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.     

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.     

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.     

RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice, and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group, and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group, ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method, and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1, p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method, and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG, TC and LDL-C, and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner, inhibited the intimal plaque formation and vascular medial proliferation, and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG, TC, HDL-C and LDL-C, and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group, ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs, while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）, but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice, and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Effects of p38 MAPK on SETD4 expression and location in p38 +/+ and p38 -/- cells treated with sodium arsenite

AIM: To investigate the expression and location of SET domain-containing 4(SETD4) protein in p38 ^+/+ and p38 ^-/- cells treated with sodium arsenite(NaAsO₂). METHODS: The expression and location of SETD4 were detected in different cells with or without NaAsO₂ treatment by Western blotting and immunofluorescence technique. RESULTS: The expression of SETD4 was detectable in both murine- and human-derived cells. Its distribution was found to be located in the whole cell, mainly in the cytoplasm. Further investigation also suggested that the protein expression of SETD4 was reduced in both p38 ^+/+ and p38 ^-/- cells 6 h after NaAsO₂ treatment. Moreover, SETD4 protein was translocated from cytoplasm to nucleus in p38 ^+/+ cells treated with NaAsO₂, which was unobvious in p38 ^-/- cells. CONCLUSION: SETD4 protein is expressed in various cells derived from different species and tissues, and it is mainly located in cytoplasm. NaAsO₂ treatment influences the expression of SETD4, and induces the translocation of SETD4 protein to nucleus, which might be involved in the p38 MAPK signal pathway.     

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.     

Effect of CD137-CD137 ligand interaction on expression of NFATc1 in apolipoprotein E-deficient mice

AIM: To observe the effects of CD137-CD137 ligand(CD137L) interaction on the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) in apolipoprotein E-knockout (ApoE^-/-) mice. METHODS: Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE^-/- mice. In vivo, the expression levels of NFATc1 in mouse plaques and lymphocytes were detected by immunohistochemical method and flow cytometry, respectively. In vitro, the expression of NFATc1 at mRNA and protein levels in cultured lymphocytes of ApoE^-/- mice was measured by RT-PCR and flow cytometry, respectively. RESULTS: In vivo, after CD137-CD137L signaling pathway was stimulated, the expression of NFATc1 was significantly increased in the atherosclerotic plaques and lymphocytes. In vitro, the expression of NFATc1 at mRNA and protein levels in cultured leukocytes of ApoE^-/- mice was also significantly increased, with the maximal effect exerted by anti-CD137 monoclonal antibody (mAb) at the concentration of 20 mg/L, and 24 h after stimulation at any concentration (P<0.05). Anti-CD137L mAb significantly inhibited the expression of NFATc1 at mRNA and protein levels in the lymphocytes of ApoE^-/- mice, with the maximal effect exerted by anti-CD137L mAb at the concentration of 20 mg/L, and 24 h after stimulation (P<0.05). CONCLUSION: CD137-CD137L interaction can regulate the expression of NFATc1 in ApoE^-/- mice.     

Effects of butylphthalide on atherosclerosis lesion and VCAM-1 expression in aortic wall of ApoE-/- mice

