Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory.

Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

Amygdala neuronal dyshomeostasis via 5-HT receptors mediates mood and cognitive defects in Alzheimer's disease.

Behavioral changes or neuropsychiatric symptoms (NPSs) are common features in dementia and are associated with accelerated cognitive impairment and earlier deaths. However, how NPSs are intertwined with cognitive decline remains elusive. In this study, we identify that the basolateral amygdala (BLA) is a key brain region that is associated with mood disorders and memory decline in the AD course. During the process from pre- to post-onset in AD, the dysfunction of parvalbumin (PV) interneurons and pyramidal neurons in the amygdala leads to hyperactivity of pyramidal neurons in the basal state and insensitivity to external stimuli. We further demonstrate that serotonin (5-HT) receptors in distinct neurons synergistically regulate the BLA microcircuit of AD rather than 5-HT levels, in which both restrained inhibitory inputs by excessive 5-HT1AR signaling in PV interneurons and depolarized pyramidal neurons via upregulated 5-HT2AR contribute to aberrant neuronal hyperactivity. Downregulation of these two 5-HT receptors simultaneously enables neurons to resist ß-amyloid peptides (Aß) neurotoxicity and ameliorates the mood and cognitive defects. Therefore, our study reveals a crucial role of 5-HT receptors for regulating neuronal homeostasis in AD pathogenesis, and this would provide early intervention and potential targets for AD cognitive decline.

Stimulation of an entorhinal-hippocampal extinction circuit facilitates fear extinction in a post-traumatic stress disorder model.

Effective psychotherapy of post-traumatic stress disorder (PTSD) remains challenging due to the fragile nature of fear extinction, for which ventral hippocampal CA1 (vCA1) region is considered as a central hub. However, neither the core pathway nor the cellular mechanisms involved in implementing extinction are known. Here, we unveil a direct pathway, where layer 2a fan cells in the lateral entorhinal cortex (LEC) target parvalbumin-expressing interneurons (PV-INs) in the vCA1 region to propel low gamma-band synchronization of the LEC-vCA1 activity during extinction learning. Bidirectional manipulations of either hippocampal PV-INs or LEC fan cells sufficed fear extinction. Gamma entrainment of vCA1 by deep brain stimulation (DBS) or noninvasive transcranial alternating current stimulation (tACS) of LEC persistently enhanced the PV-IN activity in vCA1, thereby promoting fear extinction. These results demonstrate that the LEC-vCA1 pathway forms a top-down motif to empower low gamma-band oscillations that facilitate fear extinction. Finally, application of low gamma DBS and tACS to a mouse model with persistent PTSD showed potent efficacy, suggesting that the dedicated LEC-vCA1 pathway can be stimulated for therapy to remove traumatic memory trace.

[Effect of mulberry leaves extracts on glucose uptake of insulin-resistant HepG2 cells and the mechanism].

The effect and mechanism of mulberry leaves extracts (MLE) on glucose uptake of insulin-resistant HepG2 cells in vitro was explored. The insulin resistant models of HepG2 were induced by high concentration of insulin for 24 h. The models were incubated in a buffer containing mulberry leaves extracts. The glucose consumption was detected by glucose assay kits and the AMP-activated protein kinase (AMPK), Akt activation was examined by Western blotting. Mulberry leaves polysaccharides, mulberry leaves flavonoids and mulberry leaves extracts advanced glucose uptake of insulin-resistant HepG2 cells; Mulberry leaves extracts enhance phosphorylation of AMPK. Mulberry leaves extracts do not change the phosphorylation status of Akt. The glucose consumptions of insulin resistant model of HepG2 were promoted by mulberry leaves extracts. MLE stimulates HepG2 cell AMPK activity acutely without changing the Akt activity.

Aberrant miR-339-5p/neuronatin signaling causes prodromal neuronal calcium dyshomeostasis in mutant presenilin mice.

