Regulation of Hypnotic Active Components of Cnidii Fructus on Melatonin Synthesis Rate-limiting Enzyme AANAT in Rats with PCPA-induced Insomnia / 中国实验方剂学杂志

Objective:To observe the effects of Cnidii Fructus hypnotic active components （CHC） on the behaviors of rats with p-chlorophenylalanine （PCPA）-induced insomnia and melatonin （MT） synthesis rate-limiting enzyme arylalkylamine <italic>N</italic>-acetyltransferase （AANAT）， and explore the protective mechanism of CHC on the pineal gland. Method:Male SD rats of SPF grade were randomly divided into a blank control group， a model group， a MT group， and high-， medium-， and low-dose CHC groups with 10 rats in each group. Except for the blank control group， other groups received 4.5% PCPA suspension at 10 mL·kg^-1， intragastric administration， for two consecutive days. After PCPA model of insomnia was established， normal and model groups were gavaged at the same volume of 2% Tween-80， MT control group （10 mg·kg^-1）， CHC was high， medium and low （60， 30， 15 mg·kg^-1）， 10 mL·kg^-1， once a day， for consecutive 7 days. Four days after administration， open field， elevated cross maze， and pentobarbital sodium-induced sleep tests were conducted， respectively. Serum MT was detected by enzyme-linked immunosorbent assay. The mRNA expression level of AANAT was determined by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. The expression of AANAT protein in the pineal gland was detected by Western blot. Result:Compared with the results in the blank control group， the total distance of open field activity and standing times and duration in the central area were increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， the proportions of open arm entry （OE%） and open arm time （OT%） were decreased （<italic>P</italic><0.05）， and the sleep latency was prolonged （<italic>P</italic><0.01） in the model group. Compared with the model group， no significant difference was observed in the low-dose CHC group， while other groups exhibited reduced total distance of activity （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated OE% （<italic>P</italic><0.05）， shortened sleep latency， and prolonged sleep time （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the serum MT in the blank control group， that in the model group was decreased （<italic>P</italic><0.01）. Compared with the model group， no significant difference was observed in the low-dose CHC group， while other groups displayed increased serum MT （<italic>P</italic><0.05）. The mRNA and protein expression of AANAT was decreased in the model group as compared with that in the blank control group （<italic>P</italic><0.01）. Compared with the model group， the MT group and the high-dose CHC group showed up-regulated expression （<italic>P</italic><0.05）. Conclusion:CHC improved the behavioral indexes of PCPA-induced insomnia， increased the synthesis and secretion of MT in pineal cells， and elevated the serum MT level， which was related to the up-regulation of the mRNA and protein expression of AANAT in the pineal gland.

Serum exosomes of rats with hepatic ischemia-reperfusion injury induce the damage of hippocampus and cerebral cortex / 生理学报

The paper was aimed to explore the role of serum exosomes induced by hepatic ischemia/reperfusion (I/R) injury in the damage of hippocampus and cerebral cortex of rats. The male Sprague-Dawley (SD) rats were randomly divided into 4 groups: sham operation group (S), hepatic I/R injury group (I/R), serum exosomes from S group treatment group (ES) and serum exosomes from I/R group treatment group (EI). In ES group and EI group, 100 μL serum exosomes from S group and I/R group were injected into the normal rats through tail vein respectively. Another three normal rats were injected intravenously with serum exosomes labeled with PKH26 red fluorescence, and then the expression of fluorescence in the brain tissues was observed by immunofluorescence microscope. The morphology and size of exosomes were observed by transmission electron microscope, the expression of exosomes markers CD63 and CD9 was detected by Western blot, and the damage of liver and brain, levels of apoptosis and oxidative stress response in hippocampus and cerebral cortex were observed by serological and histological indexes. The results showed that the exosomes were a group of round or ovoid membranous vesicles, sized in 30-100 nm. Compared with that in S group, the content of serum exosomes in I/R group was increased (P < 0.05). Moreover, serum exosomes could go through the blood-brain barrier and enter the brain tissue freely through blood circulation. The index of liver function in I/R group was significantly higher than that in S group (P < 0.05). There was no significance in the degree of brain damage, apoptosis and oxidative stress in hippocampus and cerebral cortex between S group and ES group. Compared with those in S group and ES group, the serum levels of brain injury markers, apoptosis index (AI) and oxidative stress in hippocampus and cerebral cortex increased in I/R group and EI group (P < 0.05). Whereas, compared with those in I/R group, the above indicators in EI group decreased (P < 0.05). Therefore, hepatic I/R injury can lead to the damage of hippocampus and cerebral cortex, and the increased serum exosomes induced by hepatic I/R plays an important role.

