| BackgroundBecause of the central nervous system (CNS) of adult individuals cannot have meaningful regeneration and form functional synaptic connections after injury, often resulting in permanent neurological deficit, bringing long-term damage to the patients, their families and society. CNS repair and regeneration after injury is an important research project in neuroscience.With the continuous development and progress of medical science and technology, the stem cell transplantation in the treatment of CNS injury shows a great prospect. Stem cells with a variety of differentiation potential and self-renewal capacity, are present in various tissues of adult individuals or embryos. In recent years, with development of stem cell research, bone marrow mesenchymal stem cells (BMSCs) become a hotspot in the Regenerative Medicine. These cells are relatively easy to isolate, expand, and differentiate into several cellular phenotypes in vitro and in vivo. Due to their self-renewal and potential multilineage properties, BMSCs appear to be an ideal cellular source for the repair of CNS injuries.Previous studies have tested the therapeutic potential of BMSCs in treating CNS injuries, including traumatic brain injury (TBI), stroke, and spinal cord injury (SCI) in animal models. The results of these studies suggested that transplanted BMSCs have beneficial effects on CNS injuries. For instance, BMSCs may promote functional neurological recovery, decrease apoptosis levels, foster endogenous neurogenesis, improve angiogenesis and reduce lesion size, etc. The initial assumption in exploring the mechanisms, by which BMSCs ameliorate CNS injury, was that they migrated to the injured tissues and transdifferentiated to replace the damaged neural cells. Therefore, we first focus on whether there is structural integration between stem cells and the host tissue, by manganese enhanced magnetic resonance imaging (MEMRI).Magnetic resonance imaging (MRI), as a non-invasive tool with the feature of high spatial and temporal resolution, is frequently used to assess the therapeutic effect in experimental models and to study the structural details of the inner depths of the body in vivo. Recently, it was shown that the manganese (Mn2+), as a paramagnetic ion and contrast agent, enhanced the signal intensity (SI) of T1-weighted images by shortening the relaxation time of surrounding water protons in research involving a variety of systems, especially the CNS. Compared with other contrast agents, Mn2+ has several potential advantages, as described below. First, as a calcium (Ca2+) analogue, Mn2+ enters the neural cells quickly via voltage-gate calcium channels, then a fraction of ions are transported along the microtubules and crosses synapses to reach post-synaptic neurons. Second, owing to the low clearance rate, Mn2+ provides sufficient time for performing MEMRI to obtain high spatial resolution and high signal-to-noise ratio images. Based on the physical and chemical properties of Mn2+ (vide supra), Mn2+ has been frequently used as a contrast agent for anatomic and functional imaging. The primary issue involving the use of Mn2+ in biomedical research is neurotoxicity. The optimal Mn2+ concentration for limpid MEMRI images without Mn2+-induced toxicity has not been established. Whether or not Mn2+ can significantly enhance the signal intensity at non-toxic levels for primary cultured cortical neurons is not clear. Therefore, we performed cellular MRI with Mn2+-labeled neurons to determine the optimal dose of Mn2+ which could produce significant signal intensity, and set some indices, including neuronal morphology, apoptosis, lactate dehydrogenase (LDH), and reactive oxygen species (ROS) formation, to evaluate Mn2+-induced cytotoxicity in vitro. The results showed that Mn2+ could produce significant enhancement of MEMRI signal intensity but with overt cytotoxicity as well. On the other hand, the MEMRI can not reach the distinguishability at the cellular and molecular level, therefore, the functional mechanism of BMSCs needs to study further. Meanwhile, we pay attention to the roles of BMSCs in the immunoregulation and inflammation process.Recent studies showed that transplanted BMSCs exerted their therapeutic effect without evidence of engraftment, which indicated that their regenerative and differentiating abilities might not play a role in enhancing tissue repair or limiting tissue destruction. Instead, BMSCs that are stimulated via an inflammatory signal secrete a variety of bioactive molecules, such as trophic factors and anti-inflammatory molecules, to modulate the host microenvironment, which may be the main mechanism responsible for their therapeutic effects. We recently found that BMSCs can modulate inflammation-associated