Oridonin alleviates d-GalN/LPS-induced acute liver injury by inhibiting NLRP3 inflammasome.

Acute liver injury (ALI) is a serious syndrome that is associated with high mortality, but there are few effective treatments. The activation of NLRP3 inflammasome is associated with ALI. Oridonin is a natural substance with an anti-inflammatory effect and has been reported to be an inhibitor of NLRP3. The aim of this study was to investigate the protective effect of oridonin on d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced ALI and whether the effect is mediated by NLRP3. Mice were pretreated with oridonin (5 or 10 mg/kg) for 3 days. Then, they were injected with d-GalN (400 mg/kg) and LPS (40 µg/kg). The levels of inflammatory factors were measured by RT-PCR, Western blot, and enzyme-linked immunosorbent assay. We confirmed that oridonin significantly alleviated ALI induced by d-GalN/LPS in mice. Oridonin markedly decreased the inflammatory response by reducing the levels of inflammatory cytokines. More importantly, oridonin markedly reduced the expression of NLRP3, caspase-1, IL-18, and IL-1ß. This study showed that oridonin has a protective effect on d-GalN/LPS-induced ALI, and the underlying mechanisms may be associated with the inhibition of the NLRP3 inflammatory pathways.

Metabolism and Toxicity of Emodin: Genome-Wide Association Studies Reveal Hepatocyte Nuclear Factor 4α Regulates UGT2B7 and Emodin Glucuronidation.

Emodin is the main toxic component in Chinese medicinal herbs such as rhubarb. Our previous studies demonstrated that genetic polymorphisms of UDP-glucuronosyltransferase 2B7 (UGT2B7) had an effect on the glucuronidation and detoxification of emodin. This study aimed to reveal the transcriptional regulation mechanism of UGT2B7 on emodin glucuronidation and its effect on toxicity. Emodin glucuronic activity and genome and transcriptome data were obtained from 36 clinical human kidney tissues. The genome-wide association studies (GWAS) identified that four single nucleotide polymorphisms (SNPs) (rs6093966, rs2868094, rs2071197, and rs6073433), which were located on the hepatocyte nuclear factor 4α (HNF4A) gene, were significantly associated with the emodin glucuronidation (p < 0.05). Notably, rs2071197 was significantly associated with the gene expression of HNF4A and UGT2B7 and the glucuronidation of emodin. The gene expression of HNF4A showed a high correlation with UGT2B7 (R2 = 0.721, p = 5.83 × 10-11). The luciferase activity was increased 7.68-fold in 293T cells and 2.03-fold in HepG2 cells, confirming a significant transcriptional activation of UGT2B7 promoter by HNF4A. The knockdown of HNF4A in HepG2 cells (36.6%) led to a significant decrease of UGT2B7 (19.8%) and higher cytotoxicity (p < 0.05). The overexpression of HNF4A in HepG2 cells (31.2%) led to a significant increase of UGT2B7 (24.4%) and improved cell viability (p < 0.05). Besides, HNF4A and UGT2B7 were both decreased in HepG2 cells and rats after treatment with emodin. In conclusion, emodin used long term or in high doses could inhibit the expression of HNF4A, thereby reducing the expression of UGT2B7 and causing hepatotoxicity.

In the Telencephalon, GluN2C NMDA Receptor Subunit mRNA is Predominately Expressed in Glial Cells and GluN2D mRNA in Interneurons.

N-methyl-D-aspartate receptors (NMDARs) are widely distributed in the brain with high concentrations in the telencephalon where they modulate synaptic plasticity, working memory, and other functions. While the actions of the predominate GluN2 NMDAR subunits, GluN2A and GluN2B are relatively well understood, the function of GluN2C and GluN2D subunits in the telencephalon is largely unknown. To better understand the possible role of GluN2C subunits, we used fluorescence in situ hybridization (FISH) together with multiple cell markers to define the distribution and type of cells expressing GluN2C mRNA. Using a GluN2C-KO mouse as a negative control, GluN2C mRNA expression was only found in non-neuronal cells (NeuN-negative cells) in the hippocampus, striatum, amygdala, and cerebral cortex. For these regions, a significant fraction of GFAP-positive cells also expressed GluN2C mRNA. Overall, for the telencephalon, the globus pallidus and olfactory bulb were the only regions where GluN2C was expressed in neurons. In contrast to GluN2C, GluN2D subunit mRNA colocalized with neuronal and not astrocyte markers or GluN2C mRNA in the telencephalon (except for the globus pallidus). GluN2C mRNA did, however, colocalize with GluN2D in the thalamus where neuronal GluN2C expression is found. These findings strongly suggest that GluN2C has a very distinct function in the telencephalon compared to its role in other brain regions and compared to other GluN2-containing NMDARs. NMDARs containing GluN2C may have a specific role in regulating L-glutamate or D-serine release from astrocytes in response to L-glutamate spillover from synaptic activity.

