| BackgroundInflammatory cytokines and anti-inflammatory factors are produced in neurovascular diseases and neurodegenerative diseases, including ischemic stroke, hypoxic-ischemic encephalopathy, Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS) and thereby modulating neuroinflammation and diseases progression. In addition to microglia activation, ischemic stroke also induces the infiltration of peripheral inflammatory cells such as neutrophils, monocytes and macrophage, which aggravating neurons injury and blood brain barrier (BBB) disruption as a result and leading a poor nerve function. What’s more, PD and AD are the two disease of high incidence among elderly persons and are also the most common neurodegenerative disease and threaten to human health. The disease pathogenesis and mechanisms of neurodegenerative disease are very complex and there are several hypotheses including genetics, infection, neurotoxicity, and oxidative stress, et al and which of these hypotheses plays a main role are not clear yet. Treatment of such disease will be a long-term challenge. With the development of immunohistochemical and morlecular biology, it is found that there is regional inflammation happened in the brain parenchyma with neurodegenerative disease. Microglia activation and the expression of a series of inflammatory factors are involved in the pathomechanism of neurodegenerative diseases. In conclusion, it suggests that microglia and inflammatory factors play an important role in the pathogenesis of intracranial diseases.Microglia are the main immune cells in central nervous system (CNS). Microglia will rapidly activate from its resting state when the CNS encounter pathogens, or biomacromoleculars and then mediate cerebral immune response so as to clear cell debris, microorganisms, et al. They have a unique role in maintaining the balance of CNS. The activated microglia function as a double-edged sword on neuroinflammation. On one hand, activated microglia release inflammatory mediators to resist pathogens and injuries, on the other hand, under certain circumstances, constant activated microglia massively release inflammatory mediators like nitric oxide (NO) and pro-inflammatory cytokines such as interleukin (IL)-1/6/12, tumor necrosis factor-α (TNF-α) and bring detrimental effects to neurons and then aggravate brain injury. Understanding the mechanism of microglia-mediated neuroinflammation could be helpful to develop inflammation targeting therapy for neurodegenerative diseases.Monocyte chemotactic protein-induced protein-1 (MCPIP), also known as ZC3H12A, Due to its endonuclease activity for mRNA per se, it was renamed as regulatory RNase-1 (Regnase-1). This protein contains a PIN-like domain for RNase activity, a nuclear localization signal sequence, a single CCCH-type zinc finger motif for RNA-binding and two proline-rich domains at the N- and C-terminals for protein interaction. It is a novel CCCH-type zinc finger motif-containing protein and proteins containing CCCH-type zinc finger motif represent a typical molecular mediating mRNA decay of many inflammatory factors and it suggests this type of proteins have effects in inhibiting inflammation. Recent years, MCPIP have been proved to be a negative regulate factor in inflammation. Inflammation is very important for body to resist pathogens and protect the body against attack. But excessive inflammatory cytokines can cause tissue damage so it is under closely controlled at the same time. Post-transcriptional regulation is one of the important mechanisms of inflammation regulation. It is reported that MCPIP has the ability to regulate inflammatory reaction because it can decay mRNA of different inflammatory factors directly. In addition, MCPIP can inhibit inflammatory pathways. Many studies nowadays have focus on MCPIP because of its anti-inflammatory function. The PIN-like domain of MCPIP possesses RNase activity and cleaves mRNA of IL-1β, IL-6, IL-12p40 directly. Moreover, the CCCH-type zinc finger motif of MCPIP and its upstream N-terminal conserved region (NCR) possess deuquitinating activity and MCPIP negatively regulates JNK and NF-κB signaling pathways via these domains. Taken together, MCPIP is a molecular essential for down regulate inflammation.The research about the effects of MCPIP in CNS is few both at home and abroad so far. The function and mechanism of MCPIP in CNS is not quite clear yet. In the last few years, there are only several researches which report that MCPIP have certain effect on neuroinflammation. Lipopolysaccharide (LPS) or electroacupuncture pretreatment induce significant ischemic tolerance to focal cerebral ischemia and MCPIP has been proved to participate in the ischemic tolerance. ZC3H12A deficiency significant increase in the infarct volume and cause up regulation of proinflammatory factors in mice brain after middle cerebral artery occlusion (MCAO). It suggests that MCPIP may play a protective role in inhibiting neuroinflammation but further research is needed to better define the role of MCPIP in CNS inflammation. MCPIP expresses in many kinds of immune cells such as T cells, monocytes and macrophages under stimulation by LPS, IL-1β, TNF-α and so on. What’s more, the expression of MCPIP negatively regulates inflammatory factors release in such cells in return. MCPIP is also expressed by microglia but its effect on microglia is unknown. Therefore, in this study, we examined the expression of MCPIP induced by the treatment of lipopolysaccharide (LPS) or oxygen-glucose deprivation plus reoxygenation (OGD/R) in BV-2 cells. We also knock downed the MCPIP in microglia cell line BV-2 cells and investigate its anti-inflammatory effect of MCPIP