An Experimental Study Of The Effects Of Shi-Quan-Da-Bu-Tang On Microglia In Alzhei Mer's Disease

PrefaceMore and more attention was paid on the functions of microglia (MG) in the inflammatory pathogenesis of Alzheimer's disease (AD) and other neurological diseases. Activated MG have dual roles. On one hand, they can produce a variety of pro-inflammatory cytokines which have neurotoxicity; On the other hand, they can produce some neurotrophic factors, and more importantly, they can phagocytoseβ-amyloid (Aβ). At present, most researchers focus on reducing inflammation-mediated nerve injury by inhibiting MG from activating. In AD, although non-selective blocking of the functions of activated MG can weaken the local inflammatory responses, but also inhibit the phagocytosis of Aβby MG, which may lead to the gathering and depositing of more Aβand ultimately the worsening of illness. So, how to regulate the functions of MG effectively in order to not only increase its specific phagocytosis of Aβ, but also inhibit the production of inflammatory cytokines, NO and other inflammatory mediators, is the fundamental direction of application of MG as a means of AD therapy.In recent years, MG which are differentiated from bone marrow stem cells (BMSC) have become a hot research. It has been confirmed that BMSC can enter the central nervous system by passing through the blood-brain barrier and differentiate into functional MG. The bone marrow-derived MG (BMDM) are more likely to be attracted to the neuropathic regions. In AD mouse model, more than 90 percent of MG around senile plaques were BMDM, and these cells played an important role in delaying the progression of disease through phagocytosing AβMoreover, compared with inherent MG, BMDM served as more effective antigen presenting cells and phagocytic cells. Therefore, transplantation of bone marrow cells may become a new strategy in AD therapy.The Chinese herbal medicine, Shi-Quan-Da-Bu-Tang, is composed of 10 medical plants: Astragali Radix, Cinnamomi Cortex, Angelicae Radix, Paeoniae Radix, Cnidii Rhizoma, Rehmanniae Radix, Ginseng Radix, Atractylodis Lanceae Rhizoma, Poria, and Glycyrrhizae Radix. It contains a variety of immunomodulatory substances which have anti-inflammatory, anti-oxidative and anti-allergic effects. It has been proved that Shi-Quan-Da-Bu-Tang can affect not only the acquired immune system, but also the innate immune system. This provides the foundation that we focus on exploring the possible anti-inflammatory and anti-oxidative effects of Shi-Quan-Da-Bu-Tang on MG, the representative of innate immune system in neurologic diseases.Our preliminary results supported that Shi-Quan-Da-Bu-Tang administration diminished the senile plaques in AD transgenic mouse. We speculated that this may be realized through the promotion of Aβphagocytosis by MG. In this study, using the extracts of Shi-Quan-Da-Bu-Tang, we stimulated MG in vitro to assess the effects on microglial proliferation, activation, fibrill Aβ_1-42 phagocytosis, as well as microglial neurotoxic/neurotrophic destination. In animal experiments, first we investigated whether BMSC could enter the normal brain or not, and whether Shi-Quan-Da-Bu-Tang could promote the migration of BMSC in normal brain. In addition, we adopted the method of hippocampal Aβinjection to mimic the AD environment, and then investigated whether Shi-Quan-Da-Bu-Tang could promote the homing of BMSC and alleviate the disease by enhancing the phagocytosis of Aβ, in order to search a new possible therapeutic way for AD.MethodsPrimary MG or Ra2 cells were used. After stimulating with Shi-Quan-Da-Bu-Tang, cell proliferation was detected by WST-1 assay, surface expression of the activation marker CD11b was detected by flow cytometry, phagocytosis of fibrillar Aβ_1-42 was examined by immunofluorescence staining, NO production in the supernatant was detected by Griess reaction, and several cytokines were examined by Bio-Plex^TMCytokine Assay. So the effects of Shi-Quan-Da-Bu-Tang on microglial activation and neurotoxic/neurotrophic destination were determined by in vitro studies.In in vivo experiments, mice were given EGFP bone marrow cells through bone marrow transplantation in order to establish a chimeric mouse model and to track the exogenous cells in the brain. In experiment one, the proportions of GFP-positive cells and CD11b-positive cells in mice blood were examined by flow cytometry, GFP positive cells and Iba1 positive cells in the brain were detected using immunohistochemical staining. In experiment two, immunostaining method were used not only to detect GFP-positive cells which gathering around the injected Aβin hippocampus, and in the non-lesion regions, but also to identify the phenotype of GFP-positive cells.All results were expressed as means±standard deviation. Data were analyzed by SPSS13.0 for windows. Comparisons were analyzed with one-way ANOVA, repeated measures or t-test.Results1,Effects of Shi-Quan-Da-Bu-Tang on microglialmorphology, proliferation and surface expression of CD11bAfter