| Background&ObjectiveAlzheimer’s disease (AD), also known as senile dementia, is the most common senile brain neurodegenerative diseases, mainly in the65years of age and older group, which accounts for50.75%of all dementia cases. The main pathological changes include brain death, and of tau protein abnormalities in aggregate to form neurofibrillary tangles (neurofibrillary tangles, NFTS), nerve cells aggregate to form senile plaques (senile plaques of amyloid P protein (amyloid-beta peptide, Aβ), SP, etc. The disease manifested as a full range of cognitive impairment, including memory, orientation, judgment and reasoning of the obstacles. The disease eventually led to the loss of life in patients with capacity, serious damage to the physical and mental health of the elderly, but also affect one of the constraints of family and social development.2010Alzheimer’s Association (AA) released data show that, in serious harm to human health, the four diseases-heart disease, diabetes, cerebral infarction and prostate cancer mortality rates have decreased, AD’s mortality rate has continued to rise. In the United States aged65and older age groups, the AD disease mortality rate ranks fourth. In United States alone, there are nearly5.3million patients with AD, every70seconds on average1new patients. As of2010, AD-related expenses were up to$172billion. Global dementia in2010was about3560million and doubling every20years the rate of increase is estimated that by2030, global dementia will reach6570million. By2050, global dementia will reach more than110million people, of which60%occur in developing countries, and presented a rising trend. According to the2005China’s first epidemiological survey results to estimate the prevalence of dementia of65years of age or older was7.8%, AD prevalence was4.8%,4.36times that of vascular dementia (1.1%), and with the age increase.China is an aging population base of the largest countries in the world, with the era of population aging in China, in2006, aged65and older population has reached134million. Estimate the number of dementia in China’s nearly8million, of which AD nearly6million, to the middle of the21century, the elderly population will increase to400million patients with AD will be close to20million. Thus, although China is not Alzheimer’s incidence countries with the highest, but it is the fastest growing countries in dementia largest number and the growth rate in the world. How to take effective preventive and treatment measures for Alzheimer’s disease has gradually become an urgent need to deal with the major issues of health care service system and the economic and social development.Although the pathogenesis of AD is not entirely clear, but with the depth of AD research, the role of astrocytes (astrocyte, AS) in the pathological changes of AD cause more and more attention. Most scholars believe that Aβ deposition activate the AS-induced inflammation, oxidative stress is an important pathological mechanism of AD. Glial cells is a large class of cells of nervous system of, about90%of the total number of cells of the central nervous system, in which AS is an important part of the glial cells, including almost all the features of the glial cells. Past that, the transmission and integration of information within the central nervous system by neural networks, astrocytes, only a passive supporting role, to play a support role of nutrition and to assist in metabolism. With the progress of the patch clamp and molecular biology techniques in recent years, it was found that the AC in the maintenance of neurons in the internal and external environment, survival, migration, immune regulation, signal transduction, axonal growth and functional integration has an important role in the nervous survival play an important role is closely related to the central nervous system (CNS) diseases.One of the key feature of Alzheimer’s disease is a brain inflammation, the inflammatory process is the release of cytokines by astrocytes and microglia-mediated, and participate in the immune response. Inflammation secondary response caused by Aβ deposition, but also lead to neuron degeneration. Pathological deposition of Aβ protein can activate astrocytes, and was highly expressed in astrocytes characteristic protein-GFAP protein. Excessive activation of astrocytes and microglia release inflammatory mediators (such as IL-1β and TNF-α, IL-8, MCP-1, etc.), followed by the inducible nitric oxide enzymes (inducible nitric oxide synthase, iNOS) activation, production of nitric oxide (nitric oxide, NO) and ROS (reactive oxygen species.ROS), which in turn can cluster (of reactive nitrogen species and reactive nitrogen, the role of the RNS), generated oxide nitrate. This peroxide can produce very toxic to cells, triggering apoptosis. In addition, microglia and astrocyte interaction will further increase the precursor of inflammatory cytokines, complement and oxide generation. These inflammatory mediators and the oxide will lead to further accumulation of Aβ in the inflammatory factor-mediated intracellular cascade reaction to further expand into a vicious cycle. Therefore, inhibition of nerve inflammation caused by excessive activation of astrocytes may play an important role in inhibiting the development of the AD diseaseThe etiology and pathogenesis of AD has not yet entirely clear, there is currently no effective therapy. To more effectively reduce the incidence of AD, we must grasp the primary prevention measures for disease prevention and control:add the effective prevention of AD food additives in food may also be a low cost, a good way