| Background Alzheimer disease(AD)is named after German psychiatrist and neural anatomist Alois Alzheimer,who described it in 1907.AD is a neurodegenerative disease characterized by progressive cognitive and memory decline.With the aging of the society,its incidence is increasing year by year.Alzheimer disease has already become a disease which seriously damages people's health.Although apparent improvements for the virulence genes of AD have been achieved in the past 20 years,so far,the reason of AD is still not clear.And the treatment of AD still stays in the period of symptomatic treatment.Therefore,enhancing the research for AD pathogenesis,looking for positive and effective method of AD treatment is of vital economic and social significance.The pathological change of AD is comprehensive atrophy of brain.At the afflicted brain area,there are two apparent pahthological hallmarks:senile plaques(SP)and neurofibrillary tangles(NFT).SP is extracellular accumulations ofβ-Amyloid(Aβ)peptides that are derived from the abnormal proteolytic processing of the amyloid precursor protein(APP).According to amyloid protein hypothesis,extracellular accumulated Aβactivated inflammation and oxidative damage.It is also demenstrated by pathology that senile plaques are often surrounded by activated microglia and astrocytes. NFT is intraneural accumulation of hyperphosphorlated tau,a cellular skeletin protein.SP and NFT together promote the neural degeneration.Futokadsura stem is the petiol of piper plant Kadsura.It is used to treat inflammatory diseases.The traditional function of the plant is to dispel wind-damp obstruction symdrome manifested as painful and stiff joints,tendon and muscle spasms,lower back pain,painful knees and pain from external injury.Using the method of aequorin,Enji Han found aqueous extract of futokadsura stem can inhibit the increase of intracellular Ca2+ induced by Aβ25-35.The inhibition effect increases with futokadsura stem concentration. Using the method of cell culture and RT-PCR,Enji Han also demonstrated that aqueous extract of futokadsura stem could selectively inhibit the APP gene expression in SK-N-SH cells.The inhibition effect increases with futokadsura stem concentration and the time of incubation.According to the hypothesis of amyloid protein,extracellular accumulated Aβactivates inflammation and oxidative damage,and these can cause neural degeneration.Based on these findings,we firstly want to observe if futokadsura stem has neural protective effect on dementia model rats.Then,we separated futokadsura stem,and tried to find the effective components which inhibit the expression of APP. Therefore,we assume that futokadsura stem has neural protective effect on SK-N-SH cells.What we are concerning about in this research is,whether this protective effect could happen in dementia models,and what the effective components in futokadsura stem which can inhibit APP expression are?Objective To observe whether futokadsura stem has neural protective effect on dementia model rats,firstly,Aβwas injected to lateral ventricle of rats to establish dementia rats model.After intragastric administration with aqueous extract of futokadsura stem,the expression of Aβ,inflammatory factors TNF-αand IL-6,synaptophysin and the content of NO,NOS were detected.Secondly, using chemical method,futokadsura stem was separated and the separated components were added to SK-N-SH cells.After the action of these components,the expression of APP and Aβin SK-N-SH cells,and the content of Aβin culture medium were detected.Then we tried to determine the effective components in futokadsura stem which could inhibit the expression of APP and Aβ. Methods and Results1.Establishment of dementia model rats and the effect of aqueous extract of futokadsura stem for ethology and histology of dementia model rats1.1 Aβwas injected to lateral ventricle of rats to establish dementia model rats60 trained rats were selected,and were divided into 6 groups:normal control group,model group,sham group,positive control group(ibuprofen), high dose futokadsura stem group and low dose futokadsura stem group.Each group has 10 rats.Normal control group received no treatment;other 5 groups were anesthetized with chloral hydrate(350mg/kg bw)by intraperitoneal injection. The heads of rats were set on the stereotaxis instrument.For the model group, positive control group,high and low dose futokadsura stern group,10μg Aβ(25-35)was injected to the lateral ventricle;for sham group,the same dose of sodium chloride were injected when the skull was opened.Each group began intragastric administration after 7 days,positive group was administered ibuprofen by 20mg/kg bw,high dose futokadsura stem group was administered aqueous extract of futokadsura stem by 140mg/100g bw,low dose group was administered aqueous extract of futokadsura stem by 46.1mg/100g bw. Normal control group,model group and sham group were administered the same dose of distilled water.1.2 Determination of learning and memory ability of ratsLearning and memory ability of rats from each group were determined by Morris water maze experiment.The escape latencies were recorded.The shorter the escape latencies are,the better their learning and memory ability is. At the same time,we detected the spanning platform times of rats.The more times rats spanned the platform,the better their learning and memory ability is.Morris water maze experiment results showed that,1)Compared with escape latencies of rats in model group,those in normal control group and sham group decreased obviously,but the spanning platform times increased apparently,the difference is significant(P＜0.05).There were no diffence between escape latencies of rats in normal control group and sham group.It meant that learning ability of rats decreased when Aβwas injected to their lateral ventricles.2)In the 4thand 