Artemisinin reduces PTSD-like symptoms, improves synaptic plasticity, and inhibits apoptosis in rats subjected to single prolonged stress.

Post-Traumatic Stress Disorder (PTSD) is a chronic mental disorder characterized by symptoms of panic and anxiety, depression, impaired cognitive functioning, and difficulty in social interactions. While the effect of the traditional Chinese medicine artemisinin (AR) on PTSD is unknown, its therapeutic benefits have been demonstrated by studies on models of multiple neurological disorders. This study aimed to extend such findings by investigating the effects of AR administration on a rat model of PTSD induced by a regimen of single prolonged stress (SPS). After rats were subjected to the SPS protocol, AR was administered and its impact on PTSD-like behaviors was evaluated. In the present study, rats were subjected to a multitude of behavioral tests to evaluate behaviors related to anxiety, memory function, and social interactions. The expression of hippocampal synaptic plasticity-related proteins was detected using Western blot and immunofluorescence. The ultrastructure of synapses was observed under transmission electron microscopy. The apoptosis of hippocampal neurons was examined with Western blot, TUNEL staining, and HE staining. The results showed that AR administration alleviated the PTSD-like phenotypes in SPS rats, including behavior indicative of anxiety, cognitive deficits, and diminished sociability. AR administration was further observed to improve synaptic plasticity and inhibit neuronal apoptosis in SPS rats. These findings suggest that administering AR after the onset of severe traumatic events may alleviate anxiety, cognitive deficits, and impaired social interaction, improve synaptic plasticity, and diminish neuronal apoptosis. Hence, the present study provides evidence for AR's potential as a multi-target agent in the treatment of PTSD.

Identification of Serum miR-501-3p and miR-338-3p as Novel Diagnostic Biomarkers for Breast Cancer and Their Target Genes Associated with Immune Infiltration.

Background: MicroRNAs influence the growth and metastasis of breast cancer (BC) by regulating their target genes. Our study aims to screen and identify miRNAs that are closely related to the development of breast cancer, and explore the role of these miRNAs and their target genes in breast cancer. Methods: Bioinformatics tools were applied to screen breast cancer-associated miRNAs and predict their potential target genes. Serum miRNAs were measured using RT-PCR. The correlation between miRNA expression and different clinicopathological features of BC patients was analyzed. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value. GEPIA, Kaplan-Meier Plotter, TIMER, and TISIDB databases were used to validate the expression levels and their prognostic value, as well as their target gene associated with immune infiltrating cells and immune checkpoints. Results: Breast cancer-associated serum miR-338-3p and miR-501-3p were screened and verified for the first time. Serum miR-501-3p was elevated in BC and was closely linked to the ki-67 index and histological grade. CDKN2C, as a potential target gene of miR-501-3p, was enriched in the cGMP-PKG signaling pathway. Serum miR-338-3p was reduced in BC and was strongly linked to lymph node metastasis and histological grading. ACTR2, CDH1, COL1A1, RBBP5, RRM1, and TPM3, as potential target genes of miR-338-3p, were enriched in MAPK, PI3K-Akt, and RAS signaling pathways. These target genes were found to be linked to breast cancer prognosis, immune infiltrating cells, and immune checkpoint inhibitors. Analysis of ROC curve showed that serum miR-501-3p combined with serum miR-338-3p had a high diagnostic value in breast cancer (AUC: 0.89, 95% CI: 0.821-0.958). Conclusion: Serum miR-501-3p combined with serum miR-338-3p show obvious clinical significance in the diagnosis and prognosis of breast cancer, which suggests that they may act as novel diagnostic biomarkers for breast cancer.

Amentoflavone impairs the reconsolidated fear memories through inhibition of ERK pathway.