GluN2D N-Methyl-d-Aspartate Receptor Subunit Contribution to the Stimulation of Brain Activity and Gamma Oscillations by Ketamine: Implications for Schizophrenia.

The dissociative anesthetic ketamine elicits symptoms of schizophrenia at subanesthetic doses by blocking N-methyl-d-aspartate receptors (NMDARs). This property led to a variety of studies resulting in the now well-supported theory that hypofunction of NMDARs is responsible for many of the symptoms of schizophrenia. However, the roles played by specific NMDAR subunits in different symptom components are unknown. To evaluate the potential contribution of GluN2D NMDAR subunits to antagonist-induced cortical activation and schizophrenia symptoms, we determined the ability of ketamine to alter regional brain activity and gamma frequency band neuronal oscillations in wild-type (WT) and GluN2D-knockout (GluN2D-KO) mice. In WT mice, ketamine (30 mg/kg, i.p.) significantly increased [(14)C]-2-deoxyglucose ([(14)C]-2DG) uptake in the medial prefrontal cortex (mPFC), entorhinal cortex and other brain regions, and decreased activity in the somatosensory cortex and inferior colliculus. In GluN2D-KO mice, however, ketamine did not significantly increase [(14)C]-2DG uptake in any brain region examined, yet still decreased [(14)C]-2DG uptake in the somatosensory cortex and inferior colliculus. Ketamine also increased locomotor activity in WT mice but not in GluN2D-KO mice. In electrocorticographic analysis, ketamine induced a 111% ± 16% increase in cortical gamma-band oscillatory power in WT mice, but only a 15% ± 12% increase in GluN2D-KO mice. Consistent with GluN2D involvement in schizophrenia-related neurologic changes, GluN2D-KO mice displayed impaired spatial memory acquisition and reduced parvalbumin (PV)-immunopositive staining compared with control mice. These results suggest a critical role of GluN2D-containing NMDARs in neuronal oscillations and ketamine's psychotomimetic, dissociative effects and hence suggests a critical role for GluN2D subunits in cognition and perception.

Liver X receptor α contribution to neuroinflammation and glial cells activation induced by MPTP: Implications for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder whose etiology remains unknown. The immune system has been implicated in hallmarks of PD including aggregation of α-synuclein and death of dopaminergic neurons in the substantia nigra. As a core regulator of immune response and inflammation, liver X receptors (LXRs) have been shown to have protective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. With two isoforms of LXRs (LXRα and LXRß) expressed in the brain, their roles and distributions in this tissue remain largely unexplored. Here, we used MPTP to mimic symptoms and biomedical changes seen in PD in LXRα-/- and wild-type mice to investigate the role of LXRα in the etiology and progression of PD. We found that MPTP is unable to induce motor deficits, anxiety-like behavior in LXRα-/- mice, which has been seen in WT mice. Gene ontology analysis of RNA sequencing revealed that knockout of LXRα led to enrichment of the process, including immune response and inflammation in the midbrain. In addition, MPTP did not lead to dopaminergic neuron death in the striatum and substantia nigra in LXRα-/- mice, the basal GFAP protein level, and pro-inflammatory cytokines were elevated in LXRα-/- mice. Lastly, the microglia activation and astrogliosis caused by MPTP intoxication we found in WT mice were abolished in LXRα-/- mice. To sum up, we conclude that LXRα is a critical regulator in MPTP intoxication and may play a unique role in astrogliosis seen in the neuroinflammation of PD.

Effect of different anesthetic dose of pentobarbital on respiratory activity in rabbits.

Anesthetics inhibit the respiratory muscles and even cause upper airway to collapse. Diaphragm electromyography (EMGdi) and airflow signals are usually extracted to assess the degree of respiration inhibition by anesthetics. However, the ECG interference in EMGdi affects the accuracy of its time domain and frequency domain information extraction. We studied the changes in EMGdi (left EMGdi and right EMGdi) and airflow characteristics under two pentobarbital anesthetic doses. First, we filtered out the ECG in EMGdi based on the combination of stationary wavelet transform and the positioning of ECG to obtain EMGdi without ECG interference (EMGdip). The effectiveness of filtering algorithm was verified by calculating the power spectrum before and after noise reduction. Second, root mean square (RMS), average rectified value (ARV), and fixed sample entropy (fSampEn) were used to quantify EMGdi (left EMGdi, left EMGdip and right EMGdi). Median frequency (MF) and centroid frequency (fc) of EMGdi were calculated. Tidal volume, respiratory cycle duration and peak airflow were calculated from airflow. Finally, the average and standard deviation of these parameters for all rabbits (n = 10) were compared and analyzed under two anesthesia states. Our results indicate that anesthesia induced by an increase in pentobarbital dose leads to decrease in ventilation and EMGdi amplitude. There was no significant change in diaphragm power spectrum (MF and fc) with the increase of anesthesia dose.

