[Screening of Active Fractions from Huanglian Jiedu Decoction against Primary Neuron Injury after Oxygen-Glucose Deprivation].

OBJECTIVE: To observe the protective effect of active fractions of Huanglian Jiedu Decoction (HJD) on primary cortical neuron injury after oxygen-glucose deprivation (OGD)/reperfusion (R) injury. Methods Using macroporous resin method, HJDFE30, HJDFE50, HJDFE75, and HJDFE95 with 30%, 50%, 75%, and 95% alcohol were respectively prepared. Then the content of active components in different HJD fractions was determined with reverse phase high-performance liquid chromatography (RP-HPLC). The OGD/R injury model was induced by sodium dithionite on primary cortical neurons in neonate rats. MTT assay was used to observe the effect of four fractions (HJDFE30, HJDFE50, HJDFE75, and HJDFE95) and seven index components of HJD on the neuron viability. RESULTS: RP-HPLC showed active component(s) contained in HJDFE30 was geniposide; baicalin, palmatine, berberine, and wogonside contained in HJDFE50; baicalin, berberine, baicalein, and wogonin contained in HJDFE75. The neuron viability was decreased after OGD for 20 min and reperfusion for 1 h, (P <0. 01), and significantly increased after administered with HJD, HJDFE30, HJDFE50, and HJDFE75 (P <0. 05, P <0. 01). Geniposide, baicalin, baicalein, palmatine, wogonside, and wogonin could increase the cortical neuron viability (P <0. 05, P <0. 01). CONCLUSIONS: HJDFE30, HJDFE50, and HJDFE75, as active fractions of HJD, had protective effect on primary cortical neuron injury after OGD/R. Furthermore, geniposide, baicalin, and baicalein were main active components of HJD.

Human papillomavirus prevalence and type-distribution among women in Zhejiang Province, Southeast China: a cross-sectional study.

BACKGROUND: Human papillomavirus (HPV) infection is the main etiological factor for cervical cancer and premalignant lesions of the cervix. The purposes of the present study were to determine the prevalence of type-specific HPV infections and the association of different HPV types with cervical dysplasia among women in Zhejiang province, Southeast China. METHODS: A total of 15,267 women presenting to a gynaecological outpatient clinic were enrolled in this study. Women were screened for HPV in addition to routine cervical cytology testing. Microarray hybridization and liquid-based cytology tests were used to detect HPV genotypes and cervical cytology, respectively. RESULTS: Based on the population attending a gynaecological outpatient clinic, overall prevalence of any 23 HPV type was 22.8% and multiple HPV infection was found in 4.0% of all the outpatients. HPV prevalence showed bimodal age distribution, with a peak (55.7%) at the ≤20 age group and a second one (35.5%) at >60 age group. In total samples, the five most frequent types were HPV 16 (4.4%), 58 (2.9%), 52 (2.7%), 33 (2.2%) and 11 (1.9%). Overall HPV prevalence increased with the severity of the cytologic result. Analysis through crude odds ratios (ORs) revealed that the cervical lesion risk of HPV-infected women increased to about 26-fold of uninfected women (OR 26.1, 95% CI 22.4 to 30.3). The five most risky HPV types associated with abnormal cytology were HPV 73, 16, 82, 45 and 51. CONCLUSIONS: This study provided baseline data on HPV prevalence in women attending a gynecological outpatient clinic in Zhejiang province. Our data will supply guidance for the primary screening and vaccination program for cervical cancer in this area.

[Influence of object material and inter-trial interval on novel object recognition test in mice].

OBJECTIVE: To investigate the efficacy of novel object recognition (NOR) test in assessment of learning and memory ability in ICR mice in different experimental conditions. METHODS: One hundred and thirty male ICR mice were randomly divided into 10 groups: 4 groups for different inter-trial intervals (ITI: 10 min, 90 min, 4 h, 24 h), 4 groups for different object materials (wood-wood, plastic-plastic, plastic-wood, wood-plastic) and 2 groups for repeated test (measured once a day or every 3 days, totally three times in each group). The locomotor tracks in the open field were recorded. The amount of time spent exploring the novel and familiar objects, the discrimination ratio (DR) and the discrimination index (DI) were analyzed. RESULTS: Compared with familiar object, DR and DI of novel object were both increased at ITI of 10 min and 90 min (P<0.01). Exploring time, DR and DI were greatly influenced by different object materials. DR and DI remained stable by using identical object material. NOR test could be done repeatedly in the same batch of mice. CONCLUSION: NOR test can be used to assess the learning and memory ability in mice at shorter ITI and with identical material. It can be done repeatedly.

[Protective effect of intranasal cilostazol administration on chronic injury after cerebral ischemia in mice].