Effects of insulin-like growth factor binding protein 4 on morphology and function of chick extraocular muscle / 中华实验眼科杂志

Background Researches have showed that the extraocular muscle injection of insulin-like growth factor(IGF) results in the thickness of extraocular muscle and enhancement of contractility,and lack of IGF probably is associated with pathogenesis of strabismus.If extraocular muscle injection of insulin-like growth factor binding protein 4(IGFBP-4),an inhibitor of IGF,can further ensure the effect of IGF is still under-investigation.Objective The aim of this study was to evaluate the effects of IGFBP-4 on the developing extraocular muscle.Methods IGFBP-4 of 2 μl(1 g/L)was injected into superior rectus muscles via supraorbital margin in the left eyes of 24 postnatal l-day-old chickens once every other day for five times,and the equivalent amount of normal saline solution was used in the same way in the fellow eyes as controls.The animals were sacrificed by overdose of anesthesia 2 days after the final injection and 12 animals were randomly selected for the maximal contractility test of superior rectus muscle.The other 12 pieces of superior rectus muscle of the chickens were obtained for the hematoxylin & eosin staining to evaluate the morphology of superior rectus muscle and calculate the myofiber diameter using imaging system.Results The maximal contractility was (1.602 ± 0.080) mN and(1.815±0.O10)mN in the IGFBP-4 injection group and normal saline solution group respectivily,showing a significant difference between the two groups(t =13.65,P＜0.01).The maximal contraction force in cross-sectional area was (95.500 ± 7.420) mN/cm2 in the IGFBP-4 injection group and it was significantly lower than that of the normal saline solution group(101.510±5.220)mN/cm2(t =28.81,P＜0.01).Histological examination showed that thin myofibers were obvious more in the IGFBP-4 injection group than those with saline injection.The mean diameter was (8.7 ± 0.5) μm in the IGFBP-4 injected group,and that of the normal saline group was(9.1 ±0.4) μm,there was no significant difference between them (t =0.75,P =0.46).However,significant differences were found in the number of different diameters of myofibers between the two groups(all at P＜0.05).Conclusions IGF is one of the key nutrition factors in the development of extraocular musele.The extraocular muscle injection of IGFBP-4 may cause the dysfunction and morphologic abnormality of extraocular muscle.

Cerebral lymphatic blockage aggravates apoptosis of cortical neurons after subarachnoid hemorrhage in rats in vivo / 生理学报

This present study was performed to investigate the influence of cerebral lymphatic blockage (CLB) on apoptosis of cortical neurons after subarachnoid hemorrhage (SAH) in rats in vivo. Healthy adult Wistar rats were randomly divided into normal control group, SAH group and SAH+CLB group. SAH model was made by double injection of autologous blood into the cisterna magna. On the third day after the second cisternal injection, morphological changes of cortical cells were observed by hematoxylin-eosin (HE) combined with propidium iodide (PI) staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was applied to determine in situ apoptosis in the cerebral cortex. Immunohistochemistry was conducted to detect the expression of Caspase-3 and Bcl-2 in cortical neurons. HE and PI staining showed that cortical neurons of SAH rats were partly shrinkable; the nuclei showed wavy, folded or wrinkled appearance, and some nuclei had the shape of crescent. The cortical neurons in SAH+CLB group distributed sparsely and the nuclear fragmentation, apoptotic bodies were found, surrounded by the formation of vacuoles. The numbers of TUNEL-positive cells in SAH group and SAH+CLB group were higher than that in the normal control group, while the number in SAH+CLB group was significantly higher than that in the SAH group. Caspase-3 expressions in SAH group and SAH+CLB group were higher than that in the normal control group, while the expression in SAH+CLB group was significantly higher than that in the SAH group. Bcl-2 expressions in SAH group and SAH+CLB group were higher than that in the normal control group, while the expression in the SAH+CLB group was significantly lower than that in SAH group. The results obtained suggest that CLB exacerbates the apoptosis of cortical neurons in rats after SAH by up-regulating Caspase-3 expression and down-regulating Bcl-2 expression.