Post-traumatic stress disorder (PTSD) is a pathological fear memory-related disease. The persistence of pathological fearful memories is one of the most characteristic symptoms of PTSD. However, this can be eliminated by intervening in reconsolidation. Inflammation is intimately involved in the pathophysiologic progression of PTSD. Amentoflavone (AF) has anti-inflammatory effects. However, the effect of AF on fear memory reconsolidation remains unclear. In the present series of experiments, the CFC paradigm of rats were constructed. This was followed by AF administration immediately after exposure to the conditioning chamber to observe the maintenance of fear memory. Finally, a Western blot for the amygdala was used to explore the possible molecular biological mechanisms of AF affecting animal behavior. The findings suggest that re-exposure to the conditioning chamber for retrieval of CFC memory followed by immediate intragastric AF administration in rats attenuated the fear response for at least 14 days. In addition, the Western blot results show that the CFC memory intervention effect of AF administration during the reconsolidation phase may be related to the ERK signaling pathway inhibition. In general, the administration of AF in the reconsolidation phase to inhibit neuroinflammation can block the reconsolidation process and disrupt fear memory retention in the long term, at least in part through ERK pathway.

G Protein-Coupled Estrogen Receptor 1 Knockout Deteriorates MK-801-Induced Learning and Memory Impairment in Mice.

The role of estrogen receptors in neuroprotection and cognition has been extensively studied in humans over the past 20 years. Recently, studies have shifted their focus to the use of selective estrogen receptor modulators in the treatment of mental illnesses in the central nervous system. We conducted this study to test the behavioral changes shown by G protein-coupled estrogen receptor 1 knockout (GPER1 KO) and wild-type (WT) mice with MK-801-induced schizophrenia (SZ). GPER1 KO and WT mice received intraperitoneal injections of MK-801 for 14 continuous days. Behavioral, learning and memory, and social interaction changes were evaluated by using the IntelliCage system, open-field, three-chamber social interaction, and novel object recognition tests (NORT). The protein expression levels of the NR2B/CaMKII/CREB signaling pathway were tested via Western blot analysis. The KO SZ group was more likely to show impaired long-term learning and memory function than the WT SZ group. Learning and memory functions were also impaired in the KO Con group. MK-801 administration to the GPER1-KO and WT groups resulted in memory deficiencies and declining learning capabilities. GPER1 deficiency downregulated the expression levels of proteins related to the NR2B/CaMKII/CREB signaling pathway. Our study suggested that GPER1 played an important role in cognitive, learning, and memory functions in the MK-801-induced mouse model of SZ. The mechanism of this role might partially involve the downregulation of the proteins related to the NR2B/CaMKII/CREB signaling pathway. Further studies should focus on the effect of GPER1 on the pathogenesis of SZ in vivo and in vitro.

Continuous High Frequency Deep Brain Stimulation of the Rat Anterior Insula Attenuates the Relapse Post Withdrawal and Strengthens the Extinction of Morphine Seeking.

Deep brain stimulation (DBS) modulates the neuronal activity in specific brain circuits and has been recently considered as a promising intervention for refractory addiction. The insula cortex is the hub of interoception and is known to be involved in different aspects of substance use disorder. In the present study, we investigate the effects of continuous high frequency DBS in the anterior insula (AI) on drug-seeking behaviors and examined the molecular mechanisms of DBS action in morphine-addicted rats. Sprague-Dawley rats were trained to the morphine-conditioned place preference (CPP, day 1-8) followed by bilaterally implanted with DBS electrodes in the AI (Day 10) and recovery (Day 10-15). Continuous high-frequency (HF) -DBS (130 Hz, 150 µA, 90 µs) was applied during withdrawal (Day 16-30) or extinction sessions. CPP tests were conducted on days 16, 30, 40 during withdrawal session and several rats were used for proteomic analysis on day 30. Following the complete extinction, morphine-CPP was reinstated by a priming dose of morphine infusion (2 mg/kg). The open field and novel objective recognition tests were also performed to evaluate the DBS side effect on the locomotion and recognition memory. Continuous HF-DBS in the AI attenuated the expression of morphine-CPP post-withdrawal (Day 30), but morphine addictive behavior relapsed 10 days after the cessation of DBS (Day 40). Continuous HF-DBS reduced the period to full extinction of morphine-CPP and blocked morphine priming-induced recurrence of morphine addiction. HF-DBS in the AI had no obvious effect on the locomotor activity and novel objective recognition and did not cause anxiety-like behavior. In addition, our proteomic analysis identified eight morphine-regulated proteins in the AI and their expression levels were reversely changed by HF-DBS. Continuous HF-DBS in the bilateral anterior insula prevents the relapse of morphine place preference after withdrawal, facilitates its extinction, blocks the reinstatement induced by morphine priming and reverses the expression of morphine-regulated proteins. Our findings suggest that manipulation of insular activity by DBS could be a potential intervention to treat substance use disorder, although future research is warranted.

