New Mechanism for the Apoptosis of Human Neuroblastoma Cells by the Interaction between Fluorene-9-Bisphenol and the G Protein-Coupled Estrogen Receptor 1.

Fluorene-9-bisphenol (BHPF) is an emerging contaminant. Presently, there is no report on its interaction with G protein-coupled estrogen receptor 1 (GPER). By using an integrated toxicity research scenario that combined theoretical study with experimental methods, BHPF was found to inhibit the GPER-mediated effect via direct receptor binding. Molecular dynamics simulations found that Trp2726.48 and Glu2756.51 be the key amino acids of BHPF binding with GPER. Moreover, the calculation indicated that BHPF was a suspected GPER inhibitor, which neither can activate GPER nor is able to form water channels of GPER. The role of two residues was successfully verified by following gene knockout and site-directed mutagenesis assays. Further in vitro assays showed that BHPF could attenuate the increase in intracellular concentration of free Ca2+ induced by G1-activated GPER. Besides, BHPF showed an enhanced cytotoxicity compared with G15, indicating that BHPF might be a more potent GPER inhibitor than G15. In addition, a statistically significant effect on the mRNA level of GPER was observed for BHPF. In brief, the present study proposes that BHPF be a GPER inhibitor, and its GPER molecular recognition mechanism has been revealed, which is of great significance for the health risk and assessment of BHPF.

Low-frequency transcranial magnetic stimulation protects cognition in mice with chronic unpredictable mild stress through autophagy regulation.

Although transcranial magnetic stimulation (TMS) has been approved for the treatment of major depression, few studies have analyzed the ability of low-frequency TMS (LF-TMS) to treat depressive symptoms. Our study confirmed that LF-TMS protects the cognitive function,which can play a certain reference role in the future clinical treatment. The effectiveness of high-frequency TMS therapy has been well documented. However, the use of low-frequency TMS (LF-TMS) in the treatment of depression is rarely reported. This study aims to evaluate whether LF-TMS can reverse depression-induced cognitive impairment. We created a mouse model of depression using the chronic unpredictable mild stress (CUMS) paradigm. Male C57BL/6J mice,6-8 weeks old,were randomly divided into four groups: a CON (control) group, a CUMS group, a CUMS+LF-TMS group, and a CUMS+LF-TMS+RAP (rapamycin) group. The CUMS was maintained for 28 days. LF-TMS (1 Hz) and Rap were administered for 28 days from the first day of CUMS. The mice in all groups except the control demonstrated evidence of anhedonia, anxiety, and cognitive decline on behavioral tests during the four weeks of CUMS.All the experiments were carried out under a 12-h light/dark cycle (lights on at 7 a.m.) except for the dark/light cycle reversal stimulation of CUMS. LF-TMS at 20 Mt, 1 Hz for 1 min alleviated damage to spatial cognition and synaptic plasticity in the hippocampus. Western blot analysis showed that LF-TMS reduced the down-regulation of autophagy signals in the CUMS+LF-TMS group, and enhanced the expression of synaptic plasticity-related factors, thereby improving the spatial cognitive impairment resulting from the CUMS. We concluded that LF-TMS can effectively relieve depressive behavior and cognitive dysfunction in mice subjected to CUMS by regulating autophagy signals and synaptic proteins.

Co-treatment with natural HMGB1 inhibitor Glycyrrhizin exerts neuroprotection and reverses Parkinson's disease like pathology in Zebrafish.

