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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Oxymatrine ameliorates high fat-induced insulin resistance in ApoE-/- mice

ATM: To investigate the effect of oxymatrine (OXY) on high fat-induced insulin resistance in mice, and to investigate the mechanism. METHODS: ApoE^-/-mice with high-fat diet for 16 weeks were divided into insulin resistance group, and OXY groups at concentrations of 25, 50 and 100 mg/kg. C57BL/6J mice served as normal control group. The mice in OXY groups were gavaged with OXY for 8 weeks. Glucose tolerance test in the mice was performed. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), fatty acid (FFA) and fasting insulin (FINS) in the plasma were measured. The mRNA expression of insulin receptor (INSR), insulin receptor substrate-2 (IRS-2), glucose transporter 2 (GLUT2) in the liver tissues was examined by RT-qPCR. The protein levels of GLUT2, INSR, IRS-2, p-INSR, p-IRS-2, PI3K, p-PI3K, serine/threonine protein kinase (AKT) and p-AKT were examined by Western blot.RESULTS: OXY reduced the levels of FBG, TC, TG, FFA and FINS, and attenuated insulin resistance. Compared with insulin resistance group, the mRNA expression of INSR, IRS-2 and GLUT2 significantly increased in OXY groups (P<0.05). The protein levels of p-INSR/INSR, p-IRS-2/IRS-2, p-PI3K/PI3K, p-AKT/AKT and GLUT2 also increased in OXY groups (P<0.05).CONCLUSION: OXY ameliorates high fat-induced insulin resistance in mice via PI3K/AKT pathway.     

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.     

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.     

Correlation between progression of atherosclerosis accelerated by emotional stimulation and imbalance of SDF-1/CXCR4 in mice

AIM To observe effects of emotional stimulation on expression of stromal cell-derived factor-1（SDF-1） and CXC chemokine receptor 4 （CXCR4） in plasma, platelets, aortas, hippocampus and bone marrow of apolipoprotein E gene knockout （ApoE^-/-） mice, and to reveal the possible mechanism of the aggravated atherosclerotic plaque vulnerability by emotional stimulation. METHODS Thirty 8-week-old male ApoE^-/- mice were randomly divided into normal control group, high fat group, and emotional stimulation group. Ten 8-week-old inbred C57BL/6J mice served as blank control group. After 12 weeks of intervention, the serum levels of SDF-1 and CXCR4 were investigated by ELISA. The protein levels of SDF-1 and CXCR4 in platelets, aortas, hippocampus and bone marrow were determined by Western blot. The pathological damage of aortas was observed by oil red O staining. RESULTS Compared with blank control group, normal control group and high fat group, the mice subjected to emotional stimulation showed more serious atherosclerosis in aortas detected by oil red O staining, and increased levels of SDF-1 and CXCR4 in the plasma and aortas were also observed （P<0.05）. The results of Western blot showed that the protein levels of SDF-1 and CXCR4 in platelets, aortas and hippocampus were increased in the mice subjected emotional stimulation, but the expression of SDF-1 and CXCR4 in the bone marrow was decreased （P<0.05）. CONCLUSION Imbalance of SDF-1/CXCR4 may be the key target by which emotional stimulation accelerates the progression of atherosclerosis.     

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 several herbal extractives with the effect of promoting blood flow and detoxication on atherosclerotic plaque stability in aorta of ApoE-gene knockout mice

AIM: To observe the effects of saponins of panax pseudo-ginseng, coptis chinensis extractive, giant knotweed rhizome and rhubarb extractives on the stability of vulnerable atherosclerotic plaques in the aortic roots of fat-fed ApoE knockout mice. METHODS: Eighty ApoE knockout mice were fed high-fat diet for 26 weeks. Thereafter, they were randomized to one of seven groups and treated with these herbal extractives for 13 weeks. The morphology and composition of atherosclerotic plaques in aortic roots were examined in tissue sections. Four sections were chosen and stained with Movat and HE staining, respectively, and the protein expressions of smooth muscle in plaque were determined by immunohistochemistry. The percentages of the plaque composition of foam cells, extracellular lipids, smooth muscle cells and collagen in each area were counted in the whole plaque. The number of buried caps and the ratio of the length of fibrous cap/plaque surface were also counted. The effect of the drugs on plaque stability was evaluated by using the vulnerability index 〔(foam cells＋extracellular lipids)/(collagens+smooth muscle cells)〕 and the number of buried fibrous caps. RESULTS: The vulnerability indexes in all drug treatment groups were decreased (P<0.05, P<0.01). The giant knotweed rhizome and rhubarb extractives, which had both the effect of promoting blood flow and detoxication, inhibited it the most significantly among them (P<0.01). The significant differences between the promoting blood flow group (saponins of panax pseudo-ginseng) and detoxication group (coptis chinensis extractive) were observed. Furthermore, the mean number of buried caps in plaque of giant knotweed rhizome extractive group and coptis chinensis extractive group were significantly decreased compared with that of control(P<0.01, P<0.05). CONCLUSION: Saponins of panax pseudo-ginseng, coptis chinensis extractive, giant knotweed rhizome and rhubarb extractives stabilize vulnerable plaque by changing plaque composition in clinical commended doses. Among them the herbal extractives with both the effects of promoting blood flow and detoxication (giant knotweed rhizome and rhubarb extractives) have the best effect on stabilizing vulnerable atherosclerotic plaque. Our investigation indicates that this kind of herbal extractives may be have similar effectiveness on stabilizing vulnerable atherosclerotic plaque for decreasing acute cardiovascular events, which is superior to herbs that has the effect of promoting blood flow only or detoxication only.     

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

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