NF-κB) p65 pathways. We found that GBE50 pretreatment suppressed LPS-induced morphological changes in BV2 cells. Moreover, GBE50 treatment significantly reduced the release of proinflammatory cytokines, TNF-α and IL-1β, and inhibited the associated signal transduction through the p38 MAPK and NF-κB p65 pathways. These results demonstrated the anti-inflammatory effect of GBE50 on LPS-activated BV2 microglia cells, and indicated that GBE50 reduced the LPS-induced proinflammatory TNF-α and IL-1β release by inhibiting signal transduction through the NF-κB p65 and p38 MAPK pathways. Our findings reveal, at least in part, the molecular basis underlying the anti-inflammatory effects of GBE50.

NF-κB/AP-1-targeted inhibition of macrophage-mediated inflammatory responses by depigmenting compound AP736 derived from natural 1,3-diphenylpropane skeleton.

AP736 was identified as an antimelanogenic drug that can be used for the prevention of melasma, freckles, and dark spots in skin by acting as a suppressor of melanin synthesis and tyrosinase expression. Since macrophage-mediated inflammatory responses are critical for skin health, here we investigated the potential anti-inflammatory activity of AP736. The effects of AP736 on various inflammatory events such as nitric oxide (NO)/prostaglandin (PG) E2 production, inflammatory gene expression, phagocytic uptake, and morphological changes were examined in RAW264.7 cells. AP736 was found to strongly inhibit the production of both NO and PGE2 in lipopolysaccharide- (LPS-) treated RAW264.7 cells. In addition, AP736 strongly inhibited both LPS-induced morphological changes and FITC-dextran-induced phagocytic uptake. Furthermore, AP736 also downregulated the expression of multiple inflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase- (COX-) 2, and interleukin- (IL-) 1β in LPS-treated RAW264.7 cells. Transcription factor analysis, including upstream signalling events, revealed that both NF-κB and AP-1 were targeted by AP736 via inhibition of the IKK/IκBα and IRAK1/TAK1 pathways. Therefore, our results strongly suggest that AP736 is a potential anti-inflammatory drug due to its suppression of NF-κB-IKK/IκBα and AP-1-IRAK1/TAK1 signalling, which may make AP736 useful for the treatment of macrophage-mediated skin inflammation.

Curcumin inhibits LPS-induced EMT through downregulation of NF-κB-Snail signaling in breast cancer cells

Epithelial-mesenchymal transition (EMT) is considered a critical event in cancer cell invasion and metastasis. Emerging evidence has shown that curcumin may prevent or delay the progression of cancer, an effect that may be partially due to its ability to disrupt EMT, yet this has not yet been demonstrated. In this study, we used lipopolysaccharide (LPS) to trigger EMT in MCF-7 and MDA-MB-231 breast cancer cell lines and showed that curcumin inhibited LPS-induced morphological changes, decreased the expression of LPS-induced markers of EMT such as vimentin, and increased the expression of E-cadherin, resulting in the inhibition of invitro cell motility and invasiveness. We discovered that these actions were mediated through the inactivation of NF-κB-Snail signaling pathways. Our results indicate that curcumin plays an important role in the inhibition of LPS-induced EMT in breast cancer cells through the downregulation of NF-κB-Snail activity. These data provide a new perspective of the anti-invasive mechanism of curcumin, indicating that the effect is partly due to its ability to attack the EMT process.

Angiotensin type 1 receptor antagonist inhibits lipopolysaccharide‐induced stimulation of rat microglial cells by suppressing nuclear factor κB and activator protein‐1 activation

We investigated whether angiotensin (ANG) II and its receptors contribute to lipopolysaccharide (LPS)-induced microglial activation through activation of the proinflammatory transcription factors nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). Using primary microglial cell cultures, we examined whether losartan [ANG type 1 receptor (AT(1)) antagonist] alters the effects of LPS on: the production of interleukin-1 (IL-1) and nitric oxide, cell morphology, and NF-kappaB and AP-1 activities. Reverse transcription-polymerase chain reaction revealed that LPS-stimulated microglial cells exhibited marked mRNA expression for AT(1), ANG type 2 receptor (AT(2)) and the ANG II precursor angiotensinogen, whereas non-stimulated microglial cells expressed only those for AT(2) and angiotensinogen. We further demonstrated marked peptide/protein expression for AT(1) and ANG II in LPS-activated microglial cells. LPS (100 ng/mL)-stimulated microglial cells showed increased concentrations of IL-1 and nitrite (a relatively stable metabolite of nitric oxide), and increased expression of IL-1 mRNA as well as a morphological change from an amoeboid shape to a multipolar (mostly bipolar but sometimes tripolar) rod shape. These effects were all significantly inhibited by losartan treatment (10(-5) M or less). NF-kappaB and AP-1 activities were enhanced in LPS-stimulated microglial cells, effects that were significantly suppressed by losartan (10(-5) M). ANG II application enhanced the LPS-induced increases in IL-1 and nitrite concentrations, as well as the LPS-induced morphological changes and AP-1 activation, and these enhancements were inhibited by losartan (10(-5) M). These results suggest that endogenous ANG II enhances LPS-induced microglial activities through stimulation of the microglial AT(1), which itself evokes activation of the transcription factors NF-kappaB and AP-1.

Modulatory effects of [Met 5]-enkephalin on interleukin-1β secretion from microglia in mixed brain cell cultures

In the present study, functional interactions between [Met 5]-enkephalin (ME), naloxone and lipopolysaccharide (LPS) on interleukin-1β (IL-1β) immunostaining and secretion have been assessed in mixed brain cell cultures from embryonic day 17 mice. Adding ME alone or together with LPS to the culture increased the release of IL-1β after 48 h in a concentration-dependent fashion. In situ hybridization studies showed that LPS, but not ME, increased the abundance of IL-1β mRNA. The enhanced release of IL-1β caused by ME or LPS was partially blocked by naloxone. LPS induced concentration-dependent morphological changes in microglia in mixed brain cell cultures, identified by a monoclonal antibody F4/80 which is specific for macrophages/microglia. Despite increasing IL-1β release into the media, ME (10 −8 M) did not induce morphological changes in microglia. Naloxone alone also had no effect on glial morphology; however, the LPS-induced morphological changes were blocked by naloxone. Our data indicate that both exogenous and endogenous opioids regulate IL-1β production by microglial cells in the mixed brain cell cultures.