Study on thrombomodulin and von Willebrand factor in microvasculature in mice lungs with pulmonary fibrosis induced by bleomycin

AIM:To explore the distribution of thrombomodulin (TM) and von Willebrand factor (vWf) on endothelial cells of lung microvasculature as well as the phenotype change of this cell in the process of pulmonary fibrosis in C57BL/6 mice. METHODS:It was carried out with dual immunofluorescent stain and quantitative analysis of fluorescent intensity of TM and vWf in normal and pulmonary fibrosis model induced by bleomycin (BLM) administration. RESULTS:① The normal lungs showed multiple continuous linear positive staining of TM and seldom positive of vWf on the surface of endothelium of alveolar capillaries. Meanwhile, blood vessels exhibited considerable positive of vWf in endothelial cell in normal C57BL/6 mice. ② The fibrotic lungs revealed a statistically significant diminution of TM expression, and at the same time, an increase of vWf expression when comparing with normal lung sections.CONCLUSION:These results suggest that TM dominant phenotype endothelial cells, rather than vWf dominant phenotype, are the major ones of alveolar capillaries in normal C57BL/6 mice lungs. TM dominant phenotype endothelial cells changed into vWf dominant ones as pulmonary fibrosis develops. Both TM and vWf antigen might be considered as markers of endothelium injury of lung microvasculature.     

Role of Api6 in lung inflammation induced by high fat/cholesterol food in C57BL/6J mice

AIM：To investigate the function of apoptosis inhibitor 6 (Api6) in lung inflammation induced by high-fat high-cholesterol diet (HFD/HCD) in male C57BL/6J mice. METHODS：Male C57BL/6J mice (6~8 weeks old) were randomly divided into 2 groups and treated with regular diet and HFD/HCD, respectively. After 16 weeks of feeding, the lung tissues were collected and the pulmonary inflammatory status was determined by immunohistochemistry and ELISA. The mRNA and protein expression levels of Api6 were determined by real-time PCR and Western blotting. The apoptotic rate of bronchioalveolar lavage cells was examined by flow cytometry. RAW264.7 cells were cultured in vitro and the apoptosis induced by oxidized low-density lipoprotein (oxLDL) was detected by flow cytometry. RESULTS：Accumulation of macrophages and increases in both tumor necrosis factor α and monocyte chemoattractant protein 1 were observed in the lung tissues of 16-week HFD/HCD-fed C57BL/6J mice. Compared with the regular diet-fed mice, the expression of Api6 at mRNA and protein levels in the lung tissues was highly increased in the HFD/HCD-fed mice (P<0.01). Meanwhile, the apoptotic rate of bronchioalveolar lavage macrophages from the HFD/HCD-fed mice was highly inhibited (P<0.01). In vitro, 500 μg/L recombinant Api6 significantly inhibited the apoptosis of RAW264.7 cells induced by oxLDL (P<0.05). CONCLUSION：HFD/HCD feeding results in the accumulation of macrophages in the lung of C57BL/6J mice, which may partly due to the increased expression of Api6 and its anti-apoptotic role in macrophages.     

Role of mineralocorticoid receptor in bleomycin-induced pulmonary fibrosis

AIM:To investigate the role of mineralocorticoid receptor (MR) in the lungs of experimental fibrotic mice. METHODS:C57BL/6 male mice (6～8 weeks old) were randomly divided into control group, bleomycin treatment group (Bleo) and bleomycin+spironolactone treatment group (Bleo+Spiro). For induction of pulmonary fibrosis, the mice were administered bleomycin at dose of 2.5 mg/kg dissolved in 50 μL saline by the intratracheal route or given 50 μL sterile saline as control. The mice in Bleo+Spiro group were treated with spironolactone (20 mg/kg) daily by oral gavage throughout the experiment. The mice were sacrificed at 12 h, 1 d, 2 d, 3 d, 7 d, 14 d and 28 d after administration of bleomycin. HE staining and Masson’s trichrome staining were used to conduct histopathologic examination. The mRNA expression levels of collagen 1 (Col1), collagen 3 (Col3), transforming growth factor beta (TGF-β), monocyte chemoattractant protein 1 (MCP-1), and MR were examined by real-time PCR. RESULTS:The results of histological analysis revealed the classical pathological stages of bleomycin-induced lung fibrosis, including acute inflammation phase (from 12 h to 3 d), progressive fibrosis phase (14 d) and late fibrosis phase (28 d). Compared with Bleo group, the inflammatory responses of the lungs in Bleo+Spiro group were attenuated in the acute inflammation phase and the degree of fibrosis was significantly reduced at 14 d after administration of bleomycin. Treatment with spironolactone effectively down-regulated the mRNA expression of MR. The levels of MCP-1 (in the acute inflammation phase), TGF-β (at 14 d), Col1 and Col3 (at 14 d) were also significantly reduced. CONCLUSION: Blockage of MR significantly attenuates the degree of bleomycin-induced pulmonary fibrosis by regulating the production and secretion of MCP-1 and TGF-β, thus reducing the degree of inflammation and inhibiting the expression of TGF-β in the progressive fibrotic phase.     