Mushroom spine loss and calcium dyshomeostasis are early hallmark events of age-related neurodegeneration, such as Alzheimer's disease (AD), that are connected with neuronal hyperactivity in early pathology of cognitive brain areas. However, it remains elusive how these key events are triggered at the molecular level for the neuronal abnormality that occurs at the initial stage of disease. Here, we identify downregulated miR-339-5p and its upregulated target protein, neuronatin (Nnat), in cortex neurons from the presenilin-1 M146V knockin (PSEN1-M146V KI) mouse model of familial AD (FAD). Inhibition of miR-339-5p or overexpression of Nnat recapitulates spine loss and endoplasmic reticulum calcium overload in cortical neurons with the PSEN1 mutation. Conversely, either overexpression of miR-339-5p or knockdown of Nnat restores spine morphogenesis and calcium homeostasis. We used fiber photometry recording during the object-cognitive process to further demonstrate that the PSEN1 mutant causes defective habituation in neuronal reaction in the retrosplenial cortex and that this can be rescued by restoring the miR-339-5p/Nnat pathway. Our findings thus reveal crucial roles of the miR-339-5p/Nnat pathway in FAD that may serve as potential diagnostic and therapeutic targets for early pathogenesis.

EphB2 mediates social isolation-induced memory forgetting.

Social isolation in adolescence leads to lasting deficits, including emotional and cognitive dysregulation. It remains unclear, however, how social isolation affects certain processes of memory and what molecular mechanisms are involved. In this study, we found that social isolation during the post-weaning period resulted in forgetting of the long-term fear memory, which was attributable to the downregulation of synaptic function in the hippocampal CA1 region mediated by EphB2, a receptor tyrosine kinase which involves in the glutamate receptor multiprotein complex. Viral-mediated EphB2 knockdown in CA1 mimicked the memory defects in group-housed mice, whereas restoration of EphB2 by either viral overexpression or resocialization reversed the memory decline in isolated mice. Taken together, our finding indicates that social isolation gives rise to memory forgetting by disrupting EphB2-mediated synaptic plasticity, which may provide a potential target for preventing memory loss caused by social isolation or loneliness.

[Separation and purification of total flavonoids in Smilax glabra by polyamide resins].

OBJECTIVE: To investigate the process of separating and purifying total flavonoids from Smilax glabra. METHODS: With the contents of total flavonoids, astilbin and engelitin as indices, the optimum process of separating and purifying flavonoids from Smilax glabra was screened by static and dynamic adsorption tests. RESULTS: The static saturated adsorption capacity of polyamide resin to flavonoids of Smilax glabra was 144 mg/g (saturated in 2 h). The dynamic adsorption capacity was 128.125 mg/g, the optimum conditions of elution were as follows: the adsorbed resin was washed by 20BV water firstly, then by 20BV 95% ethanol. The content of obtained total flavonoids reached 71.84%, as the content of astilbin was 14.32%, and the content of engelitin was 1. 08%. CONCLUSIONS: The total flavonoids of Smilax glabra is able to be easily separated and purified by polyamide resin under the optimum conditions above.

A neuronal molecular switch through cell-cell contact that regulates quiescent neural stem cells.

The quiescence of radial neural stem cells (rNSCs) in adult brain is regulated by environmental stimuli. However, little is known about how the neurogenic niche couples the external signal to regulate activation and transition of quiescent rNSCs. Here, we reveal that long-term excitation of hippocampal dentate granule cells (GCs) upon voluntary running leads to activation of adult rNSCs in the subgranular zone and thereby generation of newborn neurons. Unexpectedly, the role of these excited GC neurons in NSCs depends on direct GC-rNSC interaction in the local niche, which is through down-regulated ephrin-B3, a GC membrane-bound ligand, and attenuated transcellular EphB2 kinase-dependent signaling in the adjacent rNSCs. Furthermore, constitutively active EphB2 kinase sustains the quiescence of rNSCs during running. These findings thus elucidate the physiological significance of GC excitability on adult rNSCs under external environments and indicate a key-lock switch regulation via cell-cell contact for functional transition of rNSCs.

[Targeted imaging ability of a biotinylated imaging probe Biotin-S-S-Rhodol for breast cancer cells in vitro].

