How would composite traditional Chinese medicine protect the brain--an example of the composite formula "Pien Tze Huang".

Chinese medicine has a long history of several thousand years. The main form of Traditional Chinese Medicine (TCM) is composite, i.e. a mixture of up to 10 medicinal products. Thus a composite prescription of 4-5 kinds of Chinese medicinal products may contain several hundred kinds of chemical composition. The active ingredients and clinical efficacy of which are difficult to characterize. We aim to review the Chinese literature of TCMs with neuroprotective effects. We illustrate with our study on Pien Tze Huang (PZH) the use of in vivo tests in the study of composite TCM. Our results show evidence that PZH might have neuropreventive effects in rats.

Permanent deficits in brain functions caused by long-term ketamine treatment in mice.

Ketamine, an injectable anesthetic, is also a popular recreational drug used by young adults worldwide. Ketamine is a non-competitive antagonist of N-methyl-d-aspartate receptor, which plays important roles in synaptic plasticity and neuronal learning. Most previous studies have examined the immediate and short-term effects of ketamine, which include learning and cognitive deficits plus impairment of working memory, whereas little is known about the long-term effects of repeated ketamine injections of common or usual recreational doses. Therefore, we aimed to evaluate the deficits in brain functions with behavioral tests, including wire hang, hot plate and water maze tests, plus examine prefrontal cortex apoptotic markers, including Bax, Bcl-2 and caspase-3, in mice treated with 6 months of daily ketamine administration. In our study, following 6 months of ketamine injection, mice showed significant deterioration in neuromuscular strength and nociception 4 hours post-dose, but learning and working memory were not affected nor was there significant apoptosis in the prefrontal cortex. Our research revealed the important clinical finding that long-term ketamine abuse with usual recreational doses can detrimentally affect neuromuscular strength and nociception as part of measurable, stable and persistent deficits in brain function.

Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine.

Ketamine, a non-competitive antagonist at the glutamatergic N-methyl-d-aspartate (NMDA) receptor, might impair memory function of the brain. Loss of memory is also a characteristic of aging and Alzheimer's disease. Hyperphosphorylation of tau is an early event in the aging process and Alzheimer's disease. Therefore, we aimed to find out whether long-term ketmaine administration is related to hyperphosphorylation of tau or not in the brains of mice and monkeys. Results showed that after 6 months' administration of ketamine, in the prefrontal and entorhinal cortical sections of mouse and monkey brains, there were significant increases of positive sites for the hyperphosphorylated tau protein as compared to the control animals receiving no ketamine administration. Furthermore, about 15% of hyperphosphorylated tau positive cells were also positively labeled by terminal dUTP nick end labeling (TUNEL) indicating there might be a relationship between hyperphosphorylation of tau and apoptosis. Therefore, the long-term ketamine toxicity might involve neurodegenerative process similar to that of aging and/or Alzheimer's disease.

Mice are prone to kidney pathology after prolonged ketamine addiction.

ICR mice were injected with ketamine for 1, 3 and 6 months and the kidneys and urinary bladders were excised and processed for histology. Starting from 1 month, all addicted mice showed invasion of mononuclear white cells, either surrounding the glomerulus or the other tubules in the kidney. The aggregation of these cells extended all the way to the pelvis and ureter. As well, in the urinary bladder, the epithelium became thin and there was submucosal infiltration of mononuclear inflammatory cells. Silver staining revealed a loss of nerve fibers amongst the muscles of the urinary bladder of the treated. Immunohistochemistry on choline acetyltransferase which is a marker for cholinergic neurons also demonstrated a decrease of those cells. We hypothesized that prolonged ketamine addiction resulted in the animals prone to urinary infection.

The usefulness of the spontaneously hypertensive rat to model attention-deficit/hyperactivity disorder (ADHD) may be explained by the differential expression of dopamine-related genes in the brain.