Up-regulation of CD36 induced by casein-induced inflammation promotes renal injury in mice

AIM: To investigate the role of CD36 in casein-induced mouse renal injury.METHODS: Eight-week-old male C57BL/6J mice and CD36 knockout (CD36KO) mice were randomly divided into C57BL/6J saline injection group, C57BL/6J casein injection group and CD36KO casein injection group (n=8 in each group). After 14 weeks of treatment with high-fat diet, the mouse serum, 24 h urine and kidney tissue samples were collected for analysis. The serum content of tumor necrosis factor-α (TNF-α) was measured by ELISA. The renal function markers in the serum and urine were determined by an automatic biochemical analyzer. The pathological changes of the kidney were observed by HE staining and Masson staining. The expression of CD36 and cytokines/chemokines (TNF-α, IL-6 and MCP-1) at mRNA and protein levels in the renal tissues were determined by real-time PCR and Western blot. The content of tissue hydrogen peroxide (H₂O₂) was measured by a commercial kit. The protein levels of Nrf2 and TGF-β1 in the renal tissues were measured by immunohistochemical staining.RESULTS: Compared with saline injection group, casein injection increased the level of TNF-α in the serum and in the kidney tissues of C57BL/6J mice (P<0.05), suggesting that casein injection successfully induced chronic inflammation in C57BL/6J mice. Casein injection also promoted the protein expression of CD36 and TGF-β1 in the renal tissues of the C57BL/6J mice, accompanied with glomerular sclerosis, proteinuria, increased serum creatinine content, increased H₂O₂ content, and decreased Nrf2 protein level and the ability of antioxidant in the kidneys (P<0.05). Furthermore, CD36 deficiency protected the mice from casein-induced renal injury, as evidenced by improved kidney pathological changes and decreased proteinuria. The content of H₂O₂ in the kidneys of casein-treated CD36 knockout mice was also lower than that in casein-treated C57BL/6J mice.CONCLUSION: Inflammatory responses promote the oxidative stress and renal injury in a CD36-dependent manner.     

Sphingosine-1-phosphate receptor 2 attenuates influenza A virus-induced viral pneumonia through PI3K/AKT/eNOS pathway

AIM: To investigate the effects of sphingosine-1-phosphate receptor 2 (S1PR2) on influenza A virus-induced viral pneumonia.METHODS: The animal model of influenza A virus pneumonia was established by infecting wild-type C57BL/6 mice and S1pr2^-/- mice with influenza virus subtype FM1 mouse lung adaptable strain through nose drops. The pathological changes of the lung tissues of wild-type mice (model group), JTE-013 (S1PR2 effective antagonist)-challenged mice and S1pr2^-/- mice were observed, and the protein concentration, total cell number, and interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels were determined in the bronchoalveolar lavage fluid (BALF) at 4 d and 6 d after virus infection. The phosphorylation levels of AKT and eNOS in the lung tissues were determined by Western blot. RESULTS: Compared with the wild-type mice of control group, the influenza A virus pneumonia in JTE treatment group and S1pr2^-/- mice were more serious, and the protein concentration, total cell number and inflammatory cytokines in the BALF were remarkably increased. Moreover, the phosphorylation levels of AKT and eNOS, the downstream targets of PI3K, were significantly increased (P<0.01). CONCLUSION: S1PR2 mediates PI3K/AKT/eNOS signaling transduction pathway to regulate NO generation, and inhibit vascular permeability and inflammatory cytokine release, thus attenuating the viral pneumonia induced by influenza A virus.     

