Effects of Wenyang Zhenshuai Granules on the Expression of Key Mitochondrial Autophagy Proteins in the Doxorubicin-Induced Model of H9c2 Cardiomyocyte Injury.

This study aimed to explore the effects of Wenyang Zhenshuai granules (WZG) on the morphology of cardiomyocytes, cell viability, and the expression of key mitochondrial autophagy proteins in the doxorubicin-induced model of H9c2 cardiomyocyte injury. Cardiomyocytes were cultured for 44 h and divided into 4 groups: intact control, doxorubicin-injured cells (DOX), doxorubicin-injured cells treated with WZG (DOX+WZG), and doxorubicin-injured cells treated with valsartan (DOX+valsartan; reference group). The morphology of cardiomyocytes was analyzed under an inverted microscope; cardiomyocyte survival rate was determined by MTT assay. The expression of the key mitochondrial autophagy proteins (PINK1, parkin, LC3-II, and prohibitin-2) was analyzed by Western blotting. WZG down-regulated the expression of the key mitochondrial autophagy proteins in DOX-injured cells, which may be one of the important mechanisms for regulating ventricular remodeling and cardiomyocyte apoptosis.

Prognostic values of STAT3 and HIF-1α in esophageal squamous cell carcinoma.

OBJECTIVE: To investigate the expressions of signal transduction and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1-alpha (HIF-1α) in esophageal squamous cell carcinoma (ESCC), and their potential roles in the pathology of ESCC. PATIENTS AND METHODS: Tumor tissues and clinical data of 202 ESCC patients treated in our hospital from January 2011 to June 2013 were collected. Expressions of STAT3 and HIF-1α in tumor tissues and normal esophageal tissues were detected by immunohistochemical S-P method. Correlation of STAT3 and HIF-1α with clinicopathological parameters and prognosis of ESCC were analyzed. RESULTS: STAT3 was positively expressed in 82/202 ESCC tissues, with a positive expression rate of 40.59%, and HIF-1α was positively expressed in 142/202 ESCC tissues, with a positive expression rate of 70.30%. Both STAT3 and HIF-1α were highly expressed in ESCC tissues than those normal esophageal tissues, showing statistically significant differences (p<0.05). The expression of STAT3 was positively correlated with that of HIF-1α in ESCC tissues (r=0.401, p<0.05). Overall survival (OS) and disease-free survival (DFS) of ESCC patients with positive STAT3 and HIF-1α expression were markedly worse than those with negative expression (p<0.05). STAT3 and HIF-1α were related to the infiltration depth (T stage) of ESCC (p<0.05). Univariate and multivariate analyses revealed that the expression of STAT3 was associated with OS and DFS (p<0.05) and was an independent prognostic factor for ESCC. CONCLUSIONS: High expressions of STAT3 and HIF-1α are closely related to ESCC. STAT3 is an independent prognostic risk factor for ESCC, and HIF-1α may be a poor prognostic survival factor for ESCC, both of which can be used as indicators to predict the prognosis of ESCC patients.

Involvement of Endogenous Brain-Derived Neurotrophic Factor in Hypothalamic-Pituitary-Adrenal Axis Activity.

Brain-derived neurotrophic factor (BDNF) appears to be highly involved in hypothalamic-pituitary-adrenal (HPA) axis regulation during adulthood, playing an important role in homeostasis maintenance. The present study aimed to determine the involvement of BDNF in HPA axis activity under basal and stress conditions via partial inhibition of this endogenous neurotrophin. Experiments were conducted in rats and mice with two complementary approaches: (i) BDNF knockdown with stereotaxic delivery of BDNF-specific small interfering RNA (siRNA) into the lateral ventricle of adult male rats and (ii) genetically induced knockdown (KD) of BDNF expression specifically in the central nervous system during the first ontogenesis in mice (KD mice). Delivery of siRNA in the rat brain decreased BDNF levels in the hippocampus (-31%) and hypothalamus (-35%) but not in the amygdala, frontal cortex and pituitary. In addition, siRNA induced no change of the basal HPA axis activity. BDNF siRNA rats exhibited decreased BDNF levels and concomitant altered adrenocortoctrophic hormone (ACTH) and corticosterone responses to restraint stress, suggesting the involvement of BDNF in the HPA axis adaptive response to stress. In KD mice, BDNF levels in the hippocampus and hypothalamus were decreased by 20% in heterozygous and by 60% in homozygous animals compared to wild-type littermates. Although, in heterozygous KD mice, no significant change was observed in the basal levels of plasma ACTH and corticosterone, both hormones were significantly increased in homozygous KD mice, demonstrating that robust cerebral BDNF inhibition (60%) is necessary to affect basal HPA axis activity. All of these results in both rats and mice demonstrate the involvement and importance of a robust endogenous pool of BDNF in basal HPA axis regulation and the pivotal function of de novo BDNF synthesis in the establishment of an adapted response to stress.

