Protective effect of HSF1 on mice with LPS-induced acute lung injury and screening of relevant differentially-expressed genes

AIM: To study the protective effect of heat shock factor1 (HSF1) on the mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI), and to screen the relevant differentially-expressed genes. METHODS: ALI mouse model was established by LPS intracheal instillation. The macroscopic and pathological changes of the lung tissue were observed, and the concentrations of total protein, TNF-α, IL-β, IL-6 and VEGF in the bronchoalveolar lavage fluid (BALF) were analyzed. Differentially-expressed genes in the lung tissues of HSF1^+/+ mice and HSF1^-/- mice with ALI induced by LPS were screened by gene chips. The key gene was verified by real-time qPCR. RESULTS: The macroscopic and pathological changes of the lung injury in HSF1^-/-+LPS mice were more serious than those in HSF1^+/++LPS mice. The concentrations of total protein, VEGF, TNF-α, IL-1β and IL-6 in the BALF of HSF1^-/-+LPS mice were significantly higher than those of HSF1^+/++LPS mice (P<0.05). Compared with the HSF1^+/+ mice, a total of 918 differentially-expressed genes were indentified in the HSF1^-/- mice, among which the expression levels of 65 genes had obvious diffe-rence, with 28 genes up-regulated, including Atg7, ccr1, cxcr2, Tbl1xr1, Mmp9, Pparg, Plcb2, Arrb2, Cntn1, Col4a6, etc, and 37 genes down-regulated, including Fgfr1, Fgfr2, Map4k4, Ddx58, Tfg, Stat3, Smad4, Lamc1, Sdc3, etc. The results of real-time qPCR showed that the mRNA level of CXCR2 in HSF1^-/-+ LPS mice was significantly higher than that in HSF1^+/++ LPS mice, which was consistent with the results of gene chips. CONCLUSION: HSF1 has protective effect on the mice with LPS-induced ALI. CXCR2 may be involved in the protective effect of HSF1 on this process.     

Mutation of Npc1 gene causes abnormal lipid metabolism to induce apoptosis of renal cells and promote fibrosis

AIM: To investigate the dysfunction of renal cell and tissue in Npc1 mutant mice, in order to provide support for the treatment of Niemann-Pick disease type C1 (NPC1) patients.METHODS: The kidneys of wild-type (Npc1^+/+) and Npc1 mutant (Npc1^-/-) mice on postnatal day 60 were isolated. HE staining was performed to examine the morphological changes of the renal tissues. Oil red O staining was used to examine the lipid deposition in the renal tissues. The apoptosis of the renal cells was detected by TUNEL staining. The expression of apoptosis-related proteins in the renal tissue was determined by Western blot, and immunofluorescence was performed to examine the expression of α-smooth muscle actin (α-SMA) and vimentin in the renal tissues. RESULTS: Compared with Npc1^+/+ mice, the morphological observation showed obvious vacuoles and no lipid deposition in the renal tissue of Npc1^-/- mice. Subsequently, TUNEL staining showed significant increase in the apoptotic cells in the renal tissue of Npc1^-/- mice (P<0.01), and the expression levels of Bax and Bad were up-regulated in the renal tissues of Npc1^-/- mice (P<0.01), but Bcl-2 was down-regulated (P<0.05). Furthermore, the expression of α-SMA and vimentin was significantly up-regulated in the renal tissues of Npc1^-/- mice (P<0.01). CONCLUSION: Npc1 gene mutation causes abnormal lipid metabolism in the renal cells, which induces the apoptosis of renal cells and promotes the fibrosis of renal tissue.     