Development and study of experimental C57BL/6 mouse lupus nephritis model induced by pristane

AIM To establish a C57BL/6 mouse lupus nephritis model induced by pristane and to explore the pathogenic mechanism of this model. METHODS Thirty C57BL/6 mice were randomly divided into 2 groups. The mice in model group were intraperitoneally injected with 0.5 mL pristane, while the mice in control group were injected with 0.5 mL normal saline. The state of health and survival of the animals were monitored during the whole experiment. The activation of macrophages, granulocytes, dendritic cells and B cells in spleen and the markers on B cells were detected by flow cytometry on the 10th day after injection. The serum levels of antinuclear antibodies (ANA) and anti-double strand DNA antibodies (anti-dsDNA) were measured every month after injection by indirect immunofluorescence assay, and proteinuria was checked mouthy by Albustix test strips. Eight months after injection, all mice were killed, and the kidneys were slided and stained with HE or PE-labeled IgG to analyze the expression of interferon-γ (IFN-γ) and interleukin-4 (IL-4). The evidence of glomerulonephritis was histopathologically observed, and the ultrastructural changes of the glomerulus were determined by transmission electron microscopy. RESULTS Ten days after the pristane injection, the populations of macrophages, granulocytes, dendritic cells and B cells were increased significantly compared with control mice (P<0.05). The expression of B7-1, B7-2 and MHC-II on the B cells was also increased (P<0.05). The serum levels of ANA and anti-dsDNA were increased from 3 to 8 months (P<0.05). Meanwhile, part of the mice showed different degrees of ascites, emaciation or even death, part of the mice had proteinuria 4 months later, and 8 months later, all of the model mice had proteinuria, with 1~20 g/L of urine protein. At the 8th month, a large number of heterotopic lymphoid tissues appeared in the abdominal cavity. The deposition of immune complexes in glomeruli and mesangial areas was serious. The expression of IFN-γ in the renal tissues was increased. Pathological damages such as lymphocyte infiltration, tissue necrosis and deposition of electron dense substances were obvious in the model mice. In control group, autoantibodies and proteinuria was not detected under the same experimental condition. Meanwhile, no evidence of glomerulonephritis in the control mice was observed. CONCLUSION The C57BL/6 mouse model of lupus nephritis induced by pristane is established successfully.     

Effect of bone marrow mesenchymal stem cells on engraftment of hematopoietic stem/progenitor cells in sensitized mice

AIM: To evaluate the effects of bone marrow-derived mesenchymal stem cells (MSCs) on engraftment of hematopoietic stem/progenitor cells in sensitized mice. METHODS: Mouse bone marrow-derived MSCs were cultured by adherent culture method. MSCs combined with or without hematopoietic stem/progenitor cells were implanted into the sensitized mouse model, which was established by allogeneic splenocyte transfusion, and were divided into 6 groups: MSC intervention groups, including sensitized mice with MSCs on day 11, sensitized mice with MSCs on day 0 and sensitized-mice with MSCs both on day 11 and day 0; control groups, including sensitized mice without MSC intervention, non-sensitized mice without MSC intervention and non-sensitized mice without MSCs or transplantation of hematopoietic stem/progenitor cells. The survivors were assessed after transplantation and hematopoietic recovery was monitored weekly including hematological change, immune function reconstruction, bone marrow cell recovery, chimera analysis and graft-versus-host disease development. RESULTS: Compared with different control groups, MSC intervention did not prolong the survival rates of the sensitized model mice after lethal irradiation. CONCLUSION: Under the experimental conditions, MSC combined with C57BL/6 bone marrow hematopoietic stem/progenitor cells fail to promote the growth of engraftment in C57BL/6 allogeneic splenocyte-sensitized BALB/c mice in vivo.     