Application of the ERK signaling pathway inhibitor PD98059 in long-term in vivo experiments.

The aim of this study was to explore methods by which the ERK signaling pathway inhibitor PD98059 (PD) could be used in long-term in vivo experiments. Forty healthy New Zealand rabbits were randomly divided into blank control, model control, PD low-dose, PD high-dose, PD blank, dimethyl sulfoxide (DMSO) control, DMSO blank, and positive control groups. The corresponding treatments were administered to each experimental group over the course of four weeks, after which, total ERK1/2 and ERK5 protein levels, protein phosphorylation, and gene expression were measured in myocardial tissues. Treatment of rabbits with Adriamycin (doxorubicin) resulted in the significant overall differences in ERK1/2 and ERK5 phosphorylation (P < 0.05). Compared with the model control group, changes in phosphorylated ERK1/2 and phosphorylated ERK5 were lowest in the PD high-dose group (P < 0.05). No significant differences in total protein and mRNA levels of myocardial ERK1/2 and ERK5 were detected between the groups after four weeks (P > 0.05). Continuous intravenous injection of PD98059 significantly reduced phosphorylation of ERK1/2 and that of ERK5. In conclusion, Adriamycin-induced myocardiopathy and abnormal ERK signaling might constitute a valuable model foruse in long-term experiments. These methods may provide a theoretical basis for related in vivo studies of long duration.

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington's disease and controls.

BACKGROUND AND PURPOSE: Huntington's disease is due to a CAG triplet repeat elongation in the huntingtin gene. Boundaries in CAG numbers have been found between healthy people with and without risk to pass the disorder to the next generation, and between people without, with a mild, or with a fully penetrant phenotype. These data have been generated in western populations and it is not clear whether they are also valid amongst Chinese. METHODS: In order to establish normative data in the huntingtin gene for Chinese people, 966 chromosomes from normal controls were tested. Further, the range of CAG repeats was examined in a cohort from six centres and a total of 368 patients with the disease were included. RESULTS: The CAG triplet repeat range in normal controls was between 9 and 35 (mean 18.9, SD 2.57). Triplets in the range between 26 and 35 were found in 2.5%. In the patient cohort, triplet repeats in the shorter allele were between 8 and 37 (mean 17.7, SD 1.6). In the longer allele, a range between 36 and 120 was found. There was a negative correlation (-0.65, r = 0.42) between age at onset and the number of triplet repeats in the larger allele. The mean age at onset was 38 years, with a range between 2 and 70 years. In 23 patients (6%) a childhood or juvenile onset was noted. CONCLUSION: These data show comparable ranges of huntingtin gene CAG triplet repeats in normal people and in patients with Huntington's disease as in western populations.

Expression of the G72/G30 gene in transgenic mice induces behavioral changes.

