Danggui-Shaoyao-San: New Hope for Alzheimer's Disease

: Danggui-Shaoyao-San (DSS), also called Toki-shakuyaku-san (TJ-23) or Dangguijakyak-san (DJS), is a well-known herbal formula ( Angelica sinensis (Oliv.) Diels., Ligusticum chuanxiong Hort. , Paeonia lactiflora pall ., Poria cocos (Schw.) Wolf , Alisma orientalis (Sam.) Juzep. , Atractylodes macrocephala Koidz.), which has been widely used in oriental countries for the treatment of various gynecological diseases. Recent studies show that DSS has an effect on free radical-mediated neurological diseases and exhibits anti-inflammatory and antioxidant activities and reduces cell apoptosis in the hippocampus. In addition, DSS mediates the modulation of central monoamine neurotransmitter systems and ameliorates dysfunction of the central cholinergic nervous system and scopolamine-induced decrease in ACh levels. DSS improves the function of the dopaminergic, adrenergic, and serotonergic nervous systems. Interestingly, DSS can alleviate cognitive dysfunction of Alzheimer's disease (AD) patients, suggesting that it is a useful therapeutic agent for AD. This paper reviews the mechanism of DSS for the treatment of AD.

prevents the reduction of dopamine (DA) metabolites, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in olfactory-bulb-lesioned mice [46]. DSS has been shown to improve memory impairment and acetylcholine and norepinephrine in the cerebral cortex and hippocampus [47]. However, in recent years, DSS has pharmacological basis on memory dysfunction, modulating metabolism of monoamine neurotransmitters, protecting the ultrastructure of the cortex changed by aging [41], and increasing the level of superoxide dismutase (SOD) [55]. DSS is beneficial for the treatment of cerebrovascular dementia that has indirect neuroprotective activity [64].
It is reported that DSS not only improves microcirculation in patients with asymptomatic cerebral infarction, but also prevents cognitive impairment from AD [53]. The aim of the present review is to focus on mechanisms underlying DSS-mediated neuroprotection for AD patients. Reduces the expression of the IL-1β, IL-6, TNF-α mRNA [4] Restores the abnormal activities NOS and levels of CP, MDA, GSH and NO induced by D-gal [5] Attenuates CUS-induced decreases in noradrenaline and dopamine [6] Reverses CUS-induced increase MDA content [6] Suppresses the downregulation of Bcl-2, upregulation of Bax, the release of mitochondrial cytochrome c into cytosol [7] and sequential activation of caspase-9 and caspase-3 [7] Reduces 6-OHDA-induced intracellular ROS production and GSH depletion [8] Inhibits mitochondrial membrane instability [8] Protects TH-immunoreactive cells and fibers in the nigrostriatal region from MPTP toxicity [9] Mechanisms underlying DSS-mediated neuroprotection DSS not only increased the expression of nuclear factor-κB (NF-κB) as well as transforming growth factor-β and enhanced the activity of SOD and glutathione peroxidase (GSH-PX), but also attenuated the progressive accumulation of type IV collagen that reflects its antioxidant activity [2]. DSS has an effect on decreasing the concentrations of the metabolites of monoamines, glutamate and glutamine, and increasing SOD activity of the mitochondrial fraction in the cortex, hippocampus and striatum. In addition, DSS significantly suppressed thiobarbituric acid-reactive-substances (TBARS) formation [3]. The impairment of learning acquisition induced by colchicine was attenuated by DSS in the brain [55].
DSS significantly reduced the escape latency and expression of the IL-1β, IL-6, TNF-α mRNA, and neuronal apoptosis by suppressing the level of the nitric oxide (NO) in the hippocampus. Furthermore, DSS shortened the response latency and decreased the error numbers in a step-down passive avoidance test [4]. The abnormal activities of nitric oxide synthase (NOS) and levels of carbonyl protein (CP), malondialdehyde (MDA), glutathione (GSH) and NO induced by D-gal were restored by ethanol extract of DSS (DE). Chronic unpredictable stress (CUS) induced increase in serum MDA content is markedly reversed in mice by DSS [6]. Moreover, by regulating the expression of Bcl-2, Bax and caspase-3, DE improved neuronal survival in the hippocampus of D-gal-treated mice [5]. Recent studies have documented that DSS suppresses the downregulation of Bcl-2, upregulation of Bax, the release of mitochondrial cytochrome c into cytosol and sequential activation of caspase-9 and caspase-3 to protect against H2O2-induced apoptosis in PC12 cells [7], and DSS also significantly protects dopaminergic neurons from 6hydroxydopamine (6-OHDA)-induced neurotoxicity and reduces 6-OHDA-induced intracellular reactive oxygen species (ROS) production and GSH depletion and inhibits mitochondrial membrane instability [8]. Furthermore, DSS protects tyrosine hydroxylase (TH)-immunoreactive cells in the nigrostriatal region from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity [9] (Table 1).

