OBJECTIVE:To study pharmacokinetics and bioequivalence of Simvastatin sustained release tablets in healthy beagle dogs.METHODS:6 beagle dogs were randomized into two groups.They received oral single dose of Simvastatin sustained release tablets 20 mg(test tablet) or Simvastatin tablets for sale(reference tablet).Plasma samples were collected at different time points.Plasma concentration of simvastatin acid was determined by LC-MS/MS and pharmacokinetic parameters were calculated.RESULTS:The pharmacokinetic parameters of reference tablet vs.test tablet were as follows:Cmax:(23.461?6.043) ng?mL-1 vs.(13.942?3.236) ng?mL-1;tmax:(2.158?0.396) h vs.(4.116?1.145 3) h;t1/2:(4.564?0.645) h vs.(8.143?0.679) h;AUC0～24 h:(118.647?31.989) ng?h?mL-1 vs.(129.977?29.853) ng?h?mL-1.CONCLUSIONS:The absorption rate of test tablet is different from that of reference tablet.The two kinds of tablets are not bioequivalent.

Objective To investigate the location of receptor interacting protein 3( RIP3) in Necroptosis and its function in this signal passage, and explore the relationship between receptor interacting protein 1 ( RIP1 ) and RIP3 in nuclear translocation. Methods Primary cerebrocortical neurons were cultured for 12 days,then pre-treated with zVAD-fmk(20μ,mol/L) for half an hour to block apoptosis. ①Extracting nuclear and cytoplasmic protein after neurons were exposed to TNF for different time ,then protein levels of RIP3 were analyzed by western blot and immunofluorescence for qualitative observation;②In the following research,the neurons were treated with Nec-1 and shRlPl ,then the protein level of RIP1 and RIP3 with western blot were analyzed, cell viability were determined by measuring LDH levels. Results ①In signaling pathways of necroptosis, the protein level of RIP3 in cytoplasmic decreased gradually with prolonged TNF exposure, to the corresponding it rolled up in nucleus and a-chieved the peak in 12 hours of TNF treatment ( Cytoplasmic 0. 45 ± 0. 03 ,0. 41 ± 0. 02,0. 73 ± 0. 03 ,0. 90 ± 0.01,1.15 ±0.04,1.30 ±0.02,0.99 ±0.03,0.63 ±0. 03;Nucleus 0. 07 ±0.02,0. 26 ±0.02,0. 57 ±0. 02,0. 68 ± 0.02,0. 80 ± 0.01,0.92 ± 0.02,1.28 ± 0.03,0. 87 ± 0.02) (P < 0.01). ②Blocking the relationship between RIP1 and RIP3 with necrostatin-1 and shRIPl , nuclear translocation of RIP3 decreased and caused a great increase in cell viability( 1.00 ±0.05,0.39 ±0.03,0.50 ±0. 03) (P<0. 01). Conclusion RIP3 mainly locates in cy-tolymph of normal cells,it translocates into nucelus as necroptosis takes place. RIP1 function with RIP3 in nuclear translocation. Block nuclear translocation of RIP3 is a potential way to protect cells.

OBJECTIVEThis study aims to investigate the expression of connexin 43 (cx43) gene during early development in zebra fish and provide a foundation for further research of cx43 gene in tooth development.

METHODSTotal RNA was extracted within 72 h after fertilization of zebra fish embryos and then reversed transcribed to generate the cDNA library. The specific fragments of the cx43 gene were then cloned and connected to the PGEMT vector. After confirming the constructed plasmid, the corresponding RNA polymerase was chosen, and the digoxin-labeled anti-sense mRNA probe of cx43 was synthesized in vitro. The cx43 gene expression of zebra fish indifferent stages was carried out by in situ hybridization. The relationship of the cx43 gene expression and anatomy of the pharyngeal teeth were compared by alizarin red staining.

RESULTSThe mRNA antisense probe of cx43 was acquired. The positive signal of sepia was observed in the different stages of zebra fish pharyngeal teeth after fertilization. After fertilization for 9 days, the expression site of cx43 in situ hybridization was overlapped in accordance with the anatomical site of the pharyngeal teeth.

