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.     

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.     

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.     

IgE-FcεRI crosslink accelerates atherosclerosis development in allergic asthmatic mice

AIM:To investigate whether allergic asthma accelerates the development of atherosclerosis in mice related to Th2 cells and interleukin-4 (IL-4), and the roles of activation of macrophages by immunoglobulin E (IgE)-Fc ε receptor I (FcεRI) crosslink during the process. METHODS:Six-week-old ApoE^-/- mice were sensitized and challenged by ovalbumin to establish the allergic asthma model, and then assigned to 3 groups:control group, asthmatic placebo group and asthmatic IL-4 monoclonal antibody (mAb) intervention group (intervention for 8 weeks). The lesion area was measured by oil red O staining. The percentages of Th2 cells in the splenocytes of the mice were analyzed by flow cytometry. The mRNA expression of IL-4 and the macrophage-related inflammatory factors, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-6, in the spleen was detected by real-time PCR. Local IgE and FcεRIα expression in the plaque was evaluated by immunofluorescence/immunohistochemical staining, and the circulating IL-4 and IgE were measured by ELISA. RESULTS:Accompanied by aggravated atherogenesis in asthmatic ApoE^-/- mice, the proportion of Th2 cells and IL-4 mRNA in the spleen, IgE and FcεRIα expression in the aortic root, and the mRNA expression of MCP-1, MIP-1α and IL-6 were markedly increased. After 8-week treatment with IL-4 mAb, the lesion area in the aortic root of asthmatic ApoE^-/- mice was markedly decreased, the elevated IgE and FcεRIα expression was significantly decreased, and the mRNA expression of macrophage-related inflammatory factors was also decreased. CONCLUSION:Allergic asthma accelerates the atherosclerosis in ApoE^-/- mice, which is associated with the increased Th2 cells and IL-4, and the activation of macrophages by IgE-FcεRI crosslink.     

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.     

Effect of atorvastatin on adventitial fibroblast phenotype differentiation in atherosclerosis of apolipoprotein E-knockout mice

AIM: To explore the effect of atorvastatin on the expression of α-SMA and TGF-β1 in the adventitia of ApoE^-/- mice with atherosclerosis, and to investigate the underlying mechanism of atorvastatin therapy. METHODS: Male ApoE^-/- mice (n=40) at 6-weeks of age were used to establish the atherosclerosis model by feeding with high fat diet. The mice were randomly divided into model group and atorvastatin group. In atorvastatin group, the mice were lavaged with atorvastatin at dose of 20 mg·kg^-1·d^-1. The mice in model group were given normal saline. C57BL/6 mice of the same age served as control group, feeding with ordinary food. The mice were respectively sacrificed at the time points of 10 and 15 weeks after feeding with different diets. The ascending aorta was removed for serial sectioning. Some sections were performed with Movat staining in order to observe the morphological changes of the tissues, and to measure the relative atherosclerotic plaque area and the thickness of the adventitia. Some sections were stained with Sirius red to identify the collagen synthesis. Immunohistochemistry assay was prepared to observe the expression of α-SMA and TGF-β1 in the adventitia at different time points. The expression of TGF-β1 at mRNA and protein levels in the thoracoabdominal aorta was measured by RT-qPCR and Western blot.RESULTS: Compared with model group, the formation of plaque in atorvastatin group significantly descended. Meanwhile the adventitial thickness and collagen synthesis also decreased. The results of immunohistochemical staining showed that compared with 10 weeks-model group, α-SMA and TGF-β1 in 15 weeks-model group was increased. The expression of α-SMA and TGF-β1 in atorvastatin group decreased significantly compared with model group. The expression of TGF-β1 at mRNA and protein levels in model group were higher than those in control group. They decreased in atorvastatin group compared with model group. Compared with 10 weeks-model group, the mRNA and protein of TGF-β1 in 15 weeks-model group were increased.CONCLUSION: Atorvastatin modulates adventitial fibroblast phenotype differentiation by suppressing expression of TGF-β1 and intervenes atherosclerotic development in ApoE^-/- mice.     

CD137 signaling molecules promote proliferation of pulmonary artery endothelial cells by aerobic glycolysis

