This is the first study to report that TET can inhibit the MAPK signaling pathway in LPS-induced microglial activation. concentrations (0.1 M, 0.5 M or 1 M) did not affect the cell viability. After TET pretreatment, the levels of IL1and TNF (both in transcription and translation) were significantly inhibited in a dose-dependent manner. Further studies indicated that phospho-p65, phospho-IKK, and phospho-ERK 1/2 expression were also suppressed by TET. Conclusions Our results indicate that TET can effectively suppress microglial activation and inhibit the production of IL1and TNF by regulating the NF-kB and ERK signaling pathways. Together with our previous studies, we suggest that TET would be a promising candidate to effectively suppress overactivated microglia and alleviate neurodegeneration in glaucoma. Introduction Microglia constitute a UNC569 unique population of immune cells in the CNS. They are distributed throughout the brain and retina, represent approximately 12% of the adult brain cells, and play a pivotal role in the innate immune response [1]. In normal conditions, microglia support synaptogenesis through the local synthesis of neurotrophic factors [2], [3] and the regulation of synaptic transmission and remodeling [4],[5]. In response to acute neurodegenerative disease, they transform from a ramified basal homeostatic phenotype to an activated phagocytic phenotype and release pro-inflammatory mediators, such as IL1 and TNF. This acute neuroinflammatory response is generally beneficial to the CNS because it tends to minimize further injury and contributes to the repair of damaged tissues [6], [7], [8], [9]. In contrast, chronic neurodegenerative diseases, including Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and glaucoma are recognized to be associated with chronic neuroinflammation. Long-term activation of microglia is the most prominent feature of chronic neuroinflammation. Sustained release of inflammatory mediators by activated microglia may induce increased oxidative and nitrosative stress, always leading to neurotoxic consequences [10]. Glaucoma is a chronic neurodegenerative disease [11]. The progressive degeneration of retinal ganglion cells (RGCs) and sustained loss of the visual field are its remarkable characteristics [12]. Recent studies suggested that activated microglia participate in the pathological course of glaucomatous optic injury with adverse consequences [13], [14], and reduced microglial activation was associated with alleviating optic nerve and retinal neurodegeneration [15]. Tetrandrine(TET) [16], a bisbenzylisoquinoline alkaloid extracted from Moore, has a variety of biologic activities and has been used to treat patients with tumors [17], hypertension [18], fungal infection [19] and silicosis [20] for decades. Recently, in vitro and in vivo studies have suggested that TET reduced UNC569 the inflammatory response in macrophages by inhibiting the production of chemokines and cytokines [21]. Other studies also reported UNC569 that TET decreased the production of TNF, IL1, IL6 and NO in activated microglia by inhibiting the NF-B signaling pathway [22], [23]. Mitogen-activated protein kinases (MAPKs), including ERK 1/2, JNK, and p38, are a group of signaling molecules, and play an important role in pro-inflammatory cytokine expression [24]. Previous studies demonstrated that the up-regulation of the MAPK signaling pathway was involved in various models of microglial activation [25], [26]. Further studies also suggested that the effective Rabbit Polyclonal to DPYSL4 inhibition of the MAPK pathway could decrease the production of pro-inflammatory cytokines and thus be beneficial for neuronal survival [27]. However, it is unclear whether TET could affect the MAPK signaling pathway in activated microglia. In this study, we investigated the inhibitory function of TET in LPS-activated microglia and clarified its possible mechanisms. Methods 2.1 Experimental procedures Tetrandrine (Sigma, European Pharmacopoeia (EP) Reference Standard, purity>99%) was dissolved in 0.1N HCl and adjusted to pH 7.3. Then, it was diluted to give a 1 mM concentrated stock solution in sterile PBS and filtrated with a nitrocellulose filter with a pore size of 0.22 m (Millipore). When in use, the stock solution was further diluted to the desired concentrations with culture medium. Cell viability assays and cell apoptosis assays were used to identify the working concentrations of TET. BV2 cells were seeded, pretreated with TET at variable concentrations for 2 hours, and LPS (Sigma, final concentration: 1 g/ml) was then added to the medium. The plates were incubated for an additional 24.
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