OBJECTIVE: To investigate performance of a biotinylated imaging probe 3a for targeted imaging of breast cancer cells. METHODS: Ultraviolet absorption spectrum and fluorescence spectrum were employed to analyze the spectral characteristics of 3a. The fluorescence spectrums of 3a treated with different concentrations of glutathione (GSH) were obtained to determine the sensibility of 3a to GSH. Flow cytometry was used to determine the cellular uptake of 3a by MCF-7 cells, MDA-MB-231 cells and Hs 578Bst cells in the presence or absence of biotin, and the imaging performance of 3a in the 3 cell lines was assessed under an inverted fluorescent microscope. The toxicity of 3a to the cells was evaluated using MTT method. RESULTS: 3a showed the strongest absorption peak at 510 nm, and its fluorescence emission signal was the strongest at 544 nm. As the concentration of GSH increased (0-6 mmol/L), 3a exhibited an increasing fluorescence signal at 544 nm. The cellular uptake of 3a was markedly higher in MDA-MB-231 cells and MCF-7 cells than in Hs 578Bst cells. The imaging studies showed that 3a had a good breast cancer cell-targeting property and produced clear images under fluorescent microscope. MTT assay demonstrated no obvious toxicity of 3a in Hs 578Bst cells even at the concentration of 20 µmol/L, but MCF-7 cells and MDA-MB-231 cells exposed to 2-20 µmol/L 3a showed a lowered cell viability. CONCLUSION: 3a is capable of targeted imaging of breast cancer cells mediated by biotin. 3a at the concentration of 2-20 µmol/L has minimal cytotoxicity to normal breast cells but can lower the viability of breast cancer cells.

[Deguelin inhibits proliferation and regulates the expression of MCM3-CDC45 in MCF-7 and H1299 cells in vitro].

OBJECTIVE: To observe the effects of deguelin on the proliferation of breast cancer MCF-7 cells and lung cancer H1299 cells in vitro and the expression of minichromosome maintenance protein 3 (MCM3) and CDC45 in the cells. METHODS: MTT assay was used to evaluate the proliferation of MCF-7 and H1299 cells exposed to different concentrations of deguelin for 48, 72 or 96 h. The growth of the cells was observed microscopically and the changes of MCM3 and CDC45 expressions in MCF-7 and H1299 cells following deguelin treatment were detected with fluorescence quantitative PCR. RESULTS: The proliferation of MCF-7 cells was significantly inhibited by exposure to 0.25, 0.5, 1, 5, 10, 30, and 50 µmol/L deguelin for 48, 72, and 96 h in a concentration- and time-dependent manner. In MCF-7 cells, the IC50 of deguelin at 48, 72, and 96 h was 9, 3, and 2 µmol/L, respectively. Deguelin treatments of H1299 cells at 0.5, 1, 5, 10, 30, 50, and 100 µmol/L also resulted in a concentration- and time-dependent inhibition of the cell growth with an IC50 at 96 h of 2 µmol/L. Optical microscopy of the cells revealed a decreased number of viable cells with obvious cell shrinkage following deguelin treatments. The expression of MCM3 and CDC45 were significantly reduced in the cells after deguelin treatments. CONCLUSION: Deguelin can inhibit the proliferation of MCF-7 and H1299 cells in vitro and down-regulate the expression of MCM3 and CDC45 in the cells.