Ferulic acid
Ferulic acid (FA), a natural antioxidant and a putative AD neuroprotective compound, is able to reverse morphological defects induced by Aβ oligomers. The antioxidant mechanism includes neutralizing ROS, recovering mitochondrial membrane potential, and blocking apoptotic pathways [12]. FA can attenuate phosphorylation of ERK1/2 activated by Aβ oligomers and modulate the expression of an antioxidative protein known as Peroxiredoxin [13]. Some studies have indicated that FA greatly attenuates these changes including elevating levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement [14]. Moreover, by scavenging free radicals, FA acts as a potent antioxidant compound and has been proposed as a potential treatment for AD [11]. FA not only attenuated impairment induced by mecamylamine (MECA) and scopolamine (SCOP)+MECA as well as central acetylcholinergic neurotoxin ethylcholine mustard aziridinium ion (AF64A), but also activated central muscarinic and nicotinic receptors and antioxidant enzymes, improved cognitive deficits, and could be a key molecule for the development of therapeutics for AD [10].

Paeoniflorin
DSS have been studied in animals and healthy human volunteers.
Paeoniflorin (PF) exerts protection against Aβ-induced neurotoxicity by increases GSH content reduced, suppressing NOS activity and NO level, and decreasing CP and MDA levels [15]. By improving spatial cognitive impairment caused by cholinergic dysfunction, PF may rely on reversal of the muscarinic M1-receptor-mediated inhibition of longterm potentiation (LTP) [16]. PF can modulate Acidsensing ion channels-(ASIC) activity and protein expression and produce protective effects for PC12 cells against MPP(+) and acidosis-induced cytotoxicity [19]. L-type Ca 2+ channels in NG108-15 cells can be blocked by PF to affect neuronal or neuroendocrine function [17]. In addition, PF is able to produce protective effect on dopaminergic neurodegeneration and significantly attenuate the MPTP-induced toxicity by inhibiting neuroinflammation by activation of an adenosine A1 receptor (A1AR) [20].

Ligustilide or Z-ligustilide
Studies suggest that ligustilide (LIG) can protect cognitive deficits such as cerebral damage or neurodegenerative disorders. LIG significantly improves behavioral performance of D-gal treated mice. In the brain of D-gal treated mice, LIG is able to reduce cleaved caspase-3 and GFAP levels and the level of MDA as well as increase the activities of Na + /K + -ATPase and expression of GAP-43 [29]. Lipopolysaccharide-induced upregulation of TLR4 mRNA expression is reduced by LIG dose-dependent in spinal astrocytes [28]. Klotho expression and the AD phenotype was inversely correlated, LIG decreases Akt and Forkhead box class O1 (FOXO1) phosphorylation and upregulates Klotho expression in the cerebral choroid plexus and serum that might contribute to the neuroprotective effect against AD [30].

