Modulation of lipopolysaccharide stimulated nuclear factor kappa b mediated inos/no production by bromelain in rat primary microglial cells

Background: Microglial cells act as the sentinel of the central nervous system. They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide [NO] and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation.

Methods: Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain [5, 10, 20, and 30 ng/ml] on the levels of NO, inducible nitric oxide synthase [iNOS], and nuclear factor kappa B [NF-KB] in microglia treated with lipopolysaccharide [LPS] [1 ng/ml]. Data were analyzed using Student's t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain

Results: Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS [1 u,g/ml] treatment in a dose-dependent manner. Bromelain [30 micro.g/ml] also significantly reduced the expression of iNOS at mRNA level and NF-KB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death

Conclusion: Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases

effect of Migri-Heal on nitric oxide production in an in vitro inflammatory model of primary microglial cells

Background: Recently, much attention has been directed towards considering activated microgelial cells as putative targets for treatment of neurological disorders. MigriHeal as a novel herbal remedy was introduced for the treatment of migraine headaches. The previous researches has shown that MigriHea extracts can decrease NO in an in vitro inflammatory model. The aim of this study was to investigate the effect of MigriHeal on NO generation from LPS- stimulated microglia cells

Materials and Methods: Neonatal rat primary microglial cells were isolated from the mixed glial cultures and the purity of the cultures was determined by immunocytochemistry. Microglial cells were pretreated with Migri-Heal and activated by 1microg/ml LPS. Subsequently, NO levels in the culture supernatants were measured by a griess reaction. Our results showed that Migri-Heal 50microg/ml significantly reduced NO level in inflamed microglia in a dose-dependent manner

Results: The results showed that different concentrations of Migri-Heal had no prominent effect on cell viability in presence of LPS as compared with the control group. In addition, the pretreatment of microglia cells with Migri-Heal can prevent from a morphological changes of the cells into the round and phagocytic shape

Conclusion: Our study demonstrated that MigriHeal might have NO scavenging properties. Integrative studies are warranted to uncover the novel pharmacological insights of this herbal remedy as an putative therapeutic approach against diseases -associated with inflammation

Study of the expressions of IL-20R1 and IL-20R2 in C57BL/6 mice astroglial cells

Astrocytes are the most abundant glial cell type. They may promote or inhibit CNS inflammation depending on which cytokines are secreted. Astrocytes also have immune roles. IL-19, IL-20, and IL-24 activate a heterodimer receptor composed of the IL-20R1 alpha-chain and the IL-20R2 beta-chain. It has long been considered that signaling by these receptor complexes affects immunological reactions, however the biological functions of IL-20R1 and IL-20R2 in the brain remain unclear. As the first step to address the role of these cytokine receptors in the brain, in this study we have researched the expressions of IL-20R1 and IL-20R2 in C57BL/6 mice astrocytes. We examined expressions of IL-20R1 and IL-20R2 proteins in mice astroglial cells and in the 1321N1 astrocytoma cell line in response to MOG, LPS and GM-CSF by flow cytometry. The effect of LPS on mRNA expression of IL-20R1 and IL-20R2 was investigated by RT-PCR. We provide, for the first time, evidence that astrocytes expressed IL-20R1 and IL-20R2 mRNA not only in response to LPS stimulation but also in unstimulated astrocytes. We did not observe the expressions of IL-20R1 and IL-20R2 proteins in mice astroglial cells and the 1321N1 astrocytoma cell line. IL-20R1 and IL-20R2 mRNA are constitutively expressed in astrocytes. Because the majority of neuropathological processes involve astrocytes and inflammatory cytokines, the results of this study, which are reported for the first time, have important implications for future research

Detection of IL-20R1 and IL-20R2 mRNA inC57BL/6 Mice Astroglial Cells and Brain Cortex Following LPS Stimulation