ALG13 participates in epileptogenesis via regulation of GABAA receptors in mouse models.

ALG13 (asparagine-linked glycosylation 13) plays crucial roles in the process of N-linked glycosylation. Mutations of the ALG13 gene underlie congenital disorders of glycosylation type I (CDG-I), a rare human genetic disorder with defective glycosylation. Epilepsy is commonly observed in congenital disorders of glycosylation type I (CDG-I). In our study, we found that about 20% of adult ALG13KO knockout mice display spontaneous seizures, which were identified in a simultaneous video and intracranial EEG recording. However, the mechanisms of ALG13 by which deficiency leads to epilepsy are unknown. Whole-cell patch-clamp recordings demonstrated that ALG13KO mice show a marked decrease in gamma-aminobutyric acid A receptor (GABAAR)-mediated inhibitory synaptic transmission. Furthermore, treatment with low-dose diazepam (a positive allosteric modulator of GABAA receptors), which enhances GABAAR function, also markedly ameliorates severity of epileptic seizures in ALG13KO mice. Moreover, ALG13 may influenced the expression of GABAARα2 membrane and total protein by changing transcription level of GABAARα2. Furthermore, protein interactions between ALG13 and GABAARα2 were observed in the cortex of wild-type mice. Overall, these results reveal that ALG13 may be involved in the occurrence of epilepsy through the regulation of GABAAR function, and may provide new insight into epilepsy prevention and treatment.

Postretrieval Microinjection of Baclofen Into the Agranular Insular Cortex Inhibits Morphine-Induced CPP by Disrupting Reconsolidation.

Environmental cues associated with drug abuse are powerful mediators of drug craving and relapse in substance-abuse disorders. Consequently, attenuating the strength of cue-drug memories could reduce the number of factors that cause drug craving and relapse. Interestingly, impairing cue-drug memory reconsolidation is a generally accepted strategy aimed at reducing the intensity of cues that trigger drug-seeking and drug-taking behaviors. In addition, the agranular insular cortex (AI) is an important component of the neural circuits underlying drug-related memory reconsolidation. GABAB receptors (GABABRs) are potential targets for the treatment of addiction, and baclofen (BLF) is the only prototypical GABAB agonist available for application in clinical addiction treatment. Furthermore, ΔFosB is considered a biomarker for the evaluation of potential therapeutic interventions for addiction. Here, we used the morphine-induced conditioned place preference (CPP) paradigm to investigate whether postretrieval microinjections of BLF into the AI could affect reconsolidation of drug-reward memory, reinstatement of CPP, and the level of ΔFosB in mice. Our results showed that BLF infused into the AI immediately following morphine CPP memory retrieval, but not 6 h postretrieval or following nonretrieval, could eliminate the expression of a morphine CPP memory. This effect persisted in a morphine-priming-induced reinstatement test, suggesting that BLF in the AI was capable of preventing the reconsolidation of the morphine CPP memory. Our results also showed that the elimination of morphine CPP memory was associated with reduced morphine-associated ΔFosB expression in the longer term. Taken together, the results of our research provide evidence to support that GABABRs in the AI have an important role in drug-cue memory reconsolidation and further our understanding of the role of the AI in drug-related learning and memory.

The novel estrogen receptor GPER1 decreases epilepsy severity and susceptivity in the hippocampus after status epilepticus.