of meaningful prevention of AD. Resveratrol (trans-of resveratrol, Res), may be an ideal food additive has been used in the United States and Japan. As an antioxidant, antimutagenic and anti-infective active ingredient, have significant anti-inflammatory, anti-aging, anti-cancer and anti-cardiovascular and cerebrovascular diseases. Its chemical name3,5,4’-trihydroxy-trans-stilbene (3,5,4’-trihydroxystilbene), is generally trans-resveratrol, it is often with the cis-isomer (cis-of resveratrol) and white veratryl alcohol glycosides (of resveratrol glucoside in) co-exist in grapes, mulberries, peanuts and other plants and food, especially with higher levels in red wine. Resveratrol and its derivatives have antibacterial, antioxidant, anti-thrombosis, cancer and many other physiological activity by the extensive attention from domestic and foreign experts in the field of food, medicine, plant physiology. The present study shows that resveratrol can effectively confront the central nervous system ischemic injury, protection of ischemic neurons, reducing infarct size and reduce ischemia and reperfusion caused by delayed neuronal apoptosis, improving cerebral ischemia caused by motor dysfunction. The protective effect of resveratrol on the nervous system has been more research, but the current study focused on neurons, astrocytes influence is rarely reported. In this study, different concentrations of resveratrol for release by astrocytes inflammatory mediators in the inflammatory damage state to explore the protective effects of resveratrol on nerve injury. Explore new possible ways for the prevention of AD. In this study, a new, stable and astrocytes in vitro culture techniques, and successfully copied the astrocyte damage caused by inflammation. Expression of NO/iNOS signaling system, and to detect the LDH leakage through the release of inflammatory mediators in astrocytes before and after contrast injury and inflammatory injury of resveratrol for the damage state of astrocytes. Against central nervous system damage and slow down the AD symptoms of the development of a preventive measure to provide new experimental evidence for resveratrol.Resveratrol anti-aging activity and neuroprotective effects provide new ideas for research and development of more resveratrol anti-AD drugs and health products. Market sales of resveratrol from natural plant extract, plant extracts face costs are high, too low and limited plant resources. So people try to develop new resveratrol biosynthetic pathway. The current study found that resveratrol bio synthetic pathway, including:chemical synthesis and biosynthesis. Chemical synthesis of the advantages of high selectivity, but due to the high cost of catalysts used price is expensive and toxic, so is not suitable for industrial production, currently only in the laboratory research stage. People’s attention in recent years has been to shift from chemical synthesis to biosynthesis.Previous studies of our laboratory initial attempt and successfully established a resveratrol plant biosynthesis in the laboratory under sterile conditions to nurture high-yield root of peanut and peanut roots were induced to mass, stabilityproduction of mixed extracts containing resveratrol, a set of laboratory bio-synthesis program to produce resveratrol from peanut germination. However, the impact of the peanut root-induced extract on cellular activity, Peanut root extract induced cell activity, have anti-inflammatory protective effect, compared with pure resveratrol, the size of their effects; these are the key issues to be studied.Therefore, this study is based on the synthesis program established in the laboratory in the past with the purpose to optimize the extraction process of the mixture. Further comparative study will be conducted between peanut root-induced extract and resveratrol to anti-inflammatory protection of astrocytes in LPS injury.Methods1. Optimized methods of astrocytes culturing and established experimental model of astrocytesInstead of the traditional differential speed adherent purified astrocytes method we take the filter membrane method to purify neonatal rat brain astrocytes, and to reduce the experimental difficulty of the operation. After identification of the neonatal rat cerebral cortex astrocytes by GFAP immunohistochemistry method purity, we make different concentrations of LPS on cells with different length of time and then take CCK-8assay for detection of cell viability.2. Protection effect of resveratrol in astrocytesAstrocytes were injured by LPS after preconditioning by different content of resveratrol (5、25、50μmol/L). Collection of cell culture medium, detection of LDH leakage, NC、TNF-α levels as well as the iNOS protein expression by Western Blot. By detection of NO and TNF-a to understand the effect of resveratrol on the release of inflammatory cytokines in astrocytes after injury. Detect the expression of iNOS protein level and LDH release to know if the protective effects of resveratrol on LPS-caused astrocyte injury in rats is related with the expression of iNOS/NO pathway.3. Induction and production of resveratrol in soilless cultivation peanutCultured peanut by Soilless culture medium peanut root yield that the establish by our laboratory. Use with biological elicitor induction medium induction of peanut roots. Collection of inducing medium, ethyl acetate extraction of the liquid medium road. Collected extracts were vacuum dried and reserve.The resveratrol in induction medium was identified by TLC and HPLC comparing with that of the standard resveratrol and determined the content of