ETHNOPHARMACOLOGICAL RELEVANCE: Parkinson's disease (PD) is the second most devastating age-related neurodegenerative diseases after Alzheimer diseases (AD) and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and aggregation of α-synuclein (α-syn). The precise etiology of PD is not yet fully understood and lacks the disease-modifying therapeutic strategies that could reverse the ongoing neurodegeneration. In the quest of exploring novel disease modifying therapeutic strategies, natural compounds from plant sources have gained much attention in recent days. Glycyrrhizin (GL) is the main active ingredient of the roots and rhizomes of licorice (Glycyrrhiza glabra L), which are generally used in the treatment of inflammatory diseases or as a tonifying herbal medicine. In Persia, GL is a conventional neuroprotective agent that are used to treat neurological disorders. The traditional use of GL in Japan is to treat chronic hepatitis B. In addition, GL is a natural inhibitor of high mobility group box 1 (HMGB1) which has exerted neuroprotective effect against several HMGB1 mediated pathological conditions. AIM OF THE STUDY: The study is aimed to evaluate therapeutic effect of GL against PD in zebrafish. MATERIAL AND METHODS: PD in zebrafish larvae is induced by administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Apoptosis was assessed with TUNEL assay. Gene expression was performed to assess the modulation in genes related to neuroinflammatory and autophagy. RESULTS: We observed that GL co-treatment increased the length of DA neurons, decreased the number of apoptotic cells in zebrafish brain, and inhibited the loss of vasculature and disorganized vasculature induced by MPTP. GL co-treatment relieved the MPTP-induced locomotor impairment in zebrafish. GL co-treatment suppressed MPTP-induced upregulated mRNA expression of inflammatory markers such as hmgb1a, tlr4b, nfκb, il1ß, and il6. GL co-treatment suppressed the autophagy related genes α-syn and atg5 whereas increased the mRNA expression level of parkin and pink1. In addition, molecular docking study reveals that GL has binding interaction with HMGB1, TLR4, and RAGE. CONCLUSION: Hence, the effect of GL co-treatment on MPTP-induced PD-like condition in zebrafish is to alleviate apoptosis and autophagy, as well as suppress inflammatory responses.

Ameliorative effect of Gastrodia elata Blume extracts on depression in zebrafish and cellular models through modulating reticulon 4 receptors and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume (G. elata), a traditional Chinese herb, known as "Tian Ma", is widely used as a common medicine and diet ingredient for treating or preventing neurological disorders for thousands of years in China. However, the anti-depressant effect of G. elata and the underlying mechanism have not been fully evaluated. AIM OF THE STUDY: The study is aimed to investigate the anti-depressant effect and the molecular mechanism of G. elata in vitro and in vivo using PC12 cells and zebrafish model, respectively. MATERIAL AND METHODS: Network pharmacology was performed to explore the potential active ingredients and action targets of G. elata Blume extracts (GBE) against depression. The cell viability and proliferation were determined by MTT and EdU assay, respectively. TUNEL assay was used to examine the anti-apoptotic effect of GBE. Immunofluorescence and Western blot were used to detect the protein expression level. In addition, novel tank diving test was used to investigate the anti-depressant effect in zebrafish depression model. RT-PCR was used to analyze the mRNA expression levels of genes. RESULTS: G. elata against depression on the reticulon 4 receptors (RTN4R) and apoptosis-related targets, which were predicted by network pharmacology. Furthermore, GBE enhanced cell viability and inhibited the apoptosis in PC12 cells against CORT treatment. GBE relieved depression-like symptoms in adult zebrafish, included increase of exploratory behavior and regulation of depression related genes. Mechanism studies showed that the GBE inhibited the expression of RTN4R-related and apoptosis-related genes. CONCLUSION: Our studies show the ameliorative effect of G. elata against depression. The mechanism may be associated with the inhibition of RTN4R-related and apoptosis pathways.

Treatment of Parkinson's disease in Zebrafish model with a berberine derivative capable of crossing blood brain barrier, targeting mitochondria, and convenient for bioimaging experiments.

Berberine is a famous alkaloid extracted from Berberis plants and has been widely used as medications and functional food additives. Recent studies reveal that berberine exhibits neuroprotective activity in animal models of Parkinson's disease (PD), the second most prevalent neurodegenerative disorders all over the world. However, the actual site of anti-PD action of berberine remains largely unknown. To this end, we employed a fluorescently labeled berberine derivative BBRP to investigate the subcellular localization and blood brain barrier (BBB) permeability in a cellular model of PD and zebrafish PD model. Biological investigations revealed that BBRP retained the neuroprotective activity of berberine against PD-like symptoms in PC12 cells and zebrafish, such as protecting 6-OHDA induced cell death, relieving MPTP induced PD-like behavior and increasing dopaminergic neuron loss in zebrafish. We also found that BBRP could readily penetrate BBB and function in the brain of zebrafish suffering from PD. Subcellular localization study indicated that BBRP could rapidly and specifically accumulate in mitochondria of PC12 cells when it exerted anti-PD effect. In addition, BBRP could suppress accumulation of Pink1 protein and inhibit the overexpression of LC3 protein in 6-OHDA damaged cells. All these results suggested that the potential site of action of berberine is mitochondria in the brain under the PD condition. Therefore, the findings described herein would be useful for further development of berberine as an anti-PD drug.