AIM:To investigate whether CD137 signaling molecules promote the proliferation of pulmonary artery endothelial cells (PAECs) by aerobic glycolysis. METHODS:The experiments of mouse PAECs were performed as follows. (1) Stimulating factors TNF-α (10 μg/L), ET-1 (10 mmol/L) and 5-HT (1 μmol/L) were used to stimulate the cells for 24 h. (2) After stimulation with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group (treatment with 5 mg/L CD137L recombinant protein to activate CD137-CD137L signaling), c-Myc inhibitor group (pretreatment with 10 μmol/L c-Myc inhibitor 10074-G5, dissolved in DMSO, for 30 min, followed by treatment with 5 mg/L CD137L recombinant protein) and DMSO group (pretreated with DMSO at the same volume to c-Myc inhibitor group for 30 min followed by CD137L recombinant protein treatment). (3) After stimulated with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group and 2-deoxyglucose (2-DG) group (pretreatment with 10 mmol/L glycolysis inhibitor 2-DG for 30 min followed by CD137L recombinant protein treatment).The expression of membrane protein and total protein of CD137 in the PAECs was detected by flow cytometry and Western blot, respectively. The protein levels of glycolytic enzymes such as hexokinase (HK2), 6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 3 (PFKFB3) and c-Myc were measured by Western blot. The enzyme activity of HK2 and PFKFB3 was detected by HK2 kit and PFK kit, respectively. Glucose oxidase method was used to measure the glucose uptake rate, and lactate colorimetric assay was conducted for analyzing lactic acid production. CCK-8 assay and EdU staining were used to detect proliferation of the PAECs. RESULTS:Compared with control group, TNF-α, ET-1 and 5-HT significantly increased the expression of CD137 membrane protein and total protein in the PAECs (P<0.05). The protein levels and enzyme activity of HK2 and PFKFB3 protein in CD137 agonist group were significantly higher than those in control group (P<0.05). Compared with control group, the lactic acid production and glucose consumption in CD137 agonist group were significantly increased. The protein level of c-Myc was significantly higher than that in control group after stimulation with CD137L recombinant protein, while c-Myc inhibitor 10074-G5 significantly inhibited the promoting effect of CD137L recombinant protein on glycolysis (P<0.05). The results of CCK-8 assay and EdU staining showed that the cell proliferation in CD137 agonist group was significantly increased compared with control group, while glycolysis inhibitor 2-DG significantly inhibited the proliferation-enhancing effect of CD137 signaling activation on the cells (P<0.05). CONCLUSION:CD137 signaling molecules may modulate the aerobic glycolysis by up-regulating c-Myc, thus promoting the proliferation of mouse PAECs.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

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.     

Expression of CXC chemokine receptor 7 in atherosclerotic ApoE-deficient mice and therapeutic impact by atorvastatin

AIM: To evaluate the expression level of CXC chemokine receptor 7 (CXCR7) in atherosclerotic apolipoprotein E-deficient (ApoE^-/-) mice induced by high-fat diet (HFD) and the effects of atorvastatin on it. METHODS: ApoE^-/- male mice (8-week-old) were used and were randomly divided into 3 groups following 1-week normal rodent diet: normal diet control (NDC) group, HFD group and HFD+statins (HFD+Sat) group. HE staining and oil red O staining were used to observe the atherosclerotic lesion burdens in the aortas. The expression of CXCR7 on the aortas was detected by Western blot and immunohistochemistry. The expression of Akt and endothelial nitric oxide synthase (eNOS) in the aorta was determined by Western blot.RESULTS: Few lesions were found in the aortas in NDC group. Apparent atherosclerotic plaque burdens were seen in HFD group and HFD+Sat group, while the atherosclerotic plaque burdens in HFD+Sat group were notably reduced compared with HFD group. The protein levels of CXCR7, eNOS and Akt in aorta in HFD group and HFD+Sat group were significantly decreased compared with NDC group, while those in HFD+Sat group were increased compared with HFD group. The protein level of p-eNOS in the aorta and the concentration of NO in the plasma in HFD group were decreased compared with NDC group and HFD+Sat group. CONCLUSION: In ApoE^-/- mice, HFD increases the lipid level and promotes the development of atherosclerosis by downregulating the expression of CXCR7, Akt and eNOS. Atorvastatin reverses the above effect of hypercholesterolemia on the expression of CXCR7, Akt and eNOS, thus playing the role in treating atherosclerosis.     

Preliminary comparison of inflammatory differential gene expression during atherosclerosis in ApoE-/- mice

AIM: To screen the expression of inflammatory genes associated with atherosclerosis (AS) in different weeks of ApoE^-/- mice using Agilent gene expression profile chip (AGEPC). METHODS: Male ApoE^-/- mice (n=60) were randomly divided into 3 groups:initial phase of AS (10 weeks old), early phase of AS (15 weeks old), and late phase of AS (25 weeks old). Homologous wild-type C57BL/6J mice were used for the control. The RNA samples of the arcus aortae from these mice were isolated. Total RNA from each sample was labeled with Cy3 and hybridized with AGEPC, and microarray detection was conducted. After washing, scaning, acquiring data, and standardized analysis, the expressed genes with default threshold of statistical significance of P≤0.05 and fold change ≥ 2.0 were selected. The expression of these genes were further verified by RT-qPCR. RESULTS: Compared with the control group, there were 895 differential genes in 10 weeks of ApoE^-/- mice, while 540 genes in 15 weeks, and 591 genes in 25 weeks, respectively. KEGG pathway and gene ontology (GO) analyses revealed that those diversely expressed genes related to inflammation were particularly arresting. Several selected genes including interleukin-12a (IL-12a), matrix metallopeptidase-12 (MMP-12), IL-1β, growth differentiation factor-15 (GDF-15) and interferon-γ (IFN-γ) were validated by RT-qPCR. Compared with the control group, the expression levels of IL-12a and MMP-12 were up-regulated while IL-1β was down-regulated in 10 weeks, the expression level of GDF-15 was up-regulated while the IL-12a and IL-1β levels were down-regulated in 15 weeks, and the levels of IL-12a, MMP-12 and GDF-15 were up-regulated in 25 weeks (P<0.05). Moreover, the increased level of IL-12a in 10 weeks, decreased level of IL-1β in 15 weeks, and increased levels of MMP-12 and GDF-15 in 25 weeks were even more statistically significant (P<0.01). CONCLUSION: The changes of inflammatory gene expression in different phases of AS suggest an important direction for medical intervention of AS.     