CONCLUSIONcx43 gene participates in tooth development and mineralization process and plays a crucial role in later mineralization.

Animals ; Connexin 43 ; Gene Expression ; Genetic Vectors ; In Situ Hybridization ; Odontogenesis ; Plasmids ; RNA, Messenger ; Tooth ; Zebrafish

OBJECTIVETo investigate the mechanism of retinoic acid (RA) signal in dental evolution, RA is used to explore the influence of the mechanism on neural crest's migration during the early stage of zebra fish embryos.

METHODSWe divided embryos of wild type and transgenic line zebra fish into three groups. 1 x 10(-7) to 6 x 10(-7) mol x L(-1) RA and 1 x 10(-7) mo x L(-1) 4-diethylaminobenzaldehyde (DEAB) were added into egg water at 24 hpf for 9 h. Dimethyl sulfoxid (DMSO) with the concentration was used as control group. Then, antisense probes of dlx2a, dlx2b, and barxl were formulated to perform whole-mount in situ hybridization to check the expressions of the genes in 48 hpf to 72 hpf embryos. We observed fluorescence of transgenic line in 4 dpf embryos.

RESULTSWe obtained three mRNA probes successfully. Compared with DMSO control group, a low concentration (1 x 10(-7) mol x L(-1)) of RA could up-regulate the expression of mRNA (barx1, dlx2a) in neural crest. Obvious migration trend was observed toward the pharyngeal arch in which teeth adhered. Transgenic fish had spreading fluorescence tendency in pharyngeal arch. However, a high concentration (4 x 10(-7) mol x L(-1)) of RA malformed the embryos and killed them after treatment. One third of the embryos of middle concentration (3 x 10(-7) mo x L(-1)) exhibited delayed development. DEAB resulted in neural crest dysplasia. The expression of barxl and dlx2a were suppressed, and the appearance of dlx2b in tooth was delayed.

CONCLUSIONRA signal pathway can regulate the progenitors of tooth by controlling the growth of the neural crest and manipulating tooth development

Animals ; Branchial Region ; Cell Differentiation ; drug effects ; Embryo, Nonmammalian ; drug effects ; embryology ; metabolism ; In Situ Hybridization ; Neural Crest ; drug effects ; Odontogenesis ; Signal Transduction ; Tooth ; drug effects ; embryology ; metabolism ; Tretinoin ; pharmacology ; Zebrafish ; embryology ; genetics ; metabolism

Objective To investigate MRI appearances of aquaporin(AQP) and its effect in different brain regions of patients with Parkinson's disease(PD).Methods A prospective study was carried out in 33 PD patients(PD group) and 23 gender-and age-matched healthy controls (control group).Clinical data of PD patients were collected.The aquaporin imaging of diffusion-weighted magnetic resonance imaging (MRDWI) with multiple b-values in different brain regions were performed to detect the apparent diffusion coefficient(AQP-ADC) values of aquaporin.The PD patients were assessed and graded by modified Hoehn-Yahr grading,then the AQP-ADC values of control group,mild PD group,moderate and severe PD group were analyzed using one-way analysis of variance.The correlation analysis was carried out to detect the relationship between AQP-ADC values in different brain regions and Hoehn-Yahr grading of PD patients.Results Compared with control group,mild PD group had significantly higher AQP-ADC values in red nucleus(RN) and globus pallidus(GP) ((0.24±0.04) vs (0.21±0.04),(0.21±0.04) vs (0.16±0.04);both P＜0.05);while the AQP-ADC values in RN and GP of moderate and severe PD group were significantly lower than that of mild PD group((0.21±0.02) vs (0.24±0.04),(0.18±0.03) vs (0.21±0.04);both P＜0.05);but there was no significant difference between moderate and severe PD group and control group(P＞0.05);and there was also no significant difference in substantianigra (SN),putamen (Pu) and thalamus (THA) among control group,mild PD group and moderate and severe PD group(P＞0.05).The correlation analysis showed that there were negative correlations between the AQP-ADC values in RN and GP and Hoehn-Yahr grading(r=-0.479 and-0.395,P＜ 0.05),while there was no correlation in SN,Pu and THA (P＞ 0.05).Conclusion The AQPADC values are increased in RN and GP of mild PD patients,and decreased in moderate and severe PD patients,while there is no significant change in SN,Pu and THA of the two groups,suggesting that the expression of AQP in different brain regions may be related to the severity and pathological stage of PD.