Protective effects of Oviductus Ranae-containing serum on oxidative stress-induced apoptosis in rat ovarian granulosa cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Oviductus Ranae (OR) is a traditional Chinese medicine derived from Rana temporaria chensinensis David, and is known to have a wide variety of pharmacological effects. AIM OF THE STUDY: To investigate the function and mechanism of OR-containing serum in protecting rat ovarian granulosa cells from hydrogen peroxide (H2O2)-induced oxidative damage. MATERIALS AND METHODS: H2O2-treated granulosa cells were pretreated with OR-containing serum, and viability and proliferation assays were carried out using Cell Counting Kit-8 (CCK-8). Apoptotic granulosa cells were observed microscopically using 4',6-diamidino-2-phenylindole (DAPI), and the apoptotic ratio was quantified via Annexin V/ propidium iodide (PI) staining combined with flow cytometry. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in the cells were measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) and rhodamine 123, respectively, and analyzed by flow cytometry. Mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, and other apoptosis-related proteins (p53, Bcl-2, Bax, caspase-9, caspase-3), were detected by western blot analysis, and the related mRNA levels were detected using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The results revealed that treatment with OR-containing serum reduced apoptosis and mitochondrial membrane damage in H2O2-treated granulosa cells. The OR-containing serum interfered with H2O2-induced intracellular generation of ROS and loss of ΔΨm, which typically lead to apoptosis. Furthermore, the OR-containing serum down-regulated pro-apoptotic proteins such as p53, Bax, caspase-9, and caspase-3, while up-regulating the anti-apoptotic protein Bcl-2. Finally, the OR-containing serum increased phosphorylation of ERK1/2, and reduced JNK and p38 phosphorylation. CONCLUSIONS: OR-containing serum protected rat ovarian granulosa cells against H2O2-induced apoptosis, by reducing ROS production and improving mitochondrial membrane potential, through down-regulation of negative regulators of proliferation, activation of ERK1/2, and inhibition of the activity of JNK and p38.

[Preparation of magnetic solid liposome nanoparticles of paclitaxel].

AIM: To study a new way to prepare high-dosage paclitaxel entrapped magnetic targeted nanoparticles and evaluate its quality. METHODS: Fe3O4 nanoparticles are prepared by co-depositing, at the same time ultrasonic is used to decrease soft agglomerate of nanoparticles and increase disperse level of it. The property of nanoparticles surface is improved to make the integrating of liposome and nanoparticle to be tighter. At last, paclitaxel entrapped magnetic solid liposome nanoparticles have been prepared by microemulsion-curing under low-temperature. The loading efficiency and encapsulating rate were determined by reverse-phase high-perfomance chromatography. RESULTS: The nanoparticles have spherical shape. Diameter of nanoparticle ranged from 150 nm to 170 nm. 98.29% of the drug is entrapped in the particle. CONCLUSION: Magnetic susceptibility of nanoparticles is high, and the nanoparticles meet with the demand of targeted delivery system.

Deguelin-induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down-regulation of fibroblast growth factor receptor 4 activity.

Deguelin, a natural component derived from leguminous plants, has been used as pesticide in some regions. Accumulating evidence show that deguelin has promising chemopreventive and therapeutic activities against cancer cells. This study shows that low concentrations of deguelin can lead to significant delay in zebrafish embryonic development through growth inhibition and induction of apoptosis. Furthermore, we identified fibroblast growth factor receptor 4 (FGFR4) as the putative target of deguelin. The candidate was initially identified by a microarray approach and then validated through in vitro experiments using hormone-responsive (MCF-7) and nonresponsive (MDA-MB-231) human breast cancer cell lines. The results show that deguelin suppressed cell proliferation and induced apoptosis in both cancer cell lines, but not in Hs 578Bst cells, by blocking PI3K/AKT and mitogen-activated protein kinases (MAPK) signaling. The FGFR4 mRNA and protein level also diminished in a dose-dependent manner. Interestingly, we found that forced FGFR4 overexpression attenuated deguelin-induced proliferative suppression and apoptotic cell death in both zebrafish and MCF-7 cell lines, p-AKT and p-ERK levels were restored upon FGFR4 overexpression. Taken together, our results strongly suggest that deguelin inhibition of PI3K/AKT and MAPK signaling in zebrafish and breast cancer cell lines is partially mediated through down-regulation of FGFR4 activity.

Yifuning postpones ovarian aging through antioxidant mechanisms and suppression of the Rb/p53 signal transduction pathway.

