Differentiation of umbilical cord mesenchymal stem cells into hepatocytes with CYP450 metabolic enzyme activity induced by a liver injury microenvironment.

The aim of this study was to observe the effect of a simulated liver tissue injury microenvironment on the directed differentiation of umbilical cord mesenchymal stem cells into hepatocytes with CYP450 metabolic activity in vitro, and to explore the mechanisms underlying this directed differentiation. Normal and damaged liver tissue homogenate supernatants (LHS and CCl4-LHS, respectively) were used as induction fluids. After induction for different durations, Western blot and RT-PCR were used to measure the protein and gene expression of the hepatocellular proteins AFP, CK18, ALB, and the CYP450 family. Simultaneously, the metabolic activity of CYP450 in hepatocytes was determined. Compared with the LHS and CCl4-LHS controls, the LHS and CCl4-LHS induction groups showed a significantly elevated protein and gene expression of AFP, CK18, ALB, CYP1A1/2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 (P < 0.05). The metabolic activity of CYP450 in hepatocytes was increased (P < 0.05). In addition, compared with the LHS group, the CCl4-LHS group induced cell differentiation more rapidly and with a higher efficiency. The results suggested that a liver injury microenvironment is conducive for the directed differentiation of umbilical cord mesenchymal stem cells into hepatocytes with metabolic enzyme activity.

Anxiolytic effects of hippocampal neurosteroids in normal and neuropathic rats with spared nerve injury.

Neurosteroids are synthesized in the nervous system from cholesterol or steroidal precursors imported from peripheral sources. These compounds are important allosteric modulators of GABAA receptors, which play a vital role in modulating hippocampal functions. Chronic pain is accompanied by increased neurosteroid production in the spinal cord and thalamus. We hypothesize that hippocampal neurosteroids participate in pain or pain-associated emotions, which we tested with high-performance liquid chromatography/tandem mass spectrometry and pharmacological behavioral tests. We observed increased levels of hippocampal neurosteroids (pregnenolone, progesterone, deoxycorticosterone, and allopregnanolone) in rats with chronic neuropathic pain (28 days after spared nerve injury). Meanwhile, the expression of the translocator protein, the upstream steroidogenesis rate-limiting enzyme, increased in the ventral but not dorsal hippocampus of neuropathic rats. In both naïve and neuropathic rats, in vivo stereotaxic microinjection of PK 11195, the translocator protein inhibitor, into the ventral hippocampus exacerbated anxiety-like behaviors. These results indicate anxiolytic effects of hippocampal neurosteroids in both normal and neuropathic rats. Neurosteroids could be considered as agents for treatment of general and pain-related anxiety disorders.

[In vivo tracking of PKH26-labeled human umbilical cord mesenchymal stem cells after transplantation into rats with liver cirrhosis].

OBJECTIVE: To observe the in vivo migration of human umbilical cord mesenchymal stem cells (hUCMSCs) labeled with the PKH26 red fluorescent dye after transplantation into rats with liver cirrhosis. METHODS: Frozen hUCMSCs were resuscitated and labeled with PKH26. Labeling efficiency and fluorescent maintenance time of the PKH26-1abeled cells were measured. Morphology of the labeled and unlabeled (control) cells was observed by microscopy. The cell growth curve was determined using the MTT method. The PKH26-1abeled hUCMSCs were transplanted via tail vein injection into healthy (control) rats and rats with liver cirrhosis. Migration of the PKH26-1abeled hUCMSCs observed 48 h later in frozen liver sections under a fluorescence microscope. RESULTS: The labeling ratio of PKH26 to hUCMSCs was 100%. Growth of the labeled cells was good. The cell morphology was not significantly different between the labeled and unlabeled cells; all cells were long and spindle-like. Cell proliferation was not impacted significantly by labeling.Fluorescence was maintained for at least 20 days, as detected by in vitro analysis. After transplantation into the rats, the PKH26-1abeled hUCMSCs were mainly distributed in the area surrounding the portal vein, the blood vessels, and the false lobule of the cirrhotic liver; a small amount ofhUCMSCs were present in the spleen and lung. CONCLUSION: PKH26 is an ideal fluorescent dye to label hUCMSCs. The PKH26 labeling technique can be used to study the migration of hUCMSCs in cirrhotic liver.

