The role of vitamin D receptor in predentin mineralization and dental repair after injury.

Dentin is a permeable and complex tubular composite formed by the mineralization of predentin that mineralization and repair are of considerable clinical interest during dentin homeostasis. The role of Vdr, a receptor of vitamin D, in dentin homeostasis remains unexplored. The aim of the present study was to assess the impact of Vdr on predentin mineralization and dental repair. Vdr-knockout (Vdr-/-) mice models were constructed; histology and immunohistochemistry analyses were conducted for both WT and Vdr-/- mice. The finding revealed a thicker predentin in Vdr-/- mice, characterized by higher expression of biglycan and decorin. A dental injury model was employed to observe tertiary dentin formation in Vdr-/- mice with dental injuries. Results showed that tertiary dentin was harder to form in Vdr-/- mice with dental injury. Over time, heightened pulp invasion was observed at the injury site in Vdr-/- mice. Expression of biglycan and decorin was reduced in the predentin at the injury site in the Vdr-/- mice by immunohistochemistry. Taken together, our results imply that Vdr plays a regulatory role in predentin mineralization and tertiary dentin formation during dentin homeostasis.

[IL-6 promotes the activation of rat astrocytes and down-regulation of the expression of Kir4.1 channel].

Objective To identify the effects of interleukin-6 (IL-6) on astrocytes activation, and the regulation of the expression of inwardly rectifying potassium 4.1 (Kir4.1) channels in astrocytes. Methods Astrocytes were separated from the cerebral cortex of newborn SD rats, and cultured in the presence of IL-6 or combined with interleukin-6 receptor antagonist (IL-6Ra). CCK-8 assay was performed to measure cell viability. The expression level of Kir4.1 channels in astrocytes was measured using quantitative real-time PCR and Western blot analysis. Results IL-6 promoted the proliferation of astrocytes in a dose- (0-30 ng/mL) and time- (0-24 hours) dependent manner. After astrocytes were treated with IL-6 (30 ng/mL) for 24 hours, the levels of Kir4.1 mRNA and protein decreased significantly, and this down-regulation could be attenuated by IL-6Ra. Conclusion IL-6 promotes the activation of astrocyte and down-regulation of the expression of Kir4.1 channel.

siRNA-induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial-mesenchymal transition.

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA-induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116-CSCs were generated, and CD44 was knocked down in HCT116-CSCs using siRNA. The proliferation, migration and invasion of HCT116-CSCs were measured, and apoptosis and cell-cycle analyses were performed. The sensitivity of HCT116-CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116-CSCs tumorigenesis in vivo. In addition, the expression of epithelial-mesenchymal transition (EMT) markers E-cadherin, N-cadherin and vimentin was quantified. siRNA-induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell-cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116-CSCs to oxaliplatin in HCT116-CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116-CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA-induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.

Codon Optimization, Soluble Expression and Purification of PE_PGRS45 Gene from Mycobacterium tuberculosis and Preparation of Its Polyclonal Antibody Protein.

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti- PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

[Insulin-like growth factor 1 (IGF-1) promotes phagocytic activity of mouse BV-2 microglial cells via activating PI3K/AKT signaling pathway].