Effects of arsenic trioxide on T-bet/GATA3 signal pathway in MRL/lpr mice

AIM: To investigate the effect of arsenic trioxide (ATO) on T-bet/GATA3 signal pathway in MRL/lpr mice.METHODS: MRL/lpr mice and C57BL/6J mice at the age of 20 weeks were chosen and then divided in 2 different sub-groups, respectively. The mice in 2 sub-groups received ATO (0.4 mg·kg^-1·d^-1) and sodium chloride (NS, volume weight-determined) by intraperitoneal injection respectively for 2 months. Afterward, the spleens were isolated from the MRL/lpr and C57BL/6J mice under pathogen-free condition and the suspensions were prepared. The mRNA level of T-bet, GATA3, IFN-γ,IL-4 and the mRNA ratio of T-bet/GATA3 were detected by RT-qPCR. The protein expression of T-bet and GATA3 was determined by Western blot. The serum levels of IFN-γ and IL-4 were measured by ELISA.RESULTS: The mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 in NS group of MRL/lpr mice were higher than those in NS group of C57BL/6J mice (P<0.05). However, the GATA3 and IL-4 were lower in NS group of MRL/lpr mice in both mRNA and protein level (P<0.05). In MRL/lpr mice, the mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 were lower in ATO group compared with NS group (P<0.05), no difference was found in GATA3 and IL-4. No difference of the indexes mentioned above between ATO group and NS group in C57BL/6J mice was observed.CONCLUSION: ATO may affect the signaling pathway of T-bet/GATA3 to down-regulate the mRNA expression and the protein secretion of IFN-γ by decreasing the expression of T-bet in MRL/lpr mice.     

Strain difference of susceptibility to caerulein-induced acute pancreatitis in mice

AIM: To study the difference of susceptibility to caerulein-induced acute pancreatitis (AP) among the mice of C57BL/6J, BALB/c and ICR strains.METHODS: Two-month-old female mice of C57BL/6J, BALB/c and ICR strains (12 mice for each strain) were divided into control group (n=6) and experimental group (n=6), respectively. The mice were intraperitoneally injected with caerulein (50 μg/ kg) in 1 h interval for 7 serial injections in total. The mice in control group were treated with saline according to the same procedure in experimental group. The blood samples were collected at 0 h, 3 h, 6 h, 9 h, 12 h and 24 h after the first injection of caerulein or saline for plasma α-amylase and lipase assays. The mice were sacrificed 24 h after AP induction, and the pancreatic tissues were harvested for further investigating the pathological changes and expression of inflammatory factors.RESULTS: After AP induction, the mice of BALB/c and ICR strains demonstrated more dramatic increase in plasma α-amylase activity and lipase activity than those of C57BL/6J mice. C57BL/6J mice showed milder morphological changes and lower expression of inflammatory factors in pancreata than those of BALB/c and ICR mice.CONCLUSION: The mice of C57BL/6J strain have less susceptibility to caerulein-induced AP than that of BALB/c and ICR mice.     

Effect of bone marrow stem cell transplantation on mdx mice at different ages

AIM: To study the effect of bone marrow stem cell transplantation on mdx mice at different ages. METHODS: The bone marrow stem cells of C57BL/6 mice (4-to-weeks age) were cultured in vitro for 3 days, then injected intravenously into the 6-week and 8-week aged mdx, which were preconditioned with 7 Gy γ ray. 12 weeks after being transplanted, the mdx mice were studied for the dystrophin protein expression on the skeletal muscle membrane. RESULTS: Three months after transplanted with bone marrow stem cells, about 16% and 7% muscles cells in 6-week and 8-week mdx mice expressed dystrophin protein， respectively. CONCLUSION: 12 weeks after transplantation with bone marrow stem cells of homologous series mice, different amounts of dystrophin protein expressed on the membrane of skeletal muscle cells were observed in different aged mdx mice. Bone marrow stem cell transplantation show more benefic effect for younger mdx mice.     

Trichostatin A promotes mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells

AIM：To investigate whether trichostatin A (TSA), a new revulsant,can induce mouse mesenchymal stem cells to differentiate into insulin-secreting cells and to explore the appropriate concentration of TSA. METHODS：The mesenchymal stem cell line from C57BL/6 mice was cultured in vitro and divided into 5 groups before treated with different concentrations of TSA, (group A: DMSO; group B~E: treated with 25 nmol/L, 50 nmol/L, 100 nmol/L and 200 nmol/L of TSA, respectively). After exposed to different cultured media for 10 d during the 2 stages, the cells were detected by the following methods: the insulin-secreting cells in each group were identified by dithizone staining and the results were calculated with immunohistochemical half quantitative analysis. The insulin secreted by insulin-secreting cells in each group was identified by immunofluorescence, and the mean fluorescence intensity of insulin was compared. The content of insulin in each group was quantified by ELISA. The appropriate concentration of TSA was determined according to the above results. RESULTS：TSA treatment for 10 d promoted the mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells which produced insulin. The immunohistochemistry and immunofluorescence imaging analysis of insulin-secreting cells showed that the insulin staining positive area, positive ratio, total density of insulin expression and mean fluorescence intensity of insulin in group B were significantly higher than those in the other TSA-treated groups. When the concentrations of TSA gradually increased, the content of insulin reduced accordingly. The content of insulin in group B was significantly higher than that in the other TSA-treated groups. CONCLUSION：TSA treatment for 10 d promotes bone marrow mesenchymal stem cells from C57BL/6 mice to differentiate into insulin-secreting cells and the appropriate concentration of TSA is 25 nmol/L.     

