Research On The Neuroprotective Effects Of Ginsenoside And Berberine And The Underlying Mechanism

Alzheimer’s disease (AD) is one of the most serious neurodegenerative diseaes that is characterized by progressive loss of memory and cognitive functions, with an incidence of10-30%in the aging people over the age of85. Extracellular senile plaques, the intracellular neurofibrillary tangles and neuronal loss are the distinctive pathological hallmarks of AD.β-amyloid peptides (Aβ) is generated from the amyloid precursor protein(APP), via sequential cleavage by β-and γ-secretase. Substantial evidence demonstrates the neurotoxicity of AP and Aβ aggregates in AD pathogenesis. Aβ aggregate impair synapses and neuronal dendrites which causes neuronal damage and apoptotic neurotoxicity.Neuroinflammation plays important role in neurodegenerative diseases such as AD, PD, MS and ALS. Aβ can directly interact with microglia via cell surface receptors, resulting in activation of the microglia. Overactivation of microglial cells may produce and release proinflammatory mediators including tumor necrosis factor-a (TNF-a), nitric oxide (NO), cyclo-oxygenase-2(COX-2), monocyte chemoattractant protein-1(MCP-1) and interleukin-6(IL-6), causing neuroinflammtion and neuropathological changes. The underlying mechanism of activated glia related neuroinflammtion involves NO, ROS and proinflammatory cytokines.In this study, we focus on the the neuroprotective effects of ginsenoside and berberine using cell model system and tried to elucidate the possible mechanism of their neuroprotection. Our sudy can provide some theoretical basis and experimental evidence for natural medicine treatment of AD. Part oneReaserch on the protective effects of ginsenoside Rbl and Re on SK-N-SH cells injured by Aβ25-35[Background]As a traditional Chinese medicine, Ginseng’s medicinal value has been generally recognized. It is reported that Ginseng has protective effects in central nervous system, cardiovascular system, and immune system. The modern pharmacological researches have shown that Ginseng can promote learning and memory with intelligent function by modulating the balance between cortical excitatory and inhibitory processes.Ginsenoside Rbl and Re are the effective extract from ginseng which have been reported to have multiple phamarcy effects.Animal experimental studies have shown that Rb1can partially restore learning and memory functions of AD rats model. It was observed that Re significantly decreased mitochondrial swelling, thereby preventing the reduction of H (+)-ATPase activity in cerebral ischemia reperfusion injury model rats. However, the mechanism underlying is not clear. In our study, a cell model of AD was established to study the neuroprotective effect of ginsenoside Rb1and Re. We also want to reveal the possible mechnism involved.[Objective]The purpose of our present study is to to investigate the neuroprotective effects of ginsenoside Rb1and Re using a cell model of AD.[Methods]Aβ25-35was added to the medium for cell culture to construct a cell model of AD, and the following data was observed. Cell survival rate of the different cell-treated groups was determined by MTT assay, and intracellular reactive oxygen species (ROS) level was tested by fluorescence probe. Then the results of different groups were compared to determine the neuroprotective effect of ginsenoside Rb1and Re, and the signaling pathways involved were studid by Western blotting to reveal the possible mechanism.[Results]1. The survival ratio of cells was significantly decreased after exposure to Aβ25-35, and the treatment of ginsenoside Rbl or Re significantly increased it.2. The treatment of ginsenoside Rbl or Re significantly upregulate cellular ROS level in Aβ25-35induced SK-N-SH cells. 3. Western blotting results indicated that the ginsenoside Rbl and Re decreased the expression of phosphorated Tau (Ser396) and active GSK-3β(Tyr216), respectively.[Conclusion]Ginsenoside Rbl and Re exert neuroprotective effects on SK-N-SH neural cells treated by neurotoxic Aβ25-35, and the possible mechanism of their neuroprotection including theupregulation of active GSK-3β and decreased Tau phosphorylation and. Part twoReaserch on the anti-neuroinflammation effect of of berberine and the underlying mechanism[Background]As an isoquinoline alkaloid which is extracted from the root of several Chinese herbs such as Huanglian and Ranunculaceae, berberine is a natural anti-infective agent that is initially used as heat-clearing, detoxifying drugs and antibiotics in clinical application. In addition, it has been reported that berberine can suppress tumor growth and tumor invasion. Besides, it has been also found that berberine can be used to treat type II diabetes in our country. Additionally, it has been reported that berberine can suppress the expression of inflammatory mediators in several cell lines, which is initially identified as berberine’s anti-inflammatory effect.Central nervous system disease is often accompanied with neuron necrosis and apoptosis with a large number of activated microglia gathered around these necrotic lesions. It is reported that in the brains of both the mouse disease models and AD patients, microglia are found closely associated with the amyloid plaques as an "activated" proinflammatory phenotype. Aβ is the proximate cause of neuroinflammation associated with AD and microglia play a key role in the A(3-dependent neuroinflammation. It is well identified that berberine has strong anti-inflammatory effect; however, the effect of berberine on Aβ25-35-dependent neuroinflammation in microglia is poorly understood.[Objective]The purpose of our studies is to examine the effects of berberine on Aβ25-35-induced neuroinflammation and find the possible mechanisms.[Methods]We used BV-2microglia cell line and primary glial cells stimulated by Aβ25-35to study the anti-neuroinflammatory effect of berberine. The data as follows were observed:the mRNA level of IL-6, MCP-1, COX-2and iNOS were detected by Real-time PCR analysis, the secretion of IL-6and MCP-1were detected by ELISA analysis, the protein levels of iNOS and COX-2were detected by western blotting. To detect the effects of berberine on the Aβ25-35-induced phosphorylation levels of the Akt, p38and ERK, western blotting analysis was manipulated. When the anti-inflammatory effect of berberine was determined, the inflammaion-related signal pathways were detected by western blotting, immunofluorescence assay and EMSA to reveal the mechanism.[Results]1. Real-time PCR and ELISA results showed that the expression of IL-6and MCP-1was upregulated by Aβ25-35in BV2cells and primary microglial cells; pretreatment with berberine of1-5μM decreases these upregulation.2. Real-time PCR and ELISA results demonstrated that the expression of iNOS and COX-2was upregulated by Aβ25-35in BV2cells and primary microglial cells, pretreatment with berberine of1-5μM could inhibit this effect of AP25-35.3. Western blotting showed that Aβ25-35could up-regulate the phosphorylation levels of Akt, p38and ERK in murie BV2cells and berberine of1-5μM inhibits their phosphorylation level.4. Western blotting showed that Aβ25-35could up-regulate NF-κB p65phosphorylation levels in murine BV2cells and this phosphorylation can be repressed by berberine treatment.5. Immunofluorescence results showed that AP25-35can induce NF-κBp65’s translocation into the nucleus in BV2cells, and berberine inhibits this translocation.6. The EMSA results showed that berberine could inhibit DNA binding activity of NF-κB p65in BV2cells.[Conclusion]Our results demonstrated that berberine could significantly inhibit the expression of IL-6and MCP-1upregulated by Aβ25-35in the transcription and translation level. Besides, berberine also could inhibit the expression of COX-2and iNOS in primary glia cells and murine BV2cells. Further studies of the mechanism found that berberine could significantly inhibit the activation of NF-KBp65increased by Aβ25-35in murine BV2cell line. Moreover, berberine could also inhibit the phosphorylation of Akt, p38and ERK in Aβ25-35-induced murine BV2cells. Taken the dates together, it indicated that the berberine’s inhibitory effect on Aβ25-35-induced inflammatory of microglia may be achieved by blocking the PI3K/Akt and MAPK pathways.