Exploring Sustainable Development Pathways for Agri-Food Supply Chains Empowered by Cross-Border E-Commerce Platforms: A Hybrid Grounded Theory and DEMATEL-ISM-MICMAC Approach.

As cross-border e-commerce platforms become increasingly integrated into the agricultural supply chain, the establishment of a sustainable supply chain ecosystem is of paramount importance. This study, grounded in the platform theory and the supply chain ecosystem theory, combines the grounded theory and the DEMATEL-ISM-MICMAC model to thoroughly analyze the complex mechanisms driving sustainable development. Utilizing the grounded theory, we construct a system of driving factors comprising five primary indicators and eighteen secondary indicators. The hybrid model reveals the interrelationships, significance, system hierarchy, and dependence-driving relationships among these factors. Notably, the driving factor system is categorized into a six-level hierarchical structure, encompassing profound elements, such as policy optimization and digital empowerment, as well as surface-level factors, such as simplification of customs procedures and consumer demand forecasting. Based on the analysis results, this research proposes a set of pathways to achieve the sustainability of the supply chain. These strategies involve improving cross-border agricultural e-commerce policy frameworks, enhancing digital-driven supply-demand coordination, strengthening logistics infrastructure and transparency, and cultivating brand influence. The study's findings not only enrich the relevant theories but also provide practical guidance for the coordinated advancement of economic, social, environmental, and resilient development. Furthermore, they are conducive to advancing the United Nations Sustainable Development Goals.

Progesterone Suppresses Cholesterol Esterification in APP/PS1 mice and a cell model of Alzheimer's Disease.

AIMS: Cholesteryl ester(CE), generated from the mitochondria associated membrane (MAM), is involved in the pathogenesis of Alzheimer's Disease (AD). In theory, the different neuroprotective effects of progesterone in AD are all linked to MAM, yet the effect on cholesterol esterification has not been reported. Therefore, this study was aimed to investigate the regulation of progesterone on intracerebral CE in AD models and the underlying mechanism. METHODS: APP/PS1 mice and AD cell model induced by Aß 25-35 were selected as the research objects. APP/PS1 mice were daily administrated intragastrically with progesterone and The Morris Water Maze test was performed to detect the learning and memory abilities. Intracellular cholesterol was measured by Cholesterol/Cholesteryl Ester Quantitation Assay. The structure of MAMs were observed with transmission electron microscopy. The expression of acyl-CoA: cholesterol acyltransferase 1 (ACAT1), ERK1/2 and p-ERK1/2 were detected with western blotting, immunohistochemistry or immunofluorescence. RESULTS: Progesterone suppressed the accumulation of intracellular CE, shortened the length of abnormally prolonged MAM in cortex of APP/PS1 mice. Progesterone decreased the expression of ACAT1, which could be blocked by progesterone receptor membrane component 1 (PGRMC1) inhibitor AG205. The ERK1/2 pathway maybe involved in the progesterone mediated regulation of ACAT1 in AD models, rather than the PI3K/Akt and the P38 MEPK pathways. SIGNIFICANCE: The results supported a line of evidence that progesterone regulates CE level and the structure of MAM in neurons of AD models, providing a promising treatment against AD on the dysfunction of cholesterol metabolism.

Progesterone attenuates Aß25-35-induced neuronal toxicity by activating the Ras signalling pathway through progesterone receptor membrane component 1.

