Objective. This study was to evaluate the effect of riluzole on methylmercury- (MeHg-) induced oxidative stress, through promotion of glutathione (GSH) synthesis by activating of glutamate transporters (GluTs) in rat cerebral cortex. Methods. Eighty rats were randomly assigned to four groups, control group, riluzole alone group, MeHg alone group, and riluzole + MeHg group. The neurotoxicity of MeHg was observed by measuring mercury (Hg) absorption, pathological changes, and cell apoptosis of cortex. Oxidative stress was evaluated via determining reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDAs), carbonyl, sulfydryl, and GSH in cortex. Glutamate (Glu) transport was studied by measuring Glu, glutamine (Gln), mRNA, and protein of glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Result. (1) MeHg induced Hg accumulation, pathological injury, and apoptosis of cortex; (2) MeHg increased ROS, 8-OHdG, MDA, and carbonyl, and inhibited sulfydryl and GSH; (3) MeHg elevated Glu, decreased Gln, and downregulated GLAST and GLT-1 mRNA expression and protein levels; (4) riluzole antagonized MeHg-induced downregulation of GLAST and GLT-1 function and expression, GSH depletion, oxidative stress, pathological injury, and apoptosis obviously. Conclusion. Data indicate that MeHg administration induced oxidative stress in cortex and that riluzole could antagonize this situation through elevation of GSH synthesis by activating of GluTs.
Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H2O2)-induced cytosolic Ca2+ ([Ca2+]i) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca2+]i rise and whole-cell current change in response to H2O2. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H2O2-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H2O2-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca2+]i and whole-cell currents to H2O2. TRPM2 overexpression also aggravated the H2O2-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca2+ overload in response to H2O2 and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.
Low-dose arginine vasopressin (AVP) has been proposed as an adjunctive vasopressor for the treatment of advanced vasodilatory shock. However, its effects on renal, hepatic, and intestinal dysfunction during sepsis remain controversial.
Fecal peritonitis was induced in 20 anesthetized, invasively monitored, mechanically ventilated female pigs. Following the time point of septic shock (defined as mean artery pressure (MAP) ≤65 mmHg), animals were randomly assigned to the following groups (n = 10): 1) a norepinephrine group with MAP between 65 and 75 mmHg; and 2) an AVP group with a constant infusion rate of 0.5 mU.kg-1.min-1.
MAP, pulmonary capillary wedge pressure, hematocrit, TNF-α, IL-6, and IL-10 were similar in the two groups during the 28-h observation period. Infusion of AVP was associated with lower total norepinephrine and fluid requirements. There was a statistically significant improvement in renal function as assessed by increased urine output and renal blood flow, and decreased serum creatinine, in the AVP group when compared with the norepinephrine group (P < 0.05). Histological analyses of the intestine, liver, and kidney showed similar light microscopical appearance of the two groups. Apoptotic cells in the liver were significantly fewer in the AVP group when compared with the norepinephrine group (P < 0.05).
An adjunctive AVP to norepinephrine infusion exhibits a favorable impact on renal function without deleterious effects on the liver and intestine in a porcine model of experimental sepsis when compared with norepinephrine infusion alone.
Blood flow; intestine; kidney; liver; sepsis; vasopressin
Porcine circovirus type 2 (PCV2) is a primary etiological agent of post-weaning multi-systemic wasting syndrome (PMWS), which is a disease of increasing importance to the pig industry worldwide. Hollow mesoporous silica nanoparticles (HMSNs) have gained increasing interest for use in vaccines.
To study the potential of HMSNs for use as a protein delivery system or vaccine carriers. HMSNs were synthesized by a sol–gel/emulsion(oil-in-water/ethanol) method, purified PCV2 GST-ORF2-E protein was loaded into HMSNs, and the resulting HMSN/protein mixture was injected into mice. The uptake and release profiles of protein by HMSNs in vitro were investigated. PCV2 GST-ORF2-E specific antibodies and secretion of IFN-γ were detected by enzyme-linked immunosorbent assays, spleen lymphocyte proliferation was measured by the MTS method, and the percentage of CD4+ and CD8+ were determined by flow cytometry.
HMSNs were found to yield better binding capacities and delivery profiles of proteins; the specific immune response induced by PCV2 GST-ORF2-E was maintained for a relatively long period of time after immunization with the HMSN/protein complex.
The findings suggest that HMSNs are good protein carriers and have high potential for use in future applications in therapeutic drug delivery.
