The implementation of an active inquiry learning centered "7E" teaching mode in the cell biology course enhances the learning effects of postgraduate students.

The primary objective of science postgraduate education is to foster students' capacity for creative thinking and problem-solving, particularly in the context of scientific research quality. In order to achieve this goal, the "7E" teaching mood has been implemented in the cell biology course for postgraduate students to promote student-centered active inquiry learning instead of breaking away from traditional indoctrination-based teaching methods. This study demonstrates that the implementation of the "7E" teaching mode, through content programming, process design, and effect evaluation, effectively meets the needs of the majority of students, fosters their interest in learning, enhances their performance in comprehensive questioning, and enhances their innovative abilities in scientific research. Consequently, this research offers a theoretical framework and practical foundation for the development of the "7E" teaching mode in postgraduate courses, aiming to cultivate highly skilled scientific professionals.

The effect of hypoxia on Hydrogen Sulfide concentration of brain tissue in AD transgenic mice and its mechanism.

OBJECTIVE: Research has shown that hydrogen sulfide (H2S) plays a protective role in many diseases of the nervous system. The aim of this study is to investigate the effect of hypoxia on endogenous H2S concentration in the cerebral cortex of Alzheimer's disease (AD) transgenic mice and its mechanism. METHODS: AD transgenic mice were raised in closed boxes and pure nitrogen was introduced to reduce the oxygen concentration to 8%-10%, establishing an animal model of hypoxia. Oxygen partial pressure was measured with an oxygen meter. The expression of cystathionine-ß-synthase (CBS) in cerebral cortex tissue was determined by Western blot, and H2S concentration was measured by a modified methylene blue method. RESULTS: (1) Hypoxia down-regulated CBS expression in cerebral cortex tissue of AD transgenic mice (p < 0.05). (2) The concentration of H2S in the cerebral cortex tissue of the hypoxic transgenic group was significantly lower than that of the Control group (p < 0.01). (3) Overexpression of CBS reversed the hypoxia-induced decrease of H2S concentration in the cerebral cortex tissue of AD transgenic mice (p < 0.01). CONCLUSIONS: Hypoxia decreased the concentration of endogenous H2S in the cerebral cortex tissue of AD transgenic mice by down-regulating the expression of CBS.

Tumor Cell-Specific and Lipase-Responsive Delivery of Hydrogen Sulfide for Sensitizing Chemotherapy of Pancreatic Cancer.

The inability of small molecule drugs to diffuse into tumor interstitium is responsible for the relatively low effectiveness of chemotherapy. Herein, a hydrogen sulfide (H2S) gas-involved chemosensitization strategy is proposed for pancreatic cancer treatment by developing a tumor-specific lipase-responsive nanomedicine based on aptamer-conjugated DATS/Dox co-loaded PCL-b-PEO micelle (DA/D@Ms-A). After receptor-mediated endocytosis and subsequent digestion of PCL blocks by intracellular lipase, the nanomedicine releases Dox and DATS, which then react with intracellular glutathione to produce H2S. The cytotoxicity result indicates that H2S can enhance Dox chemotherapy efficiency owing to the synergetic therapeutic effect of Dox and H2S. Moreover, the nanomedicine is featured with well tumor penetration capability benefitting from the targeting ability of aptamers and high in vivo biocompatibility due to the high density of PEO and biodegradable PCL. The nanomedicine capable of synergetic gas-chemotherapy holds great potential for pancreatic cancer treatment.

Protective effects of isorhamnetin on N2a cell against endoplasmic reticulum stress-induced injury is mediated by PKCε.

