Prenatal alcohol exposure disrupts the development of normal fetal respiratory function, but whether it perturbs respiratory rhythmical discharge activity is unclear. Furthermore, it is unknown whether the 5-hydroxytr...Prenatal alcohol exposure disrupts the development of normal fetal respiratory function, but whether it perturbs respiratory rhythmical discharge activity is unclear. Furthermore, it is unknown whether the 5-hydroxytryptamine 2A receptor(5-HT2AR) is involved in the effects of prenatal alcohol exposure. In the present study, pregnant female rats received drinking water containing alcohol at concentrations of 0%, 1%, 2%, 4%, 8% or 10%(v/v) throughout the gestation period. Slices of the medulla from 2-day-old neonatal rats were obtained to record respiratory rhythmical discharge activity. 5-HT2 AR protein and m RNA levels in the pre-B?tzinger complex of the respiratory center were measured by western blot analysis and quantitative RT-PCR, respectively. Compared with the 0% alcohol group, respiratory rhythmical discharge activity in medullary slices in the 4%, 8% and 10% alcohol groups was decreased, and the reduction was greatest in the 8% alcohol group. Respiratory rhythmical discharge activity in the 10% alcohol group was irregular. Thus, 8% was the most effective alcohol concentration at attenuating respiratory rhythmical discharge activity. These findings suggest that prenatal alcohol exposure attenuates respiratory rhythmical discharge activity in neonatal rats by downregulating 5-HT2 AR protein and m RNA levels.展开更多
Through investigating the effect of mild hypothermia on activity of nitric oxide snythase (NOS) in cortical neurons and glycemia levels of neonatal rats with hypoxic ischemic brain damage (HIBD). We studied the mechan...Through investigating the effect of mild hypothermia on activity of nitric oxide snythase (NOS) in cortical neurons and glycemia levels of neonatal rats with hypoxic ischemic brain damage (HIBD). We studied the mechanism of protecting hypoxic ischemic neurons of mild hypothermia. We established neonatal rat HIBD models, used NOS immunohistochemistry and glycemia determination by micromethod. The number of cortical NOS positive neurons after hypoxic ischemia was significantly decreased as compared with controls. The glycemia levels was significantly increased than that controls. No significant difference was found in number of cortical NOS positive neurons and glycemia levels between 31℃ and 34℃ mild hypothemia. The results imply that hypothermia can decrease overproduction of NO through inhibiting the increase of the activity of NOS, and increase the glycemia levels, thus protect the hypoxic ischemic neurons.展开更多
BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and...BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.展开更多
BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and iden...BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.AIM To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.METHODS First,curved tip operating scissors were used to dissect brain tissues from new born rats(2 to 3 d)and the brain tissues were cut into approximately 1 mm^(3)sections.Filter the single cell suspension through a nylon mesh(200-mesh)and culture the sections in suspensions.Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques.Second,identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation.BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells.Different NSCs specific antibodies(anti-nestin,NF200,NSE and GFAP antibodies)were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.RESULTS Brain derived cells from newborn rats(2 to 3 d)proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging.When BrdU was incorporated into the 5th generation of passaged cells,positive BrdU cells and nestin cells were observed by immunofluorescence staining.After induction of dissociation using 5%fetal bovine serum,positive NF200,NSE and GFAP cells were observed by immunofluorescence staining.CONCLUSION This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.展开更多
In the current study,we sought to investigate whether T-type Ca^(2+)channels(TCCs)in the brain are involved in generating post-anesthetic hyperexcitatory behaviors(PAHBs).We found that younger rat pups(postnatal days ...In the current study,we sought to investigate whether T-type Ca^(2+)channels(TCCs)in the brain are involved in generating post-anesthetic hyperexcitatory behaviors(PAHBs).We found that younger rat pups(postnatal days 9-11)had a higher incidence of PAHBs and higher PAHB scores than older pups(postnatal days16-18)during emergence from sevoflurane anesthesia.The power spectrum of the theta oscillations(4 Hz-8 Hz)in the prefrontal cortex was significantly enhanced in younger pups when PAHBs occurred,while there were no significant changes in older pups.Both the power of theta oscillations and the level of PAHBs were significantly reduced by the administration of TCC inhibitors.Moreover,the sensitivity of TCCs in the medial dorsal thalamic nucleus to sevoflurane was found to increase with age by investigating the kinetic properties of TCCs in vitro.TCCs were activated by potentiated GABAergic depolarization with a sub-anesthetic dose of sevoflurane(1%).These data suggest that(1)TCCs in the brain contribute to the generation of PAHBs and the concomitant electroencephalographic changes;(2)the stronger inhibitory effect of sevoflurane contributes to the lack of PAHBs in older rats;and(3)the contribution of TCCs to PAHBs is not mediated by a direct effect of sevoflurane on TCCs.展开更多