Astrocytes, which comprise 90% of overall brain mass, are involved in brain immunity. These cells represent the non-professional class of CNS-resident APCs and may promote or inhibit CNS inflammation depending on the cytokines they secrete. IL-10 family of cytokines and their receptors, IL-20R1 and IL-20R2, may have a role in shifting astrocytes to a neuroprotective or neurodegenerative function. To address the expression of IL-20R1 and IL-20R2 cytokine receptors in astrocytes and brain cortex of C57BL/6 mice. We investigated the expression of IL-20R1 and IL-20R2 in C57BL/6 mice astroglial cells and brain cortex in response to lipopolysaccharide [LPS], using reverse-transcription polymerase chain reaction [RTPCR] method. Astrocytes were able to express IL-20R1 and IL-20R2 mRNA not only in response to LPS stimulation but also in the absence of LPS. Furthermore, we found the expression of IL-20R1 and IL-20R2 mRNA in the cortex of adult C57BL/6 mice. IL-20R1 and IL-20R2 are constitutively express in the brain. Since most neuropathological processes involve astrocytes and inflammatory cytokines, these findings have important implications for future therapeutic strategies

Recognition of betaine as an inhibitor of lipopolysaccharide- induced nitric oxide production in activated microglial cells

Neuroinflammation, as a major outcome of microglia activation, is an important factor for progression of neurodegenerative disorders including Alzheimer's disease and Parkinson's disease. Microglial cells, as the first-line defense in the central nervous system, act as a source of neurotoxic factors such as nitric oxide [NO], a free radical which is involved in neuronal cell death. The aim of this study was to inhibit production of NO in activated microglial cells in order to decrease neurological damages that threat the central nervous system. An in vitro model of a newborn rat brain cell culture was used to examine the effect of betaine on the release of NO induced by lipopolysaccharide [LPS]. Briefly, primary microglial cells were stimulated by LPS and after 2 minutes, they were treated by different concentrations of betaine. The production of NO was assessed by the Griess assay while cell viability was determined by the MTT assay. Our investigations indicated that LPSinduced NO release was attenuated by betaine, suggesting that this compound might inhibit NO release. The effects of betaine on NO production in activated microglial cells after 24 h were "dose-dependent". It means that microglial cells which were treated with higher concentrations of betaine, released lower amounts of NO. Also our observations showed that betaine compound has no toxic effect on microglial cells. Betaine has an inhibitory effect on NO release in activated microglial cells and may be an effective therapeutic component to control neurological disorders

Blockade of the naloxone-induced aversion in morphine-conditioned wistar rats by L-arginine intra-central amygdale

Single injection of naloxone, a selective antagonist of morphine, prior to the drug conditioning testing was used to investigate on morphine dependence. Conditioning to morphine [2.5-10 mg/kg, s.c.] was established in adult male Wistar rats [weighing 200-250 g] using an unbiased procedure. Nitric oxide agents were microinjected into the central amygdale prior to naloxone-paired place conditioning testing. The results showed that morphine produced a significant dose-dependent place preference in animals. Naloxone [0.1-0.4 mg/kg, i.p.] injections pre-testing of the response to morphine [7.5 mg/kg, s.c.] caused a significant aversion at the higher doses [0.4 mg/kg, i.p.]. This response was reversed by microinjection of L-arginine [0.3-3 micro g/rat, intra-central amygdale] prior to naloxone on the day of the testing. The response to L-arginine was blocked by pre-injection of N[G]-nitro-L-arginine methyl ester [L-NAME] intra-central amygdale]. A single injection of naloxone on the test day of morphine place conditioning may simply reveal the occurrence of morphine dependence in rats, and that the nitric oxide in the central amygdale most likely plays a key role in this phenomenon

Collagen as adherent substratum and inducer of dorsal root ganglia outgrowth

Neurite outgrowth from dorsal root ganglion [DRG] explants is a method of evaluating neurotrophic activity of growth factors. When complete medium containing collagen was supplemented with nerve growth factor [NGF] DRG outgrowth was observed after 18 h. In the absence of NGF and in the presence of collagen, the DRG outgrowth took place after 72 h. In wells not supplemented with collagen gel in substratum, no DRG outgrowth was observed. Partially, DRG differentiation was observed in the presence of NGF. In the absence of NGF and collagen, there was no DRG outgrowth detected. It seems that, in some circumstances, cells degenerated by DRG may be an indication of an apoptosis phenomenon. Therefore, we suggested that collagen as a substratum is more effective than NGF