Recent Progress in the Research Field of Neuropharmacology in China

Abstract

In recent years, Chinese neuropharmacologists have done a lot of basic and practical work in neuropharmacology, especially in the fields of pain, drug dependence, depression, Alzheimer’s disease, schizophrenia, having obtained some exciting results that are of great significance for the development of neuropharmacology. Here I would like to review recent progress in the research fields of neuropharmacology in China.

Neuropharmacological Research on Pain

The formation and modulation of pain is a complicated process. Besides the opioid receptor system, many non-opioid receptors have also been proved to participate in the formation and transmission of pain. The imidazoline receptor is another newly found receptor relating to pain modulation. Dihydroetorphine, agmatine, and acupuncture are some new kinds of treatment for pain control.

Basic Research on Pain Modulation

The endogenous opioid system is one of the most important pain modulatory systems and has been investigated for years. The endogenous opioid receptor system can be linked to many other systems by direct molecular–molecular interactions. Indeed, protein–protein interactions associated with receptor trafficking and functional changes were considered to be a mechanism underlying opioid analgesia and tolerance. Recently, direct interaction between protachykinin and δ-opioid receptors (DORs) was proved to be responsible for sorting of DORs into large dense-core vesicles (Guan et al. 2005), resulting in surface insertion of DORs and DOR-mediated spinal analgesia. Further investigation revealed that the substance P domain of protachykinin and the third luminal domain of DOR were critical for the formation of the interaction.

In recent years, many non-opioid neurotransmitters and their receptors including dopamine, glutamate, γ-amino butyric acid (GABA) were also postulated to influence the formation and transmission of pain through interaction with the opioid receptor system. Most of these agents do not directly bind with opioid receptors, but enhance opioid analgesia and inhibit tolerance to opioids. Agmatine, the decarboxylated product of l-arginine by the enzyme l-arginine decarboxylase, is postulated to be an endogenous ligand of imidazoline receptors. The regulatory roles of agmatine in the formation of pain threshold, morphine antinociception, and tolerance were described recently (Su et al. 2003). Subcutaneous injection of agmatine or intracerebroventricular injection of l-arginine produced significant analgesic effects in mouse acetic acid writhing tests, formalin inflammatory pain, and neuropathic pain (Li et al. 1999a; Su et al. 2005; Wang et al. 2005b; Qin et al. 2006). Although agmatine or l-arginine failed to influence the tail flick latency in heat radiant tail flick tests, both of them enhanced the antinociceptive effects of morphine and inhibited morphine-induced tolerance (Li et al. 1999b; Su et al. 2005). Furthermore, l-arginine decarboxylase specific antibodies inhibited morphine antinociception and promoted the development of tolerance to morphine in animal nociceptive models (Su et al. 2005). Given that the analgesia of agmatine and its influence on opioid analgesia were blocked by idazoxan, an imidazoline/α2 adrenergic receptors-mixed antagonist, rather than by yohimbine, a selective α2 adrenergic receptor antagonist, the regulatory effects of agmatine on pain processing were supposed to rely mainly on the activation of imidazoline receptors in the central nervous system (CNS). All these results point to the participation of another non-opioid receptor, the imidazoline receptor, in the formation and transmission process of pain.

Potential Therapies for Pain Control

Dihydroetorphine

Opioid agonists have long been used as pain relievers. In continuous efforts to find more powerful opioid analgesics, dihydroetorphine (DHE) was developed. DHE showed high affinity and selectivity for μ-opioid receptor with the K _i value of 9.69 × 10⁻¹² M in inhibiting DAMGO (Which is a μ-opioid receptor agonist) binding to rat brain membrane (Wang et al. 1995). As a full opioid agonist, the analgesic effect of DHE was 6,000–12,000 times more potent than morphine (Huang et al. 1982a, 1988). In addition, DHE exhibited lower respiratory depression and lower physical dependence in animals relative to morphine (Huang et al. 1982a, b). However, DHE did produce a potent reinforcing effect, which was confirmed by self-administration and conditioned place preference tests in rats (Wang et al. 1997; Wu et al. 1998; Liu et al. 1999). Therefore, the clinical application of DHE should be under strict control weighing its efficacy and safety.

Agmatine

As mentioned above, agmatine had mild analgesia effects in some animal models, and it enhanced opioid analgesia and attenuated opioid tolerance as well as opioid dependence. Possessing these properties, agmatine, either used alone or in combination with opioid analgesics, might have potential application in pain treatment in the future.

Acupuncture

Acupuncture, by inserting needles into “acupoints” in the body, has long been used for pain treatment in China and some other Asian countries. Although our understanding of the mechanisms of acupuncture analgesia is far from complete, many researchers have tried to explain how the acupuncture works in a modern scientific way. Acupuncture or electrical stimulation in certain body sites facilitates the release of specific neuropeptides, e.g., endogenous opioid peptides, in the CNS.

In the rat nerve ligation model, 2 Hz electroacupuncture relieved the mechanical allodynia and cold-induced ongoing pain. Naloxone pretreatment blocked the analgesic effect of electroacupuncture on cold-induced ongoing pain, but not on mechanical allodynia, suggesting that both opioid-dependent and opioid-independent mechanisms are involved in acupuncture (Sun et al. 2003). Accordingly, 2 Hz electroacupuncture induced long-term depression of the nociceptive synaptic transmission in the spinal dorsal horn, which was blocked by either MK-801 or naloxone (Xing et al. 2003).