Spontaneously hypertensive rats (SHR) are considered to represent a genetic animal model for attention-deficit hyperactivity disorder (ADHD). In the present studies, we compared the locomotor activity, learning and memory functions of juvenile male SHR, with age- and gender-matched genetic control Wistar-Kyoto rats (WKY). In addition, we investigated potential differences in brain morphology by magnetic resonance imaging (MRI). In other complimentary studies of the central nervous system, we used real-time PCR to examine the levels of several dopaminergic-related genes, including those coding for the five major subtypes of dopamine receptor (D1, D2, D3, D4 and D5), those coding for enzymes responsible for synthesizing tyrosine hydroxylase and dopamine-beta-hydroxylase, and those coding for the dopamine transporter. Our data revealed that SHR were more active than WKY in the open field (OF) test. Also, SHR appeared less attentive, exhibiting inhibition deficit, but in the absence of memory deficits relative to spatial learning. The MRI studies revealed that SHR had a significantly smaller vermis cerebelli and caudate-putamen (CPu), and there was also a significantly lower level of dopamine D4 receptor gene expression and protein synthesis in the prefrontal cortex (PFC) of SHR. However, there were no significant differences between the expression of other dopaminergic-related genes in the midbrain, prefrontal cortex, temporal cortex, striatum, or amygdala of SHR and WKY. The data are similar to the situation seen in ADHD patients, relative to normal volunteers, and it is possible that the hypo-dopaminergic state involves a down regulation of dopamine D4 receptors, rather than a general down-regulation of catecholamine synthesis. In conclusion, the molecular and behavioural data that we obtained provide new information that may be relevant to understanding ADHD in man.

The effects of dexamethasone and dehydroepiandrosterone (DHEA) on cytokines and receptor expression in a human osteoblastic cell line: potential steroid-sparing role for DHEA.

Osteoporosis and associated fractures are the most common and debilitating complication of glucocorticoid use. The use of alternative anti-inflammatory agents without the deleterious skeletal effects of glucocorticoids is needed. Dehydroepiandrosterone (DHEA) may have immunomodulatory as well as positive effects on bone. For our further understanding of the mechanisms of action of DHEA, as a steroid-sparing agent, we investigated and compared the effects of dexamethasone (DEX) and DHEA on the regulation of the downstream effector pathway of osteoclastogenesis; RANKL/OPG and a range of inflammatory/pro-resorbing cytokines and receptors using a human clonal osteoblastic cell line. The cells were treated with DEX, DHEA, and androstenedione (ANDI). The mRNA expression of RANKL and OPG was determined by real-time PCR after overnight incubation. The regulation of a broad spectrum of cytokines by DEX and DHEA was also investigated using a human cytokine/growth factor and receptor gene array consisting of 268 cytokine-related cDNAs. To confirm some of the gene expression changes, protein production was measured by ELISA. RANKL expression and RANKL/OPG ratio were increased by DEX. This effect was reversed by co-treatment with both DHEA or ANDI. Several pro-inflammatory/resorptive cytokines including IL-6, IL-4, IFN-gamma, macrophage inhibitory factor (MIF) were down-regulated not only by DEX but also by DHEA. In contrast to DEX, DHEA did not lead to suppression of growth factors including vascular endothelial growth factor (VEGF), fibroblast growth factor-5 (FGF-5), insulin-like growth factor-binding protein3 (IGF-BP3). Several new target genes previously documented to influence bone formation were up-regulated by DHEA such as Notch 2, insulin receptor, thrombin receptor (PAR1). The data suggest that DHEA has immunomodulatory properties without the catabolic effects on bone remodeling, observed with glucocorticoid use. DHEA may thus prove useful as a steroid-sparing agent in the management of inflammatory disorders such as SLE or rheumatoid arthritis. Further in vivo studies are indicated.

Effects of Ginkgolide on the development of NOS and AChE positive neurons in the embryonic basal forebrain.

Extract of Ginkgo biloba (EGb) has been therapeutically used for several decades to increase peripheral and cerebral blood flow so as to prevent cardiovascular and neurovascular diseases. However, the role of EGb in neuroprotective effects has received much attention recently. In this study, we investigated the effect of EGb on the development of NOS and AChE positive neurons in the rat embryonic basal forebrain. The results showed that treated with EGb, the OD of MTT staining analysis, and the numbers, the cell sizes and circumferences of NOS and AChE positive neurons were greatly promoted. These data suggest that EGb had similar effects of the neurotrophins such as NGF and BDNF in promoting the development of NOS and AChE positive neurons in the rat embryonic basal forebrain.

