Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

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.

Transforming growth factor-beta 1 enhances discharge activity of cortical neurons

Transforming growth factor-beta 1(TGF-β1)has been extensively studied for its pleiotropic effects on central nervous system diseases.The neuroprotective or neurotoxic effects of TGF-β1 in specific brain areas may depend on the pathological process and cell types involved.Voltage-gated sodium channels(VGSCs)are essential ion channels for the generation of action potentials in neurons,and are involved in various neuroexcitation-related diseases.However,the effects of TGF-β1 on the functional properties of VGSCs and firing properties in cortical neurons remain unclear.In this study,we investigated the effects of TGF-β1 on VGSC function and firing properties in primary cortical neurons from mice.We found that TGF-β1 increased VGSC current density in a dose-and time-dependent manner,which was attributable to the upregulation of Nav1.3 expression.Increased VGSC current density and Nav1.3 expression were significantly abolished by preincubation with inhibitors of mitogen-activated protein kinase kinase(PD98059),p38 mitogen-activated protein kinase(SB203580),and Jun NH2-terminal kinase 1/2 inhibitor(SP600125).Interestingly,TGF-β1 significantly increased the firing threshold of action potentials but did not change their firing rate in cortical neurons.These findings suggest that TGF-β1 can increase Nav1.3 expression through activation of the ERK1/2-JNK-MAPK pathway,which leads to a decrease in the firing threshold of action potentials in cortical neurons under pathological conditions.Thus,this contributes to the occurrence and progression of neuroexcitatory-related diseases of the central nervous system.

Genome-wide profiling of long noncoding RNA expression patterns and CeRNA analysis in mouse cortical neurons infected with different strains of borna disease virus

Borna disease virus 1(BoDV-1)is neurotropic prototype of Bornaviruses causing neurological diseases and maintaining persistent infection in brain cells of mammalian species.Long non-coding RNA(lncRNA)is transcript of more than 200 nucleotides without proteincoding function regulating various biological processes as proliferation,apoptosis,cell migration and viral infection.However,regulatory of lncRNAs in BoDV-1 infection remains unknown.To identify differential expression profiles and predict functions of lncRNA in BoDV-1 infection,microarray data showed that 3528 lncRNAs and 2661 lncRNAs were differentially expressed in Strain V and Hu-H1 BoDV-infected groups compared with control groups,respectively.Gene Ontology(GO)and pathway analysis suggested that differential lncRNAs may be involved in regulation of metabolic,biological regulation,cellular process,endocytosis,viral infections and cell adhesion processes,cancer in both BoDV-infected strains.ENSMUST00000128469 was found down-regulated in both BoDV-infected groups compared with control groups consistent with microarray(p<0.05).ceRNA analysis indicated possible interaction networks as ENSMUST00000128469/miR-22-5p,miR-206-3p,miR-302b-5p,miR-302c-3p,miR-1a-3p/Igf1.Igf1 was found up-regulated in both BoDV-infected groups compared with control groups(p<0.05).Possible functions of predicted target mRNAs and miRNAs of ENSMUST00000128469 were involved in cell proliferation,transcriptional misregulation and proteoglycan pathways enriched in cancer.lncRNA may be involved in regulation of Hu-H1 inhibited cell proliferation and promoted apoptosis through NF-kB,JNK/MAPK signaling,BCL2 and CDK6/E2F1 pathways different from Strain V.Possible interaction networks as ENSMUST00000128469/miR-22-5p,miR-206-3p,miR-302b-5p,miR-302c-3p,miR-1a-3p/Igf1 may involve in regulation of cell proliferation,apoptosis,and cancer.

Activation of sigma-1 receptor enhances synaptosomal Ca^(2+) via L-type C^(2+) channel in cortical neuron

Sigma-1 receptors are unique receptors that are postulated to act as intracellular amplifiers for signal transduction within cells of the nervous system. The present paper studied the

SIRT1 Protects Cortical Neurons from Shear Stress Induced Mechanical Injury

Emerging evidence implicates that SIRT1,one of NAD-dependent protein deacetylase,is involved in the regulation of neuronal survival.However,the role of SIRT1 in mechanical injury is still unclear.Here,we first established a mechanical injury model of rat cortical neurons through exposing to 10 dyn/cm 2 laminar shear stress for 4 h.At the same time,we found that SIRT1 expression increased during the process of shear stress induced neuronal injury.SIRT1 overexpression in cortical neurons significantly decreased the percentage of died cells induced by shear stress.Our results demonstrate that SIRT1 may play potential neuroprotective effects in shear stress induced mechanical injury.