Changes of interleukin-6 in pulmonary hypertension mice induced by chronic hypoxic-hypercapnia

AIM: To investigated the changes of interleukin-6 (IL-6) in the pulmonary hypertension mice induced by chronic hypoxic hypercapnia. METHODS: Sixteen male C57BL/6 mice were randomly divided into 2 groups (8 mice in each group): normal control group and chronic hypoxic hypercapnia group. The mice in chronic hypoxic hypercapnia group were placed in a sealed chamber where O₂ concentration was kept at 9%~11%, and the CO₂ concentration at 5% ~6%, 8 h a day, 6 days a week for 4 weeks. The right ventricular (RV) weight, the weight of left ventricle plus ventricular septum (LV+S) were measured and right ventricular hypertrophy index was calculated. The structural changes of the pulmonary arteries were assessed by the method of histology with HE staining. The vessel wall diameter/total diameter (WT%) and the vessel wall area/total area (WA%) were analyzed by Image-Pro Plus 6.0 software. The protein expression of IL-6 in the lungs of the mice was determined by immunohistochemistry and ELISA, and the mRNA expression of IL-6 in the lungs was determined by RT-PCR. RESULTS: Compared with control group, RV/(LV+S), MT%, MA% and the expression of IL-6 at mRNA and protein levels were significantly increased in chronic hypoxic hypercapnia group. CONCLUSION: In the environment of chronic hypoxia and hypercapnia, the expression of interleukin-6 was elevated in mouse lungs, which may closely related to the development of pulmonary hypertension.     

Correlation between intracellular magnesium and expression of beta 2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice

AIM: To explore the effect of intracellular magnesium on expression of beta 2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice. METHODS: Ninety-six healthy, 4-6 weeks old and female C57BL/6 mice, weighting (12±2) g, were randomly divided into the following A, B, C, D groups with 24 mice in each group. The mice were sensitized and challenged by ovalbumin to establish the asthmatic model. A and B groups were fed with magnesium deficient diet. C and D groups were fed with normal magnesium level diet. B and D groups were injected intraperitoneally with 0.2 mL sulphate salbutamol solution after OVA provocation. A and C groups were injected intraperitoneally with 0.2 mL saline as control. Eight mice in each group were randomly taken out at 1 d, 21 d, 34 d to detect plasma Mg2+, intracellular Mg2+, the beta 2-AR mRNA and protein in lung tissue. RESULTS: No significant difference in plasma Mg2+, intracellular Mg2+, the beta 2-AR mRNA and protein in lung tissue among all groups at 1st d was observed (P>0.05, respectively). Plasma Mg2+, intracellular Mg2+, the beta 2-AR mRNA and protein in lung tissue in group C at 21st d and 34th d were significantly higher than those in group A at 21st d and 34th d ［21st d:(0.84±0.09)mmol/L vs 0.57±0.10)mmol/L, (2.39±0.14)mmol/L vs (2.11±0.08) mmol/L,(0.75±0.09)pmol/g vs (0.59±0.06)pmol/g, (88.50±8.50)pmol/g vs (60.10±7.70)pmol/g, P<0.05, respectively; 34th d:(0.67±0.10)mmol/L vs (0.51±0.09)mmol/L, (2.17±0.08)mmol/L vs (2.05±0.09)mmol/L,(0.61±0.05)pmol/g vs (0.53±0.06)pmol/g, (76.60±7.10)pmol/g vs (58.00±7.60)pmol/g, P<0.05, respectively］. Also, plasma Mg2+, intracellular Mg2+, the beta 2-AR, mRNA and protein of lung tissue in group D at 21st d and 34th d were significantly higher than those in group B at 21st d and 34th d ［21st d:(0.95±0.33)mmol/L vs (0.46±0.09)mmol/L,(2.32±0.18)mmol/L vs (1.87±0.14)mmol/L,(0.73±0.10)pmol/g vs (0.43±0.07)pmol/g, (96.90±8.00)pmol/g vs (47.90±4.90)pmol/g, P<0.05, respectively; 34th d:(0.71±0.10)mmol/L vs (0.31±0.08)mmol/L, (1.66±0.13)mmol/L vs (1.45±0.16)mmol/L,(0.40±0.07)pmol/g vs (0.33±0.05)pmol/g, (61.50±3.20)pmol/g vs (35.30±7.10)pmol/g, P<0.05, respectively］.CONCLUSION: The expression of β2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice with deficient intracellular magnesium is suppressed and C57BL/6 asthmatic mice with deficient intracellular magnesium are even easier to induce downregulation of β2-adrenergic receptor when β2-AR agonist is administered.     