AIM: To observe the protective effects of butylphthalide on atherosclerosis lesion and vascular cell adhesion molecular-1 (VCAM-1) expression in the aortic wall of ApoE^-/- mice, and to explore the possible mechanism underlying these beneficial effects.METHODS: Male ApoE^-/- mice at 6 weeks of age (n=90) were randomly divided into 3 groups. Thirty ApoE^-/- mice fed with high-fat diet and treated with saline simultaneously were defined as model group. Thirty ApoE^-/- mice fed with high-fat diet and treated with butylphthalide (100 and 200 mg·kg^-1·d^-1) were defined as treatment groups. Thirty wild-type C57BL/6J mice treated with saline were defined as control group. Fifteen mice in each group were sacrificed both at the ages of 18 and 30 weeks. The body weight, food intake and water intake were monitored weekly through the experiment. The lipid profiles were determined both at 18 and 30 weeks of age. Aortic roots were stained with hematoxylin and eosin for pathological examination. Serum ox-LDL, CRP, TNF-α and IL-6 were examined by ELISA. The expression of VCAM-1 at mRNA and protein levels was determinate by real-time PCR and Western blot in the thoracic aortas. RESULTS: Compared with control group, at 18 and 30 weeks of age, the body weight, serum lipid profiles and inflammatory factors were increased, while the atherosclerotic plaques were raised. The mRNA and protein levels of VCAM-1 were up-regulated. However, serum lipid levels in butylphthalide treatment groups (both at doses of 100 and 200 mg·kg^-1·d^-1) were decreased significantly. Serum ox-LDL, CRP, TNF-α and IL-6 were also decreased by butylphthalide treatment. Furthermore, atherosclerotic plaque areas in the aortic roots were reduced by butylphthalide treatment. In addition, the expression of VCAM-1 at mRNA and protein levels in the thoracic aortas was down-regulated by butylphthalide treatment.CONCLUSION: Butylphthalide delays the occurrence of high-fat diet-induced atherosclerosis and down-regulates the expression of VCAM-1 in the ApoE^-/- mice, which may be due to its alleviative effects on hyperlipidemia and inflammation.     

Relationship between cell calcium ion transporter ryanodine receptor 3 and atherosclerotic plaque in apolipoprotein E gene-deficient mice

AIM:To investigate the change of cell calcium ion transporter ryanodine receptor 3(RYR3) in the aorta smooth muscle cells of apolipoprotein E gene-deficient(ApoE^-/-) mice, and to elucidate the relationship between RYR3 and atherosclerotic plaque in ApoE^-/- mice. METHODS:Six-week-old ApoE^-/- mice and wild-type C57BL/6J mice were used in the experiment. The animals were sacrificed for pathological observation at the time points of 20, 27 and 33 weeks after hyperlipidic diet, respectively. Four sections of the aortic root were prepared and HE and immunohistochemical staining were performed. All the sections were analyzed with a computer image analysis system. RESULTS:Compared with the controls, the expression of RYR3 was markedly lower in ApoE^-/- mice(P<0.05). As the age of ApoE^-/- mice increasing, the expression of RYR3 decreased significantly, and was negatively correlated to the plaque area corrected by lumen area(r＝-0.652, P<0.01). CONCLUSION:Cell calcium ion transporter RYR3 participates in the pathological process of atherosclerosis, and is closely related to the formation of atherosclerotic plaques.     

Effect of atorvastatin on TRPC5 expression in atherosclerosis of apolipoprotein E-knockout mice

AIM: To observe the changes of transient receptor potential channel 5 (TRPC5) in vascular smooth muscle cells (VSMCs) of apolipoprotein E-knockout (ApoE^-/-) mice and the effect of atorvastatin interference, and to investigate the mechanism of atorvastatin therapy. METHODS: Male ApoE^-/- mice at 6 weeks of age were used to establish the atherosclerosis model by feeding with hyperlipidic diet. The mice were randomly divided into model group and atorvastatin group. The mice in atorvastatin group were lavaged with atorvastatin at 20 mg·kg^-1·d^-1, while the mice in model group received normal saline. The healthy C57BL/6J mice with the same age and the same genetic background, feeding with ordinary food, served as control group. At the time points of 14 and 24 weeks, the mice were sacrificed. The serum was collected for detecting the lipid levels. The aortic roots of the heart were taken to make paraffin sections with HE staining for measuring and comparing the relative atherosclerotic plaque area in each section. The expression of TRPC5 in VSMCs was examined with immunohistochemical staining. The mRNA levels of TRPC5 in the serum and the thoracoabdominal aorta were measured by real-time PCR. RESULTS: Compared with model group, blood lipids in atorvastatin group were significantly decreased, and the formation of plaque under aorta intima also decreased. The protein expression of TRPC5 in atorvastatin group decreased significantly compared with model group. Compared with 20-week model group, TRPC5 in 30-week model group showed increasing tendency, but has no statistical significance. Compared with 20-week atorvastatin group, TRPC5 of 30-week atorvastatin group declined. CONCLUSION: Atorvastatin suppresses TRPC5 expression, thus attenuating atherosclerotic development in ApoE^-/- mice.     