AIMS: Progesterone receptor membrane component 1 (PGRMC1) has been reported to mediate the neuroprotective effect of progesterone, but the exact mechanism has not been elucidated. Therefore, the purpose of this study was to investigate the signalling pathway downstream of PGRMC1 in progesterone-induced neuroprotection. Recognition of the mechanism of progesterone opens novel perspectives for the treatment of diseases of the nervous system. MAIN METHODS: The PGRMC1 protein level was knocked down in rat primary cortical neurons, and Aß25-35 was used to establish an Alzheimer's disease cell model. The neuroprotective effect of progesterone was assessed by Hoechst 33258 staining and a cell counting kit-8 (CCK-8) assay. Then, proteomic and bioinformatic methods were used to analyse the proteins altered in response to PGRMC1 silencing to identify target proteins and signalling pathways involved in PGRMC1-mediated progesterone-induced neuroprotection. These findings were further verified by using signalling pathway inhibitors and western blotting. KEY FINDINGS: The neuroprotective effect of progesterone was significantly attenuated with PGRMC1 silencing. The expression of many proteins in the Ras signalling pathway was significantly changed in response to PGRMC1 silencing. FTI-277 inhibited progesterone-induced neuroprotection. Progesterone increased the expression of total Ras and Grb2. SIGNIFICANCE: These findings provide new perspectives for understanding the mechanism of and role of PGRMC1 in progesterone-induced neuroprotection. The Ras signalling pathway is the signalling pathway downstream of PGRMC1 in the mediation of progesterone-induced neuroprotection.

A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.

Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

The neuroprotection of progesterone against Aß-induced NLRP3-Caspase-1 inflammasome activation via enhancing autophagy in astrocytes.

Neuroinflammation and autophagy dysfunction are known to be involved in the pathological procession of Alzheimer's disease (AD). Progesterone (PG), neuroactive steroids, exerts a characteristic neuroprotective function in improving AD syndrome. The NOD-like receptor pyrin 3 (NLRP3)-Caspase-1 inflammasome has specific relevance to AD pathological procession. However, the exact role of PG in regulating NLRP3-Caspase-1 inflammasome remains to be elucidated. We demonstrated Aß up-regulated IL-1ß expression in astrocytes by activating NLRP3-Caspase-1 inflammasome. However, pharmacological activation of autophagy by Rapamycin (RAPA) efficiently suppressed Aß-, lipopolysaccharides (LPS)-induced IL-1ß expression via regulating NLRP3-Caspase-1 inflammasome in astrocytes. Remarkably, PG significantly inhibited Aß-induced NLRP3-Caspase-1 inflammasome activation. Autophagy inhibitor 3-MA blocked the protective effects of PG in mediating NLRP3 inflammasome and IL-1ß processing. Taken together, our observations suggest that autophagy-lysosome pathway is one specific molecular mechanism in regulating Aß-induced NLRP3-Caspase-1 inflammasome activation in astrocytes, particularly uncover the potential neuroprotection of PG in regulating upstream signaling leading to the sequence events of neuroinflammation. That neuroprotective mechanism of PG in regulating NLRP3-Caspase-1 inflammasome can be a potential therapeutic target for ameliorating the pathological procession of AD.

Effects of progesterone on glucose uptake in neurons of Alzheimer's disease animals and cell models.

AIMS: Alzheimer's disease (AD) is closely related to abnormal glucose metabolism in the central nervous system. Progesterone has been shown to have obvious neuroprotective effects in the pathogenesis of AD, but the specific mechanism has not been fully elucidated. Therefore, the purpose of this study was to investigate the effect of progesterone on the glucose metabolism of neurons in amyloid precursor protein (APP)/presenilin 1 (PS1) mice and Aß-induced AD cell model. MATERIALS AND METHODS: APP/PS1 mice were treated with 40 mg/kg progesterone for 40 days and primary cultured cortical neurons were treated with 1 µM progesterone for 48 h.Then behavior tests,2-NBDG glucose uptake tests and the protein levels of glucose transporter 3 (GLUT3), GLUT4, cAMP-response element binding protein (CREB) and proliferator-activated receptor γ (PPARγ) were examined. KEY FINDINGS: Progesterone increased the expression levels of GLUT3 and GLUT4 in the cortex of APP/PS1 mice, accompanied by an improvement in learning and memory. Progesterone increased the levels of CREB and PPARγ in the cerebral cortex of APP/PS1 mice. In vitro, progesterone increased glucose uptake in primary cultured cortical neurons, this effect was blocked by the progesterone receptor membrane component 1 (PGRMC1)-specific blocker AG205 but not by the progesterone receptor (PR)-specific blocker RU486. Meanwhile, progesterone increased the expression of GLUT3, GLUT4, CREB and PPARγ, and AG205 blocked this effect. SIGNIFICANCE: These results confirm that progesterone significantly improves the glucose metabolism of neurons.One of the mechanisms of this effect is that progesterone upregulates protein expression of GLUT3 and GLUT4 through pathways PGRMC1/CREB/GLUT3 and PGRMC1/PPARγ/GLUT4.