The clinical presentation of schizophrenia involves a variety of symptoms, which in many cases include hallucinations and delusions. Experimentally revealed alterations in both pre-pulse inhibition (PPI) and latent in...The clinical presentation of schizophrenia involves a variety of symptoms, which in many cases include hallucinations and delusions. Experimentally revealed alterations in both pre-pulse inhibition (PPI) and latent inhibition (LI) are also apparent in individuals afflicted with this disorder. Many have speculated that altered synaptic connections are, in part, responsible for this subset of behavioral abnormalities. We have previously reported that neonatal chronic low-dose injections of domoic acid (DOM) produce adult rats with deficits in PPI and LI. The current study was conducted to determine whether this toxin-treatment would alter the degree of apoptosis occurring in the developing brain. Results revealed significant decreases in caspase-3 within the right prelimbic cortex (PrL) in both male and female DOM-treated rats suggesting that even modest alterations in glutamate (Glu) signaling during critical periods of central nervous system (CNS) maturation will modify ontogenetic processes in the prefrontal cortex (PFC) of the juvenile rat.展开更多
BACKGROUND: Generally speaking, anesthesia is often used in gravid body and it has been already proved that many kind of medicine can result in malformation. OBJECTIVE: To explore embryonic skeleton development and ne...BACKGROUND: Generally speaking, anesthesia is often used in gravid body and it has been already proved that many kind of medicine can result in malformation. OBJECTIVE: To explore embryonic skeleton development and neonatal learning and memory of rats anesthetized with pentobarbital sodium in gravid rats. DESIGN: A randomized control trial. SETTING: Laboratory Animal Center of Xuzhou Medical College. MATERIALS: A total of 80 adult female SD rats, of clean grade and weighing 220-240 g, were selected in this study. The main reagents were detailed as follows: pentobarbital sodium (Shanghai Xingzhi Chemical Plant, batch number: 921019); MG-2 maze test apparatus (Zhangjiagang Biomedical Instrument Factory); somatotype microscope (Beijing Taike Instrument Co., Ltd.). METHODS: ① A total of 160 SD rats of half males and females were selected in this study. All rats were copulated. The day that the plug was checked out in the vagina next day was looked as the first day of pregnancy. Gravid rats were divided randomly into four groups, including early anesthesia group, second anesthesia group, late anesthesia group and control group with 20 in each group. Rats in the early anesthesia group were injected with 25 mg/kg soluble pentobarbitone on the 7th day of pregnancy for once; rats in the second anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 7th and the 14th days of pregnancy for once; rats in the late anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 14th day of pregnancy for once; rats in the control group did not treat with anything. The time of anesthetizing was controlled in 3 to 4 hours and ether was absorbed while the time was not enough. ② Half of each group was sacrificed on day 20th of pregnancy and the fetus was taken out to be stained with alizarin red S. After stained, the fetal skeleton was examined. The learning and memorizing of one-month rats that were given birth by the rest gravid rats were tested through electric mare method. Determine their study ability according to their correct rate of 90% or above of arrival at the safe area in 20 s. After they finally learned to arrive at the safe area correctly, test them once more in 24 hours and record the correct rate of 15 times. MAIN OUTCOME MEASURES: The rate of malformation in fetus and ability of learning and memory in one-month rats. RESULTS: A total of 80 female rats were anesthetized in this experiment. Totally 490 immature rats were tested with maze testing machine and 196 fetuses were stained with alizarin red S to observe the development of their skeleton. However, one of the 80 female rats was led to death because of overdose. ① Malformation experiment: Learning ability of second anesthesia group was evidently different from the control group while the other two groups were not in the electric mare method. The fetal skeleton malformation rate of three experimental groups was 87.0%, 60.9% and 17.9%, respectively, while it was 5.6% in the control group. ② Electric mare method: Times of rats which arrived at the safe regions were respectively 49.0±31.0, 68.0±35.0, 47.0±31.0 and 44.0±21.0 in early anesthesia group, second anesthesia group, late anesthesia group and control group; and then, there was significant difference between the second anesthesia group and the control group (P < 0.05). Exact rates of memory of rats were respectively (64.36±14.35)%, (62.15±18.33)%, (54.19±12.28)% and (68.24±15.91)% in early anesthesia group, second anesthesia group, late anesthesia group and control group; and then, there were no significant differences as compared with the control group (P > 0.05). CONCLUSION: The influence of anesthesia with pentobarbital sodium is obvious in fetal skeleton development and learning and memory ability.展开更多