The G72/G30 gene complex is a candidate gene for schizophrenia and bipolar disorder. However, G72 and G30 mRNAs are expressed at very low levels in human brain, with only rare splicing forms observed. We report here G72/G30 expression profiles and behavioral changes in a G72/G30 transgenic mouse model. A human BAC clone containing the G72/G30 genomic region was used to establish the transgenic mouse model, on which gene expression studies, western blot and behavioral tests were performed. Relative to their minimal expression in humans, G72 and G30 mRNAs were highly expressed in the transgenic mice, and had a more complex splicing pattern. The highest G72 transcript levels were found in testis, followed by cerebral cortex, with very low or undetectable levels in other tissues. No LG72 (the long putative isoform of G72) protein was detected in the transgenic mice. Whole-genome expression profiling identified 361 genes differentially expressed in transgenic mice compared with wild-type, including genes previously implicated in neurological and psychological disorders. Relative to wild-type mice, the transgenic mice exhibited fewer stereotypic movements in the open field test, higher baseline startle responses in the course of the prepulse inhibition test, and lower hedonic responses in the sucrose preference test. The transcriptome profile changes and multiple mouse behavioral effects suggest that the G72 gene may play a role in modulating behaviors relevant to psychiatric disorders.

Developmental changes in BDNF protein in the song control nuclei of zebra finches.

The zebra finch song system provides an excellent model to study the mechanisms underlying the development of sex difference in brain structure and function. Only male zebra finches sing and the brain nuclei controlling song learning and production are considerably larger than in females. Sexual differentiation may in part be regulated by estrogen, but other molecules including neurotrophic factors likely also affect masculinization. Brain derived neurotrophic factor (BDNF) plays a crucial role in numerous aspects of vertebrate brain development and function, including neurogenesis, cell survival, growth of axonal projections, synaptogenesis and processes linked to learning and memory. The current study investigated the expression of BDNF protein in juvenile males and females at four ages, as well as in adults, to begin to evaluate the potential roles of endogenous BDNF in particular stages of structural and functional development of the song system. In both HVC and the robust nucleus of the arcopallium (RA), males had more BDNF+ cells than females. The number of immunopositive cells increased in males and decreased in females as they matured, in a pattern generally consistent with a role for BDNF in sensorimotor integration of song learning. In addition, in HVC (but not RA) the ratio of mature BDNF compared to its precursor proBDNF was greater in adult males than those at post-hatching day 25, indicating a region-specific shift in the relative availability of the two forms. Collectively, the data suggest that changes in BDNF protein expression across development may be associated with song system maturation, particularly during the sensorimotor integration of masculine vocalizations.

Synthesis of peptides of Carapax Trionycis and their inhibitory effects on TGF-ß1-induced hepatic stellate cells.

We previous identified the antifibrotic active ingredients from Carapax Trionycis as two peptides. Here, we synthesized these two peptides (peptide 1 and peptide 2) by a solid phase method and examined their effects on proliferation and activation of cultured hepatic stellate cells (HSC) which are the main ECM (extracellular matrix)-producing cells in fibrosis progression. We demonstrated that peptide 1 and peptide 2 significantly reduced HSC proliferation and activation in a dose dependent manner. Further, peptide 1 and peptide 2 could interfere with TGF-signaling by down-regulating Smad 3 phosphorylation. Thus, these synthetic peptides of Carapax Trionycis could inhibit proliferation and activation of HSC and might be used as a candidate for treatment of liver fibrosis.

Discovery of selective small molecular TACE inhibitors for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, inflammatory disease that afflicts 1-2% of the world population, characterized by an immune mediated inflammatory synovitis that leads to joint destruction, functional impairment, and reduced quality of life. The treatment goals of RA should be longterm substantial relief of pain, arrested joint inflammation and damage, and improved function. Current treatment can be divided into four classes, namely general analgesics and non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease modifying anti-rheumatic drugs (DMARDs) and biological agents (tumor-necrosis factor modifiers). However, gastrointestinal (GI) side effects of NSAIDs cannot be neglected, direct joint injections of glucocorticoids cannot be injected more than once every 3 months, synthetic DMARDs is far from optimal and only minority of patients achieved longterm remission, the biologics are very expensive to manufacture, need to be injected, and can cause allergic reactions. An alternative and good approach to the treatment of this disease is to lower the levels of tumour necrosis factor-α (TNF-α) in RA, which can be achieved by selectively inhibiting the tumour necrosis factor-α converting enzyme (TACE) that generate these cytokines using cheaper small molecules. This review focuses on the current status of selective small molecule inhibitors of TACE, with respect to lead compound search, inhibitors design approach, structure-activity relationship (SAR) and pharmacological studies in animals and humans. Through these methods, new hope is emerging for the treatment of RA through selective inhibition of TACE.