Establishment of an acute GVHD animal model in EL9611 erythroleukemia mice

AIM: To establish an acute graft-versus-host disease (GVHD) model in EL9611 erythroleukemia mice. METHODS: Using C57BL/6 (H-2^b) mice as the donor and BALB/c (H-2^d) mice as the recipient in allogeneic bone marrow transplantation (allo-BMT), the acute GVHD model was established. The mice were divided into leukemia group (n=10), radiation control group (leukemic mice given radiation without allo-BMT, n=4), GVHD group (leukemic mice given radiation+allo-BMT, n=10) and normal control group (n=4). In leukemia group, 2×10⁶/mouse EL9611 erythroleukemic cells were transfused via tail vein into BALB/c mice to build the erythroleukemia model. In GVHD group, 7 days after leukemic cell transfusion, the mice received total dose of 8.0 Gy γ of total body irradiation(TBI), and within 5 h, 2×10⁶ C57BL/6 bone marrow cells and 1×10⁷ C57BL/6 spleen cells per mouse were transfused via tail vein to build the acute GVHD model in EL9611 erythroleukemia mice. The clinical manifestations of posture, fur, stool and so on were observed. Pathological examination was conducted to examine the changes of liver, spleen, skin, small intestine and peripheral blood. The survival rate was also calculated. RESULTS: (1) In leukemia group, the mean survival time (MST) was (14.5±2.1) days,or (7.5±0.7) days when irradiation day was as day 0(P<0.01 compared with GVHD group). The death rate was 100% with no spontaneous remission. The dead mice showed splenohepatomegalia and high WBC count . Pathological examination showed disorganization of normal tissues and leukemic cell infiltration. (2) In radiation control group, MST was (9.0±0.7) d, with significant difference as compared with GVHD group and normal group (P<0.01). The death rate was 100%. Pathological examination showed hematopoiesis exhaustion. (3) In GVHD group, MST was (32.0±3.2) d (P<0.01 compared with other groups). The death rate was 100%, the symptoms were observed on day 10-13 after allo-BMT. Clinical manifestations and pathological examination corresponded to those of I degree to II degree of GVHD. CONCLUSION: Intravenous infusion of 2×10⁶/mouse EL9611 leukemic cell successfully establishes the EL9611 erythroleukemia animal model. Seven days after EL9611 leukemic cell transfusion, lethal dose of TBI and allo-BMT can successfully build the acute GVHD model of EL9611 leukemic mice.     

Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells

AIM: To investigate the function of aged bone marrow mesenchymal stem cells (BMSCs) fused with young BMSCs in mice. METHODS: The cell fusion model, which was made by C57BL/6 mouse BMSCs labeled with PKH26 membrane red fluorescence (young cells, age of 2-3 months, Y) and (old cells, age of 18-24 months, O), and young and old BMSCs of green fluorescent protein (GFP) transgenic C57BL/6 mouse, was established by the induction of polyethylene glycol 1500 (PEG 1500). The cell fusion rate and cell surface markers were detected by flow cytometry. The morphology and nuclear characteristics of the fused cells were observed under fluorescence microscopy. In this study, the age dependent changes in BMSCs proliferation and differentiation potential in Y group, O group, and another three fusion groups (Y-Y group, Y-O group, O-O group) were examined. The proliferation potentials in 5 groups were compared by counting cell numbers at days 2, 4, 6, and 8. The osteogenic and adipogenic differentiation potentials of the cells in 5 groups were determined by using standard differentiation procedures. RESULTS: The fusion rate of 30.45%±4.13% was obtained by PEG 1500 induction. No significant difference of the fusion rates in Y-Y, Y-O and O-O groups was observed. Fused BMSCs coincided with the common BMSCs were reactive to the BMSCs lineage-specific CD44, Sca-1 surface markers and negative for the hematopoietic stem cells (HSCs) lineage-specific surface markers such as CD34, CD117, CD31, and CD45. The percentage of increasing cell numbers in Y-O group was significantly higher than that in O-O group at days 2, 4, 6, and 8. The positive rate of the area stained with Alizarin red, which represents osteogenic differentiation potential of BMSCs, was significantly higher in Y-O group than that in O-O group ［(25.46%±1.52%) vs (13.85%±1.69%), P<0.01］. In Y-O group, the higher rate of the positive area stained with oil red O, which represents adipogenic differentiation potential of BMSCs, was observed as compared to that in O-O group ［(12.99%±2.61%) vs (6.03%±1.71%), P<0.05］. CONCLUSION: Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells, particularly the proliferation and differentiation potentials.     