Ligustilide or Z-ligustilide
Reducing the expression of upregulation of TLR4 mRNA Lipopolysaccharide-induced [28] Reducing the level of MDA as well as increasing the activity of Na(+)-K(+)-ATPase [29] Raising the expression of GAP-43 and reducing cleaved caspase-3 and GFAP levels [29] Decreasing Akt and Forkhead box class O1 phosphorylation and upregulating Klotho expression [30] Increasing BDNF and phosphorylated cAMP-responsive element binding protein (p-CREB) levels and γ-aminobutyric acid (GABA) expression [31] Improving Nrf2 nuclear translocation and increasing Nrf2 and HO-1 protein expression [32] Up-regulating erythropoietin and inhibiting RTP801 expression [33] Increasing SOD activity and reducing malondialdehyde levels [34] Increasing the Bcl-2 expression and decreasing in Bax and caspase-3 immunoreactivities [35] Increasing the activities of the antioxidant enzyme GSH-PX [35] TMPZ Inhibiting the expressions of nitrotyrosine and iNOS to mediate the free radical-scavenging activity [36] Suppressing prostaglandin E(2) production and lipopolysaccharide/interferon-gamma-induced inflammation in cultured glial cells [37] Reducing proinflammatory mediator production and cerebral ischemia/reperfusion-induced inflammatory cell activation [37] Reducing cerebral I/R-induced internucleosomal DNA fragmentation, caspase-3, caspase-8 and caspase-9activation, and cytochrome c release [38] Suppressing the consequent production of monocyte chemoattractant protein 1 (MCP-1) [38] Tetramethylpyrazine Tetramethylpyrazine (TMPZ) is an active ingredient isolated from a commonly used Ligusticum chuanxiong Hort., which markedly inhibits the expressions of nitrotyrosine and iNOS to mediate the free radicalscavenging activity in ischemia-reperfusion brain injury [36]. It was reported that TMPZ markedly suppresses prostaglandin E2 production and lipopolysaccharide/ interferon-gamma-induced inflammation in cultured glial cells and reduces pro-inflammatory mediator production and cerebral ischemia/reperfusion-induced inflammatory cell activation that significantly protects the brain against ischemic injury [37]. TMPZ is able to reduce cerebral I/Rinduced internucleosomal DNA fragmentation, caspase-3, caspase-8 and caspase-9 activation, cytochrome c release, and suppresses the activation of microglia and/or recruitment of inflammatory cells to the ischemic site as well as the consequent production of monocyte chemoattractant protein 1 (MCP-1) [38].

Atractylodes macrocephalaon polysaccharides
The levels of Bcl-2 and the ratio of Bcl-2/Bax in hypoxic neurons are significantly increased by Atractylodes macrocephalaon polysaccharides (AMPS), suggesting that AMPS can improve neuronal growth and prevent mitochondrial injury and apoptosis of neurons induced by hypoxia [39].

Ligusticum chuanxiong Hort. extract
Serum TNF-α, IL-6, IL-8, NO, MIP-1α, CRP and myocardium MDA levels and serum CK, LDH and AST activities are significantly decreased while myocardium Na + /K + -ATPase, Ca 2+ /Mg 2+ -ATPase, NOS, SOD, CAT, GSH-Px and TAOC activities are increased by the Ligusticum chuanxiong Hort. extract [40]. Together, the mechanism of protecting the neuronal damage and cell apoptosis suggest that DSS could provide more useful ways for developing more comprehensive and potent therapeutic strategies for AD ( Table 2).

The effect on the neurotransmitter and the central nervous system
Alcohol poisoning decreased the activity of choline acetyltransferase (ChAT) and the content of noradrenaline (NA) in forebrain areas such as the cerebral cortex and hippocampus, which contribute to deficiencies in subcortical noradrenergic and cholinergic systems [43]. DSS treatment markedly inhibited CUS-induced decreases in NA and DA concentrations in mouse brain [6]. Moreover, DSS modulates metabolism of monoamine neurotransmitters including increasing the content of norepinephrine (NE), DA and 5-hydroxytryptamine (5-HT) in brains of aged mice while protecting the cortical ultrastructure [41], which may be the major mechanism of DSS for improving memory dysfunction induced by alcohol. In addition, DSS not only increased ACh and blood flow in intact rats, it also increased ACh in ischemic rats in the dorsal hippocampus (DH), suggesting that DSS can improve the cognition [49].
Single administration of DSS decreased the content of NE in the hippocampus, but increased the contents of DA and HVA in the cerebral cortex and the hippocampus, while repeated administration of DSS, increased the contents of NE, MHPG, DOPAC, 5-HT and 5-HIAA in the cerebral cortex, and NE and DA in the corpus striatum [44]. However, these SCOP-induced decreases in ACh levels were significantly inhibited by a single administration of DSS at 500 mg/kg. In the mouse brain, dysfunction of the central cholinergic nervous system and SCOP-induced decrease in ACh levels were ameliorated by DSS [45]. After oral administration of DSS and a 3month latency period, the step-through test was significantly prolonged and the content of monoamine neurotransmitters such as NE, DA, and 5-HT were increased in the brains of aged mice and the cortical ultrastructure was protected [48]. DSS administered in a dose-dependent manner significantly suppressed the stress-induced enhancement of hypothalamic NA turnover at a low dose level [56].
DOPAC and HVA in the olfactory bulb of olfactorylesioned mice were significantly suppressed by the administration of DSS. DSS can make NGF contents increase at 1 and 2 weeks and within 3 weeks, the expression returns to baseline [46]. Administration of DSS could decrease ChAT activity significantly in the cerebral cortex and the DH of ovariectomized mice along with a decrease of NE contents [47].
Glutamate is the major excitatory neurotransmitter in the brain and has effect on cognition and memory, but alterations of glutamatergic signaling can induce excitotoxicity [57,58,59], which is linked to several neurodegenerative disorders and cell death such as AD [64,66]. Repeated cerebral ischemia markedly decreased GluR2 flop mRNA at 1 and 3 days, but the decrease in GluR2 flop was significantly suppressed by DSS (300 mg/kg) at 3 days [64]. DSS decreases the concentration of glutamate in the cortex, hippocampus, and striatum of female and male SAMP8 and has effect on glutamate, and monoamine metabolites in the aged rat brain [3,50].
Somatomedin C/insulin-like growth factor 1 (IGF-1) level in medium from the rat corpora lutea incubated in vitro was regulated by the effect of herbal components of DSS [51]. DSS has a neuroendocrine effect on ovulation in immature female rats [52]. The Na + , K + , and Ca 2+ current components were decreased by DSS in voltageclamped NG108-15 cells and the peak heights of the Na + and Ca 2+ current components were strongly decreased in the hybrid cells [54]. DSS decreased the content of arginine vasopressin (AVP) in the pituitary and the expression of AVP mRNA in hypothalamus [42]. In addition, albiflorin can maintain the intracellular Ca 2+ concentration [86] (Fig. 1).