In addition, an electroencephalogram study in healthy volunteers showed that electroacupuncture modulated the brain activity in certain areas. The theta rhythm in the contra-lateral centro-parietal area and the beta rhythm in contralateral prefrontal cortex, ipsilateral inferior frontal and temporal lobe, and ipsilateral occipitoparietal cortex were negatively correlated with pain score after electroacupuncture (Zhang et al. 2003). The subtle structures involved in the analgesia induced by electroacupuncture varied according to the intensity of the stimulation. This showed that the opioid receptor in the thalamic nucleus submedius was responsible for the analgesia of high-intensity electroacupuncture for exciting small afferent fibers, while the analgesia induced by low-intensity electroacupuncture was mediated by the anterior pretectal nucleus (Zhu et al. 2004b). Very recently, it was demonstrated that in connexin 43 gene knockout mice, the analgesic effect of acupuncture declined, suggesting that connexin 43 is possibly related to meridians and the effect of acupuncture (Yu et al. 2007b).

Neuropharmacological Research on Drug Dependence

It is widely accepted that the mesocorticallimbic dopamine system plays an initial role in drug-induced reward and addiction. However, the establishment of addiction status also involves many other neurotransmitter and neuropeptide systems. On the one hand, plastic changes take place in these systems as a consequence of the activation of many non-opioid systems. On the other hand, adaptive changes in these systems also affect the development and maintenance of dependence. The imidazoline receptor, muscarinic receptor, histamine receptor, and NMDA receptor were proved to modulate the development of opioid dependence. The exact molecular mechanism has been explained by Chinese neuropharmacologists. Thienorphine, naltrexone microsperes, agmatine, and acupuncture were developed for the treatment of drug dependence.

Basic Research on Drug Dependence

Receptor Mechanisms

Accumulating evidence suggests that endogenous agmatine and I1-imidazoline receptors (I1R) are involved in the development of opioid dependence (Wu et al. 2007b). To learn more about the modulatory effect of agmatine in opioid dependence, a cell line was established with stably expressed MOR and imidazoline receptor antisera-selected protein (IRAS), a candidate for I1R (Wu et al. 2005a). In this cell model, agmatine with low concentrations (0.01–2.5 μM) inhibited naloxone precipitated cAMP overshooting and this inhibition was completely blocked by efaroxan, an I1R-preferential antagonist. Additionally, high concentrations of agmatine (5–100 μM) inhibited naloxone precipitated cAMP overshooting in chronic morphine-treated cells by activating I1R and then blocking L-type calcium channels in CHO cells (Wu et al. 2005a; Weng et al. 2003; Zheng et al. 2004b). Furthermore, the agmatine–IRAS system modulates gene expression by regulating the transcription factor CREB and immediate early gene product c-Fos (Wu et al. 2006a). Taking all this information into account, we have proposed that agmatine and I1R represent a new kind of modulator for opioid function. On this basis, we proposed that some non-opioid neurotransmitters and their receptors could influence the pharmacological effects of opioids at the pre-receptor, receptor and post-receptor levels. This suggests a new concept of opioid function modulator (Su et al. 2003).

The mscarinic receptor system seems to be another important neurotransmitter system associated with drug dependence. It was demonstrated that the expression of muscarinic receptors were differentially modulated under morphine-dependent or naloxone-precipitated withdrawal conditions (Zhou et al. 2002). Intrathecal injection of M2-antisense oligonucleotides decreased morphine abstinence scores (Chou et al. 2002). A recent study also showed that blocking M5-muscarinic receptors in the ventral tegmental area (VTA) inhibits the expression of heroin-induced locomotor sensitization (Liu et al. 2007a). The authors also suggested that regulation of nNOS expression in locus coeruleus, NMDA receptor expression in brainstem and FosB expression in the Nucleus Accumbens (NAc) and hippocampus might account for the muscarinic-receptor mediated effects (Zhou et al. 2002; Liu et al. 2004, 2007a).

Excitatory amino acids and sex hormone are also important factors that modulate the development of opioid dependence. During morphine withdrawal, glutamate transporter subtype 1 (GLT1), rather than excitatory amino acids carrier 1 (EAAC1), was significantly induced in the nerve terminals and expressed on surface of hippocampal neurons (Xu et al. 2003). In the rat conditioned place preference model, morphine treatment decreased the concentration of dehydroepiandrosterone in NAc as well as plasma and decreased the concentration of pregnenolone in hypothalamus (Wang et al. 2005a).

Molecular Mechanisms

There has been important progress as to the molecular mechanism for drug dependence. Recently, dysfunction of Na⁺, K⁺-ATPase activity has been proposed to be a molecular mechanism underlying drug addiction. Short-term morphine treatment stimulates whereas long-term morphine treatment inhibits Na⁺, K⁺-ATPase activity in mouse hippocampus and striatum. Strong evidences suggest that at least the cAMP-PKA pathway is employed in this effect (Wu et al. 2006b, 2007c). After chronic morphine treatment, the phosphorylation of SNAP-25, a SNARE protein essential for vesicle release, was downregulated in a PKC-dependent way. Consequently, SNARE complex formation was inhibited, allowing the alteration of exocytotic process and neural plasticity (Xu et al. 2004b).

β-Arrestins are key negative regulators and scaffolds of G protein-coupled receptor (GPCR) signaling. β-Arrestin1 and β-arrestin2 preferentially bind to the phosphorylated opioid receptors in response to agonist stimulation and result in receptor internalization and desensitization, which leads to the opioid receptors signaling terminates and is crucial to opioid tolerance and dependence. Endogenous β-arrestin1 exerted functions exclusively in the phosphorylation-dependent opioid receptor internalization, whereas endogenous β-arrestin2 was required for the phosphorylation-independent opioid receptor internalization, which indicated the different requirement for β-arrestin isoforms in the agonist induced phosphorylation-dependent and -independent opioid receptors internalization (Zhang et al. 2005a). Besides a cytosolic regulator, recent study revealed that β-arrestin1 is a nuclear messenger that regulates opioid receptors signaling. Stimulation of δ-opioid receptor induced β-arrestin1 translocation to the nucleus. The nuclear translocation of β-arrestin1 led to its accumulation and histone H4 hyperacetylation at the p27 and c-fos promoter regions, stimulating transcription of these genes (Kang et al. 2005). These results revealed an epigenetic mechanism for opioid receptors signaling from the cell membrane to the nucleus through signal-dependent histone modification and a novel function of β-arrestin1 in the nucleus as a GPCR cytoplasmic-nuclear messenger to control transcription of the target genes. Thus, transient stimulations induced by opioids activation to opioid receptors transfer into nuclear to regulate certain target genes transcription, and ultimately results in persistent alternations in behavior, such as opioids dependence and relapse.

