HMGB1 mediates synaptic loss and cognitive impairment in an animal model of sepsis-associated encephalopathy.

BACKGROUND: Microglial activation-mediated neuroinflammation is one of the essential pathogenic mechanisms of sepsis-associated encephalopathy (SAE). Mounting evidence suggests that high mobility group box-1 protein (HMGB1) plays a pivotal role in neuroinflammation and SAE, yet the mechanism by which HMGB1 induces cognitive impairment in SAE remains unclear. Therefore, this study aimed to investigate the mechanism of HMGB1 underlying cognitive impairment in SAE. METHODS: An SAE model was established by cecal ligation and puncture (CLP); animals in the sham group underwent cecum exposure alone without ligation and perforation. Mice in the inflachromene (ICM) group were continuously injected with ICM intraperitoneally at a daily dose of 10 mg/kg for 9 days starting 1 h before the CLP operation. The open field, novel object recognition, and Y maze tests were performed on days 14-18 after surgery to assess locomotor activity and cognitive function. HMGB1 secretion, the state of microglia, and neuronal activity were measured by immunofluorescence. Golgi staining was performed to detect changes in neuronal morphology and dendritic spine density. In vitro electrophysiology was performed to detect changes in long-term potentiation (LTP) in the CA1 of the hippocampus. In vivo electrophysiology was performed to detect the changes in neural oscillation of the hippocampus. RESULTS: CLP-induced cognitive impairment was accompanied by increased HMGB1 secretion and microglial activation. The phagocytic capacity of microglia was enhanced, resulting in aberrant pruning of excitatory synapses in the hippocampus. The loss of excitatory synapses reduced neuronal activity, impaired LTP, and decreased theta oscillation in the hippocampus. Inhibiting HMGB1 secretion by ICM treatment reversed these changes. CONCLUSIONS: HMGB1 induces microglial activation, aberrant synaptic pruning, and neuron dysfunction in an animal model of SAE, leading to cognitive impairment. These results suggest that HMGB1 might be a target for SAE treatment.

A role of GABAA receptor α1 subunit in the hippocampus for rapid-acting antidepressant-like effects of ketamine.

Ketamine can produce rapid-acting antidepressant effects in treatment-resistant patients with depression. Although alterations in glutamatergic and GABAergic neurotransmission in the brain play a role in depression, the precise molecular mechanisms in these neurotransmission underlying ketamine's antidepressant actions remain largely unknown. Mice exposed to FSS (forced swimming stress) showed depression-like behavior and decreased levels of GABA (γ-aminobutyric acid), but not glutamate, in the hippocampus. Ketamine increased GABA levels and decreased glutamate levels in the hippocampus of mice exposed to FSS. There was a correlation between GABA levels and depression-like behavior. Furthermore, ketamine increased the levels of enzymes and transporters on the GABAergic neurons (SAT1, GAD67, GAD65, VGAT and GAT1) and astrocytes (EAAT2 and GAT3), without affecting the levels of enzymes and transporters (SAT2, VGluT1 and GABAAR γ2) on glutamatergic neurons. Moreover, ketamine caused a decreased expression of GABAAR α1 subunit, which was specifically expressed on GABAergic neurons and astrocytes, an increased GABA synthesis and metabolism in GABAergic neurons, a plasticity change in astrocytes, and an increase in ATP (adenosine triphosphate) contents. Finally, GABAAR antagonist bicuculline or ATP exerted a rapid antidepressant-like effect whereas pretreatment with GABAAR agonist muscimol blocked the antidepressant-like effects of ketamine. In addition, pharmacological activation and inhibition of GABAAR modulated the synthesis and metabolism of GABA, and the plasticity of astrocytes in the hippocampus. The present data suggest that ketamine could increase GABA synthesis and astrocyte plasticity through downregulation of GABAAR α1, increases in GABA, and conversion of GABA into ATP, resulting in a rapid-acting antidepressant-like action. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

Kainate receptors GluK1 and GluK2 differentially regulate synapse morphology.

