To Study Anti-Alzheimer’s Disease Effect And Its Mechanism Of Effective Parts Of Gynostemma Pentaphylla On Rapid Aging Dementia Mice (SAMP8Mice)

ObjectiveTo study anti-Alzheimer’s disease (AD) effect and its mechanism of effective parts of Gynostemma pentaphylla on rapid aging dementia mice (SAMP8mice). Four parts of Gynostemma pentaphylla were isolated to screen senile dementia in vitro, identified the effective constituents of senile dementia, providing experimental evidence for treating senile dementia effect of Gynostemma pentaphyllum.Methods1. Extraction and separation of Gynostemma pentaphylla:Making dry Gynostemma pentaphylla into coarse powder, using25.0kg, impregnated with95%,70%,50%of ethanol extraction, respectively3times, soaked4～5d, and then combined extracts, the ethanol was recoveried. dissolved total Ethanol extracts into water suspensions, followed to extract petroleum ether, ethyl acetate, n-butyl alcohol extraction, and then recoveried the solvent.2. Extraction of effective part of Gynostemma pentaphylla screening in vitro:using MTT method, to study the effect of extract of effective parts of Gynostemma pentaphylla on proliferation of mouse NG-108cells in vitro by directly giving drug, to study the effect of extract of effective parts of Gynostemma pentaphylla on proliferation of mouse NG-108cells in vitro injuried by Aβ25-35fragment.3. Effect of inhibition on AD:using the method of MTT, directly giving and serum detection of Gynostemma pentaphyllum n-butanol extracts to study the mechanism which made model on NG-108cells by A β25-35fragment, through Hoechst33258staining, flow cytometry assay to obsereve NG-108cells apoptosis, Elisa method was detected to the caspase-3expression level.4. Anti-Alzheimer’s disease (AD) effect and its mechanism of effective parts of Gynostemma pentaphy11a on rapid aging dementia mice (SAMP8mice): To observe the effect of Gynostemma pentaphy11a n-butanol extracts on mice behavior. The Morris water maze method was used to seek times, escape latency, platform quadrant than as index to detect the memory behavior of mice. Elisa method was used to determin the content of SOD, MDA, GSH, Aβ and cytochrome C. Immunohistochemistry was used to detect the APP, p75NTR, pJNK, Bax, Bcl-2and cysteine aspartic proteinase-3(caspase-3) expression, Semi-Quantitative analysis was performed on APP, BACE-1, p75NTR and pJNKmRNA in the brain by RT-PCR method. Western blot method was used to detect the expression of p53protein.5. Acute toxicity test:The n-butanol extract of Gynostemma pentaphyllum, was givien to mice to determin the maximum tolerance dose of mice.Results1. Screening in vitro:the effect of Gynostemma pentaphyllum extraction on the proliferation of NG-108cells in vitro:petroleum ether and n-butanol extract of Gynostemma pentaphyllum in5～40μg/mL concentrations on the proliferation of NG-108cells were compared with noramal group, it had significant difference (P<0.05or P<0.01). Water extraction of Gynostemma pentaphyllum at5～40μg/mL concentration on NG-108cells were compared with normal group, it had no significant, but in80μg/mL concentration on NG-108cells, compared with noramal group, it had significant difference (P <0.05). Ethyl acetate had the trend of proliferation on NG-108cells, but compared with the normal group, it had no significant difference.2. Anti Alzheimer’s disease mechanism of Gynostemma pentaphyllum n-butanol extraction in vitro:①Hoechst33258dyeing:directly to drug method indicated that, when Gynostemma pentaphyllum extraction were in5and10μg/mL concentration which injuried by A025-35on NG-108cell of apoptosis compared with the control group, it had significantly difference (P<0.05),20μg/mL concentration which injuried by Aβ25-35on NG-108cell of apoptosis compared with the control group, it had no significant difference. Animal serological methods indicated that, if compared with10%blank serum,10%animal serological had significantly difference(P<0.05). Compared with20%blank serum,20%animal serological had no significantly difference.②Flow cytometry assay:directly to drug method indicated that, when Gynostemma pentaphyllum extraction were in5and10μg/mL concentration which injuried oy Aβ25-35on NG-108cell of apoptosis compared with the control group, it had significant difference (P<0.05),20μg/mL concentration which injuried by Aβ25-35on NG-108cell of apoptosis compared with the control group, it had no significant difference. Animal serological methods indicated that,10%animal serological was compared with10%blank serum, it had significantly difference (P<0.05). Compared with20%blank serum,20%animal serological had no significantly difference.3. Anti-Alzheimer’s disease effect and its mechanism of n-butanol extraction of Gynostemma pentaphylla on rapid aging dementia mice (SAMP8mice):(1)Morris water maze learning ability test:swimming ability test results showed that SAMR1and the administration group were compared with SAMP8model group, it had no significant difference (P>0.05). Positioning navigation experiment results displayed:①Incubation period:Compared with SAMP8model group, SAMR1and the administration groups had very significant difference (except high dose group)(P<0.01).②Sought number:SAMP8model group average sought number were less than other groups who were administrated, if SAMR1and the administration group were compared with SAMP8model group, it had significant difference (except high dose group)(P<0.05).③Platforms stay, the average stay length of SAMP8model group was shorter than other groups, SAMR1and the administration groups were compared with SAMP8model group, they had significant difference (except high dose group)(P<0.05).(2)N-butanol extraction of Gynostemma pentaphylla on senile dementia effects of free radical metabolism in mice:①SOD:Compared with the SAMP8model group, SAMR1group, huperzine group and low-dose group had a very significant difference (P<0.01).