Neurobiological Mechanisms

It has been reported that long-term adaptations of neurotransmitter systems induced by drug addiction mostly overlapped with the activation of learning and memory circuits. Abnormal learning and memory is considered as a crucial factor in the development and maintain of compulsive drug-seeking behavior and relapse. It has been proved that negative emotion or aversive effect during withdrawal played an important role in relapse. Further studies showed that dopamine and glutamate systems in the central nucleus of the amygdala were involved in the acquisition but not expression of conditioned place aversion in morphine-treated rats, suggesting the different neuro-mechanisms in the development of unconditioned negative emotion and the arousing of conditioned negative emotion (Zheng et al. 2006). Stress not only facilitates the development of drug addiction, but also induces relapse. Stress-induced synaptic plasticity of hippocampus was crucial to the acquisition of normal memory related to opioid addition (Yang et al. 2004). In addition, morphine-induced conditioned place preference was related to stress-induced locomotion and novelty-seeking behavior in juvenile and adult rats, which could predict the susceptibility to opioids addiction (Zheng et al. 2003, 2004c). Glutamate system, especially NMDA receptors, was found to play an opposite role in food and morphine-induced conditioned place preference. Facilitation of MK801 to food-induced conditioned place preference was related to the NMDA receptors in lateral hypothalamus, while the inhibition of MK801 to morphine-induced conditioned place preference was mainly related to the NMDA receptors in nucleus accumbens (Li et al. 2006e).

In addition, chronic opiate treatment reduces LTP in the hippocampal CA1 region of rats and this reduction can be restored by re-exposure of animals to corresponding drugs. However, subtle differences can be observed in opiates that share similar pharmacological profiles. Indeed, re-exposure of morphine restored the reduction of LTP in heroin-dependent rats, while heroin could not restore the reduction of LTP in morphine-dependent rats during opiates withdrawal, indicating differential modulations of hippocampal functions by those two kinds of opiates (Bao et al. 2007).

Potential Therapies for Drug Dependence

Thienorphine

Opioid partial agonist has been proved to be effective in reducing illicit opioid use with a good safety profile, particularly with respect to lower respiratory depression and dependence compared with full μ-agonist. Thienorphine was synthesized as a ramification of buprenorphine (Liu et al. 2005a) and was a non-selective opioid partial agonist with high inhibition potency on the lethal effect of morphine (Yu et al. 2006). Thienorphine showed a much longer antagonism on morphine-induced lethality than buprenorphine did (more than 15 days). Additionally, the bioavailability of thienorphine was much higher than that of buprenorphine through oral administration. Co-administration of thienorphine with morphine dose-dependently suppressed the development, transfer, and expression of behavioral sensitization to morphine in mice (Zhao et al. 2004). However, thienorphine showed no dependent liability and suppressed morphine-induced behaviors in the animal models of conditioned place preference and self-administration (data not published). These preliminary studies suggested that thienorphine might have possible application in the treatment of opioid dependence.

Naltrexone Microsperes

Poor compliance is the major factor that limits the clinical use of opioid receptor antagonist medication, such as naloxone, in the treatment of drug addiction. A strategy to improve patients’ adherence is to develop new formulations that maintain stable plasma drug levels. A new sustained release preparation, encapsulating naltrexone into injectable and biodegradable polymer microspheres, was developed and evaluated. In pharmacokinetic studies, intramuscular injection of this sustained release preparation of naltrexone provided a safe, complete, and sustained release of the drug for about 1 month (Yan et al. 2003). Additionally, the pharmacodynamic effects of naltrexone, such as blocking morphine-induced analgesia and dependence, last for more than 1 month after subcutaneous administration of this naltrexone preparation in mice and rats (Gong et al. 2001). Furthermore, there were no serious adverse effects other than light tissue irritation (Gong et al. 2001; Yan et al. 2003). Currently, this sustained release preparation of naltrexone is under clinical evaluation in China.

Agmatine

Exogenous agmatine inhibited physical dependence on morphine in mice, rats, beagle dogs, and rhesus monkeys (Wu et al. 2007b). In addition, when co-administered with morphine during the conditioning sessions, agmatine abolished the acquisition of morphine-induced conditioned place preference in rats in an idazoxan-reversable manner. Agmatine inhibited the expression and reinstatement of morphine-induced conditioned place preference respectively (Wei et al. 2005). The regulatory effect of agmatine and imidazoline receptor was further confirmed in other animal models, such as locomotor sensitization (Wei et al. 2007) and self-administration (data not published). Our studies also suggest the regulatory effects of agmatine are associated with its inhibition of morphine-induced changes in extracellular DA and gene expression (Wei et al. 2007).

Acupuncture

In a multi-center randomized clinical trial, in contrast to an almost 100% rate of relapse after drug withdrawal in control groups, 20 out of 611 patients kept drug free for more than 1 year after Han’s acupoint nerve stimulator (HANS) treatment (Han et al. 2003), suggesting peripheral electric stimulation might be a reasonable choice in the treatment of heroin addiction. In animal studies, 2/100 Hz electroacupuncture stimulation reversed the increased expression of nNOS in locus coeruleus (LC) and periaqueductal gray (PAG) in morphine withdrawal rats (Cui et al. 2002). Additionally, low-intensity electroacupuncture stimulation decreased the heroin-seeking behavior induced by conditional cue or small doses of heroin, correlated with the changes of FosB expression in the nucleus accumbens septi, globus pallidus, basolateral amygdaloid nucleus (Sun et al. 2006). Recently, 2/100 Hz HANS were also shown to be effective in reducing the online time of adolescent Internet addicts and inhibit Internet addiction syndrome (Wu et al. 2007a).