Objective To explore whether hypoxia-inducible factor-1α( HIF-1α )is involved in oxygenglucose deprivation (OGD)induced caspase-independent cell death in primary cortical neurons and whether their expression is infected by necrostatin-1 ( Nec-1 ).Methods ( 1 ) Primary cerebrocortical neurons were cultured for 14 days.Pretred z-VAD.Fmk (z-VAD)and Nec-1 with 0.1,1,5,10,25 and 50 μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours,then cell viability was determined by measure LDH level.(2)Pretred z-VAD before the neurons were exposed to OGD for 2 hours,then reoxygenated for 0,2,6,12,24and 48 hours.Then western blot analysis protein level of HIF-1 α ;rt-PCR check its RNA level.(3)Pretred z-VAD and Nec-1 with 25μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours.Then western blot analysis protein level of HIF-1 α; rt-PCR check its RNA level.Result ( 1 ) When cells were pretread Nec-1 with 5 μ mol/L(6.97 ± 0.06),the level of LDH was lower than cells untreated( 14.23 ± 0.08 ) (P＜ 0.05);At 25 μmol/L( 2.21 ± 0.05),the level of LDH was essentially the same as that of the control( 1.03 ±0.03 ) (P＞0.05).(2)The protein level of HIF-1 αwas different from normal (0.24 ±0.01 ) when exposed to OGD for 2 hours and reoxygenated for 2 hours (0.57 ± 0.09) and was highest after cells were exposed to OGD for 2 hours and reoxygenated for 12 hours(0.91 ± 0.08 ) (P＜ 0.05 ).The RNA level of HIF-1 α when cells were exposed to OGD was not deferent from normal (P ＞ 0.05 ).( 3 ) When cells were pretread with Nec-1 (0.32 ± 0.04 ),the protein level of HIF-1α were lower than untreated(0.83 ±0.03) (P＜0.05),but the RNA level of HIF-1α had no deference(P ＞ 0.05).Conclusion HIF-1α was involved in cell' s caspase-independent cell death;Nec-1 can protect neurons through inhibiting the expression of HIF-1α.

Objective To investigated the role of the autophagy lysosomal pathway in PD cells and the possible molecular mechanisms. Methods A dopaminergic neuronal injury model was induced by 6-OHDA in PC12 cells . Autophagosomes in PC12 cells were examined by transmission electronmicro-scopy( TEM ). The expression of LC3- Ⅱ , Cathepsin B were assayed by western blot analysis. Results TEM revealed that the autophagosomes were increased in PC12 cells after 6-OHDA treatment and appeared apoptosis. The LC3-Ⅱ (2h:52.57 ±2.27,4h:56.83 ±3.51,6h:73.43 ±5.41,12h:103.90 ±2.57,24h: 100.40 ±3.91 )and Cathepsin B expression ( model group: 113.80 ± 4.46; normal group 35.89 ± 3.40) were increased after 6-OH DA treatments (P ＜ 0.05 or P ＜ 0.01 ). Conclusion The results indicate that autophagy lysosome pathway is involved in 6-OHDA-induced cell death in PC12 cells.