Involvement of 5-HT2 serotonin receptors in cognitive defects induced by aristolochic acid I in mice.

Aristolochic acids (AAs) are a natural bioactive substance found in Chinese herbs, which are widely used for treating diseases. Many studies have demonstrated that AAs have various pharmacological function, while increasing reports indicated its toxicity. However, the role AAs in cognition remains poorly understood. This study explored the neurotoxic effect of aristolochic acid I (AAI), the most toxic component of the AAs family, on hippocampal synaptic plasticity and spatial cognition in mice. C57BL/6 mice were exposed to 5 mg/kg AAI for 4 weeks. After chronic treatment, AAI considerably increased the level of anxiety and the degree of behavioral despair in mice. Working and reference error rates were higher in the AAI exposed mice than in the control. This was further validated by the molecular docking studies, which AAI might interact with 5-HT2 serotonin receptor (5-HT2AR). Mechanism investigation indicated that AAI triggered inflammation in the hippocampus of mice through increasing the activity of Tnf-α-NF-κB-IL-6 signaling pathway. Conclusively, chronic AAI administration causes inflammation, and it possibly also serves as a potential antagonist of 5-HT2AR to influence the cognition function in C57BL/6 mice.

Identification of four novel mutations in the COL4A5 gene identified in Chinese patients with X-linked Alport syndrome.

Alport syndrome (AS) is an inherited progressive nephropathy caused by mutations in one or two of the type IV collagen novel chains (α3, α4 and α5), which are encoded by COL4A3, COL4A4 and COL4A5, respectively. To date, three genetic forms of AS have been reported, including X-linked AS, autosomal recessive AS, and autosomal dominant AS, and ~80% of patients have X-linked AS caused by mutations in COL4A5. In the present study, four novel and one previously reported COL4A5 mutations were identified using targeted next-generation sequencing in Chinese patients suspected of having AS. The results were confirmed by Sanger sequencing, which revealed two novel missense mutations resulting in the substitution of various glycine residues in a collagenous domain containing Gly-X-Y triplet sequence repeats [c.4198G>C, p.(Gly1400Arg) and c.3428G>T, p.(Gly1143Val)], a previously reported missense mutation [c.3071G>A, p.(Gly1024Glu)], a splice site mutation (c.2146+2T>A) and one frameshift mutation [c.1810delC (p.Thr605Ilefs*13)]. After analyzing the affected family members, it was shown that the identified mutations were associated with severe clinical phenotypes. These results broaden the known spectrum of mutations of the COL4A5 gene associated with AS and may have implications for genetic diagnosis, therapy and genetic counseling of affected families.

La2O3 Nanoparticles Induce Reproductive Toxicity Mediated by the Nrf-2/ARE Signaling Pathway in Kunming Mice.