OBJECTIVE: To investigate the protective effect of cilostazol administrated intranasally on chronic injury after focal cerebral ischemia in mice. METHODS: Focal cerebral ischemia in mice was induced by middle cerebral artery occlusion (MCAO). Cilostazol was administrated intranasally or intraperitoneally 1 h, 4 h and 7 h after the operation; then twice a day from the second day for 2 weeks. The neurological deficit scoring and the inclined board testing were performed within 35 d after ischemia. The survival rate, infarct volume and neuron density were assessed 35 d after ischemia. RESULT: Intranasal cilostazol at 0.3 mg/kg increased the survival rate. Intranasal cilostazol (0.3 mg/kg, 1 mg/kg) and intraperitoneal cilostazol (10 mg/kg) significantly attenuated neurological deficit, reduced infarct volume, and increased the survival neuron density in the border of ischemia region. CONCLUSION: Cilostazol administered intranasally demonstrates protective effects on chronic cerebral ischemia in mice.

GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

* Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

[Protective effects of Huanglian-Jiedu-Tang on chronic brain injury after focal cerebral ischemia in mice].

OBJECTIVE: To investigate the neuroprotective effects of Chinese herb medicine Huanglian-Jiedu-Tang (HJDT) on chronic brain injury after focal cerebral ischemia in mice. METHODS: Focal cerebral ischemia was induced by occlusion of right middle cerebral artery (MCA) for 15 min. HJDT (at dosage of 2 g/kg or 4 g/kg, qd, orally) was administered for 21 d from d 7 before ischemia until d 14 after ischemia. The sham and ischemic controls were administered with normal saline orally. The neurological deficit scoring and the inclined board testing were performed within 35 d after ischemia. The survival rate, the infarct volume and the neuron density were assessed 35 d after ischemia. RESULT: HJDT increased the survival rate at dose of 4 g/kg; significantly reduced the neurological deficits, infarct volume and cerebral atrophy at doses of 2 and 4 g/kg after ischemia; and significantly increased the neuron density in the ischemic hippocampal CA1 region, striatum and cortex at dose of 4 g/kg but only increase the density in hippocampal CA1 region at dose of 2 g/kg. CONCLUSION: Chinese herb medicine HJDT has neuroprotective effects on chronic brain injury after focal cerebral ischemia in mice.

Dendrobium polysaccharides attenuate cognitive impairment in senescence-accelerated mouse prone 8 mice via modulation of microglial activation.

Dendrobium is one of the most important traditional Chinese medicinal foods used to treat age-related disorders. However, it remains unclear whether Dendrobium affects the progression of Alzheimer's disease (AD). In the present study, we investigated the effects of Dendrobium officinale polysaccharides (DOP) on the BV2 microglial cell line and the senescence-accelerated mouse prone 8 (SAMP8) mouse strain. In vitro experiments showed that DOP pretreatment contributed to BV2 cells shifting from proinflammatory to anti-inflammatory phenotypes with enhanced Aß clearance in response to Aß insults. For the in vivo study, mice were chronically treated with DOP in drinking water from 4 to 7 months of age. The results showed that DOP remarkably attenuated cognitive decline in SAMP8 mice. DOP also inhibited the increased hippocampal microglial activation in SAMP8 mice with downregulation of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), while interleukin-10 (IL-10), neprilysin (NEP) and insulin-degrading enzyme (IDE) were upregulated. The accumulation of hippocampal Aß42 and phosphated Tau proteins in SAMP8 mice was also reduced. Taken together, our data suggest that Dendrobium has the potential to provide neuroprotection against AD-related cognitive impairment via modulation of microglial activation.

Cilostazol, a phosphodiesterase 3 inhibitor, protects mice against acute and late ischemic brain injuries.

Cilostazol, a selective inhibitor of phosphodiesterase 3, exerts neuroprotective effects on acute brain injury after cerebral ischemia in rats. However, it is unknown whether cilostazol affects the subacute or chronic ischemic injury. In the present study, we evaluated the dose- and time-dependent effects of cilostazol on acute ischemic brain injury and the long-lasting effect on the late (subacute/chronic) injury in mice with focal cerebral ischemia induced by transient middle cerebral artery occlusion. We found that pre-treatment of cilostazol (injected i.p. at 30 min before ischemia) significantly ameliorated the acute injury 24 h after ischemia, and the effective doses were 3-10 mg/kg. The post-treatment of cilostazol (10 mg/kg) was effective on the acute injury when it was injected 1 and 2 h after ischemia. In addition, for the late injury, post-treatment of cilostazol (10 mg/kg, i.p., for 7 consecutive days after ischemia) attenuated neurological dysfunctions, brain atrophy and infarct volume. It also inhibited astrocyte proliferation/glial scar formation and accelerated the angiogenesis in the ischemic boundary zone 7 and 28 days after ischemia. Thus, we conclude that cilostazol protects against not only the acute injury, but also the late injury in mice with focal cerebral ischemia; especially it can modify brain remodeling, astrogliosis and angiogenesis.