Evaluation of Correlation Between Surface Diaphragm Electromyography and Airflow Using Fixed Sample Entropy in Healthy Subjects.

In clinic, the acquisition of airflow with nasal prongs, masks, thermistor to monitor respiratory function is more uncomfortable and inconvenience than surface diaphragm electromyography (EMGdi) using electrode pads. The EMGdi with strong electrocardiograph (ECG) interference affect the extraction of its characteristic information. In this work, surface EMGdi and airflow signals of 20 subjects were collected under 5 incremental inspiratory threshold loading protocols from quiet breathing to maximum forced breathing. First, we filtered out the ECG interference in EMGdi based on the combination of stationary wavelet transform and the positioning of ECG to obtain pure EMGdi (EMGdip). Second, the Spearman's rank correlation coefficients between EMGdi and EMGdip quantified by time series fixed sample entropy (fSampEn), root mean square (RMS), and envelope were compared to verify the robustness of the fSampEn to ECG. A comparative analysis of correlation between fSampEn of EMGdi and inspiratory airflow and the correlation between envelope of EMGdip (EMGdie) and inspiratory airflow found that there was no significant difference between the two, indicating the feasibility of using fSampEn to predict airflow. Moreover, fSampEn of EMGdi was used as characteristic parameter to build a quantitative relationship with the airflow by polynomial regression analysis. Mean coefficient of determination of all subjects in any breathing state is greater than 0.88. Finally, nonlinear programming method was used to solve a universal fitting coefficient between fSampEn of EMGdi and airflow for each subject to further evaluate the possibility of using surface EMGdi to monitor and control respiratory activity.

Quantification of fat-soluble vitamins and their metabolites in biological matrices: an updated review.

Fat-soluble vitamins (FSVs) are micronutrients essential in maintaining normal physiological function, metabolism and human growth. Ongoing increased awareness regarding FSV concentrations and their impact on human growth along with disease progression warrant the need of developing selective and sensitive analytical methods. LC-MS/MS is currently the method of choice for accurate quantitation of FSVs. However, there are multiple approaches for extraction, separation and calibration of FSVs in biological matrices. This review discusses recent LC-MS/MS methods for the simultaneous quantification of FSVs in biological matrices and summarizes sample pretreatment procedures, chromatographic conditions and calibration approaches. Current challenges and clinical applications in various disease states are also highlighted.

NMDA receptors containing GluN2C and GluN2D subunits have opposing roles in modulating neuronal oscillations; potential mechanism for bidirectional feedback.

NMDA receptor (NMDAR) antagonists such as ketamine, can reproduce many of the symptoms of schizophrenia. A reliable indicator of NMDAR channel blocker action in vivo is the augmentation of neuronal oscillation power. Since the coordinated and rhythmic activation of neuronal assemblies (oscillations) is necessary for perception, cognition and working memory, their disruption (inappropriate augmentation or inhibition of oscillatory power or inter-regional coherence) both in psychiatric conditions and with NMDAR antagonists may reflect the underlying defects causing schizophrenia symptoms. NMDAR antagonists and knockout (KO) mice were used to evaluate the role of GluN2C and GluN2D NMDAR subunits in generating NMDAR antagonist-induced oscillations. We find that basal oscillatory power was elevated in GluN2C-KO mice, especially in the low gamma frequencies while there was no statistically significant difference in basal oscillations between WT and GluN2D-KO mice. Compared to wildtype (WT) mice, NMDAR channel blockers caused a greater increase in oscillatory power in GluN2C-KO mice and were relatively ineffective in inducing oscillations in GluN2D-KO mice. In contrast, preferential blockade of GluN2A- and GluN2B-containing receptors induced oscillations that did not appear to be changed in either KO animal. We propose a model wherein NMDARs containing GluN2C in astrocytes and GluN2D in interneurons serve to detect local cortical excitatory synaptic activity and provide excitatory and inhibitory feedback, respectively, to local populations of postsynaptic excitatory neurons and thereby bidirectionally modulate oscillatory power.