Neuropharmacological Research on Depression

Major depression (MD) is a serious mental illness characterized by sad mood, disinterest in daily life, sleep disturbances, difficulty in thinking and concentrating, recurrent thoughts of death and suicide. MD arises from the complex interaction of multiple-susceptibility (and likely protective) genes and environmental factors. The disease phenotypes include not only episodic and often profound mood disturbances, but also a range of cognitive, motoric, autonomic, endocrine, and sleep/wake abnormalities. Untreated or inadequately treated depression often leads to suicide. On the other hand, after medical therapy and psychotherapy or a combination of both, nearly 40–60% of the patients could be cured (Mao et al. 2007).

Despite being one of the most prevalent psychiatric conditions in the community, depression is commonly unrecognized in clinical practice. A recent survey by Asian Psychiatrical Science Summit (APNS) indicated that more than 26 million people suffer from depressive illnesses in China. And the economic cost for the disorder is high; the total estimated cost of depression is 62,000 million RMB each year (Mao et al. 2007). Traditional Chinese herbal medicine, agmatine, and acupuncture were developed for the treatment of MD.

Etiology and Pathophysiology of Depression

According to the traditional Chinese medicinal theory, the incidence of depression or associated disorders has been attributed to liver qi stagnation, a comprehensive manner, including many symptoms, such as mental stress, hypochondria , hernial pain, lumps in the breasts, and irregular menstruation (Xu et al. 2004a). In addition, great progress has been made on the neurobiological and molecular basis of MD.

Neurodegeneration and Neurogenesis During Depression

The balance between neurodegeneration and neurogenesis might be the key factor of MD. In contrast to downregulation of hippocampal neurogenesis induced by stress, antidepressant treatment has been shown to increase neuron proliferation. Antidepressants like fluoxetine (specific serotonin reuptake inhibitor), tranylcypromine (monoamine oxidase inhibitor), reboxetine (specific norepinephrine reuptake inhibitor) and rolipram (phosphodiesterase-IV inhibitor) were all proved to enhance neurogenesis. The neurogenesis action of antidepressants is closely associated with the upregulation of cAMP response element binding protein (cAMP-CREB) cascade and expression of brain-derived neurotrophic factor (BDNF) in hippocampus. In line with these reports, agmatine and xiao bu xin tang, which have antidepressant effects, were also found to increase the hippocampal neurogenesis in animal models. Thus the effect of cytoprotection may be a common pathway for the effect of antidepressants (Li et al. 2003b). However, the exact mechanisms demand more studies.

Molecular Basis for Depression

Although depression is most likely involved in the regulation of hypothalamic–pituitary–adrenal (HPA) axis, monoamine neurotransmitters, excitotoxicity, hippocampal neurons, and neurotrophic factors, no consensus has been reached so far concerning the exact molecular and cellular mechanisms of depression.

Recently, some novel receptors such as Corticotropin-Releasing Factor (CRF) receptor were also proved to relate to depression. CRF is a kind of neuropeptide that plays a key role in neuroendocrine, autonomic, and behavioral responses to stressors. Numerous reports suggest that alterations in CRF function contribute to the pathogenesis of depression. Recently, the polymorphisms of three sites (rs1876828, rs242939 and rs242941) in CRHR1 gene have been proposed to be related to depression and the effect of antidepressant in Han Chinese MD patients. The results showed that the rs242941 G/G genotype and homozygous GAG haplotype of the three single-nucleotide polymorphisms (SNPs) are associated with the therapeutic effects of fluoxetine in MD patients with high-anxiety. The results supported the idea that CRHR1 gene is likely to be involved in the antidepressant response in MD (Liu et al. 2007b).

Other studies suggest that microtubule-associated protein 4 (MAP-4) and drebrin may be involved in the antidepressant-like effects of desipramine and fluoxetine. Many genes related to antidepressant effects were identified using the differential display-polymerase chain reaction (DD-PCR) protocol. After rats were subjected to different kinds of stress for 20 days, DD-PCR was performed and differentially expressed cDNAs were further confirmed by dot-blot hybridization. cDNA homology analysis revealed that desipramine up-regulated the expression of MAP-4 mRNA and fluoxetine up-regulated the expression of drebrin A mRNA in the rat hippocampus compared with chronically stressed rats (Yang et al. 2003b).

Potential Therapies for Depression

Currently there are three main kinds of classical antidepressants in clinical practice, including tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs) and monoamine oxidase inhibitors (MAOIs). Most of them, however, have undesirable adverse effects and their mechanisms of action have not been resolved to satisfaction. Thus new antidepressants are still in urgent need.

Agmatine

Agmatine is an endogenous neurotransmitter and/or neuromodulator, which is considered as an endogenous ligand for imidazoline receptors. It was proved to have antidepressant-like effect in mice and rats, and it was also found to protect PC12 cells as well as the classical antidepressant desipramine from NMDA induced injury. Agmatine reverses the NMDA-induced intracellular Ca²⁺ overloading and the decrease of monoamines (including norepinephrine, epinephrine or dopamine) contents in PC12 cells, indicating that the antidepressant-like action of agmatine was related to its modulation of NMDA receptor, reversal of the decrease of monoamine contents, and reduction of Ca²⁺ overloading induced by NMDA. Besides, agmatine inhibited NMDA receptors and all isoforms of nitric oxide synthase (NOS). Agmatine up-regulated hippocampal neurogenesis in vivo in chronically stressed mice. Agmatine also increased the proliferation of cultured hippocampal progenitor cells significantly in a dose-dependent manner. All in all, exogenously agmatine had antidepressant effects in several animal models mediated in numerous ways (Li et al. 2003a).

Traditional Chinese Herbal Medicine

Seeking safe and effective antidepressant agents from traditional herbs may enable scientists to develop novel treatments for depressive disorders and may further reveal unknown mechanisms by which depressive symptoms can be alleviated.