To improve the cognitive ability of memory and reduce the damage of amyloid protein
The impairment of learning acquisition induced by colchicine was attenuated by DSS while the level of SOD (141 ± 3 and 135.4 ± 2.0) in the brain was increased by DSS (0.5 and 1.0 g/kg) [60]. DSS treatment tended to improve the score for orientation to place on the Mini Mental State Examination and made regional cerebral blood flow (rCBF) in the posterior cingulate significantly higher [61]. DSS significantly improved erythrocyte differentiation in iron-deficiency anemia, increased the proportion of normal erythrocytes and erythroblasts as well as erythropoietin and transferrin levels in the blood. It also possessed anemia-ameliorating efficacy [62].
Aβ is a main factor in the pathogenesis of AD [96][97][98][99][100]. DSS inhibited Aβ25-35-induced neuronal damage and lactate dehydrogenase (LDH). The Aβ25-35-induced neuronal death and lipid peroxidation were significantly reduced by DSS at concentrations of 100 and 300 µg/mL [63]. Through DSS administration, the SCOP-induced impairment including reference and working memory deficits of a rat's spatial cognition determined in the eightarmed radial maze test was decreased [65]. DSS could also improve the response in the memory-retention test in SAMP8 mice [67]. In addition, JD-30 that is extracted from DSS could improve cognitive dysfunction of mice induced by intracerebroventricular injection of Aβ, ameliorate the reduction of LTP, reduce the neuronal damage in the hippocampus, decrease the prolonged latency in the Morris water-maze test as well as the content and deposition of Aβ in the brains of SAMP8 mice [68][69].
Amyloid Precursor Protein forms ratio (APPr), independent of disease severity, was influenced by cholesterol levels suggesting that cholesterol affects APP processing in vivo [70]. The small natural molecule ferulic acid can inhibit Aβ in vitro in the early stages of Aβfibrillogenesis [76]. FA can improve hyperactivity, object recognition, and spatial, working, and reference memories through decreasing Aβ production and reducing amyloidogenic APP proteolysis [77]. FA reduced IL-1β levels and amyloid deposition in the frontal cortex [78] while pretreatment with FA could suppress increases in immunoreactivities of glial fibrillary acidic protein, the astrocyte marker, and IL-1β in the hippocampus with Aβ1-42 [79]. FA is likely to inhibit the aggregation of Aβ42 oligomers; it blocks the hydrogen bond that forms β-sheets thereby interrupting the transition of Aβ42 monomers to oligomers [80]. FA can decrease cleavage of the β-carboxyl-terminal APP fragment and reduce β-site APP cleaving enzyme 1 protein stability and activity with therapeutic potential against AD [81].
Through decreasing mitochondrial membrane potential, increasing cytochrome c release as well as activities of caspase-3 and caspase-9, Aβ 25-35 in PC12 cells can be attenuated by PF [82]. Through modulating the TNF-α-activated NF-κB signaling pathway, LIG ameliorated these neurotoxic effects of Aβ25-35 [83]. Gallic acid (GA), is a NF-κB acetyltransferase inhibitor that protects neuronal cells from Aβ-induced neurotoxicity and restores Aβ-induced cognitive dysfunction [84]. Aβ aggregation and/or the formation of Aβ-derived diffusible neurotoxin ligands were inhibited by GA [85]. Albiflorin reduced the toxicity and ROS induced by both monomeric and oligomeric Aβ species to exhibit a potent inhibitory effect on Aβ1-40 and Aβ1-42 aggregation. In addition, albiflorin can maintain the intracellular Ca 2+ concentration [86].