The regulation of dendritic spine morphology is a critical aspect of neuronal network refinement during development and modulation of neurotransmission. Previous studies revealed that glutamatergic transmission plays a central role in synapse development. AMPA receptors and NMDA receptors regulate spine morphology in an activity dependent manner. However, whether and how Kainate receptors (KARs) regulate synapse development remains poorly understood. In this study, we found that GluK1 and GluK2 may play distinct roles in synapse development. In primary cultured hippocampal neurons, we found overexpression of the calcium-permeable GluK2(Q) receptor variant increased spine length and spine head area compared to overexpression of the calcium-impermeable GluK2(R) variant or EGFP transfected, control neurons, indicating that Q/R editing may play a role in GluK2 regulation of synapse development. Intriguingly, neurons transfected with GluK1(Q) showed decreased spine length and spine head area, while the density of dendritic spines was increased, suggesting that GluK1(Q) and GluK2(Q) have different effects on synaptic development. Swapping the critical domains between GluK2 and GluK1 demonstrated the N-terminal domain (NTD) is responsible for the different effects of GluK1 and GluK2. In conclusion, Kainate receptors GluK1 and GluK2 have distinct roles in regulating spine morphology and development, a process likely relying on the NTD.

Astragaloside IV improves the pharmacokinetics of febuxostat in rats with hyperuricemic nephropathy by regulating urea metabolism in gut microbiota.

Hyperuricemic nephropathy (HN) is a common clinical complication of hyperuricemia. The pathogenesis of HN is directly related to urea metabolism in the gut microbiota. Febuxostat, a potent xanthine oxidase inhibitor, is the first-line drug used for the treatment of hyperuricemia. However, there have been few studies on the pharmacokinetics of febuxostat in HN animal models or in patients. In this study, a high-purine diet-induced HN rat model was established. The pharmacokinetics of febuxostat in HN rats was evaluated using LC-MS/MS. Astragaloside IV (AST) was used to correct the abnormal pharmacokinetics of febuxostat. Gut microbiota diversity analysis was used to evaluate the effect of AST on gut microbiota. The results showed that the delayed elimination of febuxostat caused drug accumulation after multiple administrations. Oral but not i. p. AST improved the pharmacokinetics of febuxostat in HN rats. The mechanistic study showed that AST could regulate urea metabolism in faeces and attenuate urea-ammonia liver-intestine circulation. Urease-related genera, including Eubacterium, Parabacteroides, Ruminococcus, and Clostridia, decreased after AST prevention. In addition, the decrease in pathogenic genera and increase in short-chain fatty acids (SCFA) producing genera also contribute to renal function recovery. In summary, AST improved the pharmacokinetics of febuxostat in HN rats by comprehensive regulation of the gut microbiota, including urea metabolism, anti-calcification, and short-chain fatty acid generation. These results imply that febuxostat might accumulate in HN patients, and AST could reverse the accumulation through gut microbiota regulation.

Distant coupling between RNA editing and alternative splicing of the osmosensitive cation channel Tmem63b.

Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5' end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.

Extrasynaptic CaMKIIα is involved in the antidepressant effects of ketamine by downregulating GluN2B receptors in an LPS-induced depression model.

BACKGROUND: A subanesthetic dose of ketamine provides rapid and effective antidepressant effects, but the molecular mechanism remains elusive. It has been reported that overactivation of extrasynaptic GluN2B receptors is associated with the antidepressant effects of ketamine and the interaction between GluN2B and calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) is important for GluN2B localization and activity. Here, we tested whether changes of CaMKIIα and GluN2B are involved in the antidepressant effects of ketamine. METHODS: Lipopolysaccharide (LPS) was injected intraperitoneally (i.p.) into male C57BL/6 mice. For the interventional study, mice were administrated with ketamine (10 mg/kg, i.p.) or a CaMKIIα inhibitor KN93. Behavioral alterations were evaluated by open-field, novelty-suppressed feeding, and forced-swimming tests. Physiological functions were evaluated by the body weight and fur coat state of mice. The levels of p-CaMKIIα, CaMKIIα, p-GluN2B, GluN2B, p-CREB, CREB, BDNF, GluR1, and GluR2 in the hippocampus were detected by western blotting. The interaction between GluN2B and CaMKIIα was studied using immunoprecipitation assay and small interfering RNA (siRNA) assays. The colocalizations of GluN2B/PSD95 and p-GluN2B/PSD95 were detected by immunofluorescence. The long-term potentiation (LTP) in SC-CA1 of the hippocampus was detected by electrophysiology. RESULTS: LPS injection induced depression-like behaviors, which were accompanied by significant increases in extrasynaptic p-CaMKIIα expression, extrasynaptic GluN2B localization, and phosphorylation and decreases in p-CREB, BDNF, and GluR1 expressions and LTP impairment. These changes were prevented by ketamine administration. Immunoprecipitation assay revealed that LPS induced an increase in the p-CaMKIIα-GluN2B interaction, which was attenuated by ketamine administration. SiRNA assay revealed that CaMKIIα knockdown reduced the level and number of clusters of GluN2B in the cultured hippocampal neurons. KN93 administration also reduced extrasynaptic p-CaMKIIα expression, extrasynaptic GluN2B localization, and phosphorylation and exerted antidepressant effects. CONCLUSION: These results indicate that extrasynaptic CaMKIIα plays a key role in the cellular mechanism of ketamine's antidepressant effect and it is related to the downregulation of extrasynaptic GluN2B localization and phosphorylation.