Objective To investigate the effect of insulin-like growth factor 1 (IGF-1) on the phagocytic activity of mouse BV-2 microglial cells. Methods Western blotting was performed to detect the protein levels of IGF-1 and IGF-1 receptor (IGF-1R) in the murine brain after the establishment of acute central nervous system inflammation models by intraperitoneal lipopolysaccharide (LPS) injection (10 mg/kg). The protein level of IGF-1R on BV-2 microglial cells that had been stimulated by 500 ng/mL LPS for 4, 12 and 24 hours was measured by Western blotting. To assess the phagocytic activity of microglial cells in response to IGF-1, BV-2 microglial cells were stimulated by IGF-1 at different concentrations for 24 hours after pretreated with or without wortmannin (PI3K/AKT signaling pathway blocker), and then incubated with fluorescent microbeads for 2 hours followed by measurement of phagocytosis of the fluorescent microbeads by flow cytometry. After treatment of IGF-1 (50 ng/mL), p-AKT and AKT signaling pathways in the BV-2 microglial cells were detected by Western blotting. Results Intraperitoneal LPS injection caused increased levels of IGF-1 and IGF-1R in the mouse brain. LPS upregulated the protein expression of IGF-1R on BV-2 microglial cells. The activity of BV-2 microglial cells to phagocytose fluorescent microbeads gradually increased with IGF-1 concentration rising and peaked in the IGF-1 treatment at 50 ng/mL, and gradually decreased thereafter. And IGF-1 induced the phosphorylation of AKT in BV-2 microglial cells. However, after the PI3K/AKT signaling pathway was blocked via wortmannin, the effect of IGF-1 on the activity of BV-2 microglial cells to phagocytose fluorescent microbeads was significantly alleviated. Conclusion IGF-1 can promote phagocytic activity of BV-2 cells via activating PI3K/AKT signaling pathway, which suggests a potential role of IGF-1 in regulating the cerebral inflammation.

Rat Bone Marrow-Derived Mesenchymal Stem Cells Promote the Migration and Invasion of Colorectal Cancer Stem Cells.

BACKGROUND: Colorectal cancer is one of the most common cancers and the second leading cause of cancer-related deaths worldwide. Targeting cancer stem cells (CSCs) may be a novel strategy for the treatment of colorectal cancer. Previous studies have shown that bone marrow-derived MSCs (BM-MSCs) promote tumor growth and metastasis. However, the role of rat BM-MSCs in the biological behaviors of colorectal CSCs remains unclear until now. MATERIALS AND METHODS: BM-MSCs were isolated from rats and characterized. CSCs were enriched from HCT116 cells using the microsphere culture method, and the microspheres incubated for at least 10 passages were termed HCT116-CSCs that were characterized. The effects of rat BM-MSCs on migration and invasion of HCT116-CSCs were examined using transwell migration and invasion assays and xenograft tumor growth assay. RESULTS: Rat BM-MSCs appeared typical stem cell morphology. Flow cytometry revealed positive CD29 and CD44 expression in rat BM-MSCs at passage 3, and rat BM-MSCs were found to differentiate into osteocytes following incubation in osteogenic induction medium. Microscopy, flow cytometric detection of stem cell surface markers, colony-formation assay and transwell migration and invasion assays characterized the successful preparation of HCT116-CSCs, and subcutaneous injection of HCT116-CSCs produced xenograft tumors in nude mice, while HE staining of the xenograft tumors displayed cancer specimen shapes. Transwell migration and invasion assays showed that rat BM-MSCs promoted the migration and invasion of HCT116-CSCs, and injection of rat BM-MSCs was found to promote the growth of the mouse xenograft tumor derived from HCT116-CSCs. CONCLUSION: Rat BM-MSCs promote the migration and invasion of colorectal CSCs, and colorectal CSCs may be a potential target for the therapy against colorectal cancer.

CXCR2 antagonist attenuates neutrophil transmigration into brain in a murine model of LPS induced neuroinflammation.

Sepsis-associated encephalopathy (SAE) is a devastating neurological complication of sepsis with intolerable high motility. SAE is accompanied with brain vascular injury, endothelial hyperpermeability, and neutrophil infiltration into the brain tissue, key inflammatory processes leading to further brain edema and neuronal cell apoptosis. Recent studies from us and others suggest that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is crucial for neutrophil recruitment during SAE. Here we use CXCR2 antagonist SB225002 to characterize the role of CXCR2 in brain infiltration of neutrophil in a murine model of SAE. Systemic administration of high-dose LPS (10 mg/kg) induced evident neutrophil infiltration into the cerebral cortex in wild-type mice. However, CXCR2 antagonist SB225002 markedly attenuated neutrophil infiltration into brain. The CXCR2 expression on neutrophils in the peripheral circulation was dramatically downregulated in response to this LPS dose, and endothelial CXCR2 was significantly upregulated, suggesting endothelial but not neutrophil CXCR2 plays a more important role in neutrophil infiltration into brain. Strikingly, although these CXCR2 antagonist SB225002 treated mice displayed reduced neutrophil infiltration, no change in neutrophil rolling and adhesion was observed. Furthermore, we confirmed that CXCR2 agonist CXCL1 induced a marked increase in actin stress fiber synthesis and paracellular gap formation in cultured cerebral endothelial cells, which is attenuated by SB225002. Thus, these results demonstrate a selective role for endothelial CXCR2 to regulate cerebral vascular permeability and neutrophil transmigration in high-dose LPS induced neuroinflammation, and also suggest a therapeutic potential of CXCR2 antagonist SB225002 in SAE.