The steroid hormone 17ß-estradiol (estrogen) exerts neuroprotective effects in several types of neurological disorders including epilepsy. The novel G protein-coupled estrogen receptor 1 (GPER1), also called GPR30, mediates the non-genomic effects of 17ß-estradiol. However, the specific role of GPER1 in status epilepticus (SE) remains unclear. In this report, we evaluated the effects of GPER1 on the hippocampus during SE and the underlying mechanism was studied. Our results revealed that pilocarpine-induced GPER1-KD epileptic rats exhibited a shorter latency to generalized convulsions and strikingly elevated seizure severity. Additionally, the electroencephalographic seizure activity also corresponded to these results. Fast-Fourier analysis indicated an enhancement of power in the theta and alpha bands during SE in GPER1-KD rats. In addition, epilepsy-induced pathological changes were dramatically exacerbated in GPER1-KD rats, including neuron damage and neuroinflammation in hippocampus. GPER1 might be associated with the susceptibility to and severity of epileptic seizures. In summary, our results suggested that GPER1 plays a neuroprotective role in SE, and might be a candidate target for epilepsy therapy.

GABAergic neurons in the insular cortex play an important role in cue-morphine reward memory reconsolidation.

AIMS: There have been recent reports that reconsolidation-based interventions attenuate drug reward memories in rodents. The insular cortex (IC) is an essential part of neural circuits that underlie cue-drug memory reconsolidation. GABAergic interneurons in the IC are a potent control on network excitability and play an important role in the inhibitory mediation of reward circuits. However, the function of GABAergic neurons in the IC for memory reconsolidation remains unclear; therefore, we conducted this study to clarify this. MAIN METHODS: We applied morphine-induced conditioned place preference (mCPP) paradigm and pharmacogenetic techniques to study the mediation effect of GABAergic neurons in the IC on mCPP reconsolidation. Moreover, we preliminarily explored the possible mechanisms of mediating GABAergic neurons in the IC involved in mCPP reconsolidation by assessing Arc and Erg-1 protein levels in the IC. KEY FINDINGS: We found that post-retrieval immediate activation of GABAergic neurons in the IC impaired mCPP reconsolidation. In addition, this effect was not reversed by a priming morphine injection. Further, post-retrieval inhibition and non-retrieval excitation of GABAergic neurons in the IC had no effect on mCPP. SIGNIFICANCE: Taken together, our findings suggest that GABAergic neurons in the IC are closely involved in mCPP reconsolidation. Specifically, their excitation could eliminate established mCPP and prevent the relapse risk by disruption of the reconsolidation. The underlying molecular biological mechanisms could involve reduced Arc and Erg-1 levels.

[Intra-nucleus accumbens shell injection of baclofen blocks the reconsolidation of conditioned place preference in morphine-addicted mice].

Preclinical studies suggest that the GABAB receptor is a potential target for treatment of substance use disorders. Baclofen (BLF), a prototypical GABAB receptor agonist, is the only specific GABAB receptor agonist available for application in clinical addiction treatment. The nucleus accumbens shell (AcbSh) is a key node in the circuit that controls reward-directed behavior. However, the relationship between GABAB receptors in the AcbSh and memory reconsolidation was unclear. The aim of this study was to investigate the effect of intra-AcbSh injection of BLF on the reconsolidation of morphine reward memory. Male C57BL/6J mice were used to establish morphine conditioned place preference (CPP) model and carry out morphine reward memory retrieval and activation experiment. The effects of intra-AcbSh injection of BLF on morphine-induced CPP, reinstatement of CPP and locomotor activity were observed after environmental cues activating morphine reward memory. The results showed that intra-AcbSh injection of BLF (0.06 nmol/0.2 µL/side or 0.12 nmol/0.2 µL/side), rather than vehicle or BLF (0.01 nmol/0.2 µL/side), following morphine reward memory retrieval abolished morphine-induced CPP by disrupting its reconsolidation in mice. Moreover, this effect persisted for more than 14 days, which was not reversed by a morphine priming injection. Furthermore, intra-AcbSh injection of BLF without morphine reward memory retrieval had no effect on morphine-associated reward memory. Interestingly, administration of BLF into the AcbSh had no effect on the locomotor activity of mice during testing phase. Based on these results, we concluded that intra-AcbSh injection of BLF following morphine reward memory could erase morphine-induced CPP by disrupting its reconsolidation. Activating GABAB receptor in AcbSh during drug memory reconsolidation may be a potential approach to prevent drug relapse.

