| ObjectiveAlzheimer’s disease (AD), one of the most common neurodegenerative disorders of the central nervous system, greatly threatens the health of the elderly. Although there are many theories and views about the etiology of AD, the precise mechanism remains unknown, and no utility drugs have been developed. So, to develop more effective AD therapeutic drugs with lower toxicity and lower side-effect remains an urgent task.A large number of drugs with differing targets and mechanisms of action are under development for the treatment of AD. But, the sponsor announced its intention to discontinue development of tramiprosate as a pharmaceutical. Aiming to a sole target seemed difficulty to obtain the satisfied therapeutic effects since AD is too complex. Traditional Chinese Medicine (TCM) are composed of many ingredients, they have the characteristics of multi-mechanisms, multi-pathway and multi-targets, which may consistent with AD.Danggui-Shaoyao-San (DSS) is a traditional Chinese Medicinal Prescription. The prescription now still widely used in clinical and the therapeutical effect of DSS on AD was firstly reported at1980’s in Japan. DSS was initially recorded in’Synopsis of Prescriptions of the Golden Chamber’, which was compiled by Zhang-Jing Zhang during the Han dynasty. The prescription was used to relieve menorrhalgia and other abdominal pains of women. The effects of DSS in improving the symptom of mild and moderate AD patients had been confirmed, and DSS could also ameliorate the recognizing ability in many AD model animals, such as models of aging, cerebral ischemia and chemical injury to the brain. Our previous studies showed that DSS improved the impairment of recognition in SAMP8, and that DSS contained serum reversed the long term potentiation (LTP) reduction induced by Aβ25-35. There have been many studies about the underlying mechanisms and substance at the basis of the action of DSS on AD. Our previous studies extracted several fractions from DSS. JD-30is an active fraction extracted from Danggui-Shaoyao-San under guidance of bioactive evaluation in our previous studies. Previous studies demonstrated that JD-30could ameliorate cognition deterioration and improve synaptic plasticity impairment in several Alzheimer’s disease (AD) model mouse.Through behavior evaluation, synaptic plasticity investigation, possible mechanism explore, and active component analysis, we hope to explore the possible mechanism of the neuroprotective effects of JD-30and its two major constituents (paeoniflorin and albiflorin) which may be develop to a promising new Chinese drug on AD treatment.Methods and Results1. JD-30composition analysis and quality controlThrough the methods of HPLC/MS, we determined the main effective active site of JD-30. Then we tested the different batch extraction of JD-30, determine the consistency of experimental.The results showed that main ingredients of JD-30are paeoniflorin, albiflorin, albiflorin Rl, benzoylpaeoniflorin sulfonate, paeoniflorin sulfonate, norswertianolin,4-0-ethy lpaeon iflorin, E-coniferin, syringin, oxypaeoniflora, quercetin and so on. We did an examination of the ingredients of JD-30by high pressure liquid chromatography revealed that JD-30contained more than65%saponins (60.83%paeoniflorin,8.06%albiflorin,).2. The neuroprotective effects of paeoniflorin and albiflorin(1) Neuroprotective effects of paeoniflorin and albiflorin on the Aβ25-35induced PC12cellsTo explore the possible mechanism of the neuroprotective effects of JD-30and its two major constituents (paeoniflorin and albiflorin) were investigated by using flow cytometry and CCK-8in PC12cells line.The results showed that JD-30, paeoniflorin and albiflorin had no effect on the cells survival, but JD-30and paeoniflorin could inhibit the cells apoptosis; promote the cell vitality, and more importantly, the effects of JD-30were better than paeoniflorin. Our results suggested that paeoniflorin maybe a chief active constituent of JD-30, but paeoniflorin cannot completely displace the neuroprotective effect of JD-30. Additionally, JD-30could inhibit the cell apoptosis and cell necrosis by inhibiting the aggregation of A β25-35.(2) Effects of JD-30and its major constituents on the AD model cell in vitro Firstly, we established AD cell model by PC12and SH-SY5Y cells induced by CORT, SNP, MSG, KC1, H202, Na2S204. Next, we investigated the effects of JD-30, paeoniflorin and albiflorin on the cell toxicity. The results showed that JD-30, paeoniflorin and albiflorin had no effect on the cell survival, nor affect the cell necrosis induced by CORT, SNP, MSG, KC1. JD-30and TGP (100μg/ml)had effect on the cell survival induced by H202, Na2S2O4. But paeoniflorin and albiflorin had no effect on the cell survival.(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.To evaluate the effects of PF and AF on microglial inflammatory responses, BV2cells were stimulated with LPS in the absence or presence of PF and AF, and then the amounts of TNF-α in the culture media of BV2cells were measured using AlphaLISA. Combination of PF and AF effectively and dose-dependently inhibited LPS-induced TNF-α release from BV2cells. LPS has been reported to cause activation-induced cell. However, combination of PF and AF alone did not reduce BV2cell viability at the amounts tested in BV2cell3. Effects of PF, AF and JD-30on the learning and memory in AD model miceIn this part, the effects of JD-30on the learning and memory deficits were examined in AD model mice. Two classic AD models mice were selected, one is Kunming mice which were intraperitoneal injection (ip.) injected with Scopolamine, and another is7-8month old SAMP8.