Changes in RANKL/OPG/RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene.

The RANKL/OPG/RANK pathway is the key mediator of osteoclastogenesis. Mononuclear cells may be implicated in post-menopausal osteoporosis. The effect of estrogen or raloxifene on bone resorption and the expression of RANKL/OPG/RANK in peripheral blood mononuclear cells (PBMCs) was examined. Twenty-nine women with post-menopausal osteoporosis were treated with estrogen (HRT) or raloxifene for 12 months. Bone mineral density (BMD) was measured at baseline and at 12 months at the spine and hip. Serum C-terminal telopeptide (CTX) and OPG were measured at baseline and at 1, 3, 6 and 12 months. PBMCs were isolated from 17 women and changes in RANKL, OPG and RANK mRNA were determined. The effects of estrogen or raloxifene in PBMCs in vitro were also assessed. BMD increased following treatment (lumbar spine % change mean [S.E.M.]: 4.3% [0.9], p<0.001). Serum CTX decreased (6 months: -43.7% [6.0], p<0.0001). Serum OPG declined gradually (12 months: -26.4% [4.4], p<0.001). RANKL, OPG and RANK gene expression decreased (6 months: RANKL 50.0% [24.8] p<0.001, OPG: 21.7% [28] p<0.001, RANK: 76.6% [10.2] p=0.015). Changes in OPG mRNA correlated with changes in BMD (r=-0.53, p=0.027) and CTX (r=0.7, p=0.0044). Down-regulation in RANKL, OPG, RANK mRNA and reduction in bone resorption was also seen in vitro. These results suggest that the expression of RANKL/OPG/RANK in PBMCs are responsive to the slowing in bone turnover/remodeling associated with treatment with estrogen or raloxifene. Further confirmatory studies are needed.

Genetic influences on female infidelity and number of sexual partners in humans: a linkage and association study of the role of the vasopressin receptor gene (AVPR1A).

In humans, in contrast to animals, the genetic influences on infidelity are unclear. We report here a large study of over 1600 unselected United Kingdom female twin pairs who confidentially reported previous episodes of infidelity and total lifetime number of sexual partners, as well as attitudes towards infidelity. Our findings demonstrate that infidelity and number of sexual partners are both under moderate genetic influence (41% and 38% heritable, respectively) and the genetic correlation between these two traits is strong (47%). Conversely, attitudes towards infidelity are driven by shared and unique environmental, but not genetic, influences. A genome-wide linkage scan identified three suggestive but nonsignificant linkage areas associated with infidelity and number of sexual partners on chromosomes 3, 7 and 20 with a maximum LOD score of 2.46. We were unsuccessful in associating infidelity or number of sexual partners with a locus implicated in other mammals' sexual behavior, the vasopressin receptor gene. Nonetheless, our findings on the heritability of sexual infidelity and number of sexual partners provide support for certain evolutionary theories of human sexual behavior, as well as justifying further genetic and molecular research in this domain.

Polymorphisms in the P450 c17 (17-hydroxylase/17,20-Lyase) and P450 c19 (aromatase) genes: association with serum sex steroid concentrations and bone mineral density in postmenopausal women.

The CYP 17 and CYP 19 genes encode 17alpha-hydroxylase/17,20-lyase and aromatase, respectively, both involved in sex hormone synthesis. We investigated the association between 2 common polymorphisms in 1) the promoter region (T-->C substitution) of CYP 17, and 2) exon 3 (G-->A) of CYP 19, bone mineral density (BMD) and serum androgen/estradiol, in a case-control study of 252 postmenopausal women aged 64.5 +/- 9.2 yr (mean +/- SD). There was no significant difference in serum estradiol concentrations between cases (n = 136) and controls (n = 116). The CYP 19 genotype was significantly associated with serum estradiol (P = 0.002). Women with the AA genotype had higher serum estradiol concentrations compared with those with the GG genotype (P = 0.03). In older women, those with the CYP 19 GA and GG genotypes had an increased prevalence of osteoporosis (P = 0.04) and fractures (P = 0.003). We found no significant association between CYP 17 genotype and serum androgens and estradiol concentrations. However, a significant association was seen between BMD values at the femoral neck with CYP 17 genotype in cases (P = 0.04) and in the whole study population (P = 0.012). Subjects with the CC genotype had significantly lower BMD (mean +/- SD: TT, 0.7 +/- 0.16; CC, 0.6 +/- 0.08 g/cm(2); P = 0.006). In conclusion, both CYP 17 and CYP 19 are candidate genes for osteoporosis in postmenopausal women.

Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17-beta oestradiol and raloxifene.

Oestrogen inhibits bone resorption, at least in part, by regulating the production of several cytokines, including interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) by cells of the osteoblastic lineage. The selective oestrogen receptor modulator raloxifene (RAL) acts on bone in a similar manner to oestrogen, although the mechanisms of action of RAL on osteoblasts still remain unclear. We investigated and compared the effects of 17-beta oestradiol (E(2)) and RAL on the regulation of IL-6, IL-1, RANKL and OPG in vitro in primary human osteoblastic (HOB) cells and in an immortalised clonal human bone marrow stromal cell line (HCC1) with osteoblastic characteristics. We tested E(2) and RAL at concentrations ranging from 10(-12) to 10(-6) M. IL-6, IL-1alpha and IL-1beta, OPG and RANKL were measured by ELISA. RANKL and OPG mRNA steady state level was assessed by quantitative PCR analysis. Both E(2) and RAL led to a significant reduction in IL-6 production in the HOB cells, although the effect was more marked with E(2) (P<0.05). IL-1alpha and IL-1beta also decreased significantly following treatment with E(2) and RAL in the HCC1 cells (E(2) (10(-8), 10(-7) and 10(-6) M), % reduction (means+/-S.E.M.) compared with vehicle-treated cells - IL-1alpha: 84+/-7.4, 70.8+/-2.9*, 78.2+/-4.8*; IL-1beta: 79+/-10, 72.8+/-8.2*, 66.6+/-2.8*; RAL (10(-8), 10(-7) and 10(-6) M) - IL-1alpha: 72.4+/-5*, 79+/- 5.2*, 102+/-7.7; IL-1beta: 67.9+/-3.2*, 69+/-2.5*, 73.8+/- 6.2*; *P<0.05). OPG protein concentration decreased significantly in a dose-dependent manner following treatment with E(2) and RAL (% reduction E(2) (10(-8), 10(-7) and 10(-6) M) - HOB: 72.5+/-8.4*, 80+/-6.7*, 62.8+/-8.9*; HCC1: 109+/-4, 98.8+/-6, 54.5+/-3.4*; RAL (10(-8), 10(-7) and 10(-6) M) - HOB: 81.5+/-5.5*, 62.7+/-7.4*, 55.2+/-10.9*; HCC1: 92.7+/-7.4, 67+/-12.2*, 39+/-4.5*; *P<0.05). In the HCC1 cells, RANKL protein did not change significantly following E(2). In contrast, a significant reduction in RANKL was seen with RAL at 10(-7) and 10(-6) M (66+/-6.4% and 74+/-3% respectively). There was no change in OPG mRNA expression following E(2) or RAL in the HCC1 cells, although in the HOB cells we observed a significant reduction in OPG mRNA. RANKL mRNA decreased significantly in the HCC1 cells following RAL (10(-8), 10(-7)and 10(-6) M) treatment (% change from controls: 52+/-2*, 62+/-1*, 53+/-5.8*; *P<0.05). Similar results were seen in the HOB cells with RAL at 10(-6) M (RANKL mRNA: 72+/-5.5, P<0.05). In addition, there was a significant decrease in the RANKL/OPG ratio after RAL at 10(-6) M (HOB: 65.6+/-5*, HCC1: 56.9+/-20*; *P<0.05). RANKL/OPG ratio did not change significantly in the HCC1 cells following E(2). However, in contrast to RAL, we observed an increase in the RANKL/OPG ratio in the HOB cells following treatment with E(2). In conclusion, the study shows that RAL and E(2) have divergent cell-specific effects on the regulation of cytokines. The data also suggest that, in contrast to E(2), RAL may exert its anti-resorptive actions, at least in part, via the RANKL/OPG pathway. Further in vivo studies are required to confirm this.

