Abstract:Objective This study aims to investigate the effects of high-fat diet rich in perilla oil on the insulin sensitivity-related gene expression in skeletal muscle in insulin resistant rats. Methods The insulin resistant(IR) rat models were randomly divided into 2 groups, including high fat group(HF) and perilla oil(PO) intervention group fed with 20% substitution of lard energy in the HF. The insulin sensitivity of rats was measured after 4 weeks. The α-linolenic acid(ALA) content of PO in the rat plasma were analyzed by gas chromatograph. Real-time PCR was applied to measure glucose transporter 4(GLUT4) and insulin receptor substrate-1(IRS-1) mRNA, and Western blot assay was used for detecting the expression of GLUT4 and IRS-1 in the skeletal muscle. Results At the gene and protein levels, PO remarkably reduced the level of IRS-1 and upregulated the level of GLUT4 with increasing intake of ALA and serum ALA content in IR rats. The results of hyperinsulinemic-euglycemic clamp test showed no significant difference between the two groups. Conclusions The results of our study suggest that consumption of n-3 PUFA at levels that can typically be found in the diet fed to IR rats in the form of ALA(0.556 g/d) may not improve insulin sensitivity, even though regulating the expression of GLUT4 and IRS-1 in the skeletal muscle.

Abstract:Objective To evaluate the effect of Eucommia on hyperlipidemia and related indexes in rats, and provide animal data useful for the clinical experimental studies on hyperlipidemia. Methods Seventy-two healthy male SD rats were used in this study. One group of 12 rats fed with normal diet was chosen as normal control group, and other 60 rats were fed with high fat diet for two weeks to generate rat models of hyperlipidemia. 48 of the hyperlipidemic model rats were taken and divided randomly into 4 groups, including model group, high dose Eucommia, moderate dose Eucommia, and low dose Eucommia groups. The last three groups were gavaged different dose of Eucommia, respectively. Druing this period, the other groups except the normal control group were fed with high fat diet continuously. The levels of serum TC, TG, LDL-C, and HDL-C of rats were measured on day 30 and 45. Results The serum levels of TC and LDL-C of the rats in the model group were obviously higher than those in the normal control group. The rat models of hyperlipidemia were established successfully. The three dose groups had a tendency of lowing blood lipid after 30 days. At 45 days, the levels of serum TC and LDL-C in the low and high dose groups were lower than those in the model group(P<0.01, P<0.05),(P<0.01, P<0.01). TG in the high, moderate and low dose groups were lower than that in the model group(P<0.01, P<0.01, P<0.01), but the level of the serum HDL-C was not significantly lower than that in the model group(P>0.05, P>0.05, P>0.05). Conclusions Eucommia in a dose of 0.43 g/kg, 0.86 g/kg and 1.71 g/kg administered for 30 days have a tendency to reduce the level of serum TC, TG, and LDL-C. When Eucommia is administered in a dose of 0.43 g/kg, 1.71g/kg and 3.42 g/kg for 45 days, it shows an adjuvant hypolipidemic effect.

Abstract:Objective To explore the protective effect of Xueshuantong capsule on acute myocardial ischemic injury and its antithrombotic effect in rats. Methods The rat model of acute myocardial ischemia(AMI) was developed by ligation of the left anterior descending coronary artery for 24 hours. The effects of Xueshuantong capsule, a drug of traditional Chinese medicine, on the electrocardiogram(ECG) and myocardial infarct size was observed. The serum activities of lactate dehydrogenase(LDH), creatine kinase(CK), aspartate aminotransferase(AST), creatine kinase-MB(CK-MB), and α-hydroxybutyrate dehydrogenase(α-HBDH) were detected by biochemistry. Blood was taken from the rats, and the thrombolytic effect and the platelet aggregation rate in vitro were observed. Results Xueshuantong capsule in each dose(60, 30, 15 mg/kg)group significantly reduced the myocardial infarct size, and lowered the serum contents of CK, LDH, and CK-MB. Xueshuantong capsule in a dose of 30 and 15 mg/kg significantly promoted the thrombolysis in vitro, and Xueshuantong capsule in a dose of 60, 30 and 15 mg/kg significantly inhibited the ADP-and collagen-induced platelet aggregation in vitro. Conclusions Xueshuantong capsule exerts protective effects on acute myocardial ischemic injury in rats. This effect may be partly associated with its antithrombotic effect.