Clinically,peripheral nerve reconstructions in neonates are most frequently applied in brachial plexus birth injuries.Most surgical concepts,however,have investigated nerve reconstructions in adult animal models.The i...Clinically,peripheral nerve reconstructions in neonates are most frequently applied in brachial plexus birth injuries.Most surgical concepts,however,have investigated nerve reconstructions in adult animal models.The immature neuromuscular system reacts differently to the effects of nerve lesion and surgery and is poorly investigated due to the lack of reliable experimental models.Here,we describe an experimental forelimb model in the neonatal rat,to study these effects on both the peripheral and central nervous systems.Within 24 hours after birth,three groups were prepared:In the nerve transfer group,a lesion of the musculocutaneous nerve was reconstructed by selectively transferring the ulnar nerve.In the negative control group,the musculocutaneous nerve was divided and not reconstructed and in the positive control group,a sham surgery was performed.The animal's ability to adapt to nerve lesions and progressive improvement over time were depict by the Bertelli test,which observes the development of grooming.Twelve weeks postoperatively,animals were fully matured and the nerve transfer successfully reinnervated their target muscles,which was indicated by muscle force,muscle weight,and cross sectional area evaluation.On the contrary,no spontaneous regeneration was found in the negative control group.In the positive control group,reference values were established.Retrograde labeling indicated that the motoneuron pool of the ulnar nerve was reduced following nerve transfer.Due to this post-axotomy motoneuron death,a diminished amount of motoneurons reinnervated the biceps muscle in the nerve transfer group,when compared to the native motoneuron pool of the musculocutaneous nerve.These findings indicate that the immature neuromuscular system behaves profoundly different than similar lesions in adult rats and explains reduced muscle force.Ultimately,pathophysiologic adaptations are inevitable.The maturing neuromuscular system,however,utilizes neonatal capacity of regeneration and seizes a variety of compensation mechanism to restore a functional extremity.The above described neonatal rat model demonstrates a constant anatomy,suitable for nerve transfers and allows all standard neuromuscular analyses.Hence,detailed investigations on the pathophysiological changes and subsequent effects of trauma on the various levels within the neuromuscular system as well as neural reorganization of the neonatal rat may be elucidated.This study was approved by the Ethics Committee of the Medical University of Vienna and the Austrian Ministry for Research and Science(BMWF-66.009/0187-WF/V/3 b/2015)on March 20,2015.展开更多
Hypoxic-ischemic brain damage(HIBD)is a common cause of infant death.The purpose of our research was to explore the immunoregulatory mechanism of placenta-derived mesenchymal stem cells(PD-MSCs)in HIBD treatment.Seven...Hypoxic-ischemic brain damage(HIBD)is a common cause of infant death.The purpose of our research was to explore the immunoregulatory mechanism of placenta-derived mesenchymal stem cells(PD-MSCs)in HIBD treatment.Seven-day-old rat pups were randomly divided into HIBD,PD-MSC,fibroblast,and control groups.Forty-eight hours after HIBD induction,cells at a density of 5×104 cells/10µl were injected into the cerebral tissue in the PD-MSC and fibroblast groups.The TNF-α,interleukin-17(IL-17),interferon-γ(IFN-γ),and IL-10 levels were detected through quantitative real-time polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).Regulatory T cell(Tregs)populations were detected through flow cytometry,and forkhead box P3(Foxp3)was measured through western blot analysis.Behavioral tests and gross and pathological examinations showed that PD-MSC treatment exerted significantly stronger neuroprotective effects than the other treatments.The expression levels of pro-inflammatory cytokines were substantially upregulated after HI injury.Compared with fibroblast treatment,PD-MSC treatment inhibited the production of pro-inflammatory cytokines and increased the production of IL-10 in the ischemic hemispheres and peripheral blood serum(all P<0.01).Flow cytometry results showed a notable increase in the number of Tregs within the spleen of the HIBD group.Moreover,the number of Tregs and the Foxp3 expression levels were higher in the PD-MSC treatment group than in the HIBD and fibroblast groups(all P<0.01).Our research suggests that the mechanism of PD-MSC treatment for HIBD partially involves inflammatory response suppression.展开更多
Background:Oxidative stress is involved in the development of hypoxic-ischemic brain damage(HIBD).In this study,we investigated the therapeutic efcts of placenta-derived mesenchymal stem cells(PD-MSCs)and explored the...Background:Oxidative stress is involved in the development of hypoxic-ischemic brain damage(HIBD).In this study,we investigated the therapeutic efcts of placenta-derived mesenchymal stem cells(PD-MSCs)and explored the N F-E2-related factor-2/heme oxygenase-1(Nrf2/HO-1)signaling pathway in treating HIBD.Methods:P7 rats were subjected to hypoxic-ischemic brain injury and randomly divided into four groups(control,HIBD,HIBD+PD-MSCs,and HIBD+fbroblasts).Forty-eight hours after the induction of HIBD,5×10^(5)of PD-MSCs were injected into cerebral tissue in the HIBD+PD-MSCs group,while the same dose of fibroblas ts were injected in the HIBD+fibroblasts group.Morris Water Maze,gross and pathological changes were tested at P28.The level of malondialdehyde(MDA)was detected in rats'hippocampus.RT-PCR and western blot analysis were used to evaluate the changes of Nrf2/HO-1.Results:The HIBD group showed significantly longer escape latency and a lower frequency of original platform crossing in the Morris Water M laze compared with the control group.Rats receiving PD MSCs showed significant improvement of HIBD.The pathological changes were evident after HIBD,but ameliorated in the PD-MSCs group.Compared with the control group,HO-1 and Nrf2 were up-regulated at gene and protein levels in the HI brain,beginning at 6 hours and peaking at 48 hours(P<0.05).The expression of HO-1 and Nrf2 in the PD-MSCs treatment group was more pronounced than in the HBD group(P<0.01).PD MSCs also decreased MDA production in the brain tissue.Conclusion:These results demonstrate that PD-MSCs have neuroprotective effect during the treatment of HIBD and that the mechanism may be partly due to alleviating oxidative stress.展开更多
