Effects Of Components Of Liuwei Dihuang Decoction And Danggui Shaoyao San On Microglia Activation

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects the elderly. The neuropathological hallmarks of the disease include diffused brain atrophy, intracellular neurofibrillary tangles and extra cellular amyloid plaques. Memory decline and cognitive impairment are the main clinical manifestations of AD. Considering the still growing number of the elderly, it has become an increasing burden for the society.At present, the etiology of AD is still not fully understood. However, a numbers of evidence indicate that brain inflammation induced byβ-amyloid (Aβ) deposition is closely associated with the pathogenesis of this disease. The hall mark of brain inflammation is the activation of microglia. Upon activation,the microglia can produce a variety of cytotoxic mediators,including nitric oxide, proinflammatory cytokines, excitatory amino acid, reaetive oxygen intermediates, which are toxic to neurons. Thus, drugs with the effects to inhibit microglia activation would provide neuroprotective effects. Recent reports strongly suggest that regulating microglia function may be a promising therapeutic approach for treating AD. Targeting at the prevention of the activation and generation of inflammatory cytokines of the microglia has become the focus of the development of AD treatment drugs.CA4-3, extracted from Liuwei Dihuang Decoction (LW), a traditional Chinese medicine prescription, has potent anti-inflammatory activities. Peoniflorin, extracted from Danggui Shaoyao San (DSS), has been identified as the major component. Our previous work reported that LW could not only regulate the genes of inflammatory cytokines in hippocampus and cortex, but also improve the memory abilities of SAMP8. According to the above results, we made a hypothese that LW improves the memory abilities by regulating the neuroinflammatory process. Danggui shaoyao San (DSS) could improve the congnition decline and the movement disability of AD in clinical patients and animal model such as SAMP8. DSS could protect the cortex neurons and the PC12 cells from the injury of Aβby inhibiting the Aβaggregation and solving the aggregated Aβ. Aβ. In order to study the anti-inflammatory of the CA4-3 and the peoniflorin, we choosed the Aβ25-35, Aβ1-42, LPS and IFN-γwhich all could activate the microglia. Firstly, we studied the inhibition effect of the CA4-3 and the peoniflorin on LPS activated microglia. Secondly, we studied the inhibition effect of LW on neuroinflammatory mouse induced by LPS. Thirdly, we investigated the signaling pathways of the anti-inflammatory effect of LW, the CA4-3 and the peoniflorin. The results were showed as follows:Ⅰ. The activation affect of different agents to microglia1. The cultivation of microgliaPrimary cultured rat microglia and BV-2 cells were used in our study. The doubling generation time of BV-2 is 11 hours. BV-2 cell were used when 80% of the bottom area of culture flask occupied. The activated agents such as LPS activated microglia and changed microglia appearance from arborization to ameba shape.We choose the lidocaine to separate microglia from astrocyte and oligodendroglia, which is simpler to purify microglia from mixing culture of glia cell compared with the traditional methods which called mechanical shaking and low concentration trypsin.2. The activating effects of Aβon microglia.First, the structure of unaggregated Aβand aggregated Aβwhich play a pivotal role in activating microglia was observed with transmission electron microscope. The structure of unaggregated Aβis amorphous, no obvious accumulation, low electron density, and no fibered structure. The structure of aggregated Aβis acicular or crystalline accumulation, crisscrossed fibered structure.Second, activity, cytokine and cytokine gene expression of microlgia activated by aggregated Aβwere examined. Activities of BV-2 cell were not influenced by aggregated Aβ25-35(1~10μM), but inhibited by aggregated Aβ1-42(5, 10μM). Activities of primary cultured rat microglia were obviously suppressed by aggregated Aβ25-35(25, 50μM).The expressions of IL-1β, iNOS, TNF-αmRNA were found to be elevated in Aβ25-35(25μM) activated BV-2 cell.Aggregated Aβ25-35(10~100μM) and aggregated Aβ1-42(10μM) could evoke TNF-αproduction in a dose and time-dependent manner in BV-2 cell. Aggregated Aβ1-42(10μM) could double the TNF-αsecretion. The above results indicate that aggregated