Hollow mesoporous silica nanoparticles (HMSNs); Porcine circovirus type 2 (PCV2): ORF2; Delivery; Immunization; Mice
A seventy-six-year-old lady with poor vision of the left eye due to previous retinal detachment presented with acute visual loss of her right eye secondary to central retinal artery occlusion. Clinical examination showed a pale right optic disc, macular edema, and a cherry red spot. Optical coherence tomography done four hours after onset showed right acute cystoid macular edema and diffuse inner retinal thickening. Subsequent treatment with intravenous carbonic anhydrase inhibitor resulted in some visual improvement. Central retinal artery occlusion has been known to produce diffuse intraretinal edema instead of cystoids changes. We would like to discuss a case of acute cystoid macular edema in acute central retinal artery occlusion.
The objective of this study was to investigate the use of cationized Pleurotus eryngii polysaccharide (CPEPS) as a nonviral gene delivery vehicle to transfer plasmid DNA encoding transforming growth factor beta-1 (pTGF-β1) into mesenchymal stem cells (MSCs) in vitro. Crude P. eryngii polysaccharide was purified, and then cationized by grafting spermine onto the backbone of the polysaccharide. Agarose gel electrophoresis, transmission electron microscopy, and a Nano Sense Zetasizer (Malvern Instruments, Malvern, UK) were used to characterize the CPEPS-pTGF-β1 nanoparticles. The findings of cytotoxicity analysis showed that when the nanoparticles were formulated with a CPEPS/pTGF-β1 weight ratio ≥ 10:1, a greater gel retardation effect was observed during agarose gel electrophoresis. The CPEPS-pTGF-β1 nanoparticles with a weight ratio of 20:1, respectively, possessed an average particle size of 80.8 nm in diameter and a zeta potential of +17.4 ± 0.1 mV. Significantly, these CPEPS-pTGF-β1 nanoparticles showed lower cytotoxicity and higher transfection efficiency than both polyethylenimine (25 kDa) (P = 0.006, Student’s t-test) and LipofectamineTM 2000 (P = 0.002, Student’s t-test). Additionally, the messenger RNA expression level of TGF-β1 in MSCs transfected with CPEPS-pTGF-β1 nanoparticles was significantly higher than that of free plasmid DNA-transfected MSCs and slightly elevated compared with that of Lipofectamine 2000-transfected MSCs. Flow cytometry analysis demonstrated that 92.38% of MSCs were arrested in the G1 phase after being transfected with CPEPS-pTGF-β1 nanoparticles, indicating a tendency toward differentiation. In summary, the findings of this study suggest that the CPEPS-pTGF-β1 nanoparticles prepared in this work exhibited excellent transfection efficiency and low toxicity. Therefore, they could be developed into a promising nonviral vector for gene delivery in vitro.
nonviral gene vector; transfection; plasmid DNA; spermine
Mast cells (MCs) deficient rats (Ws/Ws) were used to investigate the roles of MCs in visceral hyperalgesia. Ws/Ws and wild control (+/+) rats were exposed to T. spiralis or submitted to acute cold restraint stress (ACRS). Levels of proteinase-activated receptor 2 (PAR2) and nerve growth factor (NGF) were determined by immunoblots and RT-PCR analysis, and the putative signal pathways including phosphorylated extracellular-regulated kinase (pERK1/2) and transient receptor potential vanilloid receptor 1 (TRPV1) were further identified. Visceral hyperalgesia triggered by ACRS was observed only in +/+ rats. The increased expression of PAR2 and NGF was observed only in +/+ rats induced by T. spiralis and ACRS. The activation of pERK1/2 induced by ACRS occurred only in +/+ rats. However, a significant increase of TRPV1 induced by T. spiralis and ACRS was observed only in +/+ rats. The activation of PAR2 and NGF via both TRPV1 and pERK1/2 signal pathway is dependent on MCs in ACRS-induced visceral hyperalgesia rats.
The purpose of this study was to develop a sustained drug-release model for water-soluble drugs using silica nanoparticles.
Hollow-type mesoporous silica nanoparticles (HMSNs) were prepared using Na2CO3 solution as the dissolution medium for the first time. The water-soluble compound, silybin meglumine, was used as the model drug. The Wagner–Nelson method was used to calculate the in vivo absorption fraction.
The results of transmission electron microscopy and nitrogen adsorption revealed that the empty HMSNs had uniformly distributed particles of size 50–100 nm, a spherical appearance, a large specific surface area (385.89 ± 1.12 m2/g), and ultralow mean pore size (2.74 nm). The highly porous structure allowed a large drug-loading rate (58.91% ± 0.39%). In 0.08 M Na2CO3 solution, silybin meglumine-loaded HMSNs could achieve highly efficacious and long-term sustained release for 72 hours in vitro. The results of in vitro–in vivo correlation revealed that HMSNs in 0.08 M Na2CO3 solution had a correlation coefficient R2 value of 0.9931, while those of artificial gastric juice and artificial intestinal juice were only 0.9287 and 0.7689, respectively.