Endoplasmic reticulum stress (ERS)-induced intracellular calcium (Ca2+) overload and ROS burst plays a critical role in apoptosis. Protein kinase C epsilon (PKCε) is involved in regulating the homeostasis of Ca2+ and ROS production. isorhamnetin (Iso), as an ROS scavenger, effectively inhibit apoptosis, but the mechanism is still unclear. This study was to investigate whether Iso can inhibit ERS-induced apoptosis in N2a cells, and the protective effects are involved in PKCε-mediated Ca2+ homeostasis and inhibition of ROS. The effects of Iso against ERS injury in N2a cells were detected by cell viability, the levels of Ca2+, apoptosis and reactive oxygen species (ROS). The protein GRP78 expression levels were measured by western blot assay. The results showed that Iso can reduce ERS-induced injury by inhibiting Ca2+ overload, reducing the generation of ROS and decreasing apoptosis. In addition, Iso can promote PKCε phosphorylation, and εV1-2 (a PKCε inhibitor) drastically attenuated the protective effects of Iso against ERS injury in N2a cells. In conclusion, we firstly demonstrated that Iso can elicit protective effects against ERS injury in N2a cells and these effects are mediated at least in part via PKCε pathway.

Design, Synthesis and Evaluation of the Antidepressant and Anticonvulsant Activities of Triazole-Containing Benzo[d]oxazoles.

BACKGROUND: Epilepsy and depression are two of the common diseases seriously threatening life and health of human. A shared neurobiological substrate led to the bidirectional relationship and high comorbid occurrence of the two disorders. Recently, an increasing number of patients with epilepsy (PWE) require some form of antidepressant medication. However, most of the available antidepressants are inadequate for PWE for some reasons. So, the search for novel and increasingly effective drugs with anticonvulsant and antidepressant activities is necessary. METHODS: A series of 2-substituted-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazoles (5a-p) were designed and synthesized. Their anticonvulsant activities were evaluated using maximal electroshock shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. Their antidepressant activities were screened with the forced swimming test (FST). RESULTS: All the compounds showed anti-MES activities in different degree, among which 5g and 5j were the most promising one with ED50 value of 31.7 and 12.7 mg/kg, respectively. What's more, 5g and 5j also exhibited nice anti-scPTZ activities and low neurotoxicity. Interestingly, these compounds also showed good antidepressant activities in FST. And the efficacy of 5g were also confirmed by a tail suspension test and a open field test. The pretreatment of thiosemicarbazide (an inhibitor of γ- aminobutyric acid synthesis enzyme) significantly increased the ED50 of 5g in MES and reversed the reductions in the immobility time of 5g in FST. CONCLUSION: Triazole-containing benzo[d]oxazole is a good skeleton to develop compounds with both anticonvulsant and antidepressant activities. We have got the compound 5g, which display remarkable antidepressant and anticonvulsant activities, and the GABAergic system was involved in the action mechanism of 5g.

MeHg Suppressed Neuronal Potency of Hippocampal NSCs Contributing to the Puberal Spatial Memory Deficits.

Hippocampal neurogenesis-related structural damage, particularly that leading to defective adult cognitive function, is considered an important risk factor for neurodegenerative and psychiatric diseases. Normal differentiation of neurons and glial cells during development is crucial in neurogenesis, which is particularly sensitive to the environmental toxicant methylmercury (MeHg). However, the exact effects of MeHg on hippocampal neural stem cell (hNSC) differentiation during puberty remain unknown. This study investigates whether MeHg exposure induces changes in hippocampal neurogenesis and whether these changes underlie cognitive defects in puberty. A rat model of methylmercury chloride (MeHgCl) exposure (0.4 mg/kg/day, PND 5-PND 33, 28 days) was established, and the Morris water maze was used to assess cognitive function. Primary hNSCs from hippocampal tissues of E16-day Sprague-Dawley rats were purified, identified, and cloned. hNSC proliferation and differentiation and the growth and morphology of newly generated neurons were observed by MTT and immunofluorescence assays. MeHg exposure induced defects in spatial learning and memory accompanied by a decrease in number of doublecortin (DCX)-positive cells in the dentate gyrus (DG). DCX is a surrogate marker for newly generated neurons. Proliferation and differentiation of hNSCs significantly decreased in the MeHg-treated groups. MeHg attenuated microtubule-associated protein-2 (MAP-2) expression in neurons and enhanced the glial fibrillary acidic protein (GFAP)-positive cell differentiation of hNSCs, thereby inducing degenerative changes in a dose-dependent manner. Moreover, MeHg induced deficits in hippocampus-dependent spatial learning and memory during adolescence as a consequence of decreased generation of DG neurons. Our findings suggested that MeHg exposure could be a potential risk factor for psychiatric and neurodegenerative diseases.