Effects of extracellular vesicles from mesenchymal stem cells on oxygen-glucose deprivation/reperfusioninduced neuronal injury

BACKGROUND: Small extracellular vesicles (sEVs) from bone marrow mesenchymal stemcells (BMSCs) have shown therapeutic potential for cerebral ischemic diseases. However, themechanisms by which BMSC-derived sEVs (BMSC-sEVs) protect neurons against cerebral ischemia/reperfusion (I/R) injury remain unclear. In this study, we explored the neuroprotective effects ofBMSC-sEVs in the primary culture of rat cortical neurons exposed to oxygen-glucose deprivation andreperfusion (OGD/R) injury.METHODS: The primary cortical neuron OGD/R model was established to simulate the processof cerebral I/R in vitro. Based on this model, we examined whether the mechanism through whichBMSC-sEVs could rescue OGD/R-induced neuronal injury.RESULTS: BMSC-sEVs (20 μg/mL, 40 μg/mL) significantly decreased the reactive oxygenspecies (ROS) productions, and increased the activities of superoxide dismutase (SOD) and glutathioneperoxidase (GPx). Additionally, BMSC-sEVs prevented OGD/R-induced neuronal apoptosis in vivo, asindicated by increased cell viability, reduced lactate dehydrogenase (LDH) leakage, decreased terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining-positivecells, down-regulated cleaved caspase-3, and up-regulated Bcl-2/Bax ratio. Furthermore, Westernblot and flow cytometry analysis indicated that BMSC-sEV treatment decreased the expression ofphosphorylated calcium/calmodulin-dependent kinase II (p-CaMK II)/CaMK II, suppressed the increaseof intracellular calcium concentration ([Ca2+]i) caused by OGD/R in neurons.CONCLUSIONS: These results demonstrate that BMSC-sEVs have signifi cant neuroprotectiveeff ects against OGD/R-induced cell injury by suppressing oxidative stress and apoptosis, and Ca2+/CaMK II signaling pathways may be involved in this process.

Exosomes derived from human induced pluripotent stem cell-derived neural progenitor cells protect neuronal function under ischemic conditions

Compared with other stem cells,human induced pluripotent stem cells-derived neural progenitor cells(iPSC-NPCs)are more similar to cortical neurons in morphology and immunohistochemistry.Thus,they have greater potential for promoting the survival and growth of neurons and alleviating the proliferation of astrocytes.Transplantation of stem cell exosomes and stem cells themselves have both been shown to effectively repair nerve injury.However,there is no study on the protective effects of exosomes derived from iPSC-NPCs on oxygen and glucose deprived neurons.In this study,we established an oxygen-glucose deprivation model in embryonic cortical neurons of the rat by culturing the neurons in an atmosphere of 95%N2 and 5%CO2 for 1 hour and then treated them with iPSC-NPC-derived exosomes for 30 minutes.Our results showed that iPSC-NPC-derived exosomes increased the survival of oxygen-and glucose-deprived neurons and the level of brain-derived neurotrophic factor in the culture medium.Additionally,it attenuated oxygen and glucose deprivation-induced changes in the expression of the PTEN/AKT signaling pathway as well as synaptic plasticity-related proteins in the neurons.Further,it increased the length of the longest neurite in the oxygen-and glucose-deprived neurons.These findings validate the hypothesis that exosomes from iPSCNPCs exhibit a neuroprotective effect on oxygen-and glucose-deprived neurons by regulating the PTEN/AKT signaling pathway and neurite outgrowth.This study was approved by the Animal Ethics Committee of Sir Run Run Shaw Hospital,School of Medicine,Zhejiang University,China(approval No.SRRSH20191010)on October 10,2019.