Genetic knockout of the G protein-coupled estrogen receptor 1 facilitates the acquisition of morphine-induced conditioned place preference and aversion in mice.

Drug addiction is considered the pathological usurpation of normal learning and memory. G protein-coupled estrogen receptor 1 (GPER1) plays an important role in normal learning and memory, but the effect of GPER1 on addiction-related pathological memory has not been reported. Our study used GPER1 knockout (GPER1 KO) and wild-type (WT) mice to compare the sensitivity differences of morphine- and sucrose-induced conditioned place preference (CPP) and naloxone-induced conditioned place aversion (CPA), and differences in dopamine (DA) content in the nucleus accumbens (NAc) were determined by high performance liquid chromatography (HPLC). The results showed that GPER1 KO mice showed higher sensitivity to morphine-induced CPP and naloxone-induced CPA, and corresponding to the behavioral effect, the DA content in the NAc of GPER1 KO mice was significantly higher than that of WT mice. Interestingly, the sensitivity of GPER1 KO mice to sucrose-induced CPP did not differ from that of the WT mice, and there was no significant difference in the DA content in the NAc between the two genotypes of mice. GPER1 knockout promoted the formation of morphine addiction-related positive and aversive memory, and its molecular biological mechanism may be associated with increased DA content in the NAc. Therefore, GPER1 plays an important role in the formation of addiction-related pathological memory and may become a potential molecular target for drug addiction therapy.

The Glucagon-Like Peptide-1 Analogue Liraglutide Reduces Seizures Susceptibility, Cognition Dysfunction and Neuronal Apoptosis in a Mouse Model of Dravet Syndrome.

Dravet syndrome (DS) is a refractory epilepsy typically caused by heterozygous mutations of the Scn1a gene, which encodes the voltage-gated sodium channel Nav1.1. Glucagon-like peptide-1 (GLP-1) analogues, effective therapeutic agents for the treatment of diabetes, have recently become attractive treatment modalities for patients with nervous system disease; however, the impact of GLP-1 analogues on DS remains unknown. This study aimed to determine the neuroprotective role of liraglutide in mouse and cell models of Scn1a KO-induced epilepsy. Epileptic susceptibility, behavioral changes, and behavioral seizures were assessed using electroencephalography (EEG), IntelliCage (TSE Systems, Bad Homburg, Germany), and the open field task. Morphological changes in brain tissues were observed using hematoxylin and eosin (HE) and Nissl staining. Expression of apoptosis-related proteins and the mammalian target of rapamycin (mTOR) signaling pathway were determined using immunofluorescence and western blotting in Scn1a KO-induced epileptic mice in vitro. Scn1a KO model cell proliferation was evaluated using the Cell Counting Kit-8 assay, and the effect of liraglutide on cellular apoptosis levels was examined using Annexin V-FITC/PI flow cytometry. Apoptotic signal proteins and mTOR were assessed using reverse transcription - quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results showed that liraglutide significantly increased mRNA ((0.31 ± 0.04) *10-3 vs. (1.07 ± 0.08) * 10-3, P = 0.0004) and protein (0.10 ± 0.02 vs. 0.27 ± 0.02, P = 0.0006) expression of Scn1a in Scn1a KO-induced epileptic mice. In addition, liraglutide significantly alleviated electroencephalographic seizures, the severity of responses to epileptic seizures (96.53 ± 0.45 % vs. 85.98 ± 1.24 %, P = 0.0003), cognitive dysfunction, and epileptic-related necrotic neurons (9.76 ± 0.91 % vs. 19.65 ± 2.64 %, P = 0.0005) in Scn1a KO-induced epileptic mice. Moreover, liraglutide protected against Scn1a KO-induced apoptosis, which was manifested in the phosphorylation of mTOR (KO+NS: 1.99 ± 0.31 vs. KO+Lira: 0.97 ± 0.18, P = 0.0004), as well as the downregulation of cleaved caspase-3 (KO+NS: 0.49 ± 0.04 vs. KO+Lira: 0.30 ± 0.01, P = 0.0003) and restoration of the imbalance between BAX (KO+NS: 0.90 ± 0.02 vs. KO+Lira: 0.75 ± 0.04, P = 0.0005) and BCL-2 (KO+NS: 0.46 ± 0.02 vs. KO+Lira: 0.61 ± 0.02, P = 0.0006). Collectively, these results show that liraglutide reduces seizure susceptibility and cognitive dysfunction in the mouse model of Dravet syndrome, and exerts anti-apoptotic and neuroprotective effects in Scn1a KO mice and cells.