PURPOSE: Lanthanum oxide (La2O3) nanoparticles (NPs) have been widely used in catalytic and photoelectric applications, but the reproductive toxicity is still unclear. This study evaluated the reproductive toxicity of two different-sized La2O3 particles in the testes. MATERIALS AND METHODS: Fifty Kunming mice were randomly divided into five groups. Mice were treated with La2O3 NPs by repeated intragastric administration for 90 days (control, nano-sized with 5, 10, 50 mg/kg BW and micro-sized with 50 mg/kg BW). Mice in the control group were treated with de-ionised water without La2O3 NPs. Sperm parameters, testicular histopathology, TEM assessment, hormone assay and nuclear factor erythroid 2-related factor 2 (Nrf-2) pathway were performed and evaluated. RESULTS: The body weight of mice treated with La2O3 NPs or not had no difference; sperm parameters and histological assessment showed that La2O3 NPs could induce reproductive toxicity in the testicle. Serum testosterone and gonadotropin-releasing hormone (GnRH) in the NH (nano-sized with 50 mg/kg BW) group were markedly decreased relative to control group, and an increase of luteinizing hormone (LH) in NH group was detected . Additionally, transmission electron microscopy revealed that the ultrastructural abnormalities induced by La2O3 NPs were more severe than La2O3 MPs in the testes. Furthermore, La2O3 NPs treatment inhibited the translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) from the cytoplasm into the nucleus as well as the expression of downstream genes NAD(P)H quinone oxidoreductase1 (NQO1), hemeoxygenase 1 (HO-1) and (glutathione peroxidase) GSH-Px, thus abrogating Nrf-2-mediated defense mechanisms against oxidative stress. CONCLUSIONS: The results of this study demonstrated that La2O3 NPs improved the spermatogenesis defects in mice. La2O3 NPs inhibited Nrf-2/ARE signaling pathway that resulted in apoptosis in the mice testes.

α-asarone induces cardiac defects and QT prolongation through mitochondrial apoptosis pathway in zebrafish.

α-asarone is a natural phenylpropene found in several plants, which are widely used for flavoring foods and treating diseases. Previous studies have demonstrated that α-asarone has many pharmacological functions, while some reports indicated its toxicity. However, little is known about its cardiovascular effects. This study investigated developmental toxicity of α-asarone in zebrafish, especially the cardiotoxicity. Zebrafish embryos were exposed to different concentrations of α-asarone (1, 3, 5, 10, and 30 µM). Developmental toxicity assessments revealed that α-asarone did not markedly affect mortality and hatching rate. In contrast, there was a concentration-dependent increase in malformation rate of zebrafish treated with α-asarone. The most representative cardiac defects were increased heart malformation rate, pericardial edema areas, sinus venosus-bulbus arteriosus distance, and decreased heart rate. Notably, we found that α-asarone impaired the cardiac function of zebrafish by prolonging the mean QTc duration and causing T-wave abnormalities. The expressions of cardiac development-related key transcriptional regulators tbx5, nkx2.5, hand2, and gata5 were all changed under α-asarone exposure. Further investigation addressing the mechanism indicated that α-asarone triggered apoptosis mainly in the heart region of zebrafish. Moreover, the elevated expression of puma, cyto C, afap1, caspase 3, and caspase 9 in treated zebrafish suggested that mitochondrial apoptosis is likely to be the main reason for α-asarone induced cardiotoxicity. These findings revealed the cardiac developmental toxicity of α-asarone, expanding our knowledge about the toxic effect of α-asarone on living organisms.

Rapamycin relieves anxious emotion and synaptic plasticity deficits induced by hindlimb unloading in mice.

This study examined whether increasing autophagy could improve cognitive deficits in hindlimb unloaded (HU) mice, which was used as an animal model of synaptic plasticity impairment. Male C57BL/6 mice were randomly divided into three groups: control, HU and HU + rapamycin groups. Hindlimb unloading treatment was used to establish the animal model for 2 weeks. Rapamycin was intraperitoneally injected at a dose of 0.5 mg/kg/day along with hindlimb unloading procedure. The open field test and the elevated plus maze test showed that rapamycin considerably prevented the level of anxiety and increased exploratory behaviour in HU mice. Afterwards, long-term potentiation (LTP) recorded in the hippocampal dentate gyrus (DG) region was effectively reduced by rapamycin, which was significantly inhibited by HU procedure. In addition, rapamycin further increased the autophagy level, which was already elevated in HU mice. Meanwhile, the expression of NMDA receptor 2A and 2 B was modified by rapamycin in HU mice. Moreover, rapamycin noticeably increased the total superoxide dismutase (T-SOD) activity and reduced the malondialdehyde (MDA) as well as the level of carbonylated proteins in HU mice's hippocampus. The results show that increasing autophagy may pacificate the anxious emotion, and partly alleviate the hippocampal synaptic plasticity deficits.