cytokine release and immune cells activation during TBI-induced cerebral inflammatory responses and that the beneficial effects of BMSCs may be partially explained by the effect of TSG-6 on the NF-κB pathway. However, whether TSG-6 exerts an anti-inflammatory effect by directly affecting the resident inflammatory cells of the CNS, mainly microglial cells, remains unclear.Microglia, which are derived from primitive myeloid progenitor cells, are the resident immune cells of the CNS. As CNS-specific macrophages, they play a critical role in immune surveillance and homeostatic maintenance of the CNS. They are highly responsive to stress and injury, and become immediately and focally activated in response to brain injuries, systemic infections, and chronic diseases. Furthermore, microglia are involved in initiating inflammatory responses in the brain through secreting a variety of inflammatory mediators, including tumor necrosis factor a (TNF-α), interleukin 1 (IL-1β), IL-6 and nitric oxide (NO). Many recent studies have attributed neuronal damage to the inflammatory responses of the microglia rather than to a direct neurotoxic insult. Reactive microglia is a common feature of numerous types of brain pathology. Therefore, modulating microglia function and activity appears to be an attractive approach to treating CNS injuries.At present, some scholars abroad pay attention to the relationship between the microglia fuction and the BMSCs. Debora et al found that BMSCs can secrete CX3CL1 (a kind of chemokine) make microglia from proinflammatory state to the neuroprotective state. Zhou et al found that the BMSCs could suppress the NO production of BV2 microglial cell line after LPS stimulation. Rahmat et al found BMSCs could promote BV2 cells produce NO and IL6, reducing TNFa production. In short, the mechanism of BMSCs affecting microglia isn’t understood fully yet.This research based on previous results, further study the mechanism of BMSCs on CNS injury. Whether TSG-6 released by MSCs have a moderating effect on the activation of microglia, as well as its possible mechanisms. Our research provided the experimental basis and new ideas for the future clinical application of MSCs in the treatment of central nervous system injury.Chapter Ⅰ Determination of the detectable concentration of manganese usedin neuronal MEMRI and its effect on cortical neurons in vitroObjectiveTo explore the functional mechanism of BMSCs by the manganese enhanced magnetic resonance imaging (MEMRI). Especially to evaluate whether Mn2+ can significantly enhance the signal intensity of primary cultured cortical neurons at nontoxic level.MethodNeurons were incubated with different concentrations of Mn (control,0.01,0.05, 0.10,0.20mMol/L) and then the cells viability, lactate dehydrogenase (LDH) release assay, intracellular reactive oxygen species (ROS) level, and apoptosis were measured at the 24 hour after treatment. At the same time, the intracellular Mn2+ concentrations were analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and the cellular MRI was performed in vitro.Result(1) After the neurons were treated with Mn at low concentration (0.01 mMol/L), there was no impact on both cells viability and cytotoxicity. The percent of TUNEL-positive cells was as 9.24±0.73%, but no significant signal enhanced in MEMRI. (2) When the neurons were exposured to the higher concentrations of Mn (0.05mMol/L, 0.1mMol/L and 0.2mMol/L), the cells viability was significantly reduced to 68.30±7.97%,54.98±9.08% and 41.52±4.36% respectively in 0.05,0.10, 0.20mMol/L Mn-treated groups, which showed significant statisticly comparing with the control group (P<0.05). The intracellular ROS formation was 1.83,2.38 and 3.24 times of control group as respective (n=6, P<0.05) and the percentages of TUNEL-positive cells were as 18.15±0.81%,23.63±0.86%,35.26±3.54%, and the signal intensity of MEMRI showed 313.67±54.37,466.7±51.94, and 658.33±110.56 in the 0.05mMol/L, 0.1mMol/L and 0.2mMol/L groups respectively.ConclusionMn2+ concentration at higher than 0.05mMol/L could produce the significant enhancement of MEMRI signal intensity but with overt cytotoxicity as well.By this token, we didn’t continune to study the the synapse between BMSCs and host neurons by MEMRI. However, the therapeutic mechanism of BMSCs is still as the focus in our research. In the following study, we paied close attention to the effect of BMSCs on regulating microglia, which is the one of important therapeutic mechanisms of BMSCs during the repairing of the injured CNS.Chapter II BMSCs Inhibit Lipopolysaccharide-Induced Inflammatory Responses of BV2 Microglial Cells through TSG-6BackgroundMicroglia are the primary immunocompetent cells in brain tissue, and microglia-mediated inflammation