A Novel Retrograde AAV Variant for Functional Manipulation of Cortical Projection Neurons in Mice and Monkeys

Retrograde adeno-associated viruses(AAVs)are capable of infecting the axons of projection neurons and serve as a powerful tool for the anatomical and functional characterization of neural networks.However,few retro-grade AAV capsids have been shown to offer access to cor-tical projection neurons across different species and enable the manipulation of neural function in non-human primates(NHPs).Here,we report the development of a novel retro-grade AAV capsid,AAV-DJ8R,which efficiently labeled cortical projection neurons after local administration into the striatum of mice and macaques.In addition,intrastriatally injected AAV-DJ8R mediated opsin expression in the mouse motor cortex and induced robust behavioral alterations.Moreover,AAV-DJ8R markedly increased motor cortical neuron firing upon optogenetic light stimulation after viral delivery into the macaque putamen.These data demonstrate the usefulness of AAV-DJ8R as an efficient retrograde tracer for cortical projection neurons in rodents and NHPs and indicate its suitability for use in conducting functional interrogations.

Silencing miRNA-324-3p protects against cerebral ischemic injury via regulation of the GATA2/A1R axis

Previous studies have suggested that miR-324-3p is related to the pathophysiology of cerebral ischemia,but the mechanism underlying this relationship is unclea r.In this study,we found that miR-324-3p expression was decreased in patients with acute ischemic stroke and in in vitro and in vivo models of ischemic stro ke.miR-324-3p agomir potentiated ischemic brain damage in rats subjected to middle cerebral artery occlusion,as indicated by increased infarct volumes and cell apoptosis rates and greater neurological deficits.In a PC12 cell oxygen-glucose deprivation/reoxygenation model,a miR-324-3 p mimic decreased cell viability and expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX and rates of cell apoptosis,whereas treatment with a miR-324-3p inhibitor had the opposite effects.Silencing miR-324-3p increased adenosine A1 receptor(A1R)expression thro ugh regulation of GATA binding protein 2(GATA2).These findings suggest that silencing miR-324-3p reduces ischemic brain damage via the GATA2/A1R axis.

CREB Protein Expressed Differently in the Frontal Cortices of Datura stramonium Treated Rats: Implication for Addiction and Neurodegeneration

Background: cAMP response element-binding protein (CREB) is one of the cellular transcription factors found in neurons. CREB is also important for the survival of neurons, and has an important role in the development of drug addiction. Datura stramonium (DS) is a tropical ubiquitous plant commonly used to increase the intoxication of certain beverages for recreational purposes. The seeds of this plant are very toxic and may produce addiction on prolong usage. This research investigated the effects of administration of high doses of DS seeds on the expression of CREB protein in both male and female rats’ frontal cortices and its implication in addiction and neurodegeneration. Materials and Methods: The study was conducted with a total of 24 male and female Wistar rats weighing 200 g - 250 g. The rats were divided into three groups of 8 rats each. Each group was further divided into four sub-groups of 2 rats each. Ethanolic dried seed extract of DS was diluted in normal saline and administered intraperitoneally (i.p.) to the treatment groups. The treated sub-groups received 750 mg/kg of DS extract once in group 1, twice in group 2 and thrice in group 3 daily for 4 weeks respectively, while the control sub-groups received i.p. normal saline concurrently for the same duration of time. The rats were euthanized and an analysis of variance (ANOVA) was computed to detect a significant main difference of DS effect on CREB expression for each group, while post hoc Bonferroni Test compared CREB protein expression between male and female groups. Result: There were significant differences in the expression of CREB protein between the sub-groups and between the male and female rats of treated sub-group (p < 0.05) compared to the controls. There was a decrease in the female treated sub-groups and an increase in the male treated sub-groups compared to the respective controls. Conclusion: High doses of DS administration for a prolong time may affect the expressions of CREB protein differently in male and female treated rats which may consequently lead to addiction and neurodegeneration affecting frontal cortex neurons.

Postsynaptic Excitation of Prefrontal Cortical Pyramidal Neurons by Hypocretins/Orexins

Hypocretins/orexins are crucial for the regulation of wakefulness by the excitatory actions on multiple subcortical arousal systems. In prefrontal cortex,