Metallothionein involvement in myocardial protection of basic fibroblast growth factor

AIM: To observe whether metallothionein plays a role in cardiac protective effect of basic fibroblast growth factor on anoxic/reperfusion (A/R) injury in cultured cardiomyocytes and study the possible mechanism of cardiac protection by bFGF. METHODS: The present study made the model of myocyte A/R injury after having a 24 h incubation by bFGF (10^-10, 10^-9 and 10^-8 mol/L) and bFGF (10^-9 mol/L) +PD₀₉₈₀₅₉, respectively. We measured the levels of MT and MDA in myocytes, and the changes of LDH and protein in cultured medium. We also counted the number of viable cell in groups. RESULTS: The contents of myocardial MT were significantly increased after treatment by bFGF. The levels of MT in 10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L bFGF treated groups increased 54%, 62% and76%, respectively, compared with the A/R group, and the number of viable cell were also greatly increased, LDH and protein leakage in cultured medium and MDA contents in myocyte were dramatically decreased in bFGF treated groups. All the protection were completely disappeared with the inhibition of MT production with PD₀₉₈₀₅₉, the inhibitor of mitogen-activated protein kinase (MAPK). CONCLUSION: MT involves in the protection of bFGF in cultured cardiomyocytes. It might be related with activation of MAPKase.     

PPARγ gene and protein expression in aortic plaque of different week-old apoE-knock out mice and the relationship with the composition of aortic plaque

AIM: To investigate the relationship of PPARγ gene expression with the composition of aortic plaque in apoE-knock out mice. METHODS: PPARγ gene and protein in aortic area of 20-week-old and 40-week-old apoE-knock out mice were investigated using RT-PCR and immunoblotting. The same aged wild type mice (C57BL/6J) were served as control (n=10). The composition of aortic plaques was analyzed by Movat method and oil red O staining. The expression of antigens such as PPARγ, SM-actin and MOMA-2 in aortic plaque were compared using immunohistochemistry. The relationship of PPARγ with macrophage, smooth muscle cells (SMC), lipid, elastic fiber, collagen and proteoglycan in aortic plaque were analyzed using immunofluorescence. RESULTS: PPARγ gene and protein in aortic wall and plaque of apoE-knock out mice were more significant than that in the same aged C57BL/6J mice (P<0.05). PPARγ expression at 40-week-old apoE-knock out mice was most significant and very low in C57BL/6J mice. More PPARγ expression of gene and protein at 20-week-old C57BL/6J mice than 40-week-old C57BL/6J mice were observed. Compared with 20-week-old apoE^-/- mice, the lipid pool in aortic plaque at 40-week-old apoE^-/- mice were increased remarkably, while elastic fiber, collagen and proteoglycan in plaque were decreased and aortic remodeling was very significant. Even, upregulation of MOMA-2 and downregulation of SM-actin were also detected in latter (P<0.05). In addition to SMC of aortic tunica media, PPARγ also expressed in SMC and macrophages in the aortic plaque of apoE^-/- mice. PPARγ was very enriched in lipid pool of the plaque. CONCLUSION: PPARγ expression level decreases with aging in C57BL/6J mice, while increases with plaque progression in apoE-knock out mice. There is positive correlation between PPARγ expression and lipid composition in plaque. The observed upregulation of PPARγ gene expression in aortic plaque may be a compensatory behavior and protective mechanism in apoE-knock out mice.     