Nicotine alleviates chronic stress-induced anxiety and depressive-like behavior and hippocampal neuropathology via regulating autophagy signaling.

Recently, we reported that chronic nicotine significantly improved chronic stress-induced impairments of cognition and the hippocampal synaptic plasticity in mice, however, the underlying mechanism still needs to be explored. In the present study, 32 male C57BL/6 mice were divided into four groups: control (CON), stress (CUS), stress with chronic nicotine administration (CUS + Nic) and chronic nicotine administration (Nic). The anxiety-like behavior and neuropathological alteration of DG neurons were examined. Moreover, PC12 cells were examined with corticosterone in the presence or absence of nicotine. Both cell viability and apoptosis were determined. When treated simultaneously with an unpredictable chronic mild stress (CUS), nicotine (0.2 mg/kg/d) attenuated behavioral deficits and neuropathological alterations of DG neurons. Moreover, Western blotting showed that chronic nicotine also elevated the level of autophagy makers including Beclin-1 and LC3 II triggered by CUS. In addition, concomitant treatment with nicotine (10 µM) significantly attenuated the loss of PC12 cell viability (p < .01) and apoptosis compared to that of corticosterone treatment alone. Besides, chronic nicotine also enhanced the protein and RNA expression levels of autophagy makers triggered by corticosterone, such as Beclin-1, LC3 II and p62/SQSTM1. However, the above improvements were significantly blocked by autophagy inhibitor 3-MA. Importantly, the activation of the PI3K/Akt/mTOR signaling was carefully tested to illuminate the effects of chronic nicotine. Consequently, chronic nicotine played a role of neuroprotection in either CUS mice or corticosterone cells associating with the enhancement of the autophagy signaling, which was involved in activating the PI3K/Akt/mTOR signaling.

Nicotine Significantly Improves Chronic Stress-Induced Impairments of Cognition and Synaptic Plasticity in Mice.

The aim of this study was to examine if nicotine was able to improve cognition deficits in a mouse model of chronic mild stress. Twenty-four male C57BL/6 mice were divided into three groups: control, stress, and stress with nicotine treatment. The animal model was established by combining chronic unpredictable mild stress (CUMS) and isolated feeding. Mice were exposed to CUMS continued for 28 days, while nicotine (0.2 mg/kg) was also administrated for 28 days. Weight and sucrose consumption were measured during model establishing period. The anxiety and behavioral despair were analyzed using the forced swim test (FST) and open-field test (OFT). Spatial cognition was evaluated using Morris water maze (MWM) test. Following behavioral assessment, both long-term potentiation (LTP) and depotentiation (DEP) were recorded in the hippocampal dentate gyrus (DG) region. Both synaptic and Notch1 proteins were measured by Western. Nicotine increased stressed mouse's sucrose consumption. The MWM test showed that spatial learning and reversal learning in stressed animals were remarkably affected relative to controls, whereas nicotine partially rescued cognitive functions. Additionally, nicotine considerably alleviated the level of anxiety and the degree of behavioral despair in stressed mice. It effectively mitigated the depression-induced impairment of hippocampal synaptic plasticity, in which both the LTP and DEP were significantly inhibited in stressed mice. Moreover, nicotine enhanced the expression of synaptic and Notch1 proteins in stressed animals. The results suggest that nicotine ameliorates the depression-like symptoms and improves the hippocampal synaptic plasticity closely associated with activating transmembrane ion channel receptors and Notch signaling components. Graphical Abstract ᅟ.

Neural oscillations as a bridge between glutamatergic system and emotional behaviors in simulated microgravity-induced mice.