Calcium sensing receptor mediated the excessive generation of ß-amyloid peptide induced by hypoxia in vivo and in vitro.

Hypoxia played an important role in the pathogenesis of AD. Hypoxia increased Aß formation, then caused Alzheimer's disease. Calcium sensing receptor (CaSR) was involved in the regulation of cell growth, differentiation, hormonal secretion and other physiological function. Increasing evidence supported CaSR might play a more prominent role in susceptibility to AD, but the role of CaSR in Aß overproduction induced by hypoxia and its mechanisms remain unclear. To investigate whether CaSR mediated the overproduction of Aß induced by hypoxia, immunoblot and immunochemistry were employed to determine the expression of CaSR and BACE1 in hippocampal neurons and tissue and Ca(2+) image system was used to measure [Ca(2+)]i in hippocampal neurons. The content of Aß was detected with ELISA kits. Our research found that hypoxia increased the expression of CaSR in hippocampal neurons and tissue and [Ca(2+)]i in hippocampal neurons. Calhex 231, a selective blocher of CaSR, inhibited the increase in [Ca(2+)]i induced by hypoxia. Hypoxia or GdCl3, an agonist of CaSR, increased the expression of BACE1 in hippocampal neurons and tissue, but Calhex 231 or Xesto C (a selective inhibitor of IP3 receptor) partly prevented hypoxia-induced BACE1 overexpression. Hypoxia or GdCl3 increased the content of Aß42 and Aß40 in hippocampal tissue, however Calhex 231 or Xesto C prevented hypoxia-induced the overproduction of Aß42 and Aß40 partly. Based on the above data, we suggested that hypoxia increased [Ca(2+)]i by elevated CaSR expression to promote BACE1 expression, thereby resulting in the overproduction of Aß42 and Aß40.

Uptake of silica nanoparticles: neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells.

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 µg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular ß-amyloid 1-42 (Aß(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-ß-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3ß at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3ß. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.

Hydrogen sulfide prevents OGD/R-induced apoptosis via improving mitochondrial dysfunction and suppressing an ROS-mediated caspase-3 pathway in cortical neurons.

Hydrogen sulfide (H2S), an endogenous gaseous mediator, has been shown to have protective effects against neuronal damage caused by brain ischemia. In this study, we explored the potential effects of H2S on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal apoptosis and the possible mechanisms. We find that sodium hydrosulfide (NaHS, a donator of H2S) prevents OGD/R-induced intracellular reactive oxygen species (ROS) elevation and activation of caspase-3 in cultured mouse cortical neurons. The pretreatment of N-acetyl-l-cysteine (NAC, an ROS scavenger) also prevents OGD/R-induced activation of caspase-3. Both NaHS and NAC counteract OGD/R-induced decline in mitochondria membrane potential (MMP). Additionally, NaHS, NAC or N-Acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO, a caspase-3 inhibitor), is shown to significantly inhibit OGD/R-induced neuronal apoptosis. These data suggest that H2S can protect against OGD/R-induced neuronal apoptosis through improving mitochondria dysfunction and suppressing an ROS-activated caspase-3 signaling pathway.

Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H2O2-activated calcium signaling pathway in mouse hippocampal neurons.