Linkage and association for bone mineral density and heel ultrasound measurements with a simple tandem repeat polymorphism near the osteocalcin gene in female dizygotic twins.

In this confirmatory candidate gene study, we investigated possible linkage and association for bone density, heel ultrasound and bone turnover with the osteocalcin gene using the nearby (50-180kb) microsatellite marker D1S3737. Non-identical twin sisters aged 18-75 years at first interview were recruited for the study from the St Thomas' UK Adult Twin Registry with 1366 women being genotyped for marker D1S3737. Linkage, allelic association and joint linkage and association tests were carried out using quantitative transmission disequilibrium tests (QTDT), along with post-hoc multivariate tests of linkage and association. Phenotypes tested were bone mineral density (BMD) at the spine, left forearm and left total hip; quantitative ultrasound measurements of the heel including velocity of ultrasound (VOS) and broadband ultrasound attenuation (BUA); and bone turnover markers, urine deoxypyridinoline (DPD), serum osteocalcin, bone specific and total alkaline phosphatase (ALP). BMD and ultrasound variables showed evidence of pleiotropic linkage ( p = 0.05) and association ( p = 0.02) with the marker in postmenopausal women. Bone markers showed little or no evidence of linkage and association for any age group. Evidence for pleiotropic linkage appeared to be strongest for BUA and spine BMD in postmenopausal women. The univariate test statistic for BUA was chi(2)(1)=12.8 ( p = 0.0003), equivalent to a LOD score of 2.8. DPD showed borderline evidence of linkage to the marker for women of all ages. Multivariate model-fitting showed allele 10 to be negatively associated with BMD, VOS and BUA via a common pathway, suggesting the putative functional polymorphism affects both bone content and structure through shared underlying metabolic pathways. It is likely that the alleles are in linkage disequilibrium with functional polymorphism(s) in or nearby the osteocalcin gene, which may contribute to the onset of osteoporosis.

Mutations in the low-density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients.

It has been reported that in China, patients with heterozygous familial hypercholesterolemia (FH) may go unrecognized because they do not have xanthomata or premature coronary heart disease and their LDL cholesterol levels are lower than those in their Western counterparts. However, in the Chinese patients in Hong Kong, heterozygous FH appears to manifest in a way similar to that seen in Western countries or Japan. We studied sequence variations in the promoter and coding regions of the 18 exons of the LDL receptor gene in 30 Chinese FH patients. Eighteen mutations were identified in 21 patients scattered in the promoter and 10 exons. Eleven of them were first found in this study. We also found 6 polymorphisms with allelic frequencies different from those in whites but similar to the Japanese, indicating some isolation between white and Oriental populations. A total of 29 mutations in the LDL receptor gene are now known in the Chinese. There is no definite common mutation due to a founder effect. Meanwhile, there were no detectable LDL receptor gene mutations in 9 clinically diagnosed FH patients in whom the R3500Q mutation in apolipoprotein B had also been excluded. The gene defects leading to the FH phenotype in these patients may occur somewhere else in the apolipoprotein B or other related genes, or even in the noncoding sequences of the LDL receptor gene.

Low density lipoprotein receptor related protein gene exon 3 polymorphism association with Alzheimer's disease in Chinese.

Since apolipoprotein E4 (apoE4) is the major genetic risk for late onset Alzheimer's disease (AD), proteins that interact with apoE might be involved in AD pathogenesis. Low density lipoprotein receptor related protein (LRP) is an apoE receptor in the brain. In exon 3 of the LRP gene a polymorphism was found to be underrepresented in AD compared to normal Caucasian subjects (N). We examined this polymorphism in Chinese AD and N subjects. The polymorphism frequency in N was roughly half that reported for Caucasians. Compared to N, the frequency was significantly decreased in pathologically diagnosed, but not in clinically diagnosed AD patients. Thus, the role of the LRP exon 3 polymorphism in AD has now been demonstrated in two ethnic groups, suggesting the importance of LRP in AD pathogenesis.