This study aims to investigate if neural oscillations can play a role as a bridge between the alteration of glutamatergic system and emotional behaviors in simulated microgravity (SM) mice. Adult male C57BL/6J mice were randomly divided into two groups: SM and control groups. The animal model was established by hindlimb unloading (HU). The mice were exposed to HU continued for 14days. Weight and sucrose consumption were measured. The degree of anxious and depressive was evaluated by Open field test and Elevated plus maze test. Local field potentials were recorded in the hippocampal perforant path (PP) and dentate gyrus (DG) regions. The NMDAR2A/2B (NR2A/2B) subunits expression and glutamate level were measured by Western and high performance liquid chromatography (HPLC), respectively. After 14days, SM mice exhibited depressive-like and anxiety-like behaviors, while the expression of NR2A/2B subunits and the glutamate level were significantly decreased in the SM group. Moreover, the power distribution of theta (3-8Hz) was decreased by HU, which further significantly attenuated the identical-frequency strength of phase synchronization and the neural information flow at theta rhythm on the PP-DG pathway. The theta-gamma phase synchronization strength was also significantly reduced by HU. The data imply that the neural oscillations measurements is a sign of the emotional behaviors impairment and the glutamatergic system change induced by HU.

Repetitive transcranial magnetic stimulation effectively facilitates spatial cognition and synaptic plasticity associated with increasing the levels of BDNF and synaptic proteins in Wistar rats.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique, by which cognitive deficits can be alleviated. Furthermore, rTMS may facilitate learning and memory. However, its underlying mechanism is still little known. The aim of this study was to investigate if the facilitation of spatial cognition and synaptic plasticity, induced by rTMS, is regulated by enhancing pre- and postsynaptic proteins in normal rats. Morris water maze (MWM) test was performed to examine the spatial cognition. The synaptic plasticity, including long-term potentiation (LTP) and depotentiation (DEP), presynaptic plasticity paired-pulse facilitation (PPF), from the hippocampal Schaffer collaterals to CA1 region was subsequently measured using in vivo electrophysiological techniques. The expressions of brain-derived neurotrophic factor (BDNF), presynaptic protein synaptophysin (SYP) and postsynaptic protein NR2B were measured by Western blot. Our data show that the spatial learning/memory and reversal learning/memory in rTMS rats were remarkably enhanced compared to that in the Sham group. Furthermore, LTP and DEP as well as PPF were effectively facilitated by 5Hz-rTMS. Additionally, the expressions of BDNF, SYP and NR2B were significantly increased via magnetic stimulation. The results suggest that rTMS considerably increases the expressions of BDNF, postsynaptic protein NR2B and presynaptic protein SYP, and thereby significantly enhances the synaptic plasticity and spatial cognition in normal animals.

[The effects and mechanism of islet amyloid polypeptide on insulin secretion in INS-1 cells stimulated by glibenclamide].

OBJECTIVE: To observe the effects, and study the mechanism of islet amyloid polypeptide (IAPP) on insulin secretion in INS-1 cells stimulated by glibenclamide. METHODS: Whole cell patch clamp technique was employed to study the influences of short exposure to IAPP on electrophysiological characteristics of ATP-sensitive K+ channel (K(ATP) channel) upon sulfonylurea stimulation. Intracellular free calcium changes in this process was observed by laser scanning confocal microscope. Insulin was measured by enzyme-linked immunoassay. RESULTS: (1) Insulin secretion stimulated by 1 micomol/L glibenclamide was significantly decreased from (11.43 +/- 1.22) microg/L to (9.40 +/- 0.87) microg/L and to (7.11 +/- 1.85) microg/L after 1 micromol/L and 10 micromol/L IAPP incubation, respectively. (2) Glibenclamide-stimulated calcium influx was dose dependently inhibited by IAPP from 1 micromol/L to 10 micromol/L, with the AUC of fluorescence intensity-time reduced from 427.78 +/- 2.32 to 380.59 +/- 1.49, and to 246.53 +/- 8.41, respectively. (3) Compared with that in control cells (14.59 +/- 0.69) mV, the half activation voltage of KA, channel in response to glibenclamide was significantly increased to (28.75 +/- 0.77) mV and to (46.95 +/- 1.81) mV in cells pretreated with 1 micromol/L and 10 micromol/L IAPP, implicating an inhibitory effect of IAPP on activation of K(ATP) channel. CONCLUSION: Short-term exposure to high concentration of IAPP inhibited glibenclamide-induced closure of K(ATP) channels and decreased calcium influx, which may ultimately lead to the reduction of insulin secretion in INS-1 cells