②GSH-PX:Compared with SAMP8model group, huperzine group had very significant difference (P<0.01), low-dose group and medium-dose group had significant differences (P<0.05).③MDA:Compared with the SAMP8model group, SAMR1group and low-dose group had a very significant difference (P<0.01), medium-dose group had significant differences (P<0.05).(3)①-butanol extraction of Gynostemma pentaphylla on senile dementia effects of Aβ、APP and BACE-1:For the expression of Aβ content in serum compared with SAMP8model group, SAMR1group, huperzine group and all of drug groups had a very significant differences (P<0.01). Expression of Aβ content in the brain, compared with SAMP8model group, SAMR1group, huperzine group had very significant differences (P<0.01), medium-dose groups and low-dose group had significant differences (P<0.05).②Tissue effect of immunohisto-chemical of n-butanol extraction of Gynostemma pentaphylla on APP proteins in mouse brain:APP-immunoreactive neurons were mainly located in the parietal cortex of the brain, hippocampal CA1, CA3and parts of the inner wall of blood vessels. APP number of immunopositive neurons in the comparison group: compared with SAMP8model group, SAMR1and huperzine group had very significant differences (P<0.01), the low dose group and medium-dose group had significant differences (P<0.05).③Expression effect of n-butanol extraction of Gynostemma pentaphylla on BACE-1and APPmRNA:for BACE-1mRNA, compared with SAMP8model group, SAMR1group had very significantly differences(P<0.01), huperzine group and low-dose group had significant differences (P<0.05). For APPmRNA, compared with SAMP8group, SAMR1group had very significantly differences (P<0.01), huperzine group, medium-dose group and low-dose group had significant differences (P<0.05).(4)N-butanol extraction of Gynostemma pentaphylla on Aβ in Alzheimer’s mice which mediated p75NTR to induce apoptosis:①effects of n-butanol fraction on cytochrome c content:compared with SAMP8model group, SAMR1group, huperzine group, low-dose group and the medium-dose group had very significant difference (P<0.01).②Tissue effect in immunohistochemical of n-butanol extraction of Gynostemma pentaphylla on p75NTR, pJNK, Bax, Bcl-2and the caspase-3. p75NTR number of immunopositive neurons in the comparison group:compared with SAMP8model group, SAMR1, huperzine group and low-dose group had very significant differences (P<0.01), the medium-dose group had significant differences (P<0.05). pJNK number of immunopositive neurons in the comparison group:SAMR1, huperzine group, low-dose group and medium-dose group compared with SAMP8group, they had very significant differences (P<0.01), but high-dose group was compared with SAMP8model group, it had no significant differences. Bax number of immunopositive neurons in the comparison group:compared with SAMP8group, huperzine group and all of drug groups had very significant differences(P<0.01),Bcl-2number of immunopositive neurons in the comparison group:compared with SAMP8model group, SAMR1, huperzine group and low-dose group had very significant differences (P<0.01), but medium-dose group had no significant differences.(5)caspase-3number of immunopositive neurons in the comparison group: compared with SAMP8model group, SAMR1and huperzine group, medium-dose group and low-dose group had very significant differences (P<0.01), but high-dose group had significant differences(P<0.05).③Expression effect of n-butanol extraction of Gynostemma pentaphylla on p75NTR and pJNKmRNA:for p75NTRmRNA, compared with SAMP8group, SAMR1group and huperzine group had very significantly differences (P<0.01), medium-dose group and low-dose group had significant differences (P<0.05). For pJNKmRNA, compared with SAMP8model group, SAMR1group had very significantly differences (P<0.01), huperzine group and the low-dose group had significant differences (P<0.05).(4) Expression effect of n-butanol extraction of Gynostemma pentaphylla on P53:compared with SAMP8model group, SAMR1group, huperzine group, the medium-dose group and low-dose group had very significantly differences (P<0.01), the high-dose group had significant differences (P<0.05).(5)Acute toxicity test (MTD):The results showed that the activities, appearance, behavior, feeding of mice within14days after administration were normal. Urine, breath, no toxic reactions and death occurred. The mice growth and weight gain after administration was also normal. n-butanol fraction of Gynostemma pentaphyllum on mice by oral administration was120g/kg herbs, and it was equivalent to adults (60kg weight)daily (30g drug)240times.Conclusion(1)N-butanol extraction of Gynostemma pentaphylla in vitro can induce NG-108cells to increase proliferation, it also can protect NG-108cells which injuried by Aβ25-35fragment. Hoechst33258staining and flow cytometry assay can decrese injuried by Aβ25-35, inhibit apoptosis in NG-108cells. The protection mechanism may be related to inhibit the cysteamine aspartic acid protease-3(caspase-3) level.(2) N-butanol extraction of Gynostemma pentaphylla can anti-old sexual dementia of role, its mechanism may be related to improve learning, and memory capacity of mice. Improve the anti-oxidation capacity of mice. Inhibition APP, and BACE-1protein level, reduce Aβgenerated, also can reduce the combination of Aβand p75NTR, inhibit JNK pathway, reduced pJNK content, inhibit p53protein and increase downstream of BCL-2/Bax of expression, release the content of cytochrome C and reduce caspase-3content.(3)Acute toxicity testing show that n-butanol fraction of gynostemma pentaphyllum on mice by oral administration is120g/kg120g herbs, equivalent to240times times the adult daily amount, indicate it has low toxicity.