Like St. John’s wort (Hypericum perforatum), a herb used extensively in the treatment of mild and moderate depression in Europe, many Chinese herbs including Hypericum canariense and H. glandulosum, gingko biloba, Salvia elegans, A loysia polystachya (Griseb.) Moldenke, kudzuvine root, aceranthus sagittatus, Scrophularia ningpoensis, Plantago asiatica, ilexpubesceus, and ginseng have been studied for years. Many traditional Chinese empirical formulas, such as Xiao yao-san, Banxia-houpu-tang, xiao bu xin tang, chai hu shu gan san, si ni san and yue ju wan, have also been used successfully to manage depressive disorders (Zhang 2004). Banxia Houpu Decoction has been used for the treatment of depression-related diseases since ancient times as a traditional Chinese medicinal empirical formula consisting of Pinellia ternata, Poria cocos, Magnolia officinalis, Perilla frutescens, and Zingiber officinale. The effects of the total decoction extract and five fractions therefrom were evaluated in mice by tail suspension tests (TST) and forced swimming tests (FST). The total 90% ethanol extract of the decoction was shown to possess an antidepressant activity that was close to that of Prozac, an antidepressant agent being applied clinically. The active principles were demonstrated to be mainly in the aqueous and lipophobic parts of the decoction extract while the polyphenol fraction exhibited a moderate action (Guo et al. 2004).

Curcuma longa is a major constituent of the traditional Chinese medicine Xiaoyao-san, which has been used to effectively manage stress and depression-related disorders in China. Curcumin is the active component of curcuma longa, and its antidepressant effect was described in mouse models of behavioral despair. Curcumin influenced behavior in a rat model of depression induced by chronic unpredictable stress. Animals subjected to chronic stress protocol for 20 days resulted in performance deficits in the shuttle-box task. At the same time, several physiological effects, such as an abnormal adrenal gland weight to body weight (AG/B) ratio, increased thickness of the adrenal cortex as well as serum corticosterone levels, reduced glucocorticoid receptor (GR) mRNA expression, were also observed after chronic stress protocol. All these changes were reversed by chronic curcumin administration. In addition, chronic stress procedure induced a downregulation of BDNF levels and reduction in the ratio of pCREB to CREB levels (pCREB/CREB) in the hippocampus and frontal cortex of stressed rats, which were also blocked by chronic curcumin treatment. These results provide compelling evidence that the behavioral effects of curcumin in chronically stressed animals, and by extension humans, may be related to their modulating effects on the HPA axis and neurotrophin factor expression (Xu et al. 2005).

Icariin is a major constituent of flavonoids isolated from Epimedium brevicornum Maxim (Berberidaceae), which is used as a traditional Chinese medicine to nourish the kidney. Icariin and E. brevicornum have a wide range of pharmacological and biological activities, including regulating cardiovascular, genital, and bone marrow proliferation. Icariin was found to significantly shorten immobility time of mice in FST after oral administration for 21 days. Icariin also produced a marked reduction in immobility time in the TST when administered for 7 days. Moreover, both FST induced increases in brain monoamine oxidase (MAO) activity and serum CRF levels as well as decreases in brain neurotransmitter levels were all restored after Icariin treatment. These results suggested that icariin possessed potent antidepressant-like properties that were mediated via neurochemical and neuroendocrine systems (Pan et al. 2005).

Saponins (SCLM) extracted from a traditional Chinese medicine, Chaihu-jialonggu-muli-tang (CLM), possesses an antidepressant-like activity in behavioral models. Subchronic administration of SCLM decreased immobility time in the FST in mice and rats. In addition, MTT and LDH assays showed that SCLM or fluoxetine protected PC12 cells from the corticosterone-induced cell death. SCLM and fluoxetine attenuated the intracellular Ca²⁺ overloading induced by corticosterone treatment in PC12 cells. Pretreatment with SCLM for 48 h increased the expression of NGF mRNA in PC12 cells. In summary, SCLM possesses an antidepressant-like activity in behavioral models that might be mediated via its cytoprotective action (Zhu et al. 2006).

The protopine, isolated from a Chinese herb Dactylicapnos scandens Hutch, was identified as an inhibitor of both serotonin transporters and noradrenaline transporters in vitro assays. In 5-HTP induced head twitch response tests, protopine dose-dependently increased the number of 5-HTP-induced head twitches. Protopine also produces a dose-dependent reduction in immobility in tail suspension tests. The present results open up new possibilities for the use of protopine in the treatment of mood disorders, such as mild and moderate states of depression (Xu et al. 2006).

G. elata aqueous ethanol extract produced significant antidepressive effect in regard to ambulation, rearing and the number of grooming behaviors in open-field tests, the effect of which is not different from that of fluoxetine. G. elata aqueous ethanol extract significantly decreases the immobility duration in FST and TST. The effect of GE at the dose of 300 mg/kg was even more powerful than that of fluoxetine (Zhou et al. 2006).

The antidepressant-like effect of piperine was also investigated in mice exposed to chronic mild stress (CMS) procedures. Repeated administration of piperine reversed CMS-induced changes in sucrose consumption, plasma corticosterone levels and open field activity. Furthermore, the decreased proliferation of hippocampal progenitor cells was ameliorated and the level of BDNF in hippocampus of CMS-stressed mice was upregulated by piperine during the same time course. In addition, piperine and fluoxetine were proved to protect primary cultured hippocampal neurons in a dose-dependent manner from corticosterone induced injury in MTT and LDH assays. Piperine reversed the corticosterone-induced reduction of BDNF mRNA expression in cultured hippocampal neurons. In summary, upregulation of the progenitor cell proliferation of hippocampus and cytoprotective activity might be the mechanisms involved in the antidepressant-like effect of piperine, which may be closely related to the elevation of hippocampal BDNF level (Li et al. 2007).