Comparison of viremia formation between guinea-pigs infected with wild and attenuated (SA14-14-2) Japanese encephalitis viruses / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the viremia formation in guinea-pigs infected with wild type and attenuated Japanese encephalitis virus (JEV).</p><p><b>METHODS</b>Guniea pigs were inoculated intraperitoneally with different wild JEV strains and the attenuated vaccine strain and its parent virulent strain. Viremia was detected on different days following virus inoculation.</p><p><b>RESULTS</b>All the guinea-pigs inoculated with the wild JEV strains induced different levels of viremia (1.00-3.40 Lg pfu) on the 1st and 3rd day post inoculation. Using a virus titer of 10(4) pfu for inoculation, the animals inoculated with the SA14 parent strain induced relatively high viremia (10(2.4)-10(3.4) pfu), however no viremia coulds be detected on any tested days.</p><p><b>CONCLUSION</b>The degree of viremia in guinea pigs can be used as a new method to evaluate the attenuation of JEV.</p>

Studies on the biological and genetic characteristics of a highly neurovirulent Japanese encephalitis virus strain SA4 / 病毒学报

The biological and genetic characteristics of a highly neurovirulent JE virus strain SA4 were studied. Mice were inoculated intracerebrally with strain SA4 and SA14, and observed for 14 days, respectively. On different days, mice brains were harvested for titrations of the virus content in the brains. Full-length genome of SA4 was sequenced and compared with SA14 as well as other JE virus strains in the world. The results indicated that the mice inoculated by SA4 induced sickness and death more rapidly (24 hours faster) than those induced by the SA14. The virus titers in the brains of mice infected with SA4 were 0.5-1.0 lg PFU/mL higher than that infected with SA14. The sequence comparison indicated that the nucleotide and amino acid homology between SA4 and the other 21 JE strains were 84.6%-99.0% and 95.2%-99.7% respectively. Comparison with strain SA14 revealed that there were 17 amino acid differences between the two strains, of which 5 were in the E protein region. The results demonstrate that strain SA4 is a highly neurovirulent strain. The substitutions of the 17 amino acids in the SA4 strain can be the molecular basis for the biological characteristics of high neurovirulence.

Cerebral lymphatic blockage aggravates apoptosis of hippocampal neurons induced by cerebrospinal fluid from experimental subarachnoid hemorrhage / 生理学报

This work was performed to determine the role of cerebral lymphatic drainage pathway in the development of neural injury following subarachnoid hemorrhage (SAH). SAH and cerebral lymphatic blockage (CLB) models in adult New Zealand rabbits were used. Cerebrospinal fluid (CSF) was obtained from experimental animals 5 d after modeling and was added into cultured rat hippocampal neurons. The neurons were randomly divided into blank control, normal CSF, SAH, and SAH+CLB groups. At different points of time, lactate dehydrogenase (LDH) leakage was detected by colorimetric method. Flow cytometry was used to detect the apoptosis of neurons. Expressions of Bax and heat-shock protein 70 (Hsp70) were determined by immunohistochemical staining. LDH leakage detection revealed that, compared with blank control group, CSF from normal rabbit did not damage the neurons, whereas the leakage of LDH increased in SAH group and SAH+CLB group. The increasing effect was more obvious in SAH+CLB group than that in SAH group. Normal CSF did not induce the apoptosis of neurons, whereas neuron apoptosis was found in SAH group and the apoptosis was even more severe in SAH+CLB group. Bax and Hsp70 protein expressions were found in both SAH and SAH+CLB groups. Expression of Bax protein in SAH+CLB group was stronger than that in SAH group in a time-dependent manner. At 0.5 h and 1 h, the expression of Hsp70 protein in SAH+CLB group was stronger than that in SAH group, whereas the expression became weaker at 2 h and 4 h. These results suggest that blockage of cerebral lymphatic drainage pathway deteriorates the damage of neurons treated with CSF from SAH, indicating this pathway may act as an endogenous protective role in SAH.