in microglia under treatment by LPS. We also identified the protective effect of MCPIP on reducing the neurotoxicity of BV-2 cells induced by LPS.Purpose1. To confirm LPS or OGD/R induce MCPIP expression in BV-2 cells.2. To identify MCPIP whether inhibit inflammatory cytokines released from BV-2 cells and reduce the neurotoxicity of BV-2 cells under treatment of LPS.Methods1. BV-2 cells were treated with LPS to exam MCPIP expression.Microglia cell line BV-2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). When BV-2 cells got to about 80% confluence, the cells were treated with 1μg/ml LPS for 4,8, 12 and 24h or with different dosage of LPS (0.1,0.3, and 1μg/ml) for 4h. Experiments were repeated three times independently. Real-time PCR (RT-PCR) and Western blot assay were performed to determine the expression of MCPIP in BV2 cells after treatment of LPS.2. Establishing the hypoxia-reperfusion cells model of BV-2 cells in vitro.BV-2 cells were randomly divided into seven groups according the different time-combination of OGD/R:OGD1h/R3h、OGD1h/R24h、OGD2h/R3h、 OGD2h/R24h、OGD4h/R3h、OGD4h/R24h and normal control groups. Experiments were repeated three times.Western blot assay were performed to detect the expression of MCPIP in BV2 cells after treatment of OGD/R.3. Establishing the siRNA transfection time in BV-2 cells.MCPIP siRNA was transfected to BV-2 cells to mediate gene knockdown (siMCPIP) and the crambled sequence was used as a negative control (siControl). Lipofectamine 2000 (5μl/ml) and siRNA (50pM) were mixed and added to cells according to the manufacture’s instruction. BV-2 cells were randomly divided into two groups:cell transfection for 24h and another for 48h, each group were repeated three times independently. RT-PCR was performed to confirm the silencing efficiency of MCPIP at 48h post-transfection.4. Detection of inflammatory factors released from BV-2 cells with or without MCPIP silencing.BV-2 cells were randomly divided into two groups:cells transfected with MCPIP siRNA (siMCPIP) and cells transfected with crambled sequence (siControl). BV-2 cells were treated with LPS (1μ.g/ml) for 4h after MCPIP silencing. Experiments were performed in triplicate and repeated three times independently. Quantitative real-time polymerase chain reaction (QRT-PCR) assay was performed to observe gene expression of inflammatory factors including IL-12b, IL6, IL-1β, IL-23,immediate early response 3 (IER3), tumor necrosis factor receptor2 (TNFR2) and inducible costimulator (ICOS). The relative gene expression was analyzed using the 2-ΔΔCt method.5. Detection of the cytotoxicity of medium collected from LPS-stimulated BV-2 cells with or without MCPIP silencing.BV-2 cells were transfected with MCPIP siRNA or scrambled siRNA prior to 0.1μg/ml LPS treatment. Four hours after treatment of LPS, replaced with flesh DMEM and continue to incubate for 24h. Experiments were performed in triplicate and repeated three times independently.The cell medium was collected and added to SH-SY5Y cells for 6h. The SH-SY5Y cell viability was measured by MTT assay and the cell death rate was measured by lactate dehydrogenase (LDH) release assays.6. Statistical analysisStatistical software SPSS 13.0 analysis was used to analyze experiment data. Comparison between two groups was conducted by Student’t test if homogeneity of variance or by Wilcoxon rank sum test if heterogeneity of variance. All the experimental data are expressed by the mean ± standard deviation (X ± S). P<0.05 was considered significant.Results1. RT-PCR and Western blot showed that treatment of LPS (1μg/ml) for 4h,8h, 12hand 24h increased MCPIP mRNA level after 4h of LPS treatment and was sustained at high level for at least 24h (P<0.01). In comparison, protein expression of MCPIP induced by LPS treatment displayed time-dependent manner and peaked at 12h but reduced significantly after treatment for 24h. In addition, different dosage of LPS (0.1,0.3 and 1μg/ml) could also induce the elevation of MCPIP mRNA expression (P<0.05).2. Different time-combination of OGD/R induced the protein expression of MCPIP in BV-2 cells. Western blot showed that the groups of OGD2h/R3h and OGD2h/R24h both remarkable induced the expression of MCPIP.3. The efficiency of MCPIP knockdown was higher at 48h post-transfection (P<0.05) compared to 24h (P>0.05) and the efficiency at 48h post-transfection was about 60%. Silencing MCPIP in BV-2 cells under LPS stimulation condition increased the mRNA level of IL-12b and IL-6 (P<0.05) and decreased the IER3 (P<0.05), while the rest inflammatory factors IL-1β, IL-23, TNFR2 and ICOS were not changed significantly (P>0.05).4. We collected the cell medium from LPS-treated BV-2 cells with or without MCPIP silencing and added the cell medium to SH-SY5Y cells. The results of MTT assay and LDH assay showed that the cell medium from BV-2 cells with MCPIP silencing could induce lower cell viability (P<0.05) and higher rate of neuronal cell death (P<0.05).Conclusions1. Either LPS or OGD/R could induce the expression in BV-2 cells and the findings will provide experimental basis for further research in intracranial inflammatory diseases.2. Silencing MCPIP in BV-2 cells under LPS stimulation condition increased the mRNA level of IL-12b and IL-6 and decreased the IER3. It suggests that MCPIP is essential to regulate inflammation in CNS.3. The cell medium from BV-2 cells with MCPIP silencing under LPS stimulation condition brought higher neuronal toxicity. It indicates that MCPIP play a significant role in regulating the neurotoxicity of microglia under neuroinflammation and has protencial protective effects in neuroprotection. |
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