An activated mutant of SHP-2 tyrosine phosphatase causes murine myeloid abnormal proliferation

AIM: To investigate whether an activated mutant of SHP-2 tyrosine phosphatase is involved in abnormal proliferation of murine myeloid.METHODS: Wild-type (WT) and SHP-2^D61G/+mutant mice aged 8 weeks and 16 weeks were used. The number of peripheral blood leukocytes and the spleen sizes were measured by cell counting and weighing methods,respectively. The surface markers (Mac-1 and Gr-1 for myeloid, Ter119 for erythroid, CD3 for T-lymphocyte and B220 for B-lymphocyte) of hematopoitic cells in peripheral blood and bone marrow were detected by flow cytometry. The rate of Mac-1 or Gr-1 positive cells in the peripheral blood and the rate of Mac-1, Gr-1, Ter119, CD3 or B220 positive cells in bone marrow were analyzed. The ability of colony formation unit (CFU) of the bone marrow was also observed by CFU assay. Finally, the expression of p-Akt and p-ERK in the peripheral blood leukocytes induced by interleukin-3 (IL-3, 5 μg/L) was detected by Western blotting.RESULTS: The number of leukocytes in peripheral blood of 16-week-old mice was more (P<0.01) and the spleens were bigger in mutant SHP-2^D61G/+ mice than those in WT mice. The rate of Mac-1 and Gr-1 positive cells in peripheral blood leukocytes of 16-week-old SHP-2^D61G/+ mice were dramatically increased (P<0.05). Mac-1 and Gr-1 positive cell rates in bone marrow of SHP-2^{D61G / +} mice were much higher (P<0.05) than those in WT mice and no statistic significance was found in the erythroid or lymphocyte cells. The number of CFU-GM (represents myeloid) was increased in mutant mice. The expression of p-Akt and p-ERK in peripheral blood leukocytes of mutant mice was significantly enhanced after stimulated with IL-3.CONCLUSION: These results suggest that activated mutant SHP-2 results in the disorder of mouse myeloid proliferation via MAPK and PI3K activation.     

Role of CD163/TWEAK pathway in atherosclerosis

AIM:To investigate the effect of CD163/tumor necrosis factor-like weak inducer of apoptosis (TWEAK) pathway on atherosclerosis in mice. METHODS:APOE^-/- mice and wild-type (WT) C57BL/6 mice were divided into 4 groups (8~10 weeks, n=10):APOE^-/- +normal diet (ND) group, APOE^-/- +western diet (WD) group, WT+ND group, and WT+WD group. Detection of blood lipid levels and oil red O staining of aorta artery were performed to confirm whether the atherosclerotic model was well established in 16 weeks after feeding. The aortic tissues were harvested to measure CD163 and TWEAK protein levels by Western blot, and immunohistochemical staining was also performed to localize CD163 and TWEAK protein expression on atherosclerotic plaque in each group. The cell experiments were conducted to study whether CD163 regulated TWEAK expression in M1 macrophages and foam cells, and the possible downstream pathway was investigated. RESULTS:The blood lipid levels and aorta oil red O staining showed that the animal model of atherosclerosis was successfully established in APOE^-/- +ND group and APOE^-/- +WD group. The protein level of CD163 was significantly increased in the aortic tissue in APOE^-/- mice (P<0.05) as compared with C57BL/6 mice (P<0.05). Consistently, the protein level of TWEAK was also markedly higher in APOE^-/- +ND group and APOE^-/- +WD group than that in WT+ND group and WT+WD group (P<0.05). Immunohistochemical staining showed that CD163 was mainly expressed in the parts away from the lipid core, and TWEAK was found in all parts of the atherosclerotic plaque. CD163 significantly inhibited the protein expression of TEWAK in the M1 macrophages, and also significantly down-regulated the level of nuclear factor-κΒ (NF-κB) in the M1 macrophages and foam cells (P<0.05). CONCLUSION:The protein levels of CD163 and its ligand TWEAK are significantly increased in atherosclerotic mice. The CD163 positive macrophages are mainly located at the site far away from the lipid core, and CD163 may play an anti-atherosclerotic effect by inhibiting TWEAK/NF-κB pathway.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.     

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.     