Yifuning is a traditional Chinese medicine recipe that has been used for many years in China for its effects on treating climacteric syndrome in women. The present study aimed to demonstrate the effects and underlying molecular mechanism of Yifuning on the ovaries of aging rats. Selected aging rats were administered different doses of Yifuning (1.0 or 2.0 g/kg by lavage), and after 6 weeks the rats were sacrificed. The activit of indicators of oxidative stress in the serum were measured. The expression levels of 8-oxo-2'-deoxyguanosine (8-OHDG) and p53 in the ovaries were examined using immunohistochemistry. The expression levels of the corresponding genes and proteins were detected by reverse transcription­quantitative polymerase chain reaction and western blotting analyses, respectively. The results indicated that Yifuning significantly prevented ovarian failure, as indicated by improvements in estrous cycling, reproductive organ weights and sex hormone serum levels. Yifuning significantly increased the levels of superoxide dismutase, glutathione peroxidase, catalase and reduced malondialdehyde and hydrogen peroxide levels. Yifuning reduced DNA damage in the ovaries by reducing the expression of 8­OHDG and p53. Treatment with Yifuning significantly reduced the age­induced p19, p53, p21 and Rb activity in the ovaries. The present study demonstrates that Yifuning prevents ovarian failure and the mechanism involved is partly associated with antioxidants and suppression of the Rb/p53 signal transduction pathway.

Effects of Xie-Zhuo-Chu-Bi-Fang on miR-34a and URAT1 and their relationship in hyperuricemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xie-Zhuo-Chu-Bi-Fang (XZCBF) is an empirical formula that was developed based on the principles of traditional Chinese medicine, for the therapeutic purpose of treating hyperuricemia. XZCBF has been clinically utilized in the Department of Traditional Chinese Medicine at General Hospital of Guangzhou Military Command of PLA for many years and has exhibited favorable efficacy. The aim of the study is to evaluate the effects of XZCBF on the expression of uric acid transporter 1 (URAT1) and miR-34a in hyperuricemic mice and to determine, the correlation between the two expression levels. MATERIALS AND METHODS: A hyperuricemic animal model was created by administering adenine and allantoxanic acid potassium salt to mice. The blood uric acid levels were measured in these model mice after treatment with XZCBF for 15 days. The potential targets of miR-34a were screened. The expression levels of miR-34a and URAT1 in the renal tissues collected from the model mice were determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and their correlation was further established by immunohistochemistry and in situ hybridization. RESULTS: The uric acid levels in the model mice were significantly higher than those in the blank controls (P<0.05). These levels were significantly lower in the three groups receiving different doses of XZCBF (P<0.05), which was, in agreement with the downregulation of URAT1 and the upregulation of miR-34a in each group. The mRNA expression level of URAT1 was positively correlated with the concentration of uric acid but, negatively correlated with the expression level of miR-34a. CONCLUSIONS: The ability of XZCBF to facilitate the excretion of uric acid and to lower its level in the model group was mediated by the upregulation of miR-34a and the inhibition of URAT1 mRNA expression, which suggests that XZCBF could be an option for the treatment of hyperuricemia in mice.

Sophoridine exerts an anti-colorectal carcinoma effect through apoptosis induction in vitro and in vivo.

AIMS: To further investigate the anti-colorectal carcinoma (CRC) effect of Sophoridine (SRI) which is a quinolizidine alkaloid extracted from a traditional Chinese medicine (TCM) Sophora alopecuroides L. and detect the mechanism involved, provide some basis for the development of S. alopecuroides L. MAIN METHODS: The anti-proliferation of SRI in human colorectal cells SW480 were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The potential mechanism of anti-proliferation was also investigated using apoptosis assays. The rate of apoptosis cells was detected also. The apoptosis-related proteins cysteinyl aspartate specific protease (caspase), caspase-3, caspase-7, caspase-9, and poly-ADP-ribose-poly-merase (PARP) were determined by western blotting analysis. In animal studies, nude mice were subcutaneously injected with SW480 cells in the armpit to establish the xenograft tumors and administrated with different drugs (control, 5-Fu, SRI H, and SRI L). The general state of health of the mice and the growth of tumors were observed and the inhibitory rate was calculated. The pathology and ultrastructure of xenograft tumors treated with SRI were observed also. KEY FINDINGS: SRI significantly inhibited the growth of SW480 cells, and the administration of SRI significantly inhibited the growth of xenograft tumors without apparent toxicity. SRI's mechanism of action involved the induction of apoptosis. SIGNIFICANCE: These results suggest that SRI produces obvious anti-tumor effects in vitro and in vivo. It supports the viability of developing SRI as a novel therapeutic prodrug for CRC treatment, as well as providing a method for identifying new anti-tumor drugs in TCM.