Quantification of the Plasma Concentration of Apatinib by 2-Dimensional Liquid Chromatography.

BACKGROUND: Apatinib is a new oral micromolecular tyrosine kinase inhibitor, which is mainly used as a third-line treatment for chemotherapy-refractory advanced metastatic gastric cancer patients. However, apatinib has shown dose titration and severe adverse reactions in clinical practice. Quantification of plasma concentrations of apatinib may be an effective method to balance the clinical efficacy and adverse reactions. The purpose of this study was to develop and validate a 2-dimensional liquid chromatography method for the measurement of apatinib in plasma. METHODS: The analysis of apatinib was performed using a 2-dimensional high-performance liquid chromatography system. We precipitated the proteins with acetonitrile. The mobile phases consisted of a first-dimensional mobile phase (acetonitrile:methanol:25 mmol·L ammonium phosphate = 25:25:50, V/V/V, pH adjusted to 7.2 using phosphoric acid) and a second-dimensional mobile phase (acetonitrile:10 mmol·L ammonium phosphate = 28:72, vol/vol, pH adjusted to 3.7 using phosphoric acid). The ultraviolet detection wavelength was set at 340 nm. The temperature of the detector cell was 40°C, and the injection volume was 500 µL. RESULTS: The range of calibration curve was 15.27-1491.48 ng/mL. The accuracy and imprecision were within ±2.23% and less than 10.22%, respectively (intraday and interday). The range of recovery was 97.45%-108.92%. The intraday and interday relative SDs (reproducibility) of high-performance liquid chromatography retention times were less than 0.18% and 0.46%, respectively. In the clinical assessment, the dose range of apatinib mesylate for patients with gastric cancer was 250-500 mg every day (2-60 days), resulting in trough plasma concentrations between 272.7 and 727.8 ng/mL. CONCLUSIONS: A simple, convenient, accurate, and robust 2-dimensional liquid chromatography method was developed and verified, which successfully determined the plasma concentrations of apatinib in patients with gastric cancer.

[Safflower's origin and changes of producing areas].

Human's application of safflower (Carthamus tinctorius) has a long history, but the origin remains unclear. Safflower was introduced into China for traditional Chinese medicine, and Sichuan was major producing area. However, in recent years, the main producing area is in Xinjiang province, in contrast Sichuan safflower is difficult to find. By reading relevant document literature and the production and marketing information of safflower, and having field investigation in the main producing areas, the origin of safflower and the reasons of producing areas' changes were explored. The origin of safflower is considered as the Fertile Crescent in reasonably. The change of producing areas in China is effected by the factors of natural environment and society. The suitability of producing areas and quality of safflower still need to study further.

Alterations in the inflammatory cytokines and brain-derived neurotrophic factor contribute to depression-like phenotype after spared nerve injury: improvement by ketamine.