Abstract:Objective To compare the changes in TLR4-NF-κB signaling pathway in infant and adult mice infected with influenza virus, and to provide experimental evidence for the study of immunopathological mechanism in pediatric respiratory virus susceptibility. Methods Immunohistochemistry and RT-PCR were applied to detect the expressions of lung TLR4 and NF-κB P65 mRNA and proteins in the infant and adult mice, and to compare the changes in TLR4-NF-κB P65 signaling pathway after infection with influenza virus. Results(1) The infant model group showed the strongest expression of TLR4 protein in the lung tissue, compared with that in the normal group and adult model group showing significant differences(P<0.05).(2) The expression of NF-κB P65 protein in the lung tissue was strongest in the infant model group, and it was gradually increased over time, showing a significant difference between each time point and the next time point(P<0.05).(3) The infant model group showed the strongest expression of TLR4 mRNA in lung tissue, significantly higher than that in the normal and adult model groups(P<0.05).(4) The expression of NF-κB P65 mRNA in the lung tissue was highest in the infant model group, and significantly higher than that in the normal and the adult model groups(P<0.05), and it was gradually increased with the time. Conclusions The over-activation of TLR4-NF-κB P65 signaling pathway may be one of the immunopathological mechanisms of serious injury in the lung tissue in infant rats.

Abstract:Objective To investigate the expression levels of BDNF, trkB and ChAT mRNA and proteins in the brain of adult tree shrews(Tupaia belangeri). Methods Quantitative real-time PCR was employed to detect the expression levels of BDNF, trkB and ChAT mRNA in the hippocampus, basal ganglia and frontal cortex of adult tree shrews. The expression levels of BDNF, trkB and ChAT proteins and β-actin was used as internal standard. Results The expression level of BDNF mRNA was highest in the hippocampus of adult tree shrew, and there were significant differences between that in the hippocampus, and basal ganglia and frontal cortex(P<0.01). The expression level of trkB mRNA was higher in the frontal cortex than in the basal ganglia and hippocampus, showing a significant difference between them(P<0.05). The expression level of BDNF protein was significantly higher in the basal ganglia than in the hippocampus or frontal cortex(P<0.01). There were no significant difference(P>0.05) in the expressions of trkB protein among the hippocampus, basal ganglia and frontal cortex of the adult tree shrews. There were no significant differences in expressions of ChAT mRNA and protein among the hippocampus, basal ganglia and frontal cortex in adult tree shrews(P>0.05). Conclusions The expression levels of ChAT mRNA were consistent with that of ChAT protein in the hippocampus, basal ganglia and frontal cortex of adult tree shrews, while the expression levels of BDNF and trkB mRNA were not consistent with their proteins, which might indicate that the transcriptional regulation pattern might be more complex. Tree shrew is a valuable animal model in the study of mechanism of BDNF/trkB gene expression.