Amentoflavone Affects Epileptogenesis and Exerts Neuroprotective Effects by Inhibiting NLRP3 Inflammasome.

Brain inflammation is one of the main causes of epileptogenesis, a chronic process triggered by various insults, including genetic or acquired factors that enhance susceptibility to seizures. Amentoflavone, a naturally occurring biflavonoid compound that has anti-inflammatory effects, exerts neuroprotective effects against nervous system diseases. In the present study, we aimed to investigate the effects of amentoflavone on epilepsy in vivo and in vitro and elucidate the underlying mechanism. The chronic epilepsy model and BV2 microglial cellular inflammation model were established by pentylenetetrazole (PTZ) kindling or lipopolysaccharide (LPS) stimulation. Cognitive dysfunction was tested by Morris water maze while hippocampal neuronal apoptosis was evaluated by immunofluorescence staining. The levels of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome complexes and inflammatory cytokines were determined using quantitative real-time polymerase chain reaction, Western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay. Amentoflavone reduced seizure susceptibility, minimized PTZ-induced cognitive dysfunction, and blocked the apoptosis of hippocampal neurons in PTZ-induced kindling mice. Amentoflavone also inhibited the activation of the NLRP3 inflammasome and decreased the levels of inflammatory cytokines in the hippocampus of PTZ-induced kindling mice. Additionally, amentoflavone could alleviate the LPS-induced inflammatory response by inhibiting the NLRP3 inflammasome in LPS-induced BV2 microglial cells. Our results indicated that amentoflavone affects epileptogenesis and exerts neuroprotective effects by inhibiting the NLRP3 inflammasome and, thus, mediating the inflammatory process in PTZ-induced kindling mice and LPS-induced BV2 microglial cells. Therefore, amentoflavone may be a potential treatment option for epilepsy.

A Novel Scoring System Based on Peripheral Blood Test in Predicting Grade and Prognosis of Patients with Glioma.

PURPOSE: To explore the value of F-NLR-AGR score based on preoperative fibrinogen, neutrophil to lymphocyte ratio (NLR), and albumin to globulin ratio (AGR) in predicting the prognosis in patients with glioma. PATIENTS AND METHODS: 203 glioma patients were retrospectively analyzed. Receiver-operating characteristic (ROC) curve analysis was used to determine the optimal cut-off values for NLR, AGR, and fibrinogen. According to these cut-off values, patients with high NLR (>1.90), low AGR (<1.54), and elevated fibrinogen (>2.61 g/L) were defined as a score of 3, if none of the patients' three parameters met these standards they were given a score of 0, if any two or one parameter met these standards they were scored as 2 or 1, respectively. The correlation between F-NLR-AGR score and glioma grade was also evaluated. RESULTS: The three-year overall survival (OS) rate and the mean overall survival in patients with F-NLR-AGR=3 were lower than those of patients with F-NLR-AGR = 2, 1 or 0 [17.6% vs 35.2%, 66.9% or 83.7% (26.0 vs 39.0, 64.0 or 81.0 months), P<0.001]. Multivariate analysis revealed that age (HR=2.071; 95% CI=1.195-3.588; P=0.009), WHO grade (P<0.001), and F-NLR-AGR score (P<0.001) were independent prognostic factors for OS. Spearman's rank correlation analysis revealed that F-NLR-AGR score was positively correlated with glioma grade (r=0.278, P<0.01). CONCLUSION: Preoperative F-NLR-AGR score was correlated with glioma grading, high F-NLR-AGR score was an independent predictor of poor prognosis in glioma. Therefore, the scoring system may be applied in clinical practice to identify high-risk patients.