Predicting blood-brain barrier penetration of drugs by microemulsion liquid chromatography with corrected retention factor.

This study attempted to optimize the compositions of microemulsion mobile phase to model the drug penetration across blood-brain barrier (BBB) by microemulsion liquid chromatography (MELC). During the study, a continuous increase of the retentions along the operation time was found for all the test drugs. A correction method using methyl paraben and propranolol as the internal standards was proposed, which improved the reliability of the retention time significantly. The corrected retention factors were then used to develop the predictive model. The optimized MELC system with microemulsion consisting of 3.3% sodium dodecyl sulfate (SDS)-6.6% butanol-1.6% heptane-88.5% phosphate buffer (pH 7.0) showed superiorities to other lipophilicity measuring systems for modeling BBB penetration.

Progesterone suppresses Aß42-induced neuroinflammation by enhancing autophagy in astrocytes.

Autophagy is an intracellular catabolic mechanism essential for recycling intracellular unfolding protein and eliminating toxic protein aggregates. Several studies have shown that deficient autophagy is implicated in the development of Alzheimer's disease (AD) progression. To date, rapidly emerging evidence suggests that neurosteroid progesterone (PG) may play an important role in ameliorating AD. However, the role of PG and its neuroprotective mechanism in regulating autophagy still require further investigation. Here, we investigated the protective effects of PG against Aß-induced inflammatory responses in astrocytes and its underlying mechanism in mediating autophagy. Remarkably, Aß induced astrocyte dysfunction in autophagic activation and up-regulated inflammatory secretion. However, the autophagy inducer rapamycin (RAPA) significantly suppressed Aß-induced inflammation in astrocytes. In astrocytes, treatment with Aß caused autophagy deficiency, whereas PG significantly increased autophagy activation. Finally, PG suppressed Aß-induced neuroinflammatory production via enhancing autophagy together with regulating mTOR signaling. Taken together, these results show that autophagy is a vital mechanism against Aß-induced neuroinflammatory responses in astrocytes and demonstrate the potential neuroprotective mechanism of PG in suppressing neuroinflammatory responses by enhancing autophagy. Therefore, uncovering the neuroprotective mechanism of PG may provide new insight into novel therapies for the amelioration of AD.

Increased opioid receptor binding and G protein coupling in the accumbens and ventral tegmental area of postnatal day 2 rats.

In some regions of the developing rat brain such as the nucleus accumbens (Acb), mu opioid (MOP) receptor specific binding in the perinatal period exceeds that in the adult. To investigate the significance of these developmental changes, MOP and nociceptin/orphanin FQ (NOP) receptor binding and G protein coupling as determined by GTPgammaS binding experiments were examined in mesolimbic regions of postnatal day 2 (P2) pups and compared to those of their dams. Acb of the P2 pup exhibited 2-fold greater MOP receptor specific binding than that of the dam. In the ventral tegmental area (VTA), NOP specific binding was about 2-fold higher in the P2 pup. A correlation was found between MOP and NOP binding and their coupling to G protein on dam and P2 pup brain sections. However, the magnitude of increases in MOP and NOP receptor G protein coupling to G protein in P2 pups exceeded the 2-fold differences in binding between pups and dams. Furthermore, the amplitude of the MOP receptor G protein coupling in female P2 Acb was greater than increases in male P2 pup Acb. Differences in MOP and NOP binding and G protein coupling in other mesolimbic regions between P2 pups and dams were rarely observed. The data indicate that greater binding and G protein coupling of MOP and NOP receptors occur in discrete, mesolimbic regions of P2 pups when compared to their dams. It may be of significance that these brain regions, Acb and VTA, are undergoing maturation on P2.

Determination of total tiopronin in human plasma by LC-ESI-MS using tris (2-carboxy-ethyl) phosphine as reducing reagent and methyl acrylate as derivatization reagent for the thiol group.