Abstract:Objective To establish a rabbit model of facial skin lifting and to evaluate the skin lifting effects of absorbable soft tissue plastic belt, and to provide experimental basis for clinical application of this plastic belt. Methods 36 Japanese white rabbits were randomly divided into model control group(n=6) and plastic belt lifting group(n=30). The model control group received only facial skin resection surgery, while the plastic belt lifting group had facial skin resection and plastic belt implantation. The changes of rabbit general status, skin lifting effects, performance of plastic belt and pathological changes of subcutaneous tissue after implantation were determined during the 4 weeks after surgery. Results The rabbit skin wound was healed within 3-7 days after surgery without infection. Meanwhile, the plastic belts did not show shifting or rupture, and only whitening around the perforations was observed in the two groups. Compared with the model control group, the MA and MB distances in the plastic belt lifting group were significantly lower(P<0.01), while the biological tension of plastic belts in the facial skin lifting rabbit models was significantly increased with the extension of time after implantation(P<0.01), and the biological tension was 18.62 N at 4 weeks after transplantation. In addition, the tensile intensities of perforations and plate in the two groups were significantly reduced at 4 weeks postoperatively(P<0.01), the tensile intensity of perforations and plate in the two groups were maintained at 35.07 N and 53.31 N, respectively, and the perforations/plate tensile intensity ratio of the two groups remained unchanged during 4 weeks after transplantation(P>0.05). Moreover, the molecular weight(Mw), peak molecular weight(Mp), Z molecular weight(Mz) and viscosity were gradually decreased along with the time passing after implantation(P<0.01), and its dispersion Mz/Mw ratio was also gradually decreased from 2 weeks after implantation(P<0.01), and no obvious pathological changes were found after subcutaneous implantation of the plastic belts. Conclusions We have successfully established a facial skin lifting rabbit model, and the plastic belt can obviously lift the facial skin fascia system and keep intact more than 4 weeks in the body. Therefore, this plastic belt can be applied for anti-wrinkle facial soft tissue lifting against the skin damage caused by aging.

Abstract:Objective To investigate the protective effects and the mechanisms of 17β-estradiol on the propofol-induced neuroapoptosis in primary cultured rat cortical neurons. Methods The neurons were cultured for 7 days and then divided into three groups:vehicle-control group(treated with equal volume of 20% intralipid), propofol-treated group(treated with 500μmol/L propofol), and propofol plus 17β-estradiol treated group(treated with 500μmol/L propofol and 0.1μmol/L 17β-estradiol). 12 hours after the treatment, neuroapoptosis was detected by Hoechst 33258 staining and TUNEL assay, and the levels of Bcl-2, Bax and cleaved caspase-3 proteins were detected by Western blot. Results Compared with the vehicle-control group, the neuroapoptosis increased greatly(P<0.01), Bcl-2 level reduced(P<0.01), Bax and cleaved caspase-3 levels increased greatly(P<0.01), and Bcl-2/Bax ratio reduced significantly(P<0.01). Compared with the propofol-treatment group, the neuroapoptosis decreased greatly(P<0.01), Bcl-2 level increased(P<0.01), Bax and cleaved caspase-3 levels reduced greatly(P<0.01), and Bcl-2/Bax ratio increased greatly(P<0.01). Conclusions 17β-estradiol can protect cortical neurons against propofol-induced cortical neuroapoptosis by regulating the expression of Bcl-2 and Bax.

Abstract:Objective To study the type variation of microglial activation in spinal dorsal horn of rats after sciatic nerve injury. Methods Healthy adult male Sprague-Dawley rats were randomly divided into the control and experimental groups, 24 rats in each group. The experimental group underwent ligation of sciatic nerve trunk to generate nerve injury in the rats. The pain behavior in the rats was measured at the 1th, 7th and 14th postoperative days, and the changes of microglial activation in the rat lumbar spinal cord dorsal horn was detected by immunofluorescence staining. qRT-PCR assay was used to validate the activation trends of M1 and M2 types of microglia cells. Results No significant changes were found in the microglial cells in the spinal cord dorsal horn of rats in the sham-operation group during 14 days after operation. In the sciatic nerve ligation group at 1 day after operation, no significant change was observed in the number of microglial cells, but the expression of marker of M1 microglia was significantly increased. At 7 and 14 days after operation, the number of microglial cells and the expression of M1 microglia marker in the spinal cord dorsal horn were increased significantly. Conclusions Microglia activation in the spinal dorsal horn starts at the first day after sciatic nerve injury, and lasts at least for two weeks after the operation. M1 microglia activation dominates during this period.