resveratrol.4. Protection effect of Peanut root extracts in astrocytesThe peanut root extract dilution to different concentrations, to detect the cytotoxicity. Select non-toxic concentration for subsequent experiments. Astrocytes were damage by LPS after treat by different content of resveratrol. Collection of cell culture medium, detection of LDH leakage and expression of TNF-a to know the effect of peanut root extraction.Results1. Established of the astrocytes inflammation injury model.The astrocytes were purified by using the filtering with a menbrane and the astrocytes were identified by GFAP immunohistochemical and purity coefficient>95%. Observed in each group found:At the same point in time, the higher the protein concentration, the lower the survival rate with the cells; At the same concentration of LPS, the cell viability decreased with longer treatment time(P<0.05).1μg/ml LPS-24h experimental group which is closest to the cell survival by50%has advantages to be observed whether resveratrol has a protective effect in the follow-up test, or exacerbate injury. Therefore, this experiment selected1μg/ml LPS-24h as the conditions of cell damage.2. Protection effect of resveratrol in astrocytes(1) Cell survival rates:astrocytes injury by LPS, the cell survival rate dropped dramatically to51.04%; Astrocytes were treated by different content of resveratrol(5、25、50umol), cell survival rates were58.72%,76.38%,90.42%. there is a relationship between the concentration and dose.(2) LDH leakage:The results showed that LPS-injury can lead to the increase of LDH leakage (P0.05); compared with the LPS group,5、25、50μmol/L resveratrol preconditioning group of LDH leakage volume decreased (P<0.05), but especially high concentrations of the RES (50μmol/L,) treatment group, LDH leakage to reduce the most obvious, suggesting that high concentrations of RES on the role of strong protection of astrocytes.(3) TNF-a expression levels:ELISA assay results showed that LPS-treated cells, TNF-a release increased significantly; compared with the LPS group,25、50μmol/L, the RES-treated cells release both decreased (P0.05), but5μmol/L, the RES-treated cells TNF-a release and LPS-treated cells had no significant difference (P>0.05). Among them,50μmol/L RES treated with TNF-a release was most obvious. Tips, astrocytes、TNF-a emission, high concentrations of RES have a more significant inhibitory effect.(4) NO expression level:LPS treatment for24h, NO release was significantly increased (P<0.05); compared with the LPS group, the NO release of25、50μmol/L, the RES-treated cells was significantly reduced (P<0.05).5μmol/L RES had no significant effect on NO release of treated cells was no significant difference between cells with the LPS group (P>0.05), suggesting that high concentrations of RES astrocytes release of NO inhibition.(5) iNOS expression level:Western blot analysis showed that LPS treated iNOS protein expression and the negative control group significantly increased compared to the low level of negative control cells, expression of iNOS protein; compared with the LPS group:5、25、50μmol/L, the RES pretreatment group, iNOS protein expression were decreased. In particular, the expression of iNOS to50μmol/L RES treatment group decreased significantly. Prompted to LPS-induced NO release and iNOS protein expression, upregulation, and the high concentration of RES was significantly inhibited iNOS/NO pathway.3. Induction and production of resveratrol in soilless cultivation peanutIn this study, we used soilless culture cultivation programs and biological of elicitors. Resveratrol were positive induction medium by TLC identification. HPLC quantitative analysis showed that resveratrol content564.457μg/g DW. The resveratrol content of wild peanut51.23μg/g DW, transgenic tomato and transgenic lettuce resveratrol content was53ug/g DW,56.40μg/g DW.4. Protection effect of Peanut Root Extracts in astrocytes(1) Screening of crude extracts of non-toxic concentration:With the concentration of10"2g/L、10-3g、10μg/L of crude extract treated cells, the cell survival rate was no difference with the control group(P>0.05). Therefore, the concentration of these three groups as the experimental concentrations.(2) LDH leakage:experiment results show that extract with concentration of10-2g/L、10-3g/L、10-4g/L of can reduce the leakage of LDH compared with group of LPS injured(P<0.05).(3) TNF-a expression:ELIS A showed significantly increased TNF-a release in LPS-treated cells; compared with the LPS group,10-2g/L、10-3g/L crude extract concentration treated cells have a lower TNF-a expression levels compared with LPS treatment group (P<0.05),10-4g/L concentration group and LPS group have no significant differences (P>0.05).Prompt:10-2g/L,10-3g/L concentration of the crude extract has a protective effect on the inflammatory injury of the astrocytes.Conclusion1. Successfully established the astrocytes AD astrocytes inflammation damage cell model.2. LPS-induced inflammatory damage can increase the astrocytes of neurotransmitter release, including TNF-a and NO, and to increase iNOS expression of astrocytes and LDH leakage.5-50umol/L resveratrol on LPS-induced inflammatory damage of astrocytes have a protective effect, and this effect showing the concentration-effect relationship.3. The result proved that we succeed screening the optimum elicitor and the best induction time, the content of resveratrol could achieve564.457μg/g DW.4. Extract with concentration of10-2g/L、10-3g/L has a protective effect for Inflammatory injury induced by LPS in astrocytes. |
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