Bioactive components in the fruits of Ziziphus jujuba Mill. against the inflammatory irritant action of Euphorbia plants.

Chinese jujube (also known as Chinese date) is the fruit of Ziziphus jujuba Mill. (Rhamnaceae). As a famous folk medicine, it is used as antidote in traditional Chinese formula, Shi Zao Decoction, to relieve the drastic inflammatory irritant nature of Euphorbia species. The irritant activities may cause serious adverse effects in clinical practices. This study aimed to investigate the active components of Z. jujuba through the inhibitory effects on the inflammatory cells activated by Euphorbia kansui and prostratin, a phorbol ester isolated from Euphorbia fischeriana. Peritoneal macrophage of rat and splenic lymphocyte (splenocyte) of mouse were selected to evaluate these actions in vitro. Nitric oxide (NO) release of macrophage and the proliferation of splenocyte were examined through Griess method and MTT assay. TNF-α, as an important pro-inflammatory cytokines, was detected with enzyme-linked immunosorbent assay (ELISA) method. Six fractions extracted from Z. jujuba were evaluated and fraction F (triterpene acids fraction) was demonstrated to be the most active part, and then, 21 compounds isolated from Z. jujuba were tested at the concentrations range from 1 µg/ml to 100 µg/ml. The results show that 7 compounds of them are likely to be active compounds concerning to their pronounced inhibitory action on the activated inflammatory cells. These effects might be helpful to attenuate the irritant action of Euphorbiaceae plants and protect the gastrointestinal tissue from potent inflammatory injury, which should be beneficial to some diseases, like inflammatory bowel disease.

Developmental changes in the sexually dimorphic expression of secretory carrier membrane protein 1 and its co-localisation with androgen receptor protein in the zebra finch song system.

The song system of zebra finches differs dramatically between the sexes in terms of both structure and function. Only males sing and the brain regions regulating the learning and production of this behaviour are far more developed in males than females. Mechanisms regulating sexual differentiation likely include both direct genetic and hormonal processes. Expression of both mRNA and the protein product for secretory carrier membrane protein 1 (SCAMP1), a sex chromosome gene, are increased in the brains of juvenile males compared to females. Here we investigated developmental changes in SCAMP1 containing cells in song nuclei and co-localisation with androgen receptor (AR) protein from post-hatching day 25 through adulthood. Almost all SCAMP1 cells co-expressed AR and approximately half of the AR cells expressed SCAMP1 in the HVC and robust nucleus in the arcopallium (RA) of both sexes and in the Area X of males (which could not be clearly defined in females). In HVC and RA, more single and double-labelled cells were detected in males than females overall, and the sex differences increased as animals matured. The results suggest the potential for interaction of these two proteins in regulating development of brain and/or behaviour.

New hope for the treatment of osteoarthritis through selective inhibition of MMP-13.

Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients, and is characterized by progressive loss of articular cartilage that eventually leads to a complex process involving degradation of various components of the cartilage matrix, chief among them are the cartilage-specific type II collagen (CII) and aggrecan. While the loss of aggrecan is thought to be an early and reversible process, degradation of CII is considered to be irreversible and a key step in the loss of structural and functional integrity of cartilage. Among the various matrix metalloproteinases (MMPs), MMP-13 is specifically expressed in the cartilage of human OA patients and is not present in normal adult cartilage. It is the major collagenase in OA cartilage and has the highest activity against CII. However, the clinical utility of broad-spectrum MMP inhibitors developed for treatment of OA has been restricted by dose- and duration-dependent musculoskeletal side effects in humans. Consequently, selectively inhibiting the MMP-13 would seem to be an attractive therapeutic objective. This review mainly focuses on selective MMP-13 inhibitors development in terms of OA since the late 90s, in terms of synthetic compounds of low molecular mass incorporating specific zinc-binding groups, non-zinc-binding groups. In addition, dual inhibitors of MMP-13 and aggrecanase are also reviewed. Special emphasis is placed on logistic concerns for lead compound search as well as the structure-activity relationship (SAR) in this field. Through these methods, new hope is emerging for the treatment of OA through selective inhibition of MMP-13.