Objective To investigate the effect of sodium hydroxybutyrate(GHB) on expression of hippocampal N-methyl-D-aspartate receptor 1 subunit (NR1) mRNA in the neonatal rat with hypoxic-ischemic insult. Methods One hundred ...Objective To investigate the effect of sodium hydroxybutyrate(GHB) on expression of hippocampal N-methyl-D-aspartate receptor 1 subunit (NR1) mRNA in the neonatal rat with hypoxic-ischemic insult. Methods One hundred eighty seven-day(7d) old Sprague Dawley rat pups of either sex weighting 10 g～14 g were randomly assigned to sham group(S), normal saline group(C),GHB 50 mg/kg group(γ50),GHB 100 mg/kg group(γ100)and GHB 200 mg/kg group(γ200),n=6.The procedure for the induction of hypoxia-ischemia was done according to the way described by Rice:under ether anesthesia,pups were subjected to left carotid artery ligated followed by 2 h of hypoxic exposure(O2:N2=8﹪:92﹪).The pups in S group were only aparted left carotid artery which was not obturated and no hypoxia.GHB 50 mg/kg, 100 mg/kg, 200 mg/kg was administered i.p immediately after hypoxia,then a time every 8h inγ50、γ100 and γ200 group respectively.The same volume normal saline was administered i.p in the same way in S and C group.Reverse transcriptase polymerase chain reaction(RT-PCR) was used to analyze the expression of NR1 mRNA semi-quantificationally in the left hippocampus at 2 h、6 h、12 h、1 d、3 d、7 d after insult in S、C、γ50、γ100 and γ200 group. Results The difference of expression of NR1 mRNA between S and C group wasn’t significant.At any time after insult, The differences of expression of NR1 mRNA among C and three GHB group were not significant too. Conclusion Hypoxia-ischemia and GHB didn’t significantly affect the expression of NR1 mRNA in neonatal rat hippocampus.展开更多
Objective To investigate the effects and underlying mechanisms of Panax quinquefolium saponin(PQS)on energy deficiency in hypoxia-reperfusion(H/R)induced cardiomyocytes.Methods The H/R injury involved hypoxia for 3 h ...Objective To investigate the effects and underlying mechanisms of Panax quinquefolium saponin(PQS)on energy deficiency in hypoxia-reperfusion(H/R)induced cardiomyocytes.Methods The H/R injury involved hypoxia for 3 h and then reperfusion for 2 h.Cardiomyocytes recruited from neonatal rat ventricular myocytes(NRVMs)were randomly divided into control,H/R,H/R+compound C(C.C),H/R+PQS,and H/R+C.C+PQS groups.BrdU assay,lactase dehydrogenase(LDH)leakage and early apoptosis rate were evaluated to assess cell damages.Contents of high energy phosphate compounds were conducted to detect the energy production.Protein expression levels of adenosine monophosphate-activated protein kinase a(AMPKα),glucose transporter 4(GLUT4),phosphate fructose kinase 2(PFK2),fatty acid translocase/cluster of differentiation 36(FAT/CD36),and acetyl CoA carboxylase 2(ACC2)in the regulatory pathways were measured by Western blotting.Immunofluorescence staining of GLUT4 and FAT/CD36 was used to observe the mobilization of metabolic transporters.Results PQS(50 mg/L)pretreatment significantly alleviated H/R-induced inhibition of NRVMs viability,up-regulation of LDH leakage,acceleration of early apoptosis,and reduction of energy production(P<0.05).Compared with the H/R group,up-regulated expression of AMPKα,GLUT4,PFK2,FAT/CD36 and ACC2 were observed,and more GLUT4 and FAT/CD36 expressions were detected on the membrane in the H/R+PQS group(P<0.05).These effects of PQS on H/R-induced NRVMs were eliminated in the H/R+C.C+PQS group(P<0.05).Conclusion PQS has prominent advantages in protecting NRVMs from H/R-induced cell damages and energy metabolic disorders,by activation of AMPKα-mediated GLUT4-PFK2 and FAT/CD36-ACC2 pathways.展开更多
Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs(miRNAs) play multiple roles in biological processes...Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs(miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34 c-5 p on cardiac hypertrophy and the mechanism involved. The expression of miR-34 c-5 p was proved to be elevated in heart tissues from isoprenaline(ISO)-infused mice. ISO also promoted miR-34 c-5 p level in primary cultures of neonatal rat cardiomyocytes(NRCMs). Transfection with miR-34 c-5 p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor(Anf) and β-myosin heavy chain(β-Mhc) in NRCMs. In contrast, treatment with miR-34 c-5 p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34 c-5 p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34 c-5 p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34 c-5 p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4 B(ATG4 B) was identified as a direct target of miR-34 c-5 p, and miR-34 c-5 p was certified to interact with 3’untranslated region of Atg4 b mRNA by dual-luciferase reporter assay. miR-34 c-5 p reduced the expression of ATG4 B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34 c-5 p abolished the detrimental effects of ISO by restoring ATG4 B and increasing autophagy. In conclusion, our findings illuminate that miR-34 c-5 p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4 B and autophagy. It suggests that regulation of miR-34 c-5 p may offer a new way for handling hypertrophy-related cardiac dysfunction.展开更多