Study on the phenotypic characteristics of Japanese encephalitis virus strains isolated from different years / 病毒学报

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.

Construction and application of subgenomic replicon vectors of Japanese encephalitis virus / 病毒学报

Based on the infectious clone of JEV vaccine SA14-14-2, the subgenomic replicons pCTCJEV, pCTMJEV with large deletions in the structural region were constructed. Then they were transfected into BHK-21 cell, the RNA replication of JEV subgenome can be monitored by RT-PCR and the non-structural protein can be found expressed in the cell by IFA. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive-strand and the negative-strand JEV RNA, we inserted an enhanced green fluorescence protein (EGFP) gene into the 3'-UTR of pCTCJEV, pCTMJEV under the control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA. After transfection, the EGFP fluorescence could be seen under the fluorescence microscope 1 day later,and maintained for more than a week with no apparent cytopathic effect. The constructed JEV replicons would provide valuable tools to provide a possible vector for a long-lasting RNA virus expression system.

Construction of infectious Japanese encephalitis virus clone based on the cDNA template of the attenuated live vaccine production strain SA14-14-2 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct infectious Japanese encephalitis virus (JEV) based on the in vitro-ligated cDNA template of the vaccine strain SA14-14-2, and identify the virus.</p><p><b>METHODS</b>Full-length genomic cDNA of JEV SA14-14-2 strain was ligated and then RNA was transcribed in vitro, the infective virus was obtained by transfecting the RNA into Vero cells and identified.</p><p><b>RESULTS</b>The infective clone of JEV was constructed, the virulence was weaker than the wild virus.</p><p><b>CONCLUSION</b>It was possible to construct infectious clone from the production strain of live attenuated Japanese B encephalitis vaccine.</p>

Research on Culex tritaeniorhynchus and Culex pipiens quinquefasciatus intrathoracically infected with attenuated Japanese encephalitis virus SA14-14-2 vaccine strain / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To determine if the attenuated Japanese encephalitis (JE) virus SA14-14-2 vaccine strain interacts efficiently with Culex tritaeniorhynchus and Culex pipiens quinquefasciatus, and further to acquire a new knowledge of its characteristics and safety for human beings.</p><p><b>METHODS</b>Laboratory colonies of the two species of mosquitoes were set up and were inoculated intrathoracically with the attenuated vaccine virus and wild JE virus (Nak), both of which were used with different dilution from 10(-1) to 10(-9). Subsequently, the virus titers in the mosquitoes were detected by the plaque assay.</p><p><b>RESULTS</b>Inoculated with the vaccine strain, two species of mosquitoes were infected with the titers ranged from 10(0)-10(-3), and the maximum titers in Culex tritaeniorhynchus and Culex pipiens quinquefasciatus were 4.48 logPFU/ml and 5.63 logPFU/ml, respectively. Inoculated with wild JE virus, Culex pipiens quinquefasciatus was infected with titers ranged from 10(0)-10(-5), and the maximum titer in the mosquitoes was 6.59; Culex tritaeniorhynchus was infected with titers ranged from 10(0)-10(-4) and the maximum titer was 5.74 logPFU/ml.</p><p><b>CONCLUSION</b>By intrathoracic infection, the attenuated JE virus SA14-14-2 vaccine strain can replicate in both species of mosquitoes.</p>