Aβ25-35 and aggregated Aβ1-42 have the potency to activate microglia.3. The activating effects of LPS on microglia. LPS is a major of constituent of the cell wall of Gram-negative bacteria. Microglia as a kind of macrophage in the CNS was activated by LPS.LPS (5μg/ml) suppressed the activity of BV-2 cell obviously, but there is no affect when LPS concentration is lower than 2.5μg/ml. LPS (0~5μg/ml) have no effect on activity of primary cultured rat microglial.The expressions of IL-1β, NOS, TNF-αmRNA in activated BV-2 cell were found to be increased by LPS (1μg/ml or 2.5μg/ml).The TNF-αproduction induced by LPS (0.5~5μg/ml) on the BV-2 cell is dose and time-dependent, but only time-dependent on primary cultured rat microglia.4. The co-activation effects of IFN-γand Aβon microgliaIFN-γis a strong activated agent to macrophages. We investigated the effects of IFN-γon the microglia activated by aggregated Aβ. IFN-γ(10~50pg/ml) and aggregated Aβ25-35(20~100μM) suppressed the activity of BV-2 cell obviously. There are no differences between the BV-2 cell activity suppressed by IFN-γ(50pg/ml) and IFN-γ(50pg/ml) with Aβ25-35(20~100μM). These results indicate that there is no co-activation effects of IFN-γand Aβon microglia.The TNF-αproduction of BV-2 cell could be stimulated by IFN-γ(25~100pg/ml) or IFN-γwith Aβ25-35. IFN-γ(25 pg/ml) accompanied with Aβ25-35 (20, 50μM) induced more TNF-αproduction than IFN-γ(25pg/ml) or Aβ25-35(20, 50μM) in BV-2 cell.Ⅱ. The effect of the components extracted from LW and DSS on activated microglia1. The influence of serum-containing LW on activated microgliaOur previous work reported that LW could regulate the gene of inflammatory cytokines in hippocampus and cortex of SAMP8. In this study, using the BV-2 cell activated by LPS, we found that serum-containing LW (2.5~10%) suppressed the TNF-αproduction in BV-2 cell.2. The effects of the components extracted from LW on microgliaThe components, B-B, D-b, CA-30, and CA4-3 extracted from LW, suppressed the activity of BV-2 cell significantly. The components CA4-3 was found to inhibit the LPS-stimulated release of inflammatory cytokine TNF-αin the BV-2 cell.3. The effects of the components extracted from DSS on microgliaSome previous research found that DSS possessed effect of ameliorating the decline of learning and memory in many AD models. In present study, we found that the JD-30 and peoniflorin, the components extracted from DSS, suppressed the activity and inhibited the LPS-stimulated release of inflammatory cytokine TNF-αin BV-2 cell significantly.Ⅲ. Establishment of neuroinflammatory mouse model and anti-inflammatory effect of LW1. Establishment of neuroinflammatory mouse modelWe detected behavioral changes and memory impairment similar with clinical AD characters after injecting Aβ25-35(10μM) into the lateral cerebral ventricle in mouse, but without any changes in neuroinflammatory cytokines. The inflammatory cytokines in serum and brain did not increase when LPS (5μg/mouse) injected into the lateral cerebral ventricle in mouse. The cytokines production and cytokine gene expressions were increased remarkably in serum, hippocampus and cortex of LPS intraperitoneal injection mouse.2. The anti-inflammatory effect of LW on neuroinflammatory mouseOral administration of LW (5~20g/kg) could significantly inhibited serum TNF-αcytokine production in model mouse. Oral administration of LW (10, 20g/kg) could modestly inhibited the expressions of IL-1β, iNOS, TNF-αmRNA of hippocampus and cortex in LPS treated mouse.Ⅳ. The mechanism study of anti- neuroinflammatory of LW and DSS Western-Blotting analyses revealed CA4-3 and peoniflorin inhibited LPS-induced NF-κB expression and NF-κB-mediated reporter gene expression. At the same time, CA4-3 and peoniflorin inhibited phosphorylation of p38 kinase in LPS-stimulated BV-2 cell.Oral administration of LW could significantly inhibited phosphorylation of p38 kinase in hippocampus and cortex in model mouse.In conclusion, through the screening system, we found out two novel active components CA4-3 and peoniflorin extracted from LW and DSS,respectively. CA4-3 and peoniflorin exhibit beneficial effect on neuroinflammatory model in vivo and in vitro. We investigated the novel effect of LW and DSS on improving the decline of the learning and memory ability in AD patients and AD animal models, and tried to confirm a new mechanism that modulating neuroinflammation which might contribute to cognitive enhancement.