基金the Natural Science Foundation of Henan Province in China,No.102102310156the Foundation of Xinxiang Technology Bureau in China,No.ZG14004
文摘Prenatal alcohol exposure disrupts the development of normal fetal respiratory function, but whether it perturbs respiratory rhythmical discharge activity is unclear. Furthermore, it is unknown whether the 5-hydroxytryptamine 2A receptor(5-HT2AR) is involved in the effects of prenatal alcohol exposure. In the present study, pregnant female rats received drinking water containing alcohol at concentrations of 0%, 1%, 2%, 4%, 8% or 10%(v/v) throughout the gestation period. Slices of the medulla from 2-day-old neonatal rats were obtained to record respiratory rhythmical discharge activity. 5-HT2 AR protein and m RNA levels in the pre-B?tzinger complex of the respiratory center were measured by western blot analysis and quantitative RT-PCR, respectively. Compared with the 0% alcohol group, respiratory rhythmical discharge activity in medullary slices in the 4%, 8% and 10% alcohol groups was decreased, and the reduction was greatest in the 8% alcohol group. Respiratory rhythmical discharge activity in the 10% alcohol group was irregular. Thus, 8% was the most effective alcohol concentration at attenuating respiratory rhythmical discharge activity. These findings suggest that prenatal alcohol exposure attenuates respiratory rhythmical discharge activity in neonatal rats by downregulating 5-HT2 AR protein and m RNA levels.
文摘Through investigating the effect of mild hypothermia on activity of nitric oxide snythase (NOS) in cortical neurons and glycemia levels of neonatal rats with hypoxic ischemic brain damage (HIBD). We studied the mechanism of protecting hypoxic ischemic neurons of mild hypothermia. We established neonatal rat HIBD models, used NOS immunohistochemistry and glycemia determination by micromethod. The number of cortical NOS positive neurons after hypoxic ischemia was significantly decreased as compared with controls. The glycemia levels was significantly increased than that controls. No significant difference was found in number of cortical NOS positive neurons and glycemia levels between 31℃ and 34℃ mild hypothemia. The results imply that hypothermia can decrease overproduction of NO through inhibiting the increase of the activity of NOS, and increase the glycemia levels, thus protect the hypoxic ischemic neurons.
文摘BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.
基金Project of Sichuan Department of Science and Technology,No.2016PJ552the Project of Luzhou Department of Science and Technology,No.2016-R-70(18/24)+1 种基金the Project of Southwest Medical University of Science and Technology,No.15073 and 2015-YJ021Orthopaedic diseases(Shang Antong)special research Project of Sichuan Medical Association,No.20220206070192.
文摘BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.AIM To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.METHODS First,curved tip operating scissors were used to dissect brain tissues from new born rats(2 to 3 d)and the brain tissues were cut into approximately 1 mm^(3)sections.Filter the single cell suspension through a nylon mesh(200-mesh)and culture the sections in suspensions.Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques.Second,identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation.BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells.Different NSCs specific antibodies(anti-nestin,NF200,NSE and GFAP antibodies)were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.RESULTS Brain derived cells from newborn rats(2 to 3 d)proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging.When BrdU was incorporated into the 5th generation of passaged cells,positive BrdU cells and nestin cells were observed by immunofluorescence staining.After induction of dissociation using 5%fetal bovine serum,positive NF200,NSE and GFAP cells were observed by immunofluorescence staining.CONCLUSION This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.
基金supported by the National Natural Science Foundation,Beijing,People’s Republic of China(81671058 and 81730031 to YW and 81401089 to MD)the National Research Foundation of Korea grants funded by the Republic of Korea(2019R1I1A1A01057744 to YK)the Foundation of Shanghai Municipal Science and Technology Commission(19ZR1407500 to FS)。
文摘In the current study,we sought to investigate whether T-type Ca^(2+)channels(TCCs)in the brain are involved in generating post-anesthetic hyperexcitatory behaviors(PAHBs).We found that younger rat pups(postnatal days 9-11)had a higher incidence of PAHBs and higher PAHB scores than older pups(postnatal days16-18)during emergence from sevoflurane anesthesia.The power spectrum of the theta oscillations(4 Hz-8 Hz)in the prefrontal cortex was significantly enhanced in younger pups when PAHBs occurred,while there were no significant changes in older pups.Both the power of theta oscillations and the level of PAHBs were significantly reduced by the administration of TCC inhibitors.Moreover,the sensitivity of TCCs in the medial dorsal thalamic nucleus to sevoflurane was found to increase with age by investigating the kinetic properties of TCCs in vitro.TCCs were activated by potentiated GABAergic depolarization with a sub-anesthetic dose of sevoflurane(1%).These data suggest that(1)TCCs in the brain contribute to the generation of PAHBs and the concomitant electroencephalographic changes;(2)the stronger inhibitory effect of sevoflurane contributes to the lack of PAHBs in older rats;and(3)the contribution of TCCs to PAHBs is not mediated by a direct effect of sevoflurane on TCCs.