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

AIM:To investigate the role of apolipoprotein E(ApoE) in cholesterol efflux mediated by ATP-binding cassette transporter A1(ABCA1) and ATP-binding cassette transporter G1(ABCG1). METHODS:RAW 264.7 cells were seeded in either 6-well or 24-well plates, and then incubated with 20 mg/L low-density lipoprotein receptor gene knockout(LDLr^-/-) mouse lipoprotein 20 mg/L ApoE gene knockout(ApoE^-/-) mouse lipoprotein or culture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blotting, respectively. RESULTS:The ApoE^-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addition, ApoE^-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A-I(ApoA-I) and high-density lipoprotein(HDL) compared with LDLr^-/- mouse lipoprotein treatment. ApoE^-/- mouse lipoprotein treatment inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr^-/- mouse lipoprotein treatment. CONCLUSION:Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.     

Generation and characterization of a series of monoclonal antibodiesagainst recombinant human augmenter of liver regeneration

AIM:To generate monoclonal antibodies against human augmenter of liver regeneration (rhALR). METHODS:After BALB/C mice were immunized by the purified rhALR, the cells of spleen were fused with the cells of SP2/0; The titer and speciality were respectively fathomed from ascites or foster fluid by ELISA and Western-blot test. RESULTS:2 hybridoma cell lines were successfully obtained. The McAbs titer from ascites and foster fluid are respectively about 10^-3-10^-5 and 10^-2-10^-3. It is evident that the two McAbs were directed at different epitopes. CONCLUSIONS:The McAbs have higher speciality. It is significantly useful of the value that how hALR distribute in tissue organs, how the hALR signals the metabolism in the body and the control distribution of the hALR on cell growth on the translational level and so on is researched.     

Effects of PKC and ROCK on vasodilation induced by nifedipine

AIM: To investigate the effects of Rho-associated kinase (ROCK) and protein kinase C (PKC) on the relaxation of isolated rat aortic rings induced by nifedipine and the mechanisms. METHODS: The changes of tension in vascular rings induced by nifedipine under the basic condition and pre-contracted by norepinephrine (NE, 10^-6 mol/L) or KCl (60 mmol/L) were observed. The effects of ROCK and PKC on the vasodilation induced by nifedipine were studied using the vascular ring perfusion device. RESULTS: Nifedipine (10^-10 mol/L, 10^-9 mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L) had no significant relaxation effect on isolated aortic rings under basic condition. Nifedipine induced dose-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings pre-contracted by 10^-6 mol/L NE and 60 mmol/L KCl (P<0.05). No obvious difference between endothelium-intact group and endothelium-denuded group was observed. After incubation of the PKC inhibitor staurosporine (STA, 10^-8 mol/L) and PKC agonist phorbol 12-myristate 13-acetate (PMA, 10^-7 mol/L), STA increased the relaxation induced by nifedipine, while PMA reduced the effect of nifedipine on blood vessels (P<0.05). After the incubation of the ROCK inhibitor fasudil (10^-6 mol/L) and ROCK agonist angiotensin Ⅱ (Ang-Ⅱ, 10^-9 mol/L), fasudil increased the relaxation induced by nifedipine, while Ang-Ⅱ reduced the effect of nifedipine on blood vessels (P<0.05). The relaxation induced by nifedipine was not statistically inhibited by BaCl₂ (10^-4 mol/L), tetraethylammonium (10^-3 mol/L), glibenclamide (10^-5 mol/L) and 4-aminopyridine (10^-3 mol/L). In calcium-free and high-potassium solution, pre-treatment with nifedipine (10^-9 mol/L, 5×10^-8 mol/L and 10^-6 mol/L) inhibited calcium-induced contraction of the aortic rings (P<0.05). However, nifedipine pre-treatment did not affect the contraction induced by NE in Ca²⁺-free medium. CONCLUSION: Nifedipine exhibits vasodilatation effect in a dose-dependent manner and the vasodilatation activity is endothelium-independent. The vasodilatation effect of nifedipine may be related to the inhibition of extracellular calcium influx, and inhibition of PKC and ROCK enhances the vasodilatation effect of nifedipine.