Human umbilical mesenchymal stem cell-derived exosome attenuates cardiac fibrosis in diabetic mice

AIM: To explore the protective effects of exosome secreted by human umbilical mesenchymal stem cells on cardiac fibrosis in diabetic mouse model. METHODS: Male C57BL/6 mice at 6~8 weeks of age were divided into 3 groups randomly:control group, diabetes mellitus (DM) group and DM+exosome group. To develop mouse DM mo-del, the mice were fed with high-fat diet for 5 weeks, followed by intraperitoneal injection of 45 mg/kg streptozocin once a week for 5 weeks. It was considered as a successful DM model that the blood glucose of the mice was ≥ 16.7 mmol/L. The mice in DM+exosome group were injected with exosome via tail vein. The mice in other 2 groups were injected with saline at the same volume. The heart function was evaluated by color Doppler echocardiography for small animals. The blood samples were collected from abdominal aortas. The blood glucose and non-esterified fatty acids were measured by biochemical colorimetric assay. HE staining was performed to observe the structural changes of myocardial fibers, and Masson staining was used to observe the cardiac fibrosis. RESULTS: The results of echocardiography showed that left ventricular end-diastolic dimension (LVIDd) and left ventricular end-systolic dimension (LVIDs) of diabetic mice were larger than those in control mice (P<0.05 and P<0.01, respectively). The ejection fraction (EF) and fractional shortening (FS) decreased in the diabetic mice (P<0.01). Exosome treatment significant decreased the LVIDs (P<0.01), but increased the EF and FS (P<0.01). The blood glucose and non-esterified fatty acids were significantly increased in the diabetic mice. The injection of the stem cell exosome significantly decreased the blood glucose and non-esterified fatty acids (P<0.01). HE staining observation showed that cardiomyocyte hypertrophy and fragmentation of cardiomyocyte in DM group were more se-rious than those in control group. Masson staining showed that the area of fibrosis in DM group was larger than that in control group (P<0.01), but that in DM+exosome group was reduced (P<0.01). CONCLUSION: Exosome secreted by human umbilical mesenchymal stem cells protects the DM model mice from cardiac fibrosis.     

Gefitinib prevents bleomycin-induced lung fibrosis in mice

AIM: To evaluate the effect of epidermal growth factor receptor- tyrosine kinase inhibitor (EGFR-TKI) on lung fibrosis induced by bleomycin in mice. METHODS: Forty BALB/c female mice were randomly divided into 4 groups: the mice in control group were given vehicle orally with administering of saline intratracheally; the mice in Ge200 group were given gefitinib (a tyrosine kinase inhibitor) at dose of 200 mg/kg orally with administering of saline intratracheally; the mice in BLM group were given vehicle orally with administering of bleomycin intratracheally; the mice in BLM+Ge20 group was given gefitinib at dose of 20 mg/kg orally with administering of bleomycin intratracheally. All animals were sacrificed by abdominal aortic bleeding 14 days after treatments. The left lung was stained with hematoxylin, eosin and Masson's trichrome respectively for the pathological examination. Total EGFR and phosphorylated EGFR were detected by immunohistochemistry. The tissues of right lung were sampled and the contents of hydroxyproline (HYP) were measured to quantitate the lung fibrosis. RESULTS: Gefitinib prevented lung fibrosis induced by bleomycin with significantly reducing lung collagen accumulation and the level of HYP in BLM+Ge20 group (P<0.05). The phosphorylation of EGFR in lung mesenchymal cells and epithelial cells was inhibited by treating gefitinib after intratracheal administration of bleomycin (P<0.05). Furthermore, in those mice that did not receive bleomycin treatment (Ge200 group), gefitinib neither induced the lung fibrosis nor increased the expression of p-EGFR. CONCLUSION: These findings suggest that, in the preclinical setting, gefitinib has a protective effect on lung fibrosis induced by bleomycin. Gefitinib at high dose (200 mg/kg) dose not induces lung fibrosis.     