Molecular characteristic analysis for the VP1 region of coxsackievirus A6 strains isolated in Jiujiang area, China, from 2012 to 2013.

BACKGROUND: Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the most common causative agents causing hand, foot, and mouth disease (HFMD). However, coxsackievirus A6 (CV-A6), previously largely ignored, became the predominant pathogen in China in 2012. The objective of this study is to investigate the genetic characteristics and molecular epidemiology of HFMD caused by CV-A6 to guide the diagnosis and treatment of the disease, as well as disease prevention. MATERIAL AND METHODS: A total of 138 suspected HFMD cases were enrolled in this study and analyses based on complete VP1 nucleotide sequences were performed to determine the evolutionary trajectory of emerging CV-A6. RESULTS: Among 138 samples in Jiujiang, 125 (90.58%) were positive for enterovirus, the most frequently presented serotypes were CV-A6 (77, 61.60%), CV-A16 (28, 22.40%), EV-A71 (6, 4.80%) and untyped enteroviruses (14, 11.20%). Seventy-seven CV-A6 positive specimens were analyzed for the complete VP1 sequences by sequencing and 36 representative isolates were selected to perform nucleotide sequence similarity analysis. The results showed that 36 strains isolated from HFMD patients were clustered closely to the mainland China and were far from prototype strain CV-A6/Gdula (AY421764) and other international subtypes. Moreover, phylogenetic analysis of the VP1 gene revealed that 36 circulating strains were not significantly concentrated in one branch, but were widely distributed in each branch. CONCLUSIONS: Continuous surveillance of HFMD etiological agents other than EV-A71 and CV-A16 is necessary. CV-A6 is emerging as the most common pathogen causing HFMD. Closely monitoring the magnitude and trend of CV-A6 epidemic and the trend of pathogenic spectrum changes can provide scientific basis for this disease prevention and control to the department of disease control.

Quantitative assessment of HLA-DQ gene polymorphisms with the development of hepatitis B virus infection, clearance, liver cirrhosis, and hepatocellular carcinoma.

Hepatitis B is one of the most common infectious diseases, which leads to public health problems in the world, especially in Asian counties. In recent years, extensive human genetic association studies have been carried out to identify susceptible genes and genetic polymorphisms to understand the genetic contributions to the disease progression of HBV infection. HLA-DQ gene variations have been reported to be associated with HBV infection/clearance, disease progression and the development of hepatitis B-related complications, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the results are either inconclusive or controversial. Therefore, to derive a more precise estimation of the association, a meta-analysis was performed. Our data revealed that the HLA-DQ alleles rs2856718-G, rs7453920-A and rs9275319-G were significantly associated with decreased risk of HBV infection and HBV natural clearance. Logistic regression analyses showed that HLA-DQ alleles rs9275572-A significantly increased HBV infection clearance, and decreased HBV natural clearance. However, rs2856718-G and rs9275572-A were not associated with development of cirrhosis. The HLA-DQ polymorphisms (rs2856718 and rs9275572) were associated with a decreased HBV-related HCC risk in all genetic models, but rs9272105-A increased the risk of HBV-related HCC. In addition, no significant association was observed between HLA-DQ rs9275319-G polymorphism and HBV-related HCC. These stratified analyses were limited due to relatively modest size of correlational studies. In future, further investigation on a large population and different ethnicities are warranted. Our findings contribute to the personalized care and prognosis in hepatitis B.