A concise review of quantification methods for determination of vitamin K in various biological matrices.

Vitamin K is an essential nutrient in the body and involved in numerous physiological and pathophysiological functions. Both the lack and surplus of vitamin K can put human health at risk. Therefore, it becomes necessary to monitor vitamin K concentrations in different biomatrices through establishing sensitive and specific analytical methods. This review collectively describes an updated overview of the sample pretreatment methodologies and methods for quantitative determination of vitamin K that have been used in last two decades. High Performance Liquid Chromatography (HPLC) is commonly utilized as a standard for separation of vitamin K in combination with different detection including spectroscopic, spectrometric, fluorometric and mass spectroscopy. Recent progress in sample pretreatment technologies and quantitation methodologies have enhanced the ability to identify and quantitate vitamin K in biomatrices to further advance our understanding of the role of this vitamin in human health and disease.

The NMDA receptor GluN2C subunit controls cortical excitatory-inhibitory balance, neuronal oscillations and cognitive function.

Despite strong evidence for NMDA receptor (NMDAR) hypofunction as an underlying factor for cognitive disorders, the precise roles of various NMDAR subtypes remains unknown. The GluN2C-containing NMDARs exhibit unique biophysical properties and expression pattern, and lower expression of GluN2C subunit has been reported in postmortem brains from schizophrenia patients. We found that loss of GluN2C subunit leads to a shift in cortical excitatory-inhibitory balance towards greater inhibition. Specifically, pyramidal neurons in the medial prefrontal cortex (mPFC) of GluN2C knockout mice have reduced mEPSC frequency and dendritic spine density and a contrasting higher frequency of mIPSCs. In addition a greater number of perisomatic GAD67 puncta was observed suggesting a potential increase in parvalbumin interneuron inputs. At a network level the GluN2C knockout mice were found to have a more robust increase in power of oscillations in response to NMDAR blocker MK-801. Furthermore, GluN2C heterozygous and knockout mice exhibited abnormalities in cognition and sensorimotor gating. Our results demonstrate that loss of GluN2C subunit leads to cortical excitatory-inhibitory imbalance and abnormal neuronal oscillations associated with neurodevelopmental disorders.

Neuronal injury, but not microglia activation, is associated with ketamine-induced experimental schizophrenic model in mice.

Schizophrenia is a chronic debilitating psychiatric disorder affecting as many as 1% of the population worldwide. Unfortunately, its etiology and pathophysiology are poorly defined. Previous studies have shown that neuronal injury and microglia activation were observed in the schizophrenic patients. The present study aims to evaluate the role of neurons and microglia in ketamine-induced experimental schizophrenic model to further understand its pathophysiology. Firstly, ketamine was used to simulate the behavior abnormalities associated with schizophrenia. The effects of ketamine on mouse locomotor activity, Y-maze task, novel object recognition, and forced swimming test were studied. The results showed that ketamine (25, 50, and 100mg/kg i.p.) administered acutely or repeatedly (for 7 days) can increase the locomotor number significantly. In Y-maze task, ketamine (25, 50, and 100mg/kg) impaired spontaneous alternation after both acute and repeated treatments. In novel object recognition test, acute or chronic ketamine treatment showed no significant effect on mouse exploratory preference behavior. In forced swimming test, repeated treatment of ketamine (100mg/kg) enhanced the immobility duration. Secondly, immunohistochemical method was used to study the changes of neurons and microglia. The results showed that acute treatment of ketamine (100mg/kg) had no effect on neurons in the prefrontal cortex or hippocampus (1, 3, 5, and 7 days after the treatment). In contrast, repeated treatment of ketamine caused neuronal impairment in mouse hippocampus (3rd day, 5th day and 7th day after the final administration). The results of immunohistochemistry demonstrated that microglia in the prefrontal cortex and hippocampus were not affected after acute or repeated administration of ketamine. Finally, the neuronal impairment caused by repeated administration of ketamine was further investigated from the oxidative stress aspects. The results showed that repeated administration of ketamine increased nitric oxide (NO) and nitric oxide synthase (NOS) in prefrontal cortex, hippocampus and serum, while decreased SOD in hippocampus and serum. In summary, chronic ketamine treatment to mice successfully mimics the core behavioral deficits in schizophrenia. It is demonstrated for the first time that neuronal injury was associated with the chronic ketamine-induced experimental schizophrenic model, while microglial cells may play little role in this model. Oxidative stress may contribute to the significant neuronal injury in mouse brain induced by chronic ketamine treatment.