A quantitative method for the determination of total tiopronin (TP) in human plasma was developed by liquid chromatography with electrospray ionisation (ESI) mass spectrometric detection. After reduction with tris (2-carboxy-ethyl) phosphine (TCEP) and derivatization with methyl acrylate (MA) for the thiol group of TP, plasma samples were processed successively by deproteinization and solid phase extraction. N-acetyl-l-cysteine (NAC) was selected as internal standard undergoing the same treatment as TP. The method was validated that it could meet the need of biological analysis. The lower limit of quantitation (LLOQ) of TP in plasma was 0.02microg/mL. Finally, the method was successfully applied to a pharmacokinetic study in 20 healthy Chinese male volunteers after an oral dose of 200mg TP tablets.

Selective regulation of neurosteroid biosynthesis under ketamine-induced apoptosis of cortical neurons in vitro.

Numerous studies have suggested that ketamine administration can induce neuroapoptosis in primary cultured cortical neurons. Neurosteroids modulate neuronal function and serve important roles in the central nervous system, however the role of neurosteroids in neuroapoptosis induced by ketamine remains to be elucidated. The present study aimed to explore whether neurosteroidogenesis was a pivotal mechanism for neuroprotection against ketamine-induced neuroapoptosis, and whether it may be selectively regulated under ketamine-induced neuroapoptosis conditions in primary cultured cortical neurons. To study this hypothesis, the effect of ketamine exposure on neurosteroidogenesis in primary cultured cortical neurons was investigated. Cholesterol, a substrate involved in the synthesis of neurosteroids, was added to the culture medium, and neurosteroids were quantified using high-performance liquid chromatography-tandem mass spectrometry analysis. The data demonstrated that cholesterol blocked ketamine-induced neuroapoptosis by promoting the synthesis of various neurosteroids, and the pathway of neurosteroid testosterone conversion into estradiol was inhibited by ketamine exposure. These data suggest that endogenous neurosteroids biosynthesis is critical for neuroprotection against ketamine-induced neuroapoptosis and inhibiting the biosynthesis of neuroprotective-neurosteroid estradiol is of notable importance for ketamine-induced neuroapoptosis.

[EFFECT OF BLOOD MICROENVIRONMENT OF RATS WITH HEPATIC FIBROSIS ON DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES AND ITS MECHANISMS].

OBJECTIVE: To investigate the effect of blood microenvironment of rats with hepatic fibrosis on differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes and its mechanisms. METHODS: Eighteen male adult Sprague Dawley rats [weighing, (200±20) g] were used, liver fibrosis was induced in 12 rats by repeated intraperitoneal injections of thioacetamide. The serum was separated after successful model preparation, and the serum of 6 normal rats was collected. ELISA assay was used to detect the concentrations of epidermal growth factor (EGF), hepatocyte growth factor (HGF), oncostatin M (OSM), and basic fibroblastic growth factor (bFGF). Passage 3 HUCMSCs were divided into 3 groups: cells were cultured for 7 days in DMEM/F12 containing 10% fetal bovine serum and 5?mL/ L serum from rats with hepatic fibrosis (group A), in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from normal rats (group B), and in DMEM/F12 containing 10% fetal bovine serum (group C). The morphological changes of the cells were observed. The expressions of α-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunofluorescence. The protein levels of albumin (ALB), tryptophan 2, 3-dioxygenase (TPH2), and CYP3A4 and MAPK/ERK signal pathway protein (P-ERK) were detected using Western blot. The content of blood urea nitrogen (BUN) was measured by diacetyl m onoxime method. RESULTS: HE staining showed that the liver tissue of rats was in accordance with the change of fibrosis, indicating successful model preparation. In serum of normal rats and rats with hepatic fibrosis, the concentrations of EGF were (21.42±0.32) pg/mL and (17.57±0.31) pg/mL respectively, showing significant difference (t=14.989, P=0.000); the concentrations of OSM were (129.96±0.65) pg/mL and (98.44±1.32) pg/mL respectively, showing significant difference (t=37.172, P=0.000); the concentrations of HGF were below the detection limit and (1.03±0.12)?ng/ mL respectively; and the concentrations of bFGF were lower than the detection limit in both groups. No morphological changes of cells were observed in both groups at 7 days, and there was no significant difference between groups. At 7 days after culture, the cells in group A could express human hepatocyte biomarkers of AFP, CK18 and hepatocyte-specific-function proteins of ALB, TPH2, and CYP3 A4 while cells in groups B and C did not. Western blot showed that cells in each group could express P-ERK protein. The relative level of P-ERK protein in group A was significantly higher than that in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The BUN concentration of group A [(0.74±0.07)?mmol/ L] was significantly higher than that of groups B [(0.40±0.04)?mmol/ L] and C [(0.38±0.04) mmol/L] (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05). CONCLUSIONS: Under the condition of hepatic fibrosis, the level of HGF will increase while EGF and OSM will decrease. The formed blood microenvironment?will activate MAPK/ERK signal pathway in HUCMSCs, induce them differentiate into hepatocytes.