Rapamycin improves circadian rhythm disorder induced by Aβ31-35 in mice

AIM:To observe the effect of rapamycin (Rapa) on the circadian rhythm disorder in C57BL/6 mice and the abnormal expression of Per2 in mouse hippocampal neuronal cell line HT22 induced by amyloid β-protein 31-35 (Aβ31-35).METHODS:The 6~8-week-old male C57BL/6 mice were selected for wheel-running behavior experiment. The effect of Rapa on the circadian rhythm disorder induced by Aβ31-35 was analyzed. The HT22 cells were randomly divided into control group, Aβ31-35 group, Rapa+Aβ31-35 group and Rapa group. The cell viability was measured by CCK-8 assay. Real-time PCR was used to detect the mRNA expression of Per2, and Western blot was applied to examine the expression of Per2 protein at circadian time (CT) 16. RESULTS:Compared with control group, Aβ31-35 disturbed the circadian rhythm which exhibited significantly longer free running period. However, the disruption was significantly relieved by pretreatment with Rapa compared with Aβ31-35 treatment, which manifested significantly decreased free running period (P<0.05). Aβ31-35 decreased the viability of HT22 cells compared with control group, and Rapa pretreatment reduced the toxicity of Aβ31-35 through up-regulating the cell viability. Abnormal mRNA expressions of Per2 was induced by Aβ31-35 in the HT22 cells. Rapa pretreatment reversed the abnormal expression of Per2 at mRNA level induced by Aβ31-35. Aβ31-35 significantly decreased the protein expression of Per2 at CT16. Pretreatment with Rapa significantly improved the protein expression of Per2. CONCLUSION:Rapa attenuates the circadian rhythm disorder induced by Aβ31-35 in C57BL/6 mice, and improves the abnormal expression of Per2 induced by Aβ31-35 in HT22 cells.     

Establishment of experimental autoimmune encephalomyelitis model in mice induced by MOG and its effect on CD4+CD25+ T cells

AIM: To investigate the feasibility of inducing experimental autoimmune encephalomyelitis(EAE)model in C57BL/6 mice by TrxA-extracellular immunoglobulin domain of MOG(MOGIgd-TrxA)fusion protein produced by molecular cloning in our laboratory. Also to investigate the role of CD4+CD25+ T cells in the pathogenesis of EAE. METHODS: (1)The MOGIgd-TrxA fusion protein was induced and produced by molecular cloning and purified by metal chelate affinity chromatography and concentrated through ultrafiltration. The concentration of the protein was measured by Bradford method at last. (2)Animal experiment: C57BL/6 mice(12 mice in each group)were used. The mice in group MOG were immunized with MOGIgd -TrxA fusion protein. The mice in group GPSCH were received emulsion of spinal cord homogenate of guinea pigs(GPSCH), and mice in group TrxA or normal control group(group NC)were received the same volume emulsion of TrxA or saline/adjuvant, respectively. Clinical scores and histopathology were measured to value the models quality. (3)The percentages of CD4+CD25+ regulatory T cells in EAE mice were tested through flow cytometric analysis. RESULTS: (1)The purity of purified MOGIgd -TrxA fusion protein was about 98%, and its concentration was 2.3 g/L. (2)No significant difference between group MOG and group GPSCH in the clinical score was observed(P>0.05). Histologic sections of the brain and spinal cord taken from affected animals in both groups showed pathological change of different level throughout the central nervous system(CNS). (3)Percentages of CD4+CD25+ T cells in group MOG and group GPSCH were(4.71±1.61)%and(1.44±0.65)%,respectively, both of which were significantly lower than those in group NC(9.22±1.24)%and TrxA group(8.97±1.20)％(P<0.01). CONCLUSION: (1)The animal model of EAE in C57BL/6 mice induced by MOGIgd fusion protein produced through molecular cloning in our laboratory is stable and with high incidence. Thus, the author finds a good way to study the immune mechanisms of MS further and to search for the effective treatments as well. (2)The reduction of CD4+CD25+ T cells in EAE mice may have some relationship with the clinic.