文摘The clinical presentation of schizophrenia involves a variety of symptoms, which in many cases include hallucinations and delusions. Experimentally revealed alterations in both pre-pulse inhibition (PPI) and latent inhibition (LI) are also apparent in individuals afflicted with this disorder. Many have speculated that altered synaptic connections are, in part, responsible for this subset of behavioral abnormalities. We have previously reported that neonatal chronic low-dose injections of domoic acid (DOM) produce adult rats with deficits in PPI and LI. The current study was conducted to determine whether this toxin-treatment would alter the degree of apoptosis occurring in the developing brain. Results revealed significant decreases in caspase-3 within the right prelimbic cortex (PrL) in both male and female DOM-treated rats suggesting that even modest alterations in glutamate (Glu) signaling during critical periods of central nervous system (CNS) maturation will modify ontogenetic processes in the prefrontal cortex (PFC) of the juvenile rat.
文摘BACKGROUND: Generally speaking, anesthesia is often used in gravid body and it has been already proved that many kind of medicine can result in malformation. OBJECTIVE: To explore embryonic skeleton development and neonatal learning and memory of rats anesthetized with pentobarbital sodium in gravid rats. DESIGN: A randomized control trial. SETTING: Laboratory Animal Center of Xuzhou Medical College. MATERIALS: A total of 80 adult female SD rats, of clean grade and weighing 220-240 g, were selected in this study. The main reagents were detailed as follows: pentobarbital sodium (Shanghai Xingzhi Chemical Plant, batch number: 921019); MG-2 maze test apparatus (Zhangjiagang Biomedical Instrument Factory); somatotype microscope (Beijing Taike Instrument Co., Ltd.). METHODS: ① A total of 160 SD rats of half males and females were selected in this study. All rats were copulated. The day that the plug was checked out in the vagina next day was looked as the first day of pregnancy. Gravid rats were divided randomly into four groups, including early anesthesia group, second anesthesia group, late anesthesia group and control group with 20 in each group. Rats in the early anesthesia group were injected with 25 mg/kg soluble pentobarbitone on the 7th day of pregnancy for once; rats in the second anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 7th and the 14th days of pregnancy for once; rats in the late anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 14th day of pregnancy for once; rats in the control group did not treat with anything. The time of anesthetizing was controlled in 3 to 4 hours and ether was absorbed while the time was not enough. ② Half of each group was sacrificed on day 20th of pregnancy and the fetus was taken out to be stained with alizarin red S. After stained, the fetal skeleton was examined. The learning and memorizing of one-month rats that were given birth by the rest gravid rats were tested through electric mare method. Determine their study ability according to their correct rate of 90% or above of arrival at the safe area in 20 s. After they finally learned to arrive at the safe area correctly, test them once more in 24 hours and record the correct rate of 15 times. MAIN OUTCOME MEASURES: The rate of malformation in fetus and ability of learning and memory in one-month rats. RESULTS: A total of 80 female rats were anesthetized in this experiment. Totally 490 immature rats were tested with maze testing machine and 196 fetuses were stained with alizarin red S to observe the development of their skeleton. However, one of the 80 female rats was led to death because of overdose. ① Malformation experiment: Learning ability of second anesthesia group was evidently different from the control group while the other two groups were not in the electric mare method. The fetal skeleton malformation rate of three experimental groups was 87.0%, 60.9% and 17.9%, respectively, while it was 5.6% in the control group. ② Electric mare method: Times of rats which arrived at the safe regions were respectively 49.0±31.0, 68.0±35.0, 47.0±31.0 and 44.0±21.0 in early anesthesia group, second anesthesia group, late anesthesia group and control group; and then, there was significant difference between the second anesthesia group and the control group (P < 0.05). Exact rates of memory of rats were respectively (64.36±14.35)%, (62.15±18.33)%, (54.19±12.28)% and (68.24±15.91)% in early anesthesia group, second anesthesia group, late anesthesia group and control group; and then, there were no significant differences as compared with the control group (P > 0.05). CONCLUSION: The influence of anesthesia with pentobarbital sodium is obvious in fetal skeleton development and learning and memory ability.