The findings of in vitro–in vivo correlation indicate that HMSNs together with Na2CO3 solution could achieve an excellent linear relationship between in vitro dissolution and in vivo absorption for 72 hours, leading to a promising model for sustained release of water-soluble drugs.
hollow-type mesoporous silica nanoparticle; silybin meglumine; in vitro dissolution; in vivo absorption; in vitro-in vivo correlation
AIM: To assess the safety of Bifidobacterium longum (B. longum) JDM301 based on complete genome sequences.
METHODS: The complete genome sequences of JDM301 were determined using the GS 20 system. Putative virulence factors, putative antibiotic resistance genes and genes encoding enzymes responsible for harmful metabolites were identified by blast with virulence factors database, antibiotic resistance genes database and genes associated with harmful metabolites in previous reports. Minimum inhibitory concentration of 16 common antimicrobial agents was evaluated by E-test.
RESULTS: JDM301 was shown to contain 36 genes associated with antibiotic resistance, 5 enzymes related to harmful metabolites and 162 nonspecific virulence factors mainly associated with transcriptional regulation, adhesion, sugar and amino acid transport. B. longum JDM301 was intrinsically resistant to ciprofloxacin, amikacin, gentamicin and streptomycin and susceptible to vancomycin, amoxicillin, cephalothin, chloramphenicol, erythromycin, ampicillin, cefotaxime, rifampicin, imipenem and trimethoprim-sulphamethoxazol. JDM301 was moderately resistant to bacitracin, while an earlier study showed that bifidobacteria were susceptible to this antibiotic. A tetracycline resistance gene with the risk of transfer was found in JDM301, which needs to be experimentally validated.
CONCLUSION: The safety assessment of JDM301 using information derived from complete bacterial genome will contribute to a wider and deeper insight into the safety of probiotic bacteria.
Bifidobacterium longum; Safety assessment; Genome; Antibiotic resistance; Harmful metabolite; Virulence factor
Coxiella burnetii is the etiological agent of human Q fever. In this study, adaptive transfer of mouse bone marrow–derived dendritic cells (BMDCs) stimulated with C. burnetii antigen, phase I whole-cell antigen (PIAg), lipopolysaccharide (LPS)–removed PIAg (PIIAg), protein antigen Com1, or SecB significantly reduced coxiella burden in recipient mice compared with control mice. Mice that received PIIAg-pulsed BMDCs displayed substantially lower coxiella burden than recipient mice of PIAg-pulsed BMDCs after C burnetii challenge. The protection offered by the antigen-activated BMDCs was correlated with the increased proliferation of helper T (TH) TH1 CD4+ cells, preferential development of TH17 cells, and impaired expansion of regulatory T lymphocytes. Our results suggest that PIIAg is far superior to PIAg in activating BMDCs to confer protection against C. burnetii in vivo, whereas Com1 and SecB are protective antigens because Com1- or SecB-pulsed BMDCs confer partial protection.
Diagnostic information for psychiatric research often depends on both clinical interviews and medical records. Although discrepancies between these two sources are well known, there have been few studies into the degree and origins of inconsistencies.
We compared data from structured interviews and medical records on 1,970 Han Chinese women with recurrent DSM-IV major depression (MD). Correlations were high for age at onset of MD (0.93) and number of episodes (0.70), intermediate for family history (+0.62) and duration of longest episode (+0.43) and variable but generally more modest for individual depressive symptoms (mean kappa = 0.32). Four factors were identified for twelve symptoms from medical records and the same four factors emerged from analysis of structured interviews. Factor congruencies were high but the correlation of factors between interviews and records were modest (i.e. +0.2 to +0.4).
Structured interviews and medical records are highly concordant for age of onset, and the number and length of episodes, but agree more modestly for individual symptoms and symptom factors. The modesty of these correlations probably arises from multiple factors including i) inconsistency in the definition of the worst episode, ii) inaccuracies in self-report and iii) difficulties in coding medical records where symptoms were recorded solely for clinical purposes.
The personality trait of neuroticism is a risk factor for major depressive disorder (MDD), but this relationship has not been demonstrated in clinical samples from Asia.
We examined a large-scale clinical study of Chinese Han women with recurrent major depression and community-acquired controls.