stimulated with Shi-Quan-Da-Bu-Tang, morphological changes of primary MG from resting state to amoeboid morphology with small cell body and short processes were observed under the phase contrast microscope. Shi-Quan-Da-Bu-Tang increased the vitality and proliferation of MG with a slightly dose-dependent manner. Flow cytometric analysis showed that, in Ra2 cells which were stimulated by Shi-Quan-Da-Bu-Tang for 48 hrs, the proportion and mean fluorescence intensity of CD lib-positive cells which represented activated MG were significantly increased compared with the negative control group.2,Effects of Shi-Quan-Da-Bu-Tang on microglialphagocytosis of fibrillar Aβ_1-42In the concentration-dependent assay, after treatment with more than 200μg/ml Shi-Quan-Da-Bu-Tang for 24 hrs, the phagocytosed cell number was significantly increased, without apparent concentration-dependent manner. In the time-dependent assay, the highest proportion 60.4±4.4% of phagocytosed cells were got after 24 hrs treatment with 200μg/ml Shi-Quan-Da-Bu-Tang, not showing time-dependent manner.3,Effects of Shi-Quan-Da-Bu-Tang on the production of NOand cytokinesNO production was increased slightly with only Shi-Quan-Da-Bu-Tang treatment, but not statistically significant compared with negative control group. In the presence of fibrillar Aβ_1-42, although Shi-Quan-Da-Bu-Tang did not show the effect of reducing NO production, it had not a facilitating effect.At each time point, only Shi-Quan-Da-Bu-Tang treatment showed no effect on TNF-αproduction compared with the negative control group after statistical analysis. Shi-Quan-Da-Bu-Tang inhibited TNF-αproducted by fibrillar Aβ_1-42 treatment at the time point of 6 hrs. IL-1β, IL-6 and IL-10' could only be significantly detected at the time point of 24 hrs. Only treated with Shi-Quan-Da-Bu-Tang increased the production of IL-1β, IL-6 and IL-10 significantly compared with the negative control group, and the IL-10 level was also higher than that of fibrillar Aβ_1-42-treated group. Shi-Quan-Da-Bu-Tang did not show any effect of promotinig or inhibiting the IL-1β, IL-6 and IL-10 production by fibrillar Aβ_1-42.4,Detection of GFP-positive cells and CD11b-positive cells in mice blood of experiment one by flow cytometryMore than 79% of the white blood cells were from the transplanted bone marrow cells in chimeric mice, and there were no significant differences between the control group and experimental group. The ratio of CD11b-positive cells out of total white blood cells was increased significantly in the experimental group.5,Detection of GFP-positive cells and Iba1-positive cells in mice brain of experiment one by immunohistochemical stainingGFP-positive cells were found occasionally in the cerebellum in both groups, and Shi-Quan-Da-Bu-Tang did not show obvious effect. Compared with the control group, Shi-Quan-Da-Bu-Tang increased the number of Iba1-positive cells in hippocampus significantly. 6,Detection of GFP-positive cells in and around the hippocampal injection point of experiment two by immunofluorescence stainingLarge number of accumulated GFP-positive cells was found in and around the injection point in both groups. Moreover, in the route of injection and also in the PBS injection point, similar gathering of GFP-positive cells was found.7,Detection of GFP-positive cells in the distant regions from the injection site of experiment two by immunohistochemicalstainingBecause it was difficult to make accurate counting of GFP-positive cells around the injection sites, we then count the GFP-positive cells in the ventral 3/5 part of brain. Compared with the control group, the experimental group increased the GFP-positive cell number significantly.8,Identification of the phenotype of GFP-positive cells of experiment two by immunofluorescent stainingThe GFP-positive cells showed MG phenotype, but not neurons and astrocytes in either the experimental group or the control group and the regions either near or far from the injection point.Conclusions1,Shi-Quan-Da-Bu-Tang could cause significant morphological changes to activation pattern in MG.2,Shi-Quan-Da-Bu-Tang could promote the proliferation of MG and increased surface expression of activation marker CD11b in MG.3,Shi-Quan-Da-Bu-Tang could promote the phagocytosis of fibrillar Aβ_1-42 by MG.4,Shi-Quan-Da-Bu-Tang may play an important role in the balance of microglial neurotoxic/neurotrophic destination.5,In non-damage mice brain, Shi-Quan-Da-Bu-Tang administration could promote the proliferation of CD11b-positive cells in the blood, and increase the number of inherent MG in the brain. However, only a small number of BMDC was found in the cerebellum.6,In the AD mouse model, BMDC gathered in and around the lesions, differentiated into MG, and may play a useful role in the phagocytosis of Aβ. Shi-Quan-Da-Bu-Tang was found to increase the BMDC in the regions far from the injection point, and these BMDC also showed the phenotype of MG. The BMDC which migrated into the brain could not differentiate into neurons and astrocytes.