Huanglian-Jie-Du-Tang Extract Ameliorates Depression-Like Behaviors through BDNF-TrkB-CREB Pathway in Rats with Chronic Unpredictable Stress.

Neuroinflammation is considered as one of the common pathogeneses of depression. Huanglian-Jie-Du-Tang (HJDT) is a traditional Chinese herbal formula. The present study investigates the antidepressant-like effect of HJDT and its possible mechanism in rats. Rats were given HJDT (2, 4, and 8 g/kg, intragastrically), paroxetine (1.8 mg/kg, intragastrically), or an equivalent volume of saline for 42 days. The depression-related behaviors, including sucrose preference test (SPT), open field test (OFT), novel objective recognition task (NORT), and forced swimming test (FST), were detected. 5-Hydroxytryptamine (5-HT) and dopamine (DA) contents, microglial activation, proinflammatory cytokines, and brain derived neurotrophic factor (BDNF), tropomyosin receptor kinases B (TrkB), and cAMP-responsive element binding protein (CREB) expression were investigated. The results indicated HJDT (2 and 4 g/kg) dramatically ameliorated the depression-like behaviors. Also HJDT decreased the number of microglia and the proinflammatory cytokines in hippocampus. Western-blotting analysis displayed HJDT upregulated BDNF, TrkB, and pCREB/CREB expression in hippocampus. Particularly, pCREB DNA activity enhanced with HJDT treatment in hippocampus. But there was no difference in the 5-HT and DA contents with HJDT treatment. In conclusion, it was supposed that HJDT might be a potential Chinese medicine decoction for treating or alleviating complex symptoms of depression through BDNF-TrkB-CREB pathway.

Focal cerebral ischemia alters the spatio-temporal properties, but not the amount of activity in mice.

Cerebral ischemia induces sensorimotor and cognitive dysfunctions in rodents; however, little is known about the changes in the spatio-temporal organization of locomotor activity after ischemia. In this study, we continuously assessed the spatio-temporal properties of locomotor activity in an enclosure (40 cm x 40 cm x 65 cm, arbitrarily divided into 16 zones) with feeding and drinking supplies, and observed the spatio-temporal changes in mice with focal cerebral ischemia. Locomotor tracks were recorded from 3rd to 24th h (total 22 h) after middle cerebral artery occlusion (MCAO) or sham operation. The absolute and relative distance traveled or time spent in different regions was analyzed. We found that there was no significant difference in total traveled distances over 22 h between the two groups. Control mice moved and stayed primarily in feeding and drinking zones, frequently in peripheral but rarely in central zones. However, ischemic mice lost such a property, almost evenly moved and stayed in 16 zones. Mice in both groups were more active (traveled more distances) shortly after they entered the enclosure, while ischemic mice returned to stable levels slower. The traveled distance had a remarkable circadian variation with more locomotion in the night in control mice, but not in ischemic mice. Most of the spatial parameters (ratios) of locomotor activity were closely correlated with the ischemic infarction, neuron densities (in cortex, hippocampal CA1 region and striatum), and typical behavioral assessments (neurological scores and inclined board test). Thus, these findings indicate that focal cerebral ischemia does not alter the amount of locomotor activity in mice, but impairs the spatio-temporal properties-prolonging the initial hyperactivity and losing regionally special distribution of the activity.

Intranasal recombinant human erythropoietin protects rats against focal cerebral ischemia.

Erythropoietin (EPO) is a hematopoietic growth factor with tissue-protective properties, and can protect animals from cerebral ischemic injury. However, the central nervous effects of EPO as a glycoprotein is limited by the potential complication resulted from its erythropoietic activity and the problem of the penetration through blood-brain barrier (BBB). To avoid these limitations, in this study we administered recombinant human EPO (rhEPO) intranasally (i.n.) to evaluate its neuroprotective effect in the rats with focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). We found that rhEPO i.n. at doses of 4.8, 12 and 24 U (administered 10 min after MCAO and 1h after reperfusion) reduced infarct volume, brain swelling and cell damage in the ischemic hemispheres, and improved behavioral dysfunction 24 h after cerebral ischemia. Intraperitoneal rhEPO (5000 U/kg) also showed the protective effect, but the heat-inactivated rhEPO did not show any effect. Thus, intranasal administration of relatively small doses of rhEPO protects rats from acute injury after focal cerebral ischemia, suggesting that intranasal rhEPO may be a more effective and safer administration route for treatments of ischemic or other brain diseases.

[An improved quantitative method for evaluating neurological deficits in mice with focal cerebral ischemia].