5thday of Morris water maze experiment, rats in positive control group have lower escape latencies and higher spanning platform times compared with rats in model group.The difference is also significant(P＜0.05).This demonstrated that when treated with anti-inflammatory medicine ibuprofen,dementia model rats could have improved learning and memory ability.3)In the 4thand 5thday of Morris water maze experiment,rats in high or low dose futokadsura stem group have lower escape latencies and higher spanning platform times compared with rats in model group.The difference is significant(P＜0.05).This demonstrated that when treated with aqueous extract of futokadsura stem,dementia model rats could have improved learning and memory ability.4)However,rats in high or low dose futokadsura stem had higher escape latency and lower spanning platform times,compared with rats in normal control group.The diffence is significant(P＜0.05).This meant that even if treated with futokadsura stem, the learning and memory ability of dementia model rats could not restore to normal state.5)When rats in high and low dose futokadsura stem groups were compared,rats in high dose group had lower escape latencies and higher spanning platform times.However,the difference is not significant(P＞0.05).1.3 Histology of rats hippocampusHE staining of rats hippocampus showed that,in normal control group, cells in CA1 region of rats hippocampus were arranged in order,the structure of cells was integrated;in model group,cells in CA1 region of rats hippocampus were not arranged in order,the structure of cells was not integrated,the boundary of cells was not clear,the gap between cells was enlarged;in positive control group,high and low dose of futokadsura stem group,cells in CA1 region of rats hippocampus were arranged in order,the structure of cells was fairly integrated.2.The effect of aqueous extract of futokadsura stem on the expression of Aβ,synaptophysin in frontal lobe and hippocampus neurons and on the expression of inflammatory factors in frontal lobe gliocytes of dementia model rats.Using immuno-fluorescence staining combined with image analysis,we observed the expression of Aβ,TNF-α,IL-6 and synaptophysin in frontal lobe and hippocampus of dementia model rats which was established by lateral ventricle Aβinjection.The results of Aβexpression in hippocampus:1)Rats in model group had higher fluorescence intensity of Aβin the hippocampus than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of Aβin the hippocampus;2)Rats in positive control group had lower fluorescence intensity of Aβin the hippocampus compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05).This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of Aβin the hippocampus of dementia model rats;3)Compared with rats in model group,rats in high or low dose futokadsura stem group had lower fluorescence intensity of Aβin hippocampus,The mean positive ratios were of significant difference(P＜0.05),This meant that aqueous extract of futokadsura stern could decrease the expression of Aβin the hippocampus of dementia model rats;4)When rats in high and low dose futokadsura stem groups were compared,rats in high dose group had lower fluorescence intensity of Aβin hippocampus,but the mean positive ratios were of no significant difference.It showed that high dose futokadsura stern had similar treatment effect than low dose in decreasing the expression of Aβ.The results of TNF-α,IL-6 expression in frontal lobe:1)Rats in model group had higher fluorescence intensity of TNF-α,IL-6 in the frontal lobe gliocytes than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of TNF-α,IL-6 in the frontal lobe gliocytes;2)Rats in positive control group had lower fluorescence intensity of TNF-α,IL-6 in the frontal lobe gliocytes compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05).This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of TNF-α,IL-6 in the frontal lobe gliocytes of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had lower fluorescence intensity of TNF-α,IL-6 in frontal lobe gliocytes,The mean positive ratios were of significant difference(P＜0.05);4)Although rats in low dose futokadsura stem groups had lower fluorescence intensity of TNF-α,IL-6 in frontal lobe gliocytes,the mean positive ratios were of no significant difference(P＞0.05).It showed that aqueous extract of futokadsura stem could decrease the expression of TNF-α,IL-6 in frontal lobe gliocytes,the high dose group had better treatment effect than low dose group in decreasing the expression of TNF-α,IL-6.The detection of NO,NOS content in rats brain:1)Rats in model group had higher content of NO,NOS in the brain than rats in normal control group and sham group had.It was of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could increase the expression of NO,NOS in the brain of dementia model rats;2) Rats in positive control group had lower content of NO,NOS in the brain compared with rats in model group.It was of significant difference(P＜0.05). This meant that anti-inflammatory medicine ibuprofen could decrease the expresson of in the brain of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had lower content of NO,NOS in the brain,It was of significant difference(P＜0.05);4)Although rats in low dose futokadsura stem groups had lower content of NO,NOS in the brain,It was of no significant difference(P＞0.05).It showed that aqueous extract of futokadsura stem could decrease the expression of NO,NOS in the brain,the high dose group had better treatment effect than low dose group in decreasing the expression of NO,NOS.The results of synaptophysin(SYP)expression in rats frontal lobe and hippocampus:1)Rats in model group had lower fluorescence intensity of SYP in the frontal lobe and hippocampus than rats in normal control group and sham group had.The mean positive ratios were of