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H(2)S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O(2), 5% CO(2) and 93% N(2) at 37 °C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H(2)S) for 30 min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca(2+)](i)), and pretreatment with NaHS prevented the increase in [Ca(2+)](i). Additionally, polyethylene glycol (PEG)-catalase (a H(2)O(2) scavenger) but not PEG-SOD (an O(2)(-) scavenger) conferred an inhibitory effect similar to H(2)S on the hypoxia-induced increase in [Ca(2+)](i). Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP(3)) receptor blocker xestospongin C. Taken together, these findings suggest that H(2)S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H(2)O(2))-activated Ca(2+) signaling pathway in mouse hippocampal neurons.

Hyperphosphorylation and accumulation of neurofilament proteins in transgenic mice with Alzheimer presenilin 1 mutation.

Neurofilaments (NFs) are hyperphosphorylated and accumulate in Alzheimer's disease (AD) brains. In this study, employing the transgenic mouse model, we explored the effect of presenilin 1 (PS-1) mutation on the phosphorylation and distribution of NFs. Western blot analysis showed that there was a significant increase in the phosphorylation of NF-H and NF-M subunits with a concomitant increase in phosphorylated c-Jun N-terminal protein kinase 1/2 (JNK1/2) mitogen-activated protein kinase (MAPK) in hippocampus of PS-1 transgenic mice compared to that of wild-type littermates. Immunohistochemical analysis revealed that phosphorylated NFs accumulated throughout the hippocampal neurons of the transgenic mice. These findings suggest that PS-1 mutation may induce hyperphosphorylation and accumulation of NFs via a JNK1/2-involved mechanism.

Ca2+ oscillation frequency regulates agonist-stimulated gene expression in vascular endothelial cells.

A physiological membrane-receptor agonist typically stimulates oscillations, of varying frequencies, in cytosolic Ca2+ concentration ([Ca2+]i). Whether and how [Ca2+]i oscillation frequency regulates agonist-stimulated downstream events, such as gene expression, in non-excitable cells remain unknown. By precisely manipulating [Ca2+]i oscillation frequency in histamine-stimulated vascular endothelial cells (ECs), we demonstrate that the gene expression of vascular cell adhesion molecule 1 (VCAM1) critically depends on [Ca2+]i oscillation frequency in the presence, as well as the absence, of histamine stimulation. However, histamine stimulation enhanced the efficiency of [Ca2+]i-oscillation-frequency-regulated VCAM1 gene expression, versus [Ca2+]i oscillations alone in the absence of histamine stimulation. Furthermore, a [Ca2+]i oscillation frequency previously observed to be the mean frequency in histamine-stimulated ECs was found to optimize VCAM1 mRNA expression. All the above effects were abolished or attenuated by blocking histamine-stimulated generation of intracellular reactive oxygen species (ROS), another intracellular signaling pathway, and were restored by supplementary application of a low level of H2O2. Endogenous NF-kappaB activity is similarly regulated by [Ca2+]i oscillation frequency, as well as its co-operation with ROS during histamine stimulation. This study shows that [Ca2+]i oscillation frequency cooperates with ROS to efficiently regulate agonist-stimulated gene expression, and provides a novel and general strategy for studying [Ca2+]i signal kinetics in agonist-stimulated downstream events.

Homocysteine induces tau hyperphosphorylation in rats.

Epidemiological and clinical data suggest that homocysteine (Hcy) may increase the risk of Alzheimer's disease, but the underlying mechanisms are elusive. To investigate the effect of Hcy on phosphorylation of tau, we injected Hcy into the lateral cerebral ventricle of rats. We found that level of the hyperphosphorylated tau at PHF-1 (Ser396/404) and tau-1 (Ser198/199/202) epitopes was elevated prominently at 6, 9, and 12 h after the injection, and it was recovered to normal at 24 h. Simultaneously, the level of protein phosphatase-2A catalytic subunit (PP-2Ac) was reduced markedly as compared with control. These results imply that Hcy may induce hyperphosphorylation of tau with PP-2Ac involved mechanism.