Progesterone exerts neuroprotective effects against Aß-induced neuroinflammation by attenuating ER stress in astrocytes.

The deposition of amyloid-ß (Aß) and neuroinflammation are critical pathological features of Alzheimer's disease (AD). Astrocytes are considered the principal immunoregulatory cells in the brain. Neurosteroid progesterone (PG) exerts neuromodulatory properties, particularly its potential therapeutic function in ameliorating AD. However, the role of PG and the neuroprotective mechanism involving in the regulation of neuroinflammation in astrocytes warrant further investigation. In this study, we found that Aß significantly increased the processing of neuroinflammatory responses in astrocytes. The processing is induced by an increase activity of PERK/elF2ɑ-dependent endoplasmic reticulum (ER) stress. Additionally, the inhibition of ER stress activation by Salubrinal significantly suppressed the Aß-induced neuroinflammatory responses in astrocytes. While the treatment of astrocytes with Aß caused an increase of neuroinflammatory responses, PG significantly inhibited Aß-induced neuroinflammatory cytokine production by suppressing ER stress activation together with attenuating PERK/elF2ɑ signalling. Taken together, these results indicate that PG exerts a neuroprotective effect against Aß-induced neuroinflammatory responses, and significantly suppresses ER stress activation, which is an important mediator of the neurotoxic events occurring in Aß-induced neuroinflammatory responses in astrocytes. These neuroprotective mechanisms may facilitate the development of therapies to ameliorate AD.

Overexpression of SIRT6 in the hippocampal CA1 impairs the formation of long-term contextual fear memory.

Histone modifications have been implicated in learning and memory. Our previous transcriptome data showed that expression of sirtuins 6 (SIRT6), a member of Histone deacetylases (HDACs) family in the hippocampal cornu ammonis 1 (CA1) was decreased after contextual fear conditioning. However, the role of SIRT6 in the formation of memory is still elusive. In the present study, we found that contextual fear conditioning inhibited translational expression of SIRT6 in the CA1. Microinfusion of lentiviral vector-expressing SIRT6 into theCA1 region selectively enhanced the expression of SIRT6 and impaired the formation of long-term contextual fear memory without affecting short-term fear memory. The overexpression of SIRT6 in the CA1 had no effect on anxiety-like behaviors or locomotor activity. Also, we also found that SIRT6 overexpression significantly inhibited the expression of insulin-like factor 2 (IGF2) and amounts of proteins and/or phosphoproteins (e.g. Akt, pAkt, mTOR and p-mTOR) related to the IGF2 signal pathway in the CA1. These results demonstrate that the overexpression of SIRT6 in the CA1 impaired the formation of long-term fear memory, and SIRT6 in the CA1 may negatively modulate the formation of contextual fear memory via inhibiting the IGF signaling pathway.