Electroacupuncture

In the mouse FST and CMS-induced rat depression model, electroacupuncture exhibits antidepressant effect and has potential synergistic effects with clomipramine (CLO, a tricyclic antidepressant). When electroacupuncture was given at ‘Bai-Hui’ (Du 20) and unilateral ‘An-Mian’ (EX 17) acupoints, the immobility time was significantly reduced. Electroacupuncture combined with CLO exhibited additive effects on the immobility time. In addition, after-rats were exposed chronically to a variety of mild unpredictable stressors, depressed mood and anhedonia were recognized by a decrease in sucrose intake in the CMS rats. CLO 5 mg/kg or electroacupuncture combined with 2.5 mg/kg CLO restored the reduction of sucrose intake induced by CMS. These results further demonstrated that electroacupuncture has synergistic antidepressant action with CLO, and the combination of these two means may provide an effective strategy for depression management (Yu et al. 2007a).

Neuropharmacological Research on Alzheimer’s Disease

As reviewed recently by Zhou and Han (2006), Alzheimer’s disease (AD) is a neuro-degenerative disease that is characterized by progressive memory impairment, cognitive impairment, and clinically observed behavior disorder, as well as senile plaques (SP), neurofibrillary tangles (NFT), neuron loss, neuraxon or synapse abnormality, and granulovacuolar degeneration in neuron pathology. With the gradually intensified aging of social population, AD is exerting a more profound effect on people’s lives and intensifying the burden of the family and the whole society. China has been an aging society since the 1990s. Currently, the aging population in China has topped 126 million and the number will rise to 280 million, 18.4% of the total population, by 2025, according to epidemiological studies (Yang and Zhou 2004). The incidence of AD in China is about 1.6% among people over 60 years old and 2.9% among those over 65 years old. With advancing age, the prevalence of AD increases. Every 5 years after the age of 65, the chance of having the disease doubles. Traditional Chinese herbal medicine, acupuncture, and some kinds of drugs targeted cholinergic system were developed for the treatment of AD in China.

Etiology and Pathophysiology of AD

Neurobiological Basis for AD

Compared with mixed dementia (MD) and vascular dementia (VD) AD has higher frequency of psychiatric symptoms such as agitation, anxiety, and panic. Atrophy is found in CT/MRI images and hypo-perfusion lesions are found only in cortical structure symmetrically with SPECT scanning in most of the patients with AD. These manifestations and characteristics will contribute to the diagnosis of patients with AD (Wei et al. 2003). In addition, the resting T2* signal obtained by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) may serve as a noninvasive quantitative marker in the diagnosis of AD (Zheng et al. 2004a).

Molecular Basis for AD

The pathogenesis of AD is complicated. Many researches in recent years revealed that the disorder of cholinergic neurotransmission and the deposition of β-amyloid protein played important roles in the pathogenesis of AD. In addition, many other factors are also associated with the development of AD. It was found that NGF, BDNF, NT3 exhibited different changes in hippocampus of rats with Aβ-induced AD, which suggests their relationship with the physiological function of cholinergic neurons and their crucial effect on neuro-degeneration in hippocampus of rats with AD (Zhang et al. 2005b; Dai et al. 2007). Many studies also showed that several signaling pathways are involved in the pathogenesis of AD (Liu et al. 2003; Jin et al. 2007; Zhang et al. 2004). For example, iNOS/NO participated in the mechanisms of Aβ-induced neurotoxicity, and downregulated anti-apoptotic PI3K/Akt/p70S6K signaling pathway induced by Aβ_25–35 in rat hippocampus might contribute to the neuronal damage in AD. ERβ signaling also played a critical role in the neuroprotective effect of estrogen and the disruption of ERβ signaling of estrogen might lead to the deposition of Aβ and ApoE directly.

Genetic Basis for AD

Gene mutation was another important factor that is related to the pathogenesis of AD. Several mutations in the gene coding for APP, PS-1, PS-2 are sufficient to cause familial AD through increasing the production of β-amyloid. Thus compared with Aβ_1–40-injected rat model, the double transgenic PS1/APP mice can simulate the specific pathogenesis and progressive changes of AD, even though no neuron loss is found in this model (Li et al. 2006b). Apolipoprotein E is another important genetic risk factor for AD and is significantly associated with late-onset AD and early-onset AD (He et al. 2005; Chen et al. 2006b). In addition, the ACT-51 polymorphisms are likely to be a susceptibility factor for development of AD in Chinese Han population (Zhou et al. 2005). The forward and reverse suppression subtracted cDNA library in hippocampus of senescence-accelerated mouse (SAM) was successfully constructed, which would become the abundance material used to screen and decide the hippocampal differential expressed genes of SAMP8 and SAMR1 (Chen et al. 2006a).

Potential Therapies for AD

Although the etiology and pathophysiology of AD have not been fully elucidated yet, many drugs have been developed on the basis of pathophysiology of this disease such as cholinesterase inhibitors, cerebral vasodilator, calcium antagonist, Aβ deposition inhibitors, β, γ-secretase inhibitors, and anti-inflammatory drug. All these drugs can improve memory and cognition in AD patients as well as prevent or delay the progression of AD to some extent. For example, both vasodilator Buflomedil HCL and antioxidant Vitamine C can maintain learning and memory as well as upregulate expression of anti-oxidase of Alzheimer rat induced by haloperidol (Huang et al. 2006).