Abstract:Objective To establish a rabbit model of lumbar laminectomy and bone grains replantation and provide experimental evidence for the clinical application. Methods Eighteen healthy male New Zealand rabbits were selected and randomly divided into two groups:the control group(n=6) and experimental group(n=12). The rabbits of control group were given general anesthesia, and taking the L5 spinous process as the center to perfom left L5 laminectomy, using a micro lancet forceps to slowly bite the lamina and ligamentum flavum for fenestration and exposed to an approximately 0.8 cm x 0.3 cm sized bone window and then sutured the skin. The rabbits of experimental group were exposed to an approximately 0.8 x 0.3 cm sized bone window as well, and bone fragments were cut into small grains. Then the small bone grains were embedded in medical collagen sponge, to form an arch shape, and replanted them to the site of epidural fenestration. CT scan and histological changes were observed at 4, 8 and 12 weeks after operation. Results At 8 weeks after operation, CT examination showed that in the experimental group, a thin bone plate was formed by the bone grains. At 12 weeks after operation, the bone plate became thicker and was connected with the vertebral bone, and with continuous bone trabeculae. The spinal canal and volume were not obviously changed, and no spinal cord compression was observed. The rabbits of control group showed segment lamina defects, a small scar protruding into the spinal canal, and the vertebral canal was not completely reconstructed. Conclusions The bone grains replantation can effectively promote bone reconstruction in the laminectomized rabbits, and the formed bone plate can prevent epidural scar from intruding into the spinal canal, and can reduce the postlaminectomy adhesion.

Abstract:Objective To analyze the differences between the semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays for detecting XDH/XO mRNA expression in various organ tissues of rhesus monkey, and provide useful reference in methodology of experimental studies. Total RNA was extracted from the myocardium, kidney, testis, skin, and liver tissues, respectively, for detecting XDH/XO mRNA expression in rhesus monkey by semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays. The sensitivity and specificity of the two assays were compared with each other using the same primer sequences and reference genes. Results The expressionof XDH/XO mRNA in different organ tissues were detected by both the two PCR assays. The sensitivity of quantitative fluorescence real-time RT-PCR for the XDH/XO mRNA expression in the liver tissue was 39 times higher than that by semi-quantitative RT-PCR. Conclusions Both the quantitative and semi-quantitative fluorescence RT-PCR assays can be used to detect the expression of XDH/XO mRNA in different organ tissues of rhesus monkey. The sensitivity of quantitative fluorescence real-time PCR assay is more sensitive than that of the semi-quantitative RT-PCR assay.

Abstract:Objective The current study was aimed to establish an electrically amygdala kindling model of refractory epilepsy in Sprague-Dawley rats, and to study their changes of electrocorticagram(ECoG). Materials and methods Male Sprague-Dawley rats were used in the experiment. Two-polar electrode was implanted into the right amygdala stereotactically. Two weeks after the surgery, electrical stimulations were given to elicit the grade 4 seizure in all animals with the fast kindling protocol. Rekindling was administrated after the first kindling. The after discharge threshold(ADT) and the number of stimulus were reassessed in the two kindling protocols. ECoG and the behavior of the animals were recorded during the whole experiment. Result The changes of ECoG and behavior:Animals showed stage 1-3 seizure when the ADT was assessed. While during the kindling period, the animals showed generalized convulsion with stage 4-5 seizure. ECoG showed sharp waves, spike waves, sharp-slow waves and spike-slow waves. Statistical analysis showed that the ADT was not significantly increased at two weeks after kindling(from 83.33±22.29μA to 84.17±16.76μA, P=0.923). The number of stimulus given to elicit the stage 4 convulsion was not significantly increased as well(from 4.41±2.27 to 5.58±3.96, P=0.231). Conclusions Rapid kindling model of epilepsy in rats is an effective epilepsy model, which is stable for 2 weeks.