Elevated levels of neuroticism increased the risk for lifetime MDD (with an odds ratio of 1.37 per SD), contributed to the comorbidity of MDD with anxiety disorders, and predicted the onset and severity of MDD. Our findings largely replicate those obtained in clinical populations in Europe and US but differ in two ways: we did not find a relationship between melancholia and neuroticism; we found lower mean scores for neuroticism (3.6 in our community control sample).
Our findings do not apply to MDD in community-acquired samples and may be limited to Han Chinese women. It is not possible to determine whether the association between neuroticism and MDD reflects a causal relationship.
Neuroticism acts as a risk factor for MDD in Chinese women, as it does in the West and may particularly predispose to comorbidity with anxiety disorders. Cultural factors may have an important effect on its measurement.
Major depressive disorder; Anxiety disorders; Neuroticism
Studies of Vibrio cholerae in the environment and infected patients suggest that the waning of cholera outbreaks is associated with rise in the density of lytic bacteriophage. In accordance with mathematical models, there are seemingly realistic conditions where phage predation could be responsible for declines in the incidence of cholera. Here, we present the results of experiments with the El Tor strain of V. cholerae (N16961) and a naturally occurring lytic phage (JSF4), exploring the validity of the main premise of this model: that phage predation limits the density of V. cholerae populations. At one level, the results of our experiments are inconsistent with this hypothesis. JSF4-resistant V. cholerae evolve within a short time following their confrontation with these viruses and their populations become limited by resources rather than phage predation. At a larger scale, however, the results of our experiments are not inconsistent with the hypothesis that bacteriophage modulate outbreaks of cholera. We postulate that the resistant bacteria that evolved play an insignificant role in the ecology or pathogenicity of V. cholerae. Relative to the phage-sensitive cells from whence they are derived, the evolved JSF4-resistant V. cholerae have fitness costs and other characters that are likely to impair their ability to compete with the sensitive cells in their natural habitat and may be avirulent in human hosts. The results of this in vitro study make predictions that can be tested in natural populations of V. cholerae and cholera-infected patients.
cholera; bacteriophage; population dynamics; evolution; Vibrio cholerae
The title compound, C11H12N2O2, shows an S configuration, in which the pyrrolidinone ring is twisted with respect to the phenyl plane, making a dihedral angle of 70.73 (7)°. In the crystal, molecules are linked by N—H⋯O hydrogen bonds, building up a layer parallel to (001).
D-Ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation.
Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. C57BL/6J mice intraperitoneally injected with D-ribose for 30 days showed high blood levels of glycated proteins and AGEs. Administration of high doses D-ribose also accelerated AGE formation in the mouse brain and induced impairment of spatial learning and memory ability according to the performance in Morris water maze test.
These data demonstrate that D-ribose but not D-glucose reacts rapidly with proteins and produces significant amounts of AGEs in both cultured cells and the mouse brain, leading to accumulation of AGEs which may impair mouse spatial cognition.
Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS) in pigs. Currently, there is considerable interest in the immunology of PCV2; in particular, the immunological properties of the capsid protein. This protein is involved in PCV2 immunogenicity and is a potential target for vaccine development. In this study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2.
One monoclonal antibody (mAb; 8E4), against the capsid protein of PCV2, was generated and characterized in this study. 8E4 reacted with the genotype PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains by an immunoperoxidase monolayer assay (IPMA) and a capture ELISA. Furthermore, the mAb had the capacity to neutralize PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains. One critical amino acid that determined a conformational neutralizing epitope was identified using mAb 8E4 and PCV2 infectious clone technique. Amino acid residues 47-72 in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of mAb 8E4 was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position 59 (A59R) in the capsid protein of PCV2a (CL, LG and JF2) strains, inhibited completely the immunoreactivity of three PCV2a strains with mAb 8E4.
It is concluded that the alanine at position 59 in the capsid protein of PCV2a (CL, LG and JF2) strains is a critical amino acid, which determines one neutralizing epitope of PCV2a (CL, LG and JF2) strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2 strains, and development of a useful vaccine for control of PCV2-associated disease.
In the title compound, C10H10N2O2, all non-H atoms are approximately co-planar with an r.m.s. deviation of 0.016 Å. In the crystal, molecules are linked into inversion dimers by pairs of N—H⋯O hydrogen bonds. Chains along  are buiilt up by π–π interactions [centroid–centroid distance = 3.602 (1) Å] between the benzene and piperazine rings of adjacent molecules.
Porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem in the swine industry. Different genotypes (PCV2a, PCV2b and PCV2d) of the virus are present in the clinical cases in China, and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2. In this study, four strains of different genotypes were isolated, two were ordinary strains and another two were mutation strains, which there are one and two amino acids elongation in the capsid protein (Cap) of PCV2, respectively. Representative strains of different genotypes of the virus were constructed by infectious molecular clone and biological characterization of the rescued viruses were identified in vitro.