Drugs Targeted Cholinergic System

Therapeutic options for AD have so far focused on modifying neurotransmitter systems, in particular the cholinergic system. So cholinesterase inhibitors are typical drugs used clinically. Clinical trials demonstrated that huperzine A and donepezil were effective not only in alleviating the symptoms but in improving cognitive function and self-care ability of AD patients (Yang et al. 2003a; Peng et al. 2005). NMF is a novel chemical whose structure consists of both Metrifonate and nicotine. In vitro experiments demonstrated that NMF increased the viability and survival rate of mouse cortical neurons, and protected neurons against KA-induced neurotoxicity. So this may be a promising drug for AD therapy although the mechanism of action and in vivo effects still need further study (Wu et al. 2005b). The injured cholinergic neurons in AD rats could be protected by a single or combined transplantation of neural stem cells modified with gene of NGF or GDNF (Ruan et al. 2002). Two weeks after transplantation, the spatial learning and memory of AD rats was almost restored. Immunization with Aβ42 and its subunits also effectively ameliorate impairment of spatial learning and memory in APPSWE transgenic mice (Li et al. 2006c). Anti-Aβ₄₂ or anti-K7Aβ_36–42 vaccine immunized rat sera counteracted cytotoxicity of Aβ₄₂ in vitro (Li et al. 2004).

Chinese Traditional Herbal Medicine

In recent years, researches on the inhibitors of protein and gene levels, immunotherapy, application of Chinese traditional drugs, and neurenergen have thrown light on the future of the treatment of Alzheimer disease. Pharmaceutical development on anti-AD drugs from natural products and Chinese medicinal herbs is of theoretical and practical importance. Many in vivo and in vitro experiments were carried out to search for effective anti-AD Chinese traditional herbal medicine. Clausenamide is the active principle extracted from the leaves of Clansena lansium (Laur) Skeels. Extracellular recording of the population spike (PS) of hippocampal dentate gyrus following application of low-frequency stimulation (1/30 Hz, 1.0 mA) showed that (−)-7-OH-Clau, the metabolite of Clausenamide, potentiated the basal synaptic transmission in the dentate of the rat hippocampus (Zhu et al. 2004a). Panaxnotoginsengsaponins (PNS) effectively protected NG108-15 cells from apoptosis induced by Aβ, promoted the growth of neural cellular axon, and enhanced synaptic function by slowing down excessive activity of SOD and decreasing NO amount (Lu et al. 2004). In addition, PNS inhibited the internal flow of Ca²⁺ and the toxicity of excitatory amino acids. On the basis of subacute injury induced by intravenous injection of d-galactose and bilateral cerebral Meynert basal nuclei injured by ibotenic acid, PNS played a protective role in pathological lesion of cholinergic neuron in AD rats by increasing the survival rate and quality of cells as well as increasing the content and activity of ChAT (Zhong et al. 2006a). Gingkgolide was also found to enhance learning and memory of AD rats established by injecting Oksdaic acid (OA) into CA1 area of hippocampal. The effect of gingkgolide was related to the reduced loss of cholinergic neurons, the deposition of senile plaque and the formation of neurofibrillary tangle by enhancing the microcirculation of brain (Wu et al. 2004). Additionally, gingkgolide combined with insulin treatment exerted a stronger improving effect on the learning of AD rats, possibly by reducing phosphorylation of Tau in neurons and inhibiting hyperplasia of the glial cells in the CA1 area of hippocampal (Wang et al. 2006a). Picrosides also improved learning and memory of AD rats. The increased activity of SOD and decreased concentration of MDA in the brain of experimental mice suggest that this result may contribute to its anti-oxidative effect (Li et al. 2006d).

Acupuncture

Some researches found that Chinese traditional acupuncture had good clinical effects on AD (Fu et al. 2005; Li et al. 2002; Luo et al. 2006). The effect of acupuncture at shenmen (HT7) on brain function of AD patients was observed by brain functional MRI. The result showed that different areas of cerebral cortices activated by acupuncture had close relationship with the sensory-motor central nervous system and cognitive function. Compared with acupuncture or DGSYS of TCM alone, acupuncture plus DGSYS of TCM exhibited the best therapeutic effect on intelligence, gnosia and routine self-care ability in AD patients. Electrotherapy could also repair the formation and inhibit the degeneration of synapses on hippocampal neurons in AD rats.

Neuropharmacological Research on Schizophrenia

Schizophrenia is a complex disorder affecting approximately 1% of the population worldwide. The peak for schizophrenia onset occurs in one’s twenties for both males and females. Epidemiologic evidences, together with recent linkage and association studies, have clearly demonstrated the high heritability of schizophrenia (up to 80%). Schizophrenia is characterized by positive symptoms (including delusions, hallucinations, disorganized speech) and negative symptoms (including affective flattening, impoverishment of speech and language, social withdrawal) in nature. At the same time, there are cognitive deficits including attention deficits, impairment of executive functions (planning, abstract thinking, ruling flexibility), inhibition of inappropriate actions and irrelevant sensory information, short-term and long-term memory deficits. Cognitive deficits associated with working memory and executive function are becoming increasingly recognized as the keypoint for the diagnosis and improvement of this mental disease. Great progress has been made in China as to the genetic basis of schizophrenia, and some new animal models have been established.

Etiology and Pathogenesis of Schizophrenia

It is generally acknowledged that schizophrenia has a multifactorial etiology. Recent studies have mainly focused on dopaminergic dysfunction, neurodevelopmental disorder, and genetic susceptibility. Dopaminergic dysfunction has long been proved to be the main reason for schizophrenia, although the exact role remains unclear. It is assumed that hypofunction of the cortical and prefrontal dopamine systems contributes to the negative symptoms as well as cognitive disorders, while the hyperactivity of the subcortical and limbic dopamine systems causes positive symptoms. In recent years, schizophrenia has been supposed to arise not only from the loss of neurons but also from abnormally elevated levels of synaptic pruning during development. This excessive pruning may account for the mild abnormalities in social and cognitive function before the onset of the full positive, negative, and cognitive deficits that define the disorder in adulthood. Animal models support this neurodevelopmental hypothesis by demonstrating that early insult produced some of the behavioral, morphological, and neurochemical alterations resembling those observed in patients with schizophrenia (Wong et al. 2003).