Relationship between HLA-DQ Gene Polymorphism and Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infection is the predominant risk factor for chronic hepatitis B (CHB). The association between HBV infection and human leukocyte antigen- (HLA-) DQ polymorphism (rs2856718 and rs7453920) has been demonstrated in other studies; however, the results were controversial or inconclusive. Therefore, to derive a more precise estimation of the association, a meta-analysis was performed. Crude odds ratios (ORs) and their 95% confidence intervals (CIs) were used to assess the strength of association between HLA-DQ polymorphism (rs2856718 and rs7453920) and HBV infection risk. A total of 11 articles were used to evaluate the effect of the two polymorphisms on risk of HBV infection. The pooled data showed that HLA-DQ rs2856718-G polymorphism showed protection against HBV infection, and rs2856718-A was a risk factor for chronic HBV infection. The pooled risk estimates indicated that HLA-DQ rs7453920-A polymorphism was associated with decreased risk of HBV infection, and rs7453920-G serves as a risk factor in HBV infection. However, these stratified analyses were lacking credibility due to the limitation of correlational study numbers; further investigation on a large population and different ethnicities is warranted.

[IL-1ß promotes the proliferation of astrocytes and downregulates the expression of Kir4.1].

Objective To explore the impact of IL-1ß on the proliferation of astrocytes and the expression of the inwardly rectifying potassium channel 4.1 (Kir4.1) in astrocytes. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats and cultured in the presence of IL-1ß or IL-1ß combined with interleukin-1 receptor antagonist (IL-1Ra). The effect of IL-1ß on the cell cycle of astrocytes was measured with flow cytometry; the level of Kir4.1 mRNA was determined by quantitative real-time PCR. The level of Kir4.1 protein was detected by Western blotting. Results IL-1ß promoted astrocyte proliferation in a time- and dose-dependent manner. After astrocytes were treated with IL-1ß (10 ng/mL) for 24 hours, the levels of Kir4.1 mRNA and protein significantly decreased, but IL-1Ra significantly inhibited IL-1ß-induced decrease of Kir4.1. The downregulation of Kir4.1 mRNA and protein expressions could be partially reverted when IL-1ß was removed. Conclusion IL-1ß could promote the proliferation of cultured astrocytes, and IL-1ß may be a key factor influencing the expression of Kir4.1 in astrocytes.

[Glycyrrhizinic acid modulates the immunity of MRL/lpr mice and related mechanism].

Objective To investigate the immunological effect of glycyrrhizic acid (GA) on MRL/lpr mice and its underlying mechanism. Methods The research included 10 wild type C57BL/6 mice and 40 MRL/lpr mice. MRL/lpr mice were randomly assigned to four groups: MRL/lpr control, MRL/lpr treated with dexamethasone (1.5 mg/kg), MRL/lpr treated with GA (20 mg/kg) and GA (40 mg/kg), 10 mice in each group. The serum levels of interleukin 4 (IL-4) and interferon-γ (IFN-γ) were tested by ELISA. The ratio of Th1/Th2 cell subsets in spleen was analyzed by flow cytometry. The protein levels of GATA-binding protein 3 (GATA3), T-bet, p-JAK3, JAK3, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), STAT3, p-NF-κBp65, NF-κBp65, p-IκBα, IκBα in spleen were detected by Western blot analysis. Results GA decreased serum levels of IL-4 and increased IFN-γ, modulated the balance of Th1/Th2 cell subsets in spleen markedly. GA inhibited the protein levels of GATA3, p-JAK3, p-STAT3, p-NF-κB, p-IκBα and increased T-bet protein in MRL/lpr mice. Conclusion Administration of GA can ameliorate the immune function in MRL/lpr mice.

Y-27632, a Rho-associated protein kinase inhibitor, inhibits systemic lupus erythematosus.