Sex- and age-related differences in ribosomal proteins L17 and L37, as well as androgen receptor protein, in the song control system of zebra finches.

The zebra finch song system is sexually dimorphic--only males sing, and the morphology of forebrain regions controlling the learning and production of this song is greatly enhanced in males compared to females. Masculinization appears to involve effects of steroid hormones as well as other factors, perhaps including the expression of sex chromosome genes (males: ZZ, females: ZW). The present study investigated three proteins--two encoded by Z-linked genes, ribosomal proteins L17 and L37 (RPL17 and RPL37), including their co-localization with androgen receptor (AR), from post-hatching day 25 to adulthood. Extensive co-expression of AR with the ribosomal proteins was detected in the three song nuclei investigated (HVC, robust nucleus of the arcopallium (RA), and Area X) across these ages. In general, more cells expressed each of these proteins in males compared to females, and the sex differences increased as animals matured. Specific patterns differed across regions and between RPL17 and RPL37, which suggest potential roles of one or both of these proteins in the incorporation and/or differentiation of song system cells.

Chemical correlation between Gegen Qinlian dispensing granule and its four raw herbs by LC fingerprint.

Gegen Qinlian dispensing granule, a favorite composite formula, is a combination of Radix Puerariae Lobatae, Radix Scutellariae, Rhizoma Coptidis and Radix et Rhizoma Glycyrrhizae Praeparata cum Melle. To develop a method to overall evaluate correlation between the formula and its four raw herbs, LC fingerprints of the formula and its raw herbs were developed and LC-DAD-MS was employed to identify the components in the formula fingerprint. According to the characteristic fragmentation behavior of known flavonoids, alkaloids and saponins isolated from the four raw herbs as well as retention time, UV and MS data of detected compounds, a total of 23 constituents in the formula fingerprint were structurally characterized. Chemical correlation between the formula and the four crude herbs was evaluated qualitatively and quantitatively by the developed LC fingerprints. The results showed that 8 components in the formula fingerprint were addressed to Radix Puerariae Lobatae, 11 to Radix Scutellariae, 7 to Rhizoma Coptidis, 3 to Radix et Rhizoma Glycyrrhizae Praeparata cum Melle, and that the relative area ratios of the common peaks in the formula vary slightly in comparison with corresponding ratios in its crude herbs, demonstrating the chemical constituents in the formula have patterns similar to those in its crude herbs.

Management of attention deficit hyperactivity disorder: a parental perspective.

OBJECTIVES: There are few studies exploring parental perceptions of the diagnosis and overall treatment of their children with attention deficit hyperactivity disorder (ADHD). This community-based study was conducted to consider this important aspect of care. METHODS: A total of 7 226 (65%) parents responded to a community survey of 11 184 children aged 10-12 years living in northern Sydney in 2000, out of which 278 children with ADHD were identified. Their parents completed an anonymous questionnaire covering their perceptions relating to diagnosis, treatment and overall management. RESULTS: Only 66% of parents recalled the use of questionnaires or rating scales. There were 82% of children who had trialed medication and 66% of these were still taking it. Behavioural intervention had occurred in 42% of the children. Non-conventional treatments, most commonly elimination diet and/or fatty acid supplementation, had been used in 71% of the children. These were considered helpful in one-third of cases. A total of 55% of parents reported being either satisfied or very satisfied with their child's care. Parents were more likely to report satisfaction when their children were on medication and when reviews were held at least 6 monthly. Lack of educational support and teachers' understanding of ADHD were identified as ongoing issues. CONCLUSION: Parental responses suggested that adherence to recommended diagnostic guidelines was inadequate. Behavioural intervention was underutilized despite its documented positive role. Non-conventional therapies were widely used and considered helpful in one-third of the children who used them. Use of stimulant medication and frequent reviews were more likely to be associated with overall management satisfaction.

Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces.

To examine the in vivo function of presenilin-1 (PS1), we selectively deleted the PS1 gene in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, our postlearning enrichment experiments lead us to postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain.

Effect of transgenic overexpression of NR2B on NMDA receptor function and synaptic plasticity in visual cortex.

The NMDA receptor (NMDAR) is a heteromer comprised of NR1 and NR2 subunits. Mice that overexpress the NR2B subunit exhibit enhanced hippocampal LTP, prolonged NMDAR currents, and improved memory ( Tang et al., 1999). In the current study, we explored visual cortex plasticity and NMDAR function in NR2B overexpressing transgenic mice. Unlike the hippocampus, in vitro synaptic plasticity of the visual cortex was unaltered by NR2B overexpression. Consistent with the plasticity findings, NMDAR excitatory postsynaptic current (EPSC) durations from layer 2/3 pyramidal cells were similar in wild-type (wt) and transgenic (tg) mice. Furthermore, temporal summation of NMDAR EPSCs to 10, 20, and 40 Hz stimulation did not differ between cells from wt and tg mice. Finally, although in situ studies clearly demonstrate overexpression of NR2B mRNA in visual cortex, we failed to observe a significant elevation in the synaptic expression of NR2B protein. We conclude that the synaptic ratio of NR2B over NR2A in the NMDA receptor complex in the visual cortex is not significantly influenced by the transgene overexpression. These data suggest that mRNA availability is not a limiting factor for the synthesis of NR2B protein in the visual cortex, and support the hypothesis that levels of NR2A, rather than NR2B, normally determine the subunit composition of NMDARs in visual cortex.

Differential effects of enrichment on learning and memory function in NR2B transgenic mice.

It has been known that environmental enrichment leads to better learning and memory in mice. However, the molecular mechanisms are not known. In this study, we used the 10th-12th of the NR2B transgenic (Tg) lines, in which the NMDA receptor function is enhanced via the NR2B subunit transgene in neurons of the forebrain, to test the hypothesis of the involvement of NMDA receptor function in enrichment-induced better learning and memory. Consistent with our previous results, both larger long-term potentiation (LTP) in the hippocampus and superior learning and memory were observed in naive NR2B Tg mice even after the 10th-12th generation of breeding. After enrichment, wild-type mice exhibited overall improvement in their performances in contextual and cued conditioning, fear extinctions, and novel object recognition tasks. Interestingly, the same enrichment procedures could not further increase the performance of NR2B Tg mice in contextual conditioning, cued conditioning, or fear extinction, thereby indicating that enhanced NMDA receptor function can occlude these enrichment effects. However, we found that in the novel object recognition task enriched NR2B Tg mice exhibited much longer recognition memory (up to 1 week), compared to that (up to 3 days) in naive NR2B Tg mice. Furthermore, our biochemical experiments showed that enrichment significantly increased protein levels of GluR1, NR2B, and NR2A subunits of glutamate receptors in both wild-type and NR2B Tg mice. Therefore, our results suggest an interactive nature of molecular pathways involved in both environmental and genetic NMDA receptor manipulations for enhancing learning and memory.

[Two kaempferol triglycosides from pericarps of Sophora japonica L].

OBJECTIVE: To study the flavonol triglycosides in the pericarps of Sophora japonica. METHOD: Various chromatographic techniques were used to isolate and purify the constituents. The structures were elucidated by chemical evidence and spectral analysis, especially by 2D NMR experiments. RESULTS: Two kaempferol triglycosides were isolated and identified as kaempferol 3-O-beta-D-sophoroside-7-O-alpha-L-rhamnoside and kaempferol 3-O-(2"-O-beta-D-glucosyl)-beta-D-rutinoside. CONCLUSION: Both of them were reported in S. japonica for the first time.