Evidence from the family, twin, and adoption studies clearly indicates that genetic susceptibility plays a key role in the etiology of schizophrenia. The interaction of multiple susceptibility genes with environmental factors yields a range of phenotypes in the schizophrenia spectrum. Recent studies have identified linkages at the following loci: 8p, 22q, 2q, 3p, 6p, 1q, 11q, 5q, 13q, and 20p. A large number of schizophrenia susceptibility genes that encode proteins implicated in the regulation of synaptic plasticity, neurotransmission, neuronal migration, cell adherence, signal transduction, energy metabolism, and neurite outgrowth have been identified. Most of these genes had been proved to play important roles in the etiology of schizophrenia in Chinese Han population, such as DTNBP1 (Li et al. 2005), NRG1 (8p21–22) (Li et al. 2006a; Yang et al. 2003c), G72/G30 (Ma et al. 2006; Wang et al. 2004), and DISC1 (1q42) (Liu et al. 2006). However, minimal evidence supported that COMT (Fan et al. 2005) and RGS4 (Guo et al. 2006) are susceptibility factors for schizophrenia in Chinese Han population. In addition, a series of genes has been discovered, which may be involved in the susceptibility to schizophrenia in Chinese Han population. For example, synapsin II (3p25) (Chen et al. 2004), GRID1 (Guo et al. 2007), GRM3 (7q21–22) (Chen et al. 2005a), GRIN2A (Tang et al. 2006), CHI3L1 (Zhao et al. 2007), DGCR14 (22q11.21) (Wang et al. 2006b), MAG (Wan et al. 2005), S100B (Liu et al. 2005b), CHL1 (Chen et al. 2005b), and IL-10 (Yu et al. 2004) genes have been reported to be associated with schizophrenia. The association of these genes with schizophrenia offered an unprecedented opportunity to understand the pathogenesis and pathophysiology of this disease.

Epidemiological studies have also revealed that severe maternal malnutrition, exposure to influenza virus, repeated psychological stress, obstetrical complications (hypoxia/ischemia), and exposure to adverse intrauterine events are possible environmental risk factors.

Potential Therapies for Schizophrenia

Antipsychotics for schizophrenia include mainly two groups: the first-generation or typical antipsychotics and the second-generation or atypical antipsychotics. Typical antipsychotics include phenothiazines and butyrophenones. This kind of drug controls the positive psychotic symptoms effectively, but may induce extrapyramidal motor deficits as well as exacerbate negative symptoms and fail to influence cognitive deficits. Most atypical antipsychotics, such as risperidone, olanzapine, quetiapine, ziprasidone, and aripiprazole, have been found to produce greater effect than the first-generation antipsychotics as far as overall antipsychotic efficacy. In particular, these drugs relieved negative symptoms dramatically, minimized extrapyramidal side effects and even improved the cognitive deficits. Atypical antipsychotics have already become the first choice for schizophrenia treatment at different levels, at different stages and also for the prevention of recurrence. The therapeutic effects of many atypical antipsychotics have been confirmed in many clinical researches (Wang et al. 2005c; Lu et al. 2007; Wei et al. 2006; Kuang et al. 2006; Cao et al. 2006). A new kind of atypical antipsychotic drug, perospirone, with the properties of both dopamine D2 receptor and serotonin receptor antagonist, has been reported to be an effective and safe drug for schizophrenia (Fang et al. 2006).

Although atypical antipsychotics are breakthroughs in controlling negative symptoms and to some extent in improving cognitive dysfunction for schizophrenia, the side effects hinder their clinical application. Clinical researches showed that risperidone, ziprasidone, and olanzapine (but not quetiapine and clozapine) induced dose-limiting extrapyramidal motor deficits except quetiapine and clozapine. Risperidone increased, while clozapine, olanzapine and quetiapine decreased the level of serum prolactin (PRL) in the patients. Chlorpromazine resulted in severe cardiac arrhythmia and weight gain. In addition, there were also some disturbances of glucose and lipid metabolism in those patients undertaking long-term treatment with risperidone, clozapine, and chlorpromazine (Jiao et al. 2006).

In addition, Chinese traditional herbal medicines have contributed a lot to the treatment of schizophrenia. It has been reported that L-stepholidine (l-SPD) showed a definite effect on both the positive and negative symptoms of schizophrenia, and the efficiency of L-SPD was superior to perphenazine (Wu et al. 2003). The meiluoniuhuangninggong tablet combined with risperidone has been reported to be effective and safe in the treatment of schizophrenia (Zhang et al. 2006). Therefore, Chinese traditional herbal medicines, given in a western biomedical context, may be beneficial for treatment of schizophrenia when combined with other antipsychotics.

New Animal Models for Schizophrenia

Animal models are important tools for the study of mechanisms underlying human diseases and in developing new drugs. In recent years, with the development of neurobiology and genetic technology, great progress has been made for schizophrenia animal models. Based on the etiological hypothesis of schizophrenia, many animal models including neurodevelopmental or neurotransmissional disruption and hereditary models for schizophrenia have been developed. The conventional schizophrenia animal models of neurotransmissional disruption through systemic injection of the NMDA receptor antagonist MK-801 or phencyclidine (PCP) were related to dopaminergic dysfunction. These animal models have been improved in different laboratories, such as using different mouse strains or regulating the concentration and the time for PCP administration (Qiao et al. 2006). Recently some schizophrenia animal models have been established in China through electrical stimulation on VTA in rats, which were associated with dopaminergic hyperactivity and simulated both the positive and negative symptoms of schizophrenia (Zhong et al. 2006b). Understanding the association between gene polymorphisms and schizophrenia may also provide potential genetic relevant models. Based on the rapid achievement in animal models, more antipsychotics will be developed in the near future.

In summary, great progress has been made in the research field of neuropharmacology in China. However, the etiology and pathophysiology of pain, drug dependence, depression, Alzheimer’s disease, and schizophrenia are very complicated. At present, there is no ideal animal model that can simulate all the pathophysiological changes for all these diseases. In addition, there are still not enough effective drugs for the therapy of these diseases. Thus it is still necessary to further explore the pathogenesis of these diseases and develop new drugs for them.