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.     

Inhibitory effect of Wendan decoction on atherosclerotic plaque formation in ApoE-/- mice by regulating expression of membrane proteins involved in reverse cholesterol transport

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on reverse cholesterol transport. METHODS Eight-week-old apolipoprotein E gene knockout （ApoE^-/-） mice with high-fat diet and daily drug gavage were randomly divided into model group, simvastatin group, and low-, middle- and high-dose Wendan decoction groups, with 15 mice in each group. The C57BL/6 mice of the same age served as control group. The mice were weighed once every week. After 10 weeks, the mice were anesthetized with chloral hydrate. The serum were collected for lipid level examination. The atherosclerotic plaque buildup in aortic root and whole aorta was observed by HE staining and oil red O staining, respectively. The levels of proteins related to cholesterol transport, ATP-binding cassette transporter A1 （ABCA1） and caveolin-1 in the aorta, and scavenger receptor class B type I （SR-BI） and CD36 in the liver, were quantified by Western blot. RESULTS Wendan decoction at middle dose inhibited the increase in the body weight of ApoE^-/- mice fed with high-fat diet （P<0.05）. Wendan decoction at different doses significantly reduced the serum levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the ApoE^-/- mice （P<0.05 or P<0.01）, but had no effect on serum high-density lipoprotein cholesterol level （P>0.05）. Wendan decoction at different doses inhibited the formation of atherosclerotic plaques in whole aorta of the ApoE^-/- mice （P<0.05 or P<0.01）. Middle- and high-dose Wendan decoction significantly inhibited the formation of atherosclerotic plaques in the aortic root （P<0.05）. Bedsides, Wendan decoction at different doses increased the protein level of ABCA1 and decreased the protein level of caveolin-1 in the aorta of the ApoE^-/- mice （P<0.01）. Middle- and high-dose Wendan decoction increased the liver protein level of SR-BI in the ApoE^-/- mice （P<0.01）. However, Wendan decoction at different doses had no effect on the liver protein level of CD36 in the ApoE^-/- mice （P>0.05）. CONCLUSION Wendan decoction reduces the body weight, serum lipid levels and formation of atherosclerotic plaques in ApoE^-/- mice fed with high-fat diet, and its mechanism is related to up-regulation of ABCA1 protein level in the aorta and SR-BI protein level in the liver as well as down-regulation of caveolin-1 protein level in the aorta.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

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.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Effect of pioglitazone on balance of regulatory and effector T cells of uremic apolipoprotein E knockout mice in vitro

AIM: To investigate the effects of pioglitazone on the quantity and function-related factors of regulatory and effector T cells (Treg and Teff ) of uremic apolipoprotein E knockout mice in vitro with or without the stimulation of atherosclerotic plaque-specific antigen oxidized low-density lipoprotein (oxLDL). METHODS: Uremic apolipoprotein E knockout mouse model was established by 2-step surgical procedure. After intervention with different concentrations (2 μmol/L and 20 μmol/L) of pioglitazone and PPARγ antagonist GW9662 (5 μmol/L) on splenocytes of uremic mice for 12 h in the presence or absence of oxLDL (2 mg/L), the levels of CD4⁺ CD25⁺ Foxp3⁺ Treg and IFNγ⁺ CD4⁺ Teff were determined by flow cytometry. The mRNA expressions of Foxp3 and IFNγ was detected by real-time fluorescent quantitative PCR. RESULTS: In vitro, oxLDL induced a Treg/Teff imbalance in splenocytes from the uremic mice. Pioglitazone upregulated the level of Treg and mRNA expression of Foxp3 in the presence of oxLDL, which was not antagonized by GW9662. Meanwhile, pioglitazone downregulated the level of Teff and mRNA expression of IFNγ in the presence or absence of oxLDL, which was reversed by GW9662. CONCLUSION: oxLDL induces a Treg/Teff imbalance in uremic apolipoprotein E knockout mice. Pioglitazone modulates the Treg/Teff imbalance probably through PPARγ-independent and-dependent mechanisms.     

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.