Rapid effects of dexamethasone on activation of ERK and p38 in HO-8910cells

AIM: To investigate the effects of synthetical glucocorticoid dexamethasone(Dex) on the activation of two members of mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated protein kinase1/2(ERK1/2) and p38 MAPK (p38) in human ovarian cancer cell line HO-8910. METHODS: The activation of ERK1/2 and p38 was determined by Western blot. RESULTS: Inhibition of activation of ERK1and ERK2 by10^-7 mol/L Dex occurred at 5 min, with maximum up to 41% and 54% respectively at 30min (P<0.01), and sustained until 4 h. On the contrary, p38 activity was rapidly stimulated by 10^-7 mol/L Dex, with maximum to 84% at15 min (P<0.01), and sustained till1h. Furthermore, these effects increased with the concentration of Dex(10^-10-10^-6 mol/L). RU486, an antagonist of glucocorticoid receptor (GR), did not affect these effects. CONCLUSION: Dex can rapidly inhibit ERK1/2 and stimulate p38 activation in a GR-independent manner in HO-8910cells, which might play a role in Dex-mediated growth inhibition in these cells.     

Role of P-selectin in intestinal tumorigenesis in ApcMin/+ mice

AIM: To investigate the role of P-selectin in intestinal tumorigenesis in Apc^Min/+ mice (C57BL/6J-Apc^Min/J). METHODS: Female P-selectin knockout mice (B6.129S7-Selp^tm1Bay /J, that was P-selectin ^-/-) were mated with male Apc^Min/+ mice. The offspring was genotyped for Min ^+/- and P-selectin null mutantions, which were Apc^Min/+ P-selectin ^-/- mice. The tumor number and gross tumor volume in the small and large intestines of the Apc^Min/+ P-selectin ^-/- mice and Apc^Min/+ mice were determined. RESULTS: P-selectin deficiency in Apc^Min/+ mice resulted in significant decreases in the tumor number and gross tumor volume in the small intestine and total intestine. CONCLUSION: Deletion of P-selectin significantly inhibits the tumorigenesis in mouse intestines. In addition, the results also suggest that P-selectin may be a marker for colon cancer.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

Renal function and pathological changes in Niemann-Pick disease type C1 mice

AIM: To investigate the renal function and pathological changes in Npc1 mutant (Npc1^-/-) mice. METHODS: Different genotypes of Niemann-Pick disease type C1 (Npc1) mice were identified by PCR. Subsequently, the renal function of Npc1^-/- and Npc1^+/+ mice at postnatal day 60 (P60) was evaluated by measuring the activity and content of important indicators in the serum including ALT, AST, LDH, urea, UA and Cr. Furthermore, β-galactosidase staining and Masson staining were performed to examine the aging and fibrosis of the renal tissues, respectively. RESULTS: Compared with the Npc1^+/+ mice, the body weight and kidney weight had a significant reduction (P<0.01) in the Npc1^-/- mice. The results of hepatic and renal functions showed that the activities of ALT, AST and LDH, and contents of urea, UA and Cr had marked increases (P<0.05) in the Npc1^-/-mice. Moreover, the results of senescence-associated β-galactosidase staining in the renal tissues demonstrated accelerated aging in the Npc1^-/- mice (P<0.01), and these results were confirmed by Masson staining, which clearly showed the formation of collagen fibers (P<0.01).CONCLUSION: Mutation of the Npc1 gene results in abnormal lipid metabolism, which accelerates kidney senescence by promoting fibrosis in the renal tissue and subsequently causes reduction in renal function.     

Age-associated changes of p38 MAPK and JNK signal pathways in cardiac fibroblasts treated with transforming growth factor beta 1

AIM:To investigate the effect of aging on p38 mitogen-activated protein kinase(MAPK) and c-Jun N-terminal kinase(JNK) signal pathways in rat cardiac fibroblasts(CFs). METHODS:Cardiac fibroblasts obtained from neonatal and aged rats were cultured and randomly divided into 4 groups:neonatal PBS control group(N1 group), neonatal TGF-β₁ treatment group(N2 group), aged PBS control group(A1 group) and aged TGF-β₁ treatment group(A2 group). Proliferation of CFs was detected by MTT coloricmetric assay. The expression levels of total p38 MAPK, JNK, phospho-p38 and phospho-JNK were measured by Western blotting. RESULTS:The proliferative capacity of aged CFs was significantly decreased as compared with neonatal CFs after stimulated with TGF-β₁. In response to TGF-β₁, the expression levels of phospho-p38 and phospho-JNK were significantly increased in N2 group and A2 group as compared with N1 group and A1 group, respectively. The levels of total p38 and nonphosphorylated JNK in N2 group were similar to those in A2 group. Compared with N2 group, the levels of phospho-p38 and phospho-JNK markedly decreased in A2 group. CONCLUSION:These data indicate that p38 MAPK and JNK signal pathways are impaired in aged CFs.     