(1) Effects of PF, AF and JD-30on the spatial learning and memory in SMAP8Seven months SAMP8mice were separated into5groups at random, each group of6-10mice (10males) were divided into4cages by gender. JD-30was dissolved in distilled water at14mg/ml, and PF to8mg/ml, AF to8mg/ml, combination of PF and AF (P/A,8+1mg/kg). The mice in drug treated groups were given intragastric administration of JD-30, PF, AF or PF+AF (0.1ml/10g body weight) once a day during the test. SAMP8as negative control and the age-matched SAMR1(10males) as positive control were given an equal volume of distilled water. The mice were weighed every3days. After8weeks of drug administration, the Morris water-maze test was performed and the drugs given lasted until the whole test wasfinished.In the Morris water-maze training session, SAMR1rapidly learned the location of the platform, but SAMP8had significant longer escape latency on every test days. This increase was significantly shortened by combination of PF and AF (P/A,80+10mg/kg) administration from2to5day. Similarly, on the day of the probe trial following the final day of training, latency (the first time that the mice crossed the former plat form) was increased more in SAMP8than in SAMR1. The latency was decreased by P/A administration, the above results indicate that combination of PF and AF improves spatial learning and memory deficits in SAMP8.(2) Effects of PF, AF and JD-30on the spatial learning and memory in AD model mice induced by ip. ScopConsistent with previous papers, aggregated Scop (ip. lmg/kg) injection induced spatial learning and memory impairment in mice. In the training trial session of the Morris water maze performance, both the control group and the model group mice rapidly learned the location of the platform. These results suggest that intraperitoneal injection of Scop did not caused impairment in it, PF, AF and JD-30can’t improved spatial learning and memory deficits in Scop treated mice.4. Effects of PF, AF and JD-30on the neuron and microglia in SAMP8The decrease and damage of neuron and microglia are the direct factors in AD, so we investigated the effects of PF, AF and JD-30on the damage of neuron and microglia, and investigated the possible mechanism in vitro.Nissl’s staining results showed that, except for SAMP8positive group, the other groups showed intact and well-arranged pyramidal neurons of hippocampal. In contrast, SAMP8showed reduced staining of pyramidal neurons, which were significantly swollen and arranged abnormally. Combination of PF and AF (P/A,8+1mg/kg) diminished the loss and swelling of pyramidal neurons in the hippocampal, neurons recovered their characteristic shape and arrangement, and the number of CA3region neurons approached the normal number of SAMR1.Immunofluorescence staining showed that groups of PF and AF (P/A,8+1mg/kg),PF, AF, TGP had a less number of microglia, mostly exist in inactive state.5. Effects of PF, AF and JD-30on the adenosine A1R and A2aR mRNA expression in the cortex and hippocampal of SAMP8Compared with SAMR1, SAMP8had significantly lower hippocampal A1R and A2a mRNA expression. Compared with SAMP8, both PF and AF administration groups had significantly higher hippocampalAIR mRNA expression.5. Effects of PF and AF on the mechanism of the balance of adenosine Aland A2a receptorsThere are four types of adenosine receptors:A1R, A2aR, A2bR, A3R. They belong to the G-proteincoupled receptor (GPCR) family and all have been cloned and characterized from several mammalian species including humans Neuromodulation by adenosine is exerted through the activation of high-affinity A1and A2aRs, which are probably of physiological importance, and of low affinity A2bRs, which may be relevant in pathological conditions. The A3R is a high-affinity receptor in humans, but has a low density in most tissues.Effects of PF and AF on the binding of [3H] DPCPX and [3H] CGS21680, the data indicated that PF and AF failed to displace the binding of DPCPX and [3H] CGS21680, even at the highest concentration of1OOmmo1-1.The interaction between PF and AF and adenosine receptors (A1R, A2aR). has been studied using the molecular dynamic simulation. We tested whether PF and AF bind directly to adenosine A1and A2a receptors. The results showed that PF and AF both unite with A1R to form hydrogen bond. Besides that PF reacted with TYR4-Double Hydrogen bond, ASN62-Hydrogen bond, ASN246-Double Hydrogen bond, HIS256-Pi Pi reaction, THR262-Hydrogen bond, ILE266-Hydrogen bond. AF reacted with ILE61-Hydrogen bond, ASN62-Hydrogen bond, GLU164-Hydrogen bond, LYS257-Hydrogen bond, THR262-Hydrogen bond, TYR263-Double Hydrogen bond. PF and AF are combined with A2aR, effecting hydrogen bond and PHE168-Pi Pi reaction. Also, PF reacted with SER67-Hydrogen bond, GLU169-Hydrogen bond, ASN253-Hydrogen bond. AF reacted with ALA63-Hydrogen bond, LEU267-Hydrogen bond.Conclusion1. JD-30contained more than65%saponins (60.83%paeoniflorin,8.06%albiflorin,).2. PA and AF maybe two chief active constituents of JD-30, but they cannot completely displace the neuroprotective effect of JD-30in the AD model cells.3. Combination of PF and AF (P/A,8+1mg/kg) can improves spatial learning and memory deficits in SAMP8, and decrease and damage of neuron and microglia.4. Molecular mimicry study indicates that PF could activate adnosine A1receptor and inhibit adenosine A2a receptor at the same time.In summary, paeoniflorin and albiflorin are the two major constituents of JD-30. Combination of PF and AF is a neuroprotective agent against the damage and senescence by the different way, making it a potential therapeutic agent for AD treatment. |
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