The purpose of the present study was to evaluate whether Rho-kinase inhibition (Y-27632) modulated the expressions of nuclear factor kappaB (NF-κB) in systemic lupus erythematosus. 20 wild type mice and 20 MRL/lpr mice were applied for the research. The animals were randomly assigned to wild type, wild type+Y-27632 group, MRL/lpr group and MRL/lpr+Y-27632 group. 5mg/kg Y-27632 was intravenously injected to inhibit the ROCK expressions.Y-27632 significantly decreased the serum levels of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α) and increased IL-10 level in serum of MRL/lpr mice. Flow cytometry (FCM) studies also showed that Y-27632 remarkably increased Regulatory cells(Treg) cell percentage in spleen cells. Western blot analysis demonstrated Y-27632 downregulated the expressions of ROCK1, ROCK2, upregulated the expression of forkhead/winged helix transcription factor(Foxp3), and inhibited the phosphorylations of NF-κBp65 and IκBα. The findings showed that the inhibition of ROCK was beneficial for the prevention of systemic lupus erythematosus, which possibly by suppressing NF-κB activation.

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p < 0.05), (ii) elevation in ΔΨm (p < 0.05), (iii) increased OCR and ATP formation (p < 0.05), (iv) increased intracellular NO levels (p < 0.05), (v) increased mitochondrial ROS production (p < 0.05), and (vi) increased susceptibility to rotenone (p < 0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p < 0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

[Selectively activating melanocortin 4 receptor acts against rat sepsis-induced acute liver injury via HMGB1/TLR4/NF-κB signaling pathway].

Objective To observe the effect of selective activation of melanocortin 4 receptor (MC4R) on the rats with sepsis-induced acute liver injury. Methods Sixty-four male SD rats were randomly grouped into sham operation group (PBS treatment after sham operation), cecal ligation and puncture (CLP) group (sepsis model was established by CLP), Ro27-3225 treatment group (Ro27-3225 treatment after CLP), and Ro27-3225 sham operation control group (Ro27-3225 treatment after sham operation), 16 rats for each group (ten rats were used to observe general condition and 72-hour survival after operation. Then, six rats were used to collect blood and liver samples). These groups were intraperitoneally injected with PBS or Ro27-3225 (180 µg/kg) 30 minutes after operation. Heart rate (HR), mean arterial pressure (MAP), aspartate transaminase (AST) and alanine transaminase (ALT) were measured 24 hours after operation. After execution of the rats, pathological changes of liver tissues were observed by HE staining. The levels of Toll-like receptor 4 (TLR4), high mobility group box 1 (HMGB1) and caspase-3 mRNA in liver tissues were analyzed by reverse transcription PCR. The expression of nuclear factor-κB (NF-κB) p65 in hepatocytes was detected by immunohistochemical staining, which was followed by analysis of nuclear positive rate of NF-κB p65. Results Compared with the sham operation group, CLP group showed decreased 72-hour survival and MAP, significantly increased levels of AST and ALT, hepatic cords disorder, hepatocyte swelling, and diffuse inflammatory cell infiltration; the levels of TLR4, HMGB1 and caspase-3 mRNA in liver tissues remarkably increased, and the positive rate of NF-κB p65 in hepatocytes went up as well. However, compared with the CLP group, the Ro27-3225 treatment group was found with obviously increased 72-hour survival and MAP, inhibited levels of AST and ALT, attenuated damage of liver tissues, decreased levels of TLR4, HMGB1 and caspase-3 mRNA in liver tissues, and significantly downregulated positive rate of NF-κB p65 in hepatocytes. Conclusion Sepsis causes liver injury. Selectively activating MC4R can reduce sepsis-induced acute liver injury in rats, which may act via inhibiting HMGB1/TLR4/NF-κB signaling pathway to relieve inflammation response.

Ciliary neurotrophic factor-treated astrocyte-conditioned medium increases the intracellular free calcium concentration in rat cortical neurons.