Relationship of p21WAF1 gene polymorphisms with protein expression in breast carcinomas

AIM: To investigate the relationship between p21^WAF1gene polymorphisms and protein expression in breast carcinoma. METHODS: Polymerase chain reaction single-strand conformation polymorphisms technique (PCR-SSCP) and immunohistochemical assay of S-P immunostaining technique were used to study polymorphisms of p21^WAF1 and protein expression respectively on the specimen of paraffin-embedded tissues in 100 cases of breast carcinomas and 40 benign breast diseases as control. RESULTS: Two p21^WAF1 gene polymorphisms were found in 18% (18/100) of breast carcinomas and 5% (2/40) of control samples. The difference between the two groups was statistically significant (χ²=3.94, P＜0.05). The positive immunohistochemical reaction of p21^WAF1 protein were found in 50% (50/100) of breast carcinomas and 12.5% (5/40) of control samples. The difference between the two groups was statistically significant (χ²=16.84, P＜0.01). The positive immunohistochemical reaction of p21^WAF1 protein were found in 100% (18/18) of breast carcinomas with p21^WAF1 gene polymorphisms and 39% (32/82) of no p21^WAF1 gene polymorphisms. The difference between two groups was statistically significant (χ²=21.95, P＜0.01). The p21^WAF1 gene polymorphisms were correlated with the protein expression in breast carcinomas (r=0.576, P＜0.01). CONCLUSION: p21^WAF1 gene polymorphisms may create the different copies of mRNA and may make relevant protein molecules.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.     

Role of p38 MAPK signaling pathway in inflammatory effects on cerulein-induced pancreatic cells

AIM: To investigate the effect of p38 MAPK signal pathway on cerulein-treated pancreatic acinar AR42J cells.METHODS: AR42J cells were divided into control group, cerulein group (treated with 10^-8 mol/L of cerulein), and SB203580 group (treated with 10 μmol/L of SB203580 and 10^-8mol/L of cerulein).The cells were harvested 3 h after treatment.Secretion rate of amylase was measured.The translocation of p-p38 MAPK to nuclei was imaged by immunofluorescence.The protein expression levels of p-p38 MAPK and TNF-α were detected by Western blotting.The activation of NF-κB was measured by electrophoretic mobility assay.RESULTS: Compared with control group, cerulein resulted in increases in the secretion rate of amylase and protein level of TNF-α (P<0.01), as well as the expression levels of p-p38 MAPK and NF-κB (P<0.01).Cerulein induced nuclear translocation of p-p38 MAPK.Compared with cerulein group, the secretion rate of amylase and protein level of TNF-α in SB203580 group decreased significantly (P<0.01).The expression of p-p38 MAPK and NF-κB also decreased greatly (P<0.05).Nuclear translocation of p-p38 MAPK was inhibited by SB203580.CONCLUSION: The p38 MAPK pathway involves in cerulein-induced pancreatic inflammatory response via regulating NF-κB.     