The effect of DSS on the hypothalamus-pituitarygonadal axis
Multiple factors lead to AD. Estrogen is a potent factor that may play an important role in the preservation of vascular disease and improve blood flow in regions of the brain affected by AD [73]. DSS increases the latency in step-down test especially in female animals. In the MWM test, SAMP8 mice that were administered DSS orally spent less time finding the platform. DSS could increase the estradiol (E2), NO, and glycine, and ameliorate deterioration of cognition in SAMP8 mice, especially in females [71]. After oral administration of DSS for 3 weeks, melatonin level was significantly increased at night. Phosphorylation of CREB can be significantly increased by DSS. DSS also had an effect on the beta-adrenergic receptors binding in the pineal glands [72]. In vivo, both DSS and Keishibukuryogan can stimulate the production of estradiol-17 β, progesterone, and testosterone by preovulatory follicles, but DSS stimulation was more effective than Keishibukuryogan [74]. Further research show that DSS could stimulate the cerebral cortex to produce nicotinic acetylcholine receptors, which directly impacts the brain to accelerate the process of neuroendocrine regulation of ovulation. In addition, Levistilide A is a main component in Rhizoma Chuanxiong when paired with DSS, could significantly increase its bioavailability compared with LA alone and Rhizoma Chuanxiong alone [75].

The effect of DSS on metabolic risk factors of AD
Several other studies have also reported that hypercholesterolemia is a potentially modifiable environmental risk factor for AD [92]. Hypercholesterolemia within the membrane is likely to impact APP trafficking, APP metabolism, activities of β, γ and α secretases and Aβ synthesis [93,94]. In addition, other studies have showed an aggregated relative risk of approximately 2.2 that links type 2 diabetes with AD [95]. Type 2 diabetes leads to cognitive decline and increases the risk of late-onset AD. However, hypercholesterolemia and type 2 diabetes are an important risk factor linking AD.
Dietary vitamin E and FA are beneficial for hypercholesterolemia. In particular, FA was added to the vitamin E-rich Western diet that could increase activities of antioxidant enzymes (SOD, catalase, and GSH-PX) and paraoxonase [87]. GA has potent anti-oxidative and anti-obesity activities and can protect against hypercholesterolemia [88]. Moreover, DSS treatment can effectively lower the higher renal levels of Advanced Glycation End Products (AGEs) in streptozotocin (STZ)diabetic rats [90]. DSS reduces the activities of SOD and GSH-Px in the kidneys of STZ-diabetic rats [91]. FA can also significantly decrease blood glucose and increase serum adiponectin levels for the FA-treated OLETF rats [89]. However, DSS has effects on metabolic risk factors of AD: hypercholesterolemia and type 2 diabetes.

Conclusion
AD is a complicated brain disease that involves multiple mechanisms. Therefore, DSS might exert beneficial effects on AD. In fact, investigations clearly show that it has recently been a hot target of many research studies related to a strong relationship among the chemical, biological, pharmacological, and medical properties of these plants. Interestingly, studies indicate that the traditional formula of DSS has potential diseasemodifying properties including its well proven effects of anti-inflammation and antioxidant activities, neuroprotective/neurotrophic activities, regulation of the neurotransmitter and the central nervous systems, and modulation of amyloid precursor protein metabolism. Therefore, it might not only contribute to symptomatic improvement, but also play important roles for the treatment of neurodegenerative diseases by interfering with key factors of the disease. These encouraging findings suggest that DSS is a promising candidate for AD.

Acknowledgment
This work is supported by the Grant (No. 2013BJ10) from the Science Foundation of Heilongjiang Institute of Technology.

Conflict of Interest?
There are no conflicts of interest.