significant difference(P＜0.05).This demonstrated that dementia model rats established by lateral ventricle Aβinjection could decrease the expression of SYP in the frontal lobe and hippocampus;2)Rats in positive control group had higher fluorescence intensity of SYP in the frontal lobe and hippocampus compared with rats in model group.The mean positive ratios were of significant difference(P＜0.05). This meant that anti-inflammatory medicine ibuprofen could increase the expresson of SYP in the frontal lobe of dementia model rats;3)Compared with rats in model group,rats in high dose futokadsura stem group had higher fluorescence intensity of SYP in frontal lobe and hippocampus,The mean positive ratios were of significant difference(P＜0.05),It showed that aqueous extract of futokadsura stem could increase the expression of SYP in frontal lobe and hippocampus,4)Compared with rats in model group,rats in low dose futokadsura stem group had higher fluorescence intensity of SYP in hippocampus,The mean positive ratios were of significant difference(P＜0.05),5)Rats in low dose futokadsura stem groups did not have higher fluorescence intensity of SYP in frontal lobe,the mean positive ratios were of no significant difference(P＞0.05).It meant that low dose aqueous extract of futokadsura stem could only increase the expression of SYP in the hippocampus,not the frontal lobe of dementia model rats.3.Inhibition of APP gene transcription and protein expression in SK-N-SH cells by piperlonguminineldihydropiperlonguminine components separated from futokadsura stem 3.1 Preparation of futokadsura stem monomers componentsFutokadsura stem from Fujian province was first extracted with water,after concentration,we got the extract.Then,it was extracted with petroleum ether, acetic ether and normal butyl alcohol in turn.The acetic ether extract phase was dissolved by 95%ethanol,then subject to silica gel column chromatography,chloroform-acetone gradient elution.Chloroform-acetone(9:1) elution phase Ft.1 was repeatedly crystallized by petroleum ether-acetone, and HFT-1 was obtained.According to the data of 1HNMR and 13CNMR spectra,referring to related literature,we determined the crystal was composed of piperlonguminine(A) and dihydropiperlonguminine(B),the ratio of A to B is 1:0.8.The structures of piperlonguminine and dihydropiperlonguminine are showed below:A:piperlonguminineB:dihydropiperlonguminine3.2 Pharmacological effect of piperlonguminine/ dihydropiperlonguminine components separated from futokadsura stemSK-N-SH cells were divided into normal control group,DMSO group(1‰DMSO),aqueous extract group(15g/I),high dose of monomers components group(13.13μg/ml),middle dose of monomers components group(6.56μg/ml), low dose of monomers components group(3.28μg/ml).After 22 hours of treatment,different indexes were detected.6 reproducible experiments were performed.Using the method of MTT,SK-N-SH cells proliferation assay was performed.The results showed that,after 22 hours different treatments,no significant difference was found for SK-N-SH cells proliferation among different groups.Using the method of RT-PCR,APP gene expression was detected in SK-N-SH cells.After electrophoresis,the fluorescence intensity ratio between APP andβ-Actin was used to represent the expression of APP mRNA.The results showed that the fluorescence intensity ratios in aqueous extract group and high dose of monomers components group were apparently lower than the ratios in normal control group.The difference was significant.However,the ratios of DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of APP mRNA.Using the method of Western blot,amyloid precursor protein(APP) expression was detected in SK-N-SH cells.The fluorescence intensity ratio between APP andβ-Actin was used to represent the expression of amyloid precursor protein.The results showed that the fluorescence intensity ratios in aqueous extract group and high dose of monomers components group were apparently lower than the ratios in normal control group.The difference was significant.However,the ratios of DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of amyloid precursor protein.Using the method of immuno-fluorescence staining combined with image analysis,Aβexpression was detected in SK-N-SH cells.The results showed that the fluorescence intensity of Aβin aqueous extract group and high dose of monomers components group was apparently lower than that in normal control group.The mean positive ratios were of significant difference.However,the mean positive ratios in DMSO group,middle dose and low dose of monomers components group had no significant difference compared to the ratios of normal control group.This demonstrated that aqueous extract of futokadsura stem(15g/I)and piperlonguminine/dihydropiperlonguminine components separated from futokadsura stem(13.13μg/ml)could reduce the expression of Aβ.Conclusion1.Aβlateral ventricle injection to rats could decrease the their learning and memory ability,damage the neurons in hippocampus.It is a fairly ideal AD animal model.Aqueous extract of futokadsura stem could ameliorate the learning and memory ability of dementia model rats,protect the damaged neurons in hippocampus.2.Aqueous extract of futokadsura stem could decrease the expression of Aβin neurons,reduce the expression of inflammatory factors TNF-α,IL-6 and NO, NOS in gliocytes,ameliorate the chronic inflammatory response in the brain of Aβlateral ventricle injection AD model rats.Aqueous extract of futokadsura stem could also increase the expression of synaptophysin in the neurons of AD model rats,improve the synapse damage caused by chronic inflammatory response.3.Piperlonguminine/dihydropiperlonguminine components separated from Futokadsura stem could inhibit the expression of APP mRNA and APP in SK-N-SH. |
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