Effects of p38 MAPK gene knockout on arsenite-induced cell apoptosis

AIM: To investigate the role of p38 mitogen-activated protein kinase (MAPK) in the cell apoptosis induced by arsenite.METHODS: After the treatment with or without arsenite, p38+/+ and p38-/- cells were fixed with paraformaldehyde and stained with DAPI. The nuclear morphological changes were observed under a fluorescence microscope. p38+/+ and p38-/- cells were double-stained with Annexin V-FITC and PI, and the ratios of apoptotic cells were detected by flow cytometry.RESULTS: With the stimulation of arsenite, typical apoptotic morphological changes appeared in most p38-/- cells, yet only a few p38+/+ cells showed nucleus condensation. Consistently, the ratio of apoptotic p38-/- cells induced by arsenite significantly increased in comparison with that in the untreated cells (P<0.01), and also much higher than that in p38+/+ cells stimulated with arsenite (P<0.01).CONCLUSION: p38 gene knockout leads to a decrease in cellular resistance to stress of arsenite, which produces a proapoptotic state. p38 MAPK may play an important role on the enhancement of cell adaptive capability to harmful stresses.     

Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

Inhibitory effect of notoginsenoside monomer R1 on activation of p38 MAPK signaling pathway induced by hypoxic hypercapnia in PASMCs

AIM: To explore the mechanism of notoginsenoside monomer R₁ (R₁) against hypoxic hypercapnia-induced pulmonary vasoconstriction (HHPV) by investigating the effect of R₁ on p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs) under the condition of hypoxia and hypercapnia. METHODS: Primary cultured PASMCs, which were isolated from Sprague-Dawley rats, were incubated in logarithmic growth phase from the 2nd to 5th generation with different concentrations (8, 40 and 100 mg/L) of R₁ under the condition of 6% CO₂ plus 1% O₂ for 24 h. The expression of p38 at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. RESULTS: The results of Western blotting and RT-PCR analysis indicated that the protein and mRNA expression levels of p-p38 MAPK were significantly higher in hypoxic hypercapnia group with DMSO control than those in normoxia control group (P<0.01). In R₁ treatment groups, the levels of p-p38 MAPK protein and p38 MAPK mRNA were markedly decreased (P<0.01) in a dose-dependent manner. CONCLUSION: p38 MAPK signaling pathway may mediate hypoxic hypercapnia pulmonary vasoconstriction in rats. Notoginsenoside monomer R₁ attenuates HHPV, which may be related to blockage of p38 MAPK signal pathway.     

Antagonistic effects of cyproheptadine and anisodamine on [Ca2+]i elevation induced by TNFα in endothelial cell strains

AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani) on the changes of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by tumor necrosis factor (TNFα) in single endothelial cells, and to explore the mechanisms of TNFα mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca²⁺]_i in single endothelial cell was determined by laser scanning confocal microscopy (LSCM). RESULTS: [Ca²⁺]_i in single endothelial cell after stimulation of TNFα rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca²⁺]_i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3×10^-5, 6×10^-5 mol/L) and Ani (2×10^-5, 4×10^-5 mol·L^-1) markedly inhibited TNFα (1.2×10^-9 mol·L^-1)-induced [Ca²⁺]_i elevation. CONCLUSIONS: TNFα markedly induces elevation of [Ca²⁺]_i in single endothelial cell, it may be an important mechanism of TNFα-induced shock and tissue injury. Cyp and Ani obviously suppress TNFα-induced [Ca²⁺]_i elevation, which probably is one of the mechanisms of their antishock effects.     

Establishment of method for PD-1 immunophenotype detection in TCR Vβ repertoire of CD3+, CD4+ and CD8+ T-cell subsets

AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein 1 (PD-1) in T-cell receptor (TCR) Vβ repertoire of CD3⁺, CD4⁺ and CD8⁺ T-cell subsets, therefore to evaluate the distribution of PD-1 in T-cell repertoire from human peripheral blood (PB). METHODS:The PB samples from 10 cases of healthy individuals (HI) were collected. Using multi-colored fluorescence flow cytometry, the distribution frequency of PD-1 in TCR Vβ repertoire was detected with a wide panel of anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and 24 anti-TCR Vβ repertoire (IOTest^® Beta Mark TCR Vβ Repertoire Kit, containing 8 tubes which labeled A~H, each tube is a composite antibody of FITC and PE coupling, each cocktail contains antibodies direc-ted to 3 different Vβ subfamilies) monoclonal antibodies. RESULTS:The total number of the 24 TCR Vβ repertoire detected in CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells from 10 cases of HI was consistent with the Quick Reference Card data provided by the kit. The preliminary results showed that the frequency of Vβ usage in CD3⁺, CD4⁺ and CD8⁺ T cells was different. High usage of Vβ2, Vβ3, Vβ8, Vβ9, Vβ5.1, Vβ13.1 and Vβ13.2 was found in CD3⁺ T cells, while high usage of Vβ2, Vβ3, Vβ8, Vβ5.1, Vβ9 and Vβ13.1 in CD3⁺CD4⁺ T cells, and high usage of Vβ1, Vβ2, Vβ3, Vβ9, Vβ13.1 and Vβ13.2 in CD3⁺CD8⁺ T cells were also observed. Further analysis showed that the expression of PD-1 was detected in all 24 TCR Vβ subfamilies of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. The higher frequency of PD-1⁺ T cells was CD4⁺Vβ5.2⁺ T cells, whereas the higher frequency of PD1⁺ T cells in CD8⁺Vβ11⁺ and CD8⁺Vβ13.6⁺ T cells was detected. CONCLUSION:The method for detection of the immunosuppressive receptor PD-1 in TCR Vβ repertoire of T-cell subsets is successfully established, which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβ repertoire in the patients with leukemia.     

ROS promotes adhesion of neutrophils to bone marrow stromal cells and its mechanism

AIM:To investigate the effect of reactive oxygen species (ROS) on the adhesion of neutrophils to bone marrow stromal cells (BMSCs) and its mechanism. METHODS:Murine bone marrow neutrophils were isolated from mouse tibia and femur by density gradient centrifugation. HL60 cells were induced into human mature neutrophils (dHL60 cells) by DMSO treatment. Murine bone marrow neutrophils and dHL60 cells were labeled with CFDA-SE. The adhesion of the CFDA-SE-labeled cells to BMSC monolayer was tested by microplate reader after H₂O₂ treatment. The level of glutaredoxin 1 (Grx1) in dHL60 cells infected by lentivirus carrying Grx1 expression vector was examined by fluorescence microscopy and Western blot. The genotype of Grx1^-/- mice was identified by PCR. RESULTS:Diff-Quick staining result displayed that the purity of murine bone marrow neutrophil was higher than 90%. The adhesion of H₂O₂-pretreated neutrophils to BMSCs was higher than that of the control cells (P<0.01). The expression of Grx1 in Grx1 stably transfected dHL60 cells was significantly higher than that in the control cells. After H₂O₂ treatment, the results of in vitro adhesion assay showed that the adhesion of dHL60 cells with Grx1 over-expression to BMSCs was lower than that of the control cells (P<0.01). The results of PCR showed no Grx1 was detected at the whole gene level in Grx1^-/- mice. Compared with the neutrophils from wild-type mice, the neutrophils from Grx1^-/- mice displayed increased adhesion to BMSCs after H₂O₂ treatment. Vascular cell adhesion molecule-1 (VCAM-1) antibody pretreatment induced the adhesion rate back to non-H₂O₂-treated condition. CONCLUSION:ROS promotes the adhesion of neutrophils to BMSCs in bone marrow, which might be regulated by VCAM-1 adhesion signaling-related S-glutathionylation.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Volume-activated chloride channel exists in CD133+ lung cancer stem cells

AIM: To analyze the possibility that volume-activated chloride channel (VACC) exists in tumor stem cells. METHODS: CD133⁺ cells were purified by magnetic cell separation (MACS) system from non-small-cell lung cancer cell line A549. The purity of the cells was detected by flow cytometry. VACC current was recorded with whole-cell patch-clamp. RESULTS: The purity of CD133⁺ cells isolated from A549 by MACS was 92.14%. VACC current was recorded in the 22/40(55%) CD133⁺ cells of A549 by whole-cell patch-clamp. CONCLUSION: VACC exists in CD133⁺ lung cancer stem cells.