Objective To investigate the expressions of c-Fos,5-HT and spontaneous activities in single prolonged stress (SPS) stress rats,and to study the possible mechanism of posttraumatic stress disorder(PTSD).Methods forty-eight male SD rats were randomly divided into 6 groups:Individual living + SPS group (IS group),Individual living+control group (IC group),Enriched environment+SPS group (ES group),Enriched environment+control group (EC group),control+SPS group(CS group),control group(C group),8 rats in each group,spontaneous activities were measured before the experiment and post experiment at week 1,2.And the expressions of c-Fos,5-HT in the hippocampus were examined by immunohistochemical staining.Results (1) Spontaneous activity:there were no differences of crossing number and distance among 6 groups before experiment (P＞0.05).On SPS 14 days the crossing number and distance of IS group (12.12±9.64,(2.71 ± 1.99)m) decreased compared with the CS group(45.25±8.37,(6.37± 1.18) m,P＜0.05),and ES group (69.75± 10.05,(10.69± 1.50) m)showed significant increase compared with CS group (P＜0.05).(2)5-HT:the expression of 5-HT in the hippocampus of IS group(0.1125±0.0095) was significantly higher than that in CS group(0.6138±0.0059,P＜0.05),menwhile lower in ES group(0.0495±0.0074,P＜0.05).(3)c-Fos:compared with CS group(0.3108±0.0074),the expression of c-Fos in the hippocampus of IS group(0.3585±0.0150,P＜0.05) significantly increased,but decreased in ES group (0.2613±0.0063,P＜ 0.05).Conclusion Enriched environmental can improve the level of activities in PTSD rats,and to reduce the the expression of c-Fos and 5-HT in the hippocampal neurons.

Objective To investigate the treatment of octanol on matrix metalloproteinase-9(MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein expression,cerebral water content,infarction volume after ischemia-reperfusion in rats.Methods 150 SD rats were randomly divided into sham operated group (n=24),MCAO group (n=24),DMSO solvent control group (n=24) and octanol treatment group (n=24).A model of middle cerebral artery occlusion was induced by suture method.TTC stain was used to detect the infarction volume,dry-wet weight method to determine the brain water content.The expression of MMP-9 and TIMP-1 protein was detected by immunofiuorescence and Western blot.Results At 24 h of reperfusion after ischemia for 2 h,the octanol treatment group compared with MCAO group brain infarction volume obviously decreased(P＜0.05),water content significantly reduced ((78.16± 1.47) ％ vs (80.88±0.73) ％,P＜0.05),the number of MMP-9 positive cells obviously decreased((10.67±2.16) vs (29.00±3.40),P＜0.05),the expression of MMP-9 protein significantly reduced ((0.14±0.01) vs (0.21±0.02),P＜0.05)and the number of TIMP-1 positive cells significantly increased ((27.83 ±2.13) vs (5.67± 1.03),P＜0.05),the expression of TIMP-1 protein obviously increased((0.42±0.01) vs (0.28± 0.01),P＜0.05).The difference between MCAO group and DMSO solvent control group was not statistically significant(P ＜0.05).Conclusion Octanol may reduce brain edema,brain infarction volume.Up-regulation the expression of MMP-9 and down-regulation the expression of TIMP-1 may be one of the underlying mechanisms of the octanol neuroprotection.

Objective To study the effect of social isolation( SI) on the exploratory behavior and working memory in mice. Methods The Kunming mice of postnatal 21 days were divided into the control group,SI 2 weeks group,SI 2 weeks gregarious group,SI 4 weeks group and SI 8 weeks group,according to randomized design with ten animals each. All isolated mice were isolated for 2, 4 and 8 weeks respectively, the gregarious group were housed under normal grouped housing enviroment after isolation until adult, the mice with the relative same age were control groups. All animals were measured for exploratory behavior and working memory by performing open field and T?maze after the treatment. Results In the open field,compared to the relative control group,the central area of the total time in the SI 4 weeks group(0.07±0.04) was less than the control (0.10±0.04) obviously. The central area percentage of total time in SI 8 weeks group (0.64±0.12) were more than the control (0.43±0.08). In the T?maze,the alteration times in SI 2 weeks group (first day (5.92±0.79),second day (6.67±1.3),third day (7.42±1.08),fourth day (8.17±1.27)) were less than the control (first day (6.80±1.14); second day (7.60± 0.84);third day (8.30±0.95);forth day (9.20±1.32)). However,the alteration times of gregarious group showed no obvious change. Both the alteration times of SI 4 weeks (8.18±1.99) in the second day and that of SI 8 weeks (8.29±3.04) in the forth day were more than the control (6.60±2.11) and (7.80±2.53) respectively.Conclu?sions Working memory of SI 2 weeks rats decrease,which can be improved by the resocialization.SI 4 weeks and 8 weeks rats show the decreasing exploring ability and increasing anxiety and work memory.