Four PCV2 isolates (PCV2a/CL, PCV2b/YJ, PCV2b/JF and PCV2d/BDH) of different genotypes were isolated from the clinical cases of PMWS in China. Four infectious clones of PCV2 were constructed and the rescued viruses were harvested after transfection into PK15 cells. The rescued viruses were verified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for more than ten passages, and virus propagation reached its peak 72h post infection (PI), and the virus titers were up to 105.7 TCID50/ml. By using neutralizing 1D2 monoclonal antibody (mAb) of PCV2, the antigen capture ELISA showed that only the PCV2a/rCL and PCV2b/rJF strains has immunoreactivity with the 1D2 mAb, however, another two rescued strains (PCV2b/rYJ and PCV2d/rBDH) do not, which indicated the antigenic difference among the rescued viruses of different genotypes. In addition, here is the first report of obtaining the newly emerging PCV2 with mutation in vitro by infectious molecular clone technology.
Conclusions drawn from this study show that PCV2 has prevailing differences in genomic and ORF2 gene length and antigen in swine herds in China. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic differences resulting from different subtypes of PCV2.
Zinc (Zn) is an essential micronutrient for humans, but Zn deficiency has become serious as equally as iron (Fe) and vitamin A deficiencies nowadays. Selection and breeding of high Zn-density crops is a suitable, cost-effective, and sustainable way to improve human health. However, the mechanism of high Zn density in rice grain is not fully understood, especially how Zn transports from soil to grains. Hydroponics experiments were carried out to compare Zn uptake and distribution in two different Zn-density rice genotypes using stable isotope technique. At seedling stage, IR68144 showed higher 68Zn uptake and transport rate to the shoot for the short-term, but no significant difference was observed in both genotypes for the long-term. Zn in xylem sap of IR68144 was consistently higher, and IR68144 exhibited higher Zn absorption ratio than IR64 at sufficient (2.0 µmol/L) or surplus (8.0 µmol/L) Zn supply level. IR64 and IR68144 showed similar patterns of 68Zn accumulation in new leaves at seedling stage and in developing grains at ripening stage, whereas 68Zn in new leaves and grains of IR68144 was consistently higher. These results suggested that a rapid root-to-shoot translocation and enhanced xylem loading capacity may be the crucial processes for high Zn density in rice grains.
Zinc; Stable isotope; High Zn-density rice genotype; Translocation; Remobilization
Bifidobacteria, known as probiotic bacteria, are high-G+C Gram-positive bacteria which naturally inhabit the human gastrointestinal tract and vagina. Recently, we completely sequenced Bifidobacterium longum JDM301, which is a widely used Chinese commercial strain with several probiotic properties.
Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1+ cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1+ cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44+/CD24− was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%–33% with the CD44+/CD24−/ALDH1+ cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.
We have previously reported that inhibition of astrocytic activation contributes to the analgesic effects of intrathecal ketamine on spinal nerve ligation (SNL)-induced neuropathic pain. However, the underlying mechanisms are still unclear. c-Jun N-terminal kinase (JNK), a member of mitogen-activated protein kinase (MAPK) family, has been reported to be critical for spinal astrocytic activation and neuropathic pain development after SNL. Ketamine can decrease lipopolysaccharide (LPS)-induced phosphorylated JNK (pJNK) expression and could thus exert its anti-inflammatory effect. We hypothesized that inhibition of astrocytic JNK activation might be involved in the suppressive effect of ketamine on SNL-induced spinal astrocytic activation.
Immunofluorescence histochemical staining was used to detect SNL-induced spinal pJNK expression and localization. The effects of ketamine on SNL-induced mechanical allodynia were confirmed by behavioral testing. Immunofluorescence histochemistry and Western blot were used to quantify the SNL-induced spinal pJNK expression after ketamine administration.
The present study showed that SNL induced ipsilateral pJNK up-regulation in astrocytes but not microglia or neurons within the spinal dorsal horn. Intrathecal ketamine relieved SNL-induced mechanical allodynia without interfering with motor performance. Additionally, intrathecal administration of ketamine attenuated SNL-induced spinal astrocytic JNK activation in a dose-dependent manner, but not JNK protein expression.
The present results suggest that inhibition of JNK activation may be involved in the suppressive effects of ketamine on SNL-induced spinal astrocyte activation. Therefore, inhibition of spinal JNK activation may be involved in the analgesic effects of ketamine on SNL-induced neuropathic pain.