is associated with the pathogenesis of various neuronal disorders. Recently, many studies have shown that the BMSCs displayed a remarkable ability to modulate inflammatory and immune responses through the release of a variety of bioactive molecules, protecting the CNS. Previously, we reported that BMSCs have the ability to modulate inflammatory responses in a Traumatic brain injury (TBI) model and that the potential mechanisms may be partially attributed to upregulated TSG-6 expression. However, whether TSG-6 exerts an anti-inflammatory effect by affecting microglia is not fully understood. In this study, we investigated the anti-inflammatory effects of BMSCs and TSG-6 in an in vitro LPS-induced BV2 microglial activation model.Methods/resultsA 6-well transwell system was used to assess the effect of BMSCs on BV2 cells that were stimulated using lipopolysaccharide (LPS). A total of 5×105 BV2 cells were placed in the lower chamber with or without 100 ng/mL of LPS in the presence of one of the following treatments in the upper chamber:(1) control medium (LPS group); (2) MSCs (LPS-BMSCs group); (3) MSCs transfected with TSG-6 siRNA(LPS-BMSCs group); (4) 1.0×105 activated MSCs transfected with controlsiRNA;(5) rmTSG-6 at 1 ng/ml; (6) rmTSG-6 at 10 ng/ml; or (7) rmTSG-6 at 100 ng/ml. After 6hrs of treatment, the expression levels of TNFa, IL1β, IL6 and iNOS genes in BV2 cells were evaluated using real-time PCR. The altered expression of these genes was described as the relative ratio to the unstimulated group.We found that BMSCs significantly inhibited the expression of pro-inflammatory mediatorsby microglia activated. The relative expression between LPS and LPS-BMSCs group were 11.13±1.53 versus 2.77±0.99,5.70±0.56 versus 2.28±0.17,11.67±1.15 versus 3.23±0.42,4.73±0.23 versus 2.47±0.40 for TNFa (P<0.05), ILlβ(P<0.05), IL6(P<0.05) and iNOS(P<0.05) respectively. In addition, rmTSG-6 also could reduce these pro-inflammatory mediators in a dose dependent manner [1ng (P<0.05), 10ng(P<0.05),100ng(P<0.05)]. However, BMSCs effects on microglia were attenuated when TSG-6 expression was silenced [LPS-BMSCs versus LPS-TSG-6siRNA BMSCs:2.77±0.99 versus 6.01±0.32,2.28±0.17 versus 4.02±0.26, 3.23±0.42 versus 7.68±1.14,2.47±0.40 versus 3.55±0.37 for TNFa (P<0.05), IL1β(P<0.05), IL6(P<0.05) and iNOS(P<0.05) respectively]. To elucidate the molecular mechanism underlying the anti-inflammatory function of TSG-6 in microglia, we evaluated its effect on NF-κB and MAPK signaling pathways by western blot. We found that the activation of NF-κB(P<0.05) and Erk, p38, JNK of MAPKs pathways in LPS-stimulated BV2 cells was significantly inhibited by TSG-6. Furthermore, to obtain insights into the role of CD44 and the relationship between CD44 and TSG-6 in modulating the activities of LPS-activated microglia, we repeated some of the above-reported experiments using BV2 cells in which CD44 expression was knocked down using CD44-siRNA. BMSCs or rmTSG-6 had less effect on reducing the expression of the pro-inflammatory cytokines TNF-a (for BMSCs treated groups, the relative expression of TNF-a in control BV2 cells versus CD44-knockdown BV2 cells were:3.43±0.50 versus 8.27±0.35, P<0.05; for rmTSG-6 groups:5.48±0.87 versus 12.33±0.87, P<0.05) and IL-1β (for BMSCs treated groups? the relative expression of IL-1β in control BV2 cells versus CD44-knockdown BV2 cells were:2.63±0.32 versus 3.93±0.35, P<0.05; for rmTSG-6 groups:2.82±0.66 versus 6.33±0.25, P<0.05) in CD44-knockdown BV2 cells compared with non-transfected control cells. The magnitude of the LPS-induced increased NF-κB activity, was significantly higher in CD44-siRNA-transfected BV2 cells than in non-transfected control cells in the presence of BMSCs (P<0.05) or rmTSG-6 (P<0.05). Consistent with these observations, the western blotting results showed that BMSCs or rmTSG-6 had substantially less impact on reducing the phosphorylation levels of Erk, p38and JNK MAPKs upon LPS stimulation in CD44-knockdown BV2 cells compared with non-transfected control cells.ConclusionsThe results of this study suggested that the BMSCs can modulate microglia activation through releasing TSG-6. The TSG-6 attenuates the inflammatory cascade in activated microglia. And the presence of CD44 on the microglial cells was essential for BMSCs- and TSG-6-mediated inhibition of pro-inflammatory gene expression and of NF-κB and MAPK. activation in microglial cells. Our study indicates that novel mechanisms are responsible for the immunomodulatory effect of BMSCs on microglia. The BMSCs as well as TSG-6 might be promising therapeutic agents for the treatment of neurotraumatic injuries or neuroinflammatory diseases associated with microglial activation. |
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