No association detected between very-low-density lipoprotein receptor (VLDL-R) and late-onset Alzheimer's disease in Hong Kong Chinese.

The epsilon4 allele of apolipoprotein E (ApoE) is a risk factor in late-onset Alzheimer's disease (AD). As a receptor for ApoE, very-low-density lipoprotein receptor (VLDLR) might be involved in AD pathogenesis. A Japanese study [Okuizimi, K., et al., Nature Genet., 11 (1995) 207-209] has shown an increased 5 and decreased 8 CGG-repeat allele frequency in the 5' untranslated region of VLDLR in Japanese AD versus normal controls (N). Subsequent studies in Caucasian Americans failed to duplicate the result. We examined this polymorphism in pathologically- or clinically-diagnosed Chinese late-onset AD. Our data did not show a significant increase in the 5 CGG-repeat in AD, thus suggesting no association to VLDLR. However, our data did show that the allele frequencies for each CGG-repeat were similar in both Chinese and Japanese.

Biochemical investigation of young hospitalized Chinese children: results over a 7-year period.

During the seven years from January 1989 to December 1995, we investigated 2,269 Chinese infants and young children for metabolic disorders in Hong Kong. These young patients, all aged under 4 years and originated from southern China, were ill with no apparent cause and had clinical manifestations suggestive of inherited metabolic diseases. A spot urine and a plasma sample were obtained from each patient for biochemical analysis, including urinary organic acid identification and plasma amino acid analysis. Six cases of mucopolysaccharidosis, four multiple carboxylase deficiency, three 2-methylacetoacetyl CoA thiolase deficiency, two methymalonic aciduria, one glutaric aciduria type I, one glutaric aciduria type II, one a-oxoglutaric aciduria, and one case of orotic aciduria were detected. There were also single suspected cases of medium-chain acyl-CoA dehydrogenase deficiency and isovaleric aciduria. No primary amino acid disorder, such as phenylketouria and maple syrup urine disease, has been detected. Our results suggest that a different pattern of inherited metabolic diseases exists in the southern Chinese when compared with the Chinese in other regions of China.

Apolipoprotein E genotype and Alzheimer's disease in Hong Kong elderly Chinese.

We studied the apolipoprotein E (apoE) allele frequencies in 65 Chinese patients with late-onset Alzheimer's disease (AD) and 82 age- and sex-matched controls. The apoE epsilon 4 allele frequency was significantly higher in the AD group than in the control group (0.169 versus, p < 0.01). There were five homozygotes for epsilon 4 in the AD group but none among the controls. The odds ratio for AD was 1.6 for epsilon 4 heterozygotes. The age at onset was lower in the presence of the epsilon 4 allele and higher with the epsilon 2 allele, although neither of these differences reached statistical significance. The association between apoE alleles and AD previously reported in Caucasian populations was also present in this reports of lower prevalence of AD compared with the prevalence of multi-infarct dementia.

Nutritional status of elderly patients during recovery from chest infection and the role of nutritional supplementation assessed by a prospective randomized single-blind trial.

We examined changes in the nutritional status of elderly patients with chest infection for a period of 3 months after discharge from hospital, including the effects of nutritional supplementation on well-being and functional status as well as on nutritional indices. Eighty-one subjects admitted to an acute medical ward aged 65 years and over with chest infection were recruited consecutively, and randomized to receive supplement (500 ml of Ensure liquid daily) for 1 month, or no supplement, on discharge. Assessment at baseline, 1, 2 and 3 months included a questionnaire to determine health, mental and functional status, and anthropometric measurements. Biochemical nutritional status was assessed at baseline, 1 and 3 months, and dietary intake (24 h recall method) at 1 and 3 months. During recovery, both supplement and non-supplement groups showed improvement in various measures of well-being and biochemical status. In addition, the former group showed improvement in more anthropometric measurements, in thiamine and pyridoxine status, while the non-supplement group showed a lower level of functional ability after 3 months. Various measures of well-being and biochemical status of the water-soluble vitamins were better in the supplement groups. We conclude that nutritional supplementation may have a role in helping elderly patients to recover from chest infections.