Effect of Microenvironment on Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Hepatocytes In Vitro and In Vivo.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are considered to be an ideal cell source for cell therapy of many diseases. The aim of this study was to investigate the contribution of the microenvironment to the hepatic differentiation potential of hUCMSCs in vitro and in vivo and to explore their therapeutic use in acute liver injury in rats. We established a new model to simulate the liver tissue microenvironment in vivo using liver homogenate supernatant (LHS) in vitro. This induced environment could drive hUCMSCs to differentiate into hepatocyte-like cells within 7 days. The differentiated cells expressed hepatocyte-specific markers and demonstrated hepatocellular functions. We also injected hUCMSCs into rats with CCl4-induced acute hepatic injury. The hUCMSCs were detected in the livers of recipient rats and expressed the human hepatocyte-specific markers, suggesting that hUCMSCs could differentiate into hepatocyte-like cells in vivo in the liver tissue microenvironment. Levels of biochemistry markers improved significantly after transplantation of hUCMSCs compared with the nontransplantation group (P < 0.05). In conclusion, this study demonstrated that the liver tissue microenvironment may contribute to the differentiation of hUCMSCs into hepatocytes both in vitro and in vivo.

Elevated Neurosteroids in the Lateral Thalamus Relieve Neuropathic Pain in Rats with Spared Nerve Injury.

Neurosteroids are synthesized in the nervous system from cholesterol or steroidal precursors imported from peripheral sources. These compounds are important allosteric modulators of γ-aminobutyric acid A receptors (GABAARs), which play a vital role in pain modulation in the lateral thalamus, a main gate where somatosensory information enters the cerebral cortex. Using high-performance liquid chromatography/tandem mass spectrometry, we found increased levels of neurosteroids (pregnenolone, progesterone, deoxycorticosterone, allopregnanolone, and tetrahydrodeoxycorticosterone) in the chronic stage of neuropathic pain (28 days after spared nerve injury) in rats. The expression of the translocator protein TSPO, the upstream steroidogenesis rate-limiting enzyme, increased at the same time. In vivo stereotaxic microinjection of neurosteroids or the TSPO activator AC-5216 into the lateral thalamus (AP -3.0 mm, ML ±3.0 mm, DV 6.0 mm) alleviated the mechanical allodynia in neuropathic pain, while the TSPO inhibitor PK 11195 exacerbated it. The analgesic effects of AC-5216 and neurosteroids were significantly attenuated by the GABAAR antagonist bicuculline. These results suggested that elevated neurosteroids in the lateral thalamus play a protective role in the chronic stage of neuropathic pain.

[Determination of unconjugated neurosteroids in rat brain regions by liquid chromatography-negative atmospheric pressure ionization mass spectrometry].

AIM: To simultaneously determine three unconjugated neurosteroids, dehydroepiandrosterone (DHEA) , pregnenolone (PREG), allopregnenolone (AP), from several brain regions of the rat. METHODS: Neurosteroids were isolated separately in a two steps procedure by using ethyl acetate-n-hexane (90:10) as the first step to extract the unconjugated steroids, then the steroid fractions were further purified by SPE. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2-NFPH) and analyzed by HPLC-MS ( APCI) using selected-ion monitoring. Methyltestosterone was chosen as the internal standard. Results The linear calibration curve of DHEA was obtained in the concentration range of 0.030-2.00 microg x L(-1). The linear calibration curves of PREG and AP were obtained in the concentration range of 0.025-2.00 microg x L(-1). The concentrations of DHEA, PREG and AP in male rat brain regions were (0.70 +/- 0.23), (4.8 +/- 1.9), (1.1 +/- 0.6) ng x g(-1) for frontal cortex, (0.57 +/- 0.28), (6 +/- 3), (0.5 +/- 0.3) ng x g(-1) for hippocampus, (1.5 +/- 1.0), (9 +/- 5), (1.4 +/- 0.9) ng x g(-1) for amygdale, (0.52 +/- 0.14), (7.7 +/- 2.8), (0.5 +/- 0.6) ng x g(-1) for striatum, (2.9 +/- 1.6), (18 +/- 9), (1.6 +/- 1.3) ng x g(-1) for nucleus accumbens, (4.0 +/- 2.0), (27 +/- 12), (0.8 +/- 0.5) ng x g(-1) for pituitary gland, (1.7 +/- 1.2), ( 16 +/- 10), and (0. 8 +/- 0.7) ng x g(-1) for hypothalamus, respectively. CONCLUSION: Good linearity and accuracy were observed for each steroid. The procedure was suitable for measuring concentrations of the unconjugated steroids in rat brain simultaneously.