基金supported by the Christian Doppler Research Association and the European Research Council under the European Union’s Horizon 2020 research and innovation program(both to OCA)。
文摘Clinically,peripheral nerve reconstructions in neonates are most frequently applied in brachial plexus birth injuries.Most surgical concepts,however,have investigated nerve reconstructions in adult animal models.The immature neuromuscular system reacts differently to the effects of nerve lesion and surgery and is poorly investigated due to the lack of reliable experimental models.Here,we describe an experimental forelimb model in the neonatal rat,to study these effects on both the peripheral and central nervous systems.Within 24 hours after birth,three groups were prepared:In the nerve transfer group,a lesion of the musculocutaneous nerve was reconstructed by selectively transferring the ulnar nerve.In the negative control group,the musculocutaneous nerve was divided and not reconstructed and in the positive control group,a sham surgery was performed.The animal's ability to adapt to nerve lesions and progressive improvement over time were depict by the Bertelli test,which observes the development of grooming.Twelve weeks postoperatively,animals were fully matured and the nerve transfer successfully reinnervated their target muscles,which was indicated by muscle force,muscle weight,and cross sectional area evaluation.On the contrary,no spontaneous regeneration was found in the negative control group.In the positive control group,reference values were established.Retrograde labeling indicated that the motoneuron pool of the ulnar nerve was reduced following nerve transfer.Due to this post-axotomy motoneuron death,a diminished amount of motoneurons reinnervated the biceps muscle in the nerve transfer group,when compared to the native motoneuron pool of the musculocutaneous nerve.These findings indicate that the immature neuromuscular system behaves profoundly different than similar lesions in adult rats and explains reduced muscle force.Ultimately,pathophysiologic adaptations are inevitable.The maturing neuromuscular system,however,utilizes neonatal capacity of regeneration and seizes a variety of compensation mechanism to restore a functional extremity.The above described neonatal rat model demonstrates a constant anatomy,suitable for nerve transfers and allows all standard neuromuscular analyses.Hence,detailed investigations on the pathophysiological changes and subsequent effects of trauma on the various levels within the neuromuscular system as well as neural reorganization of the neonatal rat may be elucidated.This study was approved by the Ethics Committee of the Medical University of Vienna and the Austrian Ministry for Research and Science(BMWF-66.009/0187-WF/V/3 b/2015)on March 20,2015.
基金supported by grants from the Dongying City Technology Development Project(Grant No.2011106).
文摘Hypoxic-ischemic brain damage(HIBD)is a common cause of infant death.The purpose of our research was to explore the immunoregulatory mechanism of placenta-derived mesenchymal stem cells(PD-MSCs)in HIBD treatment.Seven-day-old rat pups were randomly divided into HIBD,PD-MSC,fibroblast,and control groups.Forty-eight hours after HIBD induction,cells at a density of 5×104 cells/10µl were injected into the cerebral tissue in the PD-MSC and fibroblast groups.The TNF-α,interleukin-17(IL-17),interferon-γ(IFN-γ),and IL-10 levels were detected through quantitative real-time polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).Regulatory T cell(Tregs)populations were detected through flow cytometry,and forkhead box P3(Foxp3)was measured through western blot analysis.Behavioral tests and gross and pathological examinations showed that PD-MSC treatment exerted significantly stronger neuroprotective effects than the other treatments.The expression levels of pro-inflammatory cytokines were substantially upregulated after HI injury.Compared with fibroblast treatment,PD-MSC treatment inhibited the production of pro-inflammatory cytokines and increased the production of IL-10 in the ischemic hemispheres and peripheral blood serum(all P<0.01).Flow cytometry results showed a notable increase in the number of Tregs within the spleen of the HIBD group.Moreover,the number of Tregs and the Foxp3 expression levels were higher in the PD-MSC treatment group than in the HIBD and fibroblast groups(all P<0.01).Our research suggests that the mechanism of PD-MSC treatment for HIBD partially involves inflammatory response suppression.
基金supported by grants of Shandong Province Natural Science Foundation(ZR2011HM007&2013GSF11812)Innovation Fund Project of Shandong University(2012ZD023&2014QY003-11)the Dongying City Technology Development Project(Grant 2011106)
文摘Background:Oxidative stress is involved in the development of hypoxic-ischemic brain damage(HIBD).In this study,we investigated the therapeutic efcts of placenta-derived mesenchymal stem cells(PD-MSCs)and explored the N F-E2-related factor-2/heme oxygenase-1(Nrf2/HO-1)signaling pathway in treating HIBD.Methods:P7 rats were subjected to hypoxic-ischemic brain injury and randomly divided into four groups(control,HIBD,HIBD+PD-MSCs,and HIBD+fbroblasts).Forty-eight hours after the induction of HIBD,5×10^(5)of PD-MSCs were injected into cerebral tissue in the HIBD+PD-MSCs group,while the same dose of fibroblas ts were injected in the HIBD+fibroblasts group.Morris Water Maze,gross and pathological changes were tested at P28.The level of malondialdehyde(MDA)was detected in rats'hippocampus.RT-PCR and western blot analysis were used to evaluate the changes of Nrf2/HO-1.Results:The HIBD group showed significantly longer escape latency and a lower frequency of original platform crossing in the Morris Water M laze compared with the control group.Rats receiving PD MSCs showed significant improvement of HIBD.The pathological changes were evident after HIBD,but ameliorated in the PD-MSCs group.Compared with the control group,HO-1 and Nrf2 were up-regulated at gene and protein levels in the HI brain,beginning at 6 hours and peaking at 48 hours(P<0.05).The expression of HO-1 and Nrf2 in the PD-MSCs treatment group was more pronounced than in the HBD group(P<0.01).PD MSCs also decreased MDA production in the brain tissue.Conclusion:These results demonstrate that PD-MSCs have neuroprotective effect during the treatment of HIBD and that the mechanism may be partly due to alleviating oxidative stress.