Although pain is frequently accompanied with depression, little is known about the risk factors contributing to individual differences to the comorbidity of pain and depression. In this study, we examined whether cytokines and brain-derived neurotrophic factor (BDNF) might contribute to the individual differences in the development of neuropathic pain-induced depression. Rats were randomly subjected to spared nerved ligation (SNI) or sham surgery. The SNI rats were divided into two groups by the data from depression-related behavioral tests. Rats with depression-like phenotype displayed higher levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1ß, IL-6) as well as imbalance of pro/anti-inflammatory cytokines compared with rats without depression-like phenotype and sham-operated rats. Levels of BDNF in the prefrontal cortex of rats with depression-like phenotype were lower than those of rats without depression-like phenotype and sham-operated rats. A single dose of ketamine ameliorated depression-like behaviors in the rats with depression-like phenotype. Interestingly, higher serum levels of IL-1ß and IL-6 in the rat with depression-like phenotype were normalized after a single dose of ketamine. These findings suggest that alterations in the inflammatory cytokines and BDNF might contribute to neuropathic pain-induced depression, and that serum cytokines may be predictable biomarkers for ketamine's antidepressant actions.

Spared Nerve Injury Increases the Expression of Microglia M1 Markers in the Prefrontal Cortex of Rats and Provokes Depression-Like Behaviors.

Pain and depression are frequently co-existent in clinical practice, yet the underlying mechanisms remain largely to be determined. Microglia activation and subsequent pro-inflammatory responses play a crucial role in the development of neuropathic pain and depression. The process of microglia polarization to the pro-inflammatory M1 or anti-inflammatory M2 phenotypes often occurs during neuroinflammation. However, it remains unclear whether M1/M2 microglia polarization is involved in the neuropathic pain induced by spared nerve injury (SNI). In the present study, the mechanical withdrawal threshold, forced swim test, sucrose preference test, and open field test were performed. The levels of microglia markers including ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation 11b (CD11b), M1 markers including CD68, inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-a (TNF-α), 8-hydroxy-2-deoxyguanosine (8-OH-dG), and M2 markers including CD206, arginase 1 (Arg1), IL-4 in the prefrontal cortex were determined on day 14 after SNI. The results showed that SNI produced mechanical allodynia and depressive-like behaviors, and also increased the expressions of microglia markers (Iba1, CD11b) and M1 markers (CD68, iNOS, IL-1ß, TNF-α, and 8-OH-dG) in the prefrontal cortex. Notably, minocycline administration reversed these abnormalities. In addition, minocycline also promoted M2 microglia polarization as evidenced by up-regulation of CD206 and Arg1. In conclusion, data from our study suggest that SNI can lead to depression-like behaviors, while M1 polarization and consequent overproduction of pro-inflammatory cytokines plays a key role in the pathogenesis of neuropathic pain. The data furthermore indicate that modulation of inflammation by inhibition of M1 polarization could be a strategy for treatment of neuropathic pain, and might prevent the induction of neuropathic pain-induced depression symptoms.

Six generations of epidermolytic palmoplantar keratoderma, associated with a KRT9 R163W mutation.

Epidermolytic palmoplantar keratoderma (EPPK) is a rare autosomal dominant skin disorder characterized by diffuse hyperkeratosis on the palms and soles. Whole-exome sequencing (WES) has become a powerful tool for the detection of rare causal variants of Mendelian disorders. However, no causal gene for EPPK in the Uygur population has been identified until now, and no treatment exists than can address the underlying pathology.WES analysis was undertaken on two individuals from a large Uygur EPPK pedigree whose disease locus mapped to 17q21.2 (chr:38994621-39893408) following previous linkage analysis. KRT9 (NM_000226.3:c.487C>T, p.Arg163Trp), and KRT15 (XM_005257346.1:c.212G>T, XP_005257403.1:p.Gly71Val) located in this region, have been identified as two candidate causative genes for EPPK in the Uygur family. Sanger sequencing was conducted on this region in other affected individuals (n = 38) from this family, non-affected individuals (n = 56) from this family and 100 unrelated controls. The missense mutation KRT9 c.487C>T, identified in this large Uygur population, is a potential causative mutation. To date, EPPK has no effective therapy, and siRNA is a potential avenue for EPPK therapy. To investigate this, full-length wild-type Keratin9 (KRT9; pKRT9-WT) and p.Arg163Trp (pKRT9-R163W) were then transfected into HaCaT cells. The small interfering RNAs targeting the KRT9 R163W mutant and wildtype KRT9 were transfected into HaCaT cells, and total RNA isolated at 72 h post-transfection. Quantitative polymerase chain reaction and western blotting were used to analyse the effects of knock-down on KRT9 mRNA and protein levels, respectively. siRNA was shown to specifically inhibit mutant KRT9 mRNA and protein expression (p < 0.01, with 95% confidence limits). Our study suggests that KRT9 is a causal gene for EPPK. This information is helpful for understanding the pathogenesis of EPPK in the Uygur population and raises the possibility of designing a novel siRNA treatment strategy for this population of EPPK patients.