The purpose of this study was to develop a quantitative and objective method for evaluating neurological deficits in mice with focal cerebral ischemia. After middle cerebral artery occlusion (MCAO), the neurological deficits were evaluated 24 h later. We measured the mean angles, dominant angles and turns in a hanged test in which the mice were sticked on the wall, and the holding angles in an inclined plane test as well, Then we determined the cerebral infarct volumes, neuron density in hippocampus, cortex and subcortical areas 24 h after MCAO. The correlations among infarct volume, neuron density and neurological deficits were analyzed. We also compared the quantitative method with two typical complex methods of behavioral assessment. The effect of [pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate] (ONO-1078), a neuroprotective agent, on ischemic injury was observed using this method. We found that the variables measured by both quantitative and typical behavioral methods significantly changed in the ischemic mice, and correlated with the infarct volumes and neuron densities. The quantitative variables well correlated with those of typical behavioral assessment, too. ONO-1078 inhibited ischemic injury and reduced the total scores of quantitative assessment. Thus, the quantitative method we developed is useful in evaluating neurological deficits of focal cerebral ischemia with the advantages of objectivity, quantification, simplicity and non-invasion, and can be used in the evaluation of neuroprotective effects of drugs.

[Light transmission measurement of focal ischemic cerebral infarction in mice]

OBJECTIVE: To evaluate the feasibility of light transmission to measure focal cerebral ischemia in mice. METHODS: Persistent focal cerebral ischemia was induced by middle cerebral artey occlusion (MCAO) in mice. The brain were removed 24 h after MCAO and coronally dissected into 1 mm sections. Using a stereomicroscope, the brain section was illuminated with a halogen lamp and computerized images were stored. Next the brain sections were stained for 30 minutes with 0.5% TTC (2, 3, 5-triphenylterzolim chloride) at 37 degrees C. Using an image analyzer (AnalyPower 1.0), the infarct volumes obtained by light transmittance and TTC staining were calculated. Integrated gray scales of sections of both hemispheres were calculated by Photoshop 5.0. RESULTS: A close correlation existed between cerebral infarct volume measured by light transmission and TTC staining (r=0.81). The mean gray scales measured by both techniques of the ischemic hemispheres as well as those of the cortex, subcortex and hippocampus were siginificantly higher than those of non-ischemic hemispheres and of control mouse hemispheres (P <0.001). Further there were no significant difference between the two hemispheres of control mice and between hemispheres of control mice and non-ischemic hemispheres of the MCAO mice. CONCLUSION: Light transmission can be used for qualitative analysis of focal cerebral ischemia.

[A novel method for continuously assessing the spatio-temporal properties of locomotor activity in mice].

OBJECTIVE: To develop a novel method for continuously assessing the spatio-temporal properties of locomotor activity of mice in an open field using a video-tracking system. METHODS: The locomotor tracks in the open field were recorded by video camera within 22 h, and analyzed by AnalyPower1.1 system that we developed recently. Total distance, distances traveled in different zones and their ratios to total distance; total time,times spent in different zones and their ratios to total time were used as indicators to assess the properties of locomotor activity. RESULTS: In free and wakeful state, the locomotor activity of mice presented obvious regional and temporal properties. Mice preferred to stay in home base (food and water zones), and frequently visited the peripheral zones but seldom the center zones within 22 h. On the other hand, mice were most active within the first 1 h, and then their activity obviously decreased. After their activity became stable, the mice showed the obvious circadian variation of the activity as they were more active in the night. CONCLUSION: The novel method we developed in this study can continuously assess the spatio-temporal properties of locomotor activity quantitatively and objectively.

Spatiotemporal properties of locomotor activity after administration of central nervous stimulants and sedatives in mice.

In the present study, we investigated the spatiotemporal properties of locomotor activity after administration of CNS sedatives (pentobarbital and diazepam) and stimulants (theophylline and caffeine) in an open field test. The absolute and relative distances traveled in central or peripheral regions within 2 h were analyzed. We found that both pentobarbital and diazepam increased total travel distances, especially within the initial 30 min, when traveling was mainly in the peripheral region. Pentobarbital induced this hyperactivity at higher doses (maximum at 30 mg/kg); while diazepam at higher doses (4 and 8 mg/kg) mainly decreased the traveled distance during 0-1 h but increased that in the periphery during 1-2 h. On the other hand, both theophylline and caffeine generally increased the traveled distance in the central region; this effect lasted longer with increasing dose. Caffeine increased the traveled distance at lower doses (maximum at 10 mg/kg) but decreased it at higher doses (30 and 100 mg/kg) during the initial 1 h. Theophylline exhibited a similar but smaller decrease at higher doses. Thus, we revealed the spatiotemporal properties that sedatives decrease central locomotion but induce a dose-related peripheral hyperactivity while stimulants induce central hyperactivity with a bell-shaped dose-response relation.