文摘Objective To investigate the effect of sodium hydroxybutyrate(GHB) on expression of hippocampal N-methyl-D-aspartate receptor 1 subunit (NR1) mRNA in the neonatal rat with hypoxic-ischemic insult. Methods One hundred eighty seven-day(7d) old Sprague Dawley rat pups of either sex weighting 10 g～14 g were randomly assigned to sham group(S), normal saline group(C),GHB 50 mg/kg group(γ50),GHB 100 mg/kg group(γ100)and GHB 200 mg/kg group(γ200),n=6.The procedure for the induction of hypoxia-ischemia was done according to the way described by Rice:under ether anesthesia,pups were subjected to left carotid artery ligated followed by 2 h of hypoxic exposure(O2:N2=8﹪:92﹪).The pups in S group were only aparted left carotid artery which was not obturated and no hypoxia.GHB 50 mg/kg, 100 mg/kg, 200 mg/kg was administered i.p immediately after hypoxia,then a time every 8h inγ50、γ100 and γ200 group respectively.The same volume normal saline was administered i.p in the same way in S and C group.Reverse transcriptase polymerase chain reaction(RT-PCR) was used to analyze the expression of NR1 mRNA semi-quantificationally in the left hippocampus at 2 h、6 h、12 h、1 d、3 d、7 d after insult in S、C、γ50、γ100 and γ200 group. Results The difference of expression of NR1 mRNA between S and C group wasn’t significant.At any time after insult, The differences of expression of NR1 mRNA among C and three GHB group were not significant too. Conclusion Hypoxia-ischemia and GHB didn’t significantly affect the expression of NR1 mRNA in neonatal rat hippocampus.
基金Supported by the National Natural Science Foundation of China(No.81273934 and No.81874410)。
文摘Objective To investigate the effects and underlying mechanisms of Panax quinquefolium saponin(PQS)on energy deficiency in hypoxia-reperfusion(H/R)induced cardiomyocytes.Methods The H/R injury involved hypoxia for 3 h and then reperfusion for 2 h.Cardiomyocytes recruited from neonatal rat ventricular myocytes(NRVMs)were randomly divided into control,H/R,H/R+compound C(C.C),H/R+PQS,and H/R+C.C+PQS groups.BrdU assay,lactase dehydrogenase(LDH)leakage and early apoptosis rate were evaluated to assess cell damages.Contents of high energy phosphate compounds were conducted to detect the energy production.Protein expression levels of adenosine monophosphate-activated protein kinase a(AMPKα),glucose transporter 4(GLUT4),phosphate fructose kinase 2(PFK2),fatty acid translocase/cluster of differentiation 36(FAT/CD36),and acetyl CoA carboxylase 2(ACC2)in the regulatory pathways were measured by Western blotting.Immunofluorescence staining of GLUT4 and FAT/CD36 was used to observe the mobilization of metabolic transporters.Results PQS(50 mg/L)pretreatment significantly alleviated H/R-induced inhibition of NRVMs viability,up-regulation of LDH leakage,acceleration of early apoptosis,and reduction of energy production(P<0.05).Compared with the H/R group,up-regulated expression of AMPKα,GLUT4,PFK2,FAT/CD36 and ACC2 were observed,and more GLUT4 and FAT/CD36 expressions were detected on the membrane in the H/R+PQS group(P<0.05).These effects of PQS on H/R-induced NRVMs were eliminated in the H/R+C.C+PQS group(P<0.05).Conclusion PQS has prominent advantages in protecting NRVMs from H/R-induced cell damages and energy metabolic disorders,by activation of AMPKα-mediated GLUT4-PFK2 and FAT/CD36-ACC2 pathways.
基金supported by grants from the National Natural Science Foundation of China (81872860,81673433,and82070268)Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y093,China)+4 种基金National Major Special Projects for the Creation and Manufacture of New Drugs (2019ZX09301104,China)National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province,2017B090903004,China)Special Program for Applied Science and Technology of Guangdong Province (2015B020232009,China)Guangdong Basic and Applied Basic Research Foundation(2020A1515011512,China)Young Teacher Training Program of Sun Yat-sen University (18ykpy26,China)。
文摘Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs(miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34 c-5 p on cardiac hypertrophy and the mechanism involved. The expression of miR-34 c-5 p was proved to be elevated in heart tissues from isoprenaline(ISO)-infused mice. ISO also promoted miR-34 c-5 p level in primary cultures of neonatal rat cardiomyocytes(NRCMs). Transfection with miR-34 c-5 p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor(Anf) and β-myosin heavy chain(β-Mhc) in NRCMs. In contrast, treatment with miR-34 c-5 p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34 c-5 p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34 c-5 p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34 c-5 p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4 B(ATG4 B) was identified as a direct target of miR-34 c-5 p, and miR-34 c-5 p was certified to interact with 3’untranslated region of Atg4 b mRNA by dual-luciferase reporter assay. miR-34 c-5 p reduced the expression of ATG4 B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34 c-5 p abolished the detrimental effects of ISO by restoring ATG4 B and increasing autophagy. In conclusion, our findings illuminate that miR-34 c-5 p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4 B and autophagy. It suggests that regulation of miR-34 c-5 p may offer a new way for handling hypertrophy-related cardiac dysfunction.