[Extraction of intraocular foreign body at or near the ciliary body under endoscopic vitrectomy].

OBJECTIVE: To study the feasibility and effectiveness of the extraction of intraocular foreign body (IOFB) at or near the ciliary body by using endoscopic vitrectomy. METHODS: It was a retrospective case series study. Clinical materials and data of 15 patients (16 eyes) with IOFB at or near the ciliary body from July 2002 to December 2010 were analyzed retrospectively. Vitrectomy and IOFB extraction under endoscope were conducted in all cases. RESULTS: Thirty six IOFB (18 magnetic and 18 nonmagnetic IOFBs) were extracted successfully. The size of IOFB ranged from smaller than 1 mm×1 mm×1 mm to 9 mm×6 mm×4 mm. Among them, 31 IOFB were extracted from ciliary body or nearby and 5 IOFB were located at other places. The cornea was opaque before the operation in 11 eyes. Postoperative visual acuity was improved in 12 eyes. Vision was improved from finger-counting and below to 0.1-0.3 in 4 eyes. Intraocular pressure was normal in 14 eyes. The retina was reattached in 12 eyes. The cornea was clearer than before in 8 eyes. After the end of the fellow-up, vision was remained in finger-counting and below in only 7 eyes. CONCLUSIONS: There was almost no blind spot by using endoscope to observe the ciliary body, periphery retina tear or other pathological changes, the IOFB at or near ciliary body and the whole retina in the vitrectomy. This makes the extraction of IOFB at or near the ciliary body and the conduct of cyclophotocoagulation more accurate and simple.

A new triterpenoid from Panax ginseng exhibits cytotoxicity through p53 and the caspase signaling pathway in the HepG2 cell line.

A new triterpenoid, 20(R),22(xi),24(S)-dammar-25(26)-ene-3beta,6 alpha,12 beta,20,22,24-hexanol (1), and three known triterpenoids, beta-D-glucopyranoside,(3beta,12 beta)-12,20-dihydroxydammar-24-en-3-yl,6-acetate (2), 20(R)-ginsenoside Rg3 (3), and 20(R)-ginsenoside Rh2 (4), were isolated from the leaves of Panax ginseng. Their structures were determined by chemical analysis and spectral methods (IR, 1D and 2D NMR, HR-ESI-MS). Compounds 1-4 were exhibited various degrees of cytotoxicity in the human hepatoma cell line, HepG2. Compound 1 had the highest cytotoxic potency, with an IC50 value of 20.1 microM, by stimulating p53-mediated cell cycle arrest at the G1 to S phase transition, leading to apoptosis via activation of the caspase signaling pathway.

[Cardiac enzymes related to high-altitude hypoxic adaptation in Tibetan chicken].

AIM: The purpose of this experiment was to investigate the cardiac physiological characteristics for adaptation to high-altitude hypoxia in chickens. METHODS: Tibetan, Dwarf Recessive White and Shouguang chickens were fed at low-and high-altitude, and measurements were made in heart weights, lactic acid (LA), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) at the age of 10 weeks. RESULTS: The results showed that Tibetan chickens at high-altitude had lower heart weight and LA content, and similar LDH activity, and higher SDH activity when compared to Dwarf Recessive White and Shouguang chickens. CONCLUSION: It was concluded that the cardiac mechanisms of high-altitude hypoxic adaptation in Tibetan chickens were increasing neither heart weight, nor level of anaerobic metabolism, but the higher SDH activity was significant to the adaptation. The SDH was a symbol enzyme for hypoxic adaptation in Tibetan chicken.