Abstract:Objective To introduce an improved extraction method of prefrontal cortical and striatal synaptosomes from SHR rat. Methods Synaptosomes were prepared from SHR rat brain tissue by Percoll density gradient centrifugation. Transmission electron microscopy was used to assess the morphology and structural integrity of the synaptosomes. Results The obtained synaptosomes showed oval structures surrounded by an intact membrane. Presynaptic components contained one or more mitochondria and a large number of synaptic vesicles. The synaptic clefts were clearly visible, and prominent part of the characteristic compact structure was clear, complete and with higher electron-density. The synaptosome presynaptic membrane, synaptic cleft, and postsynaptic membrane were well preserved,and the synaptosomes were densely distributed, showing typical morphological characteristics of synaptosomes. Conclusions The results of our study improved the traditional preparation method and provide a less time-consuming, highly productive protocol for preparation of structurally typical and intact synaptosomes, suitable for further research on neuroscience and neurological diseases.

Abstract:Objective To develop a multiplex polymerase chain reaction(mPCR) assay for detection of four pathogenic dermatophytes[Trichophyton mentagrophytes(Tm), Microsporum gypseum(Mg), Microsporum canis(Mc), and Arthroderma simii(As)]in laboratory animals, which could be used rapidly and simultaneously for direct detection of those four pathogens. Methods We designed 5 specific primers according to 18S-28S rRNA sequences of the four pathogenic dermatophytes reported in Genbank. The four mPCR assays were established through optimizing the concentration of primers, dNTP, TaqDNA polymerase and the annealing temperature. After verifying the specificity and sensibility, this method was used to detect 15 hair samples with artificial infection and 260 samples taken from laboratory animals. Results This mPCR technique can distinguish the four dermatophytes by producing 192 bp(Tm),460 bp(Mg),290 bp(Mc)and 602 bp(As)fragments. The sensibility for detection of the four dermatophytes was 5.9 pg/μL, 6.6 pg/μL, 9.5 pg/μL and 5.1 pg/μL, respectively. The results of 15 artificial infection samples were accurate, and the results of 260 hairs samples were negative for the four fungi. Conclusions Our results suggest that the mPCR assay developed in this study can efficiently detect the four dermatophytes, is a useful and rapid technique for rapid detection of the pathogenic dermatophytes in laboratory animals.

Abstract:Objective To develop a real-time RT-PCR assay(rRT-PCR) for efficient detection of duck hepatitis virus type 1(DHV-I). Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences. One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay(rRT-PCR). Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates. The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China. Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.

Abstract:Inteleukin-37 is a member of IL-1 family and originally named as IL-1F7. It has five different subtypes(IL-37a-e). It has been reported that IL-37 suppresses pro-inflammatory cytokines production of a variety of immune cells, and further regulate innate immune response. In addition, IL-37 has protective effects on colitis, arthritis, pancreatitis and other inflammatory diseases that induced by exogenous stimuli. In a word, IL-37 is a novel anti-inflammatory cytokine and plays an important role in a variety of inflammation-related diseases.

Abstract:Tissues and organs generate angiogenesis under the stimulation of angiogenic factors in physiological or pathological conditions. Multiple signal pathways including VEGF, Notch, Wnt/β-catenin, Ang1(2)/tie2 and PIK-Akt etc. have effects on various stages of angiogenesis. VEGF exerts irreplaceable effects on the whole process of angiogenesis through multiple signal pathways. Over the past few years, new progress has been made in the researches of mechanisms regulating angiogenesis through VEGF-related signal pathways both at home and abroad. These findings provide us new theoretical basis for clarification of the pathogenesis of many diseases and clinical drug development. In this article we will summarize the recent research progress in this field, hoping to provide new possibilities for the treatment of angiogenesis-related diseases.