Ciliary neurotrophic factor (CNTF) is involved in the activation of astrocytes. A previous study showed that CNTF-treated astrocyte-conditioned medium (CNTF-ACM) contributed to the increase of the calcium current and the elevation of corresponding ion channels in cortical neurons. On this basis, it is reasonable to assume that CNTF-ACM may increase the intracellular free calcium concentration ([Ca2+]i) in neurons. In the present study, the effects of CNTF-ACM on [Ca2+]i in rat cortical neurons were determined, and on this basis, the aim was to investigate the potential active ingredients in ACM that are responsible for this biological process. As expected, the data indicated that CNTF-ACM resulted in a clear elevation of [Ca2+]i in neurons. Additionally, the fibroblast growth factor-2 (FGF-2) contained in the CNTF-ACM was found to participate in the upregulation of [Ca2+]i. Taken together, CNTF induces the production of active factors (at least including FGF-2) released from astrocytes, which finally potentiate the increase of [Ca2+]i in cortical neurons.

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKß3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKß3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKß3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

[Lipopolysaccharide induces astrocyte activation and downregulates the expression of Kir4.1 channel].

OBJECTIVE: To explore the influence of lipopolysaccharide (LPS) on the activation of astrocytes and the expression of the inwardly rectifying K(+) channel Kir4.1. METHODS: Astrocytes were separated from the cerebral cortex of newborn SD rats and cultured in the presence of LPS or LPS combined with interleukin-1 receptor antagonist (IL-1ra). The cell vitality was detected by MTT assay; the expression of glial fibrillary acidic protein (GFAP) was analyzed by immunocytochemistry; the production of IL-1ß was tested by ELISA; the expression of Kir4.1 and IL-1ß mRNAs were determined by quantitative real-time PCR. RESULTS: LPS promoted the activation of astrocytes in a time- and dose-dependent manner. LPS significantly increased the production of IL-1ß and the expression of IL-1ß mRNA, while decreased the expression of Kir4.1 mRNA in cultured astrocytes. Compared with the LPS group, IL-1ra could effectively reversed the above two results. CONCLUSION: The cultured astrocytes could be activated by LPS; LPS-induced downregulation of Kir4.1 mRNA in cultured astrocytes might be related with the inflammatory cytokine IL-1ß.

[Expression and significance of PD-1 on CD4(+); and CD8(+); T cells from the peripheral blood of patients with systemic lupus erythematosus].

OBJECTIVE: To detect the expression of programmed death-1 (PD-1) on CD4(+); and CD8(+); T cells from the peripheral blood of patients with systemic lupus erythematosus (SLE) and analyze its clinical implications. METHODS: The expression of PD-1 on the CD4(+); and CD8(+); T cells were examined in 51 SLE patients and 38 healthy controls (HCs) by flow cytometry. The proportions of PD-1 positive CD4(+); and CD8(+); T cells were compared not only between inactive or active SLE patients and HCs, but also between patients with and without lupus nephritis. The correlations of PD-1 expression with clinical manifestations and laboratory findings were analyzed. RESULTS: The proportions of CD4(+); PD-1(+); T cells significantly increased in active SLE patients as compared with HCs and inactive SLE ones (all P<0.05). The proportions of CD8(+); PD-1(+); T cells were significantly higher in active and inactive SLE patients than those in HCs (all P<0.05). The numbers of CD4(+); PD-1(+); and CD8(+); PD-1(+); T cells in SLE patients with nephritis were significantly higher than those in patients without nephritis (P<0.01). In SLE patients, the numbers of CD4(+);PD-1(+); and CD8(+);PD-1(+); T cells were significantly higher in ones with positive anti-dsDNA, anti-Sm, and anti-nucleosome antibodies than in corresponding negative ones; the proportions of CD4(+); PD-1(+); and CD8(+); PD-1(+); T cells were positively correlated with SLEDAI score and the amount of proteinuria, but were negatively correlated with complement C3. CONCLUSION: The aberrations of CD4(+); PD-1(+); and CD8(+); PD-1(+); T cells were observed in patients with SLE. Increased numbers of CD4(+); PD-1(+); and CD8(+); PD-1(+); T cells were correlated with increased disease activity and the production of anti-self antibodies.