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.     

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.     

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.     

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.     

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.     

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.     

Effects of amifostine on formation of abdominal aortic aneurysm in mice induced by benzo[a] pyrene

AIM: To study the role of amifostine on the formation of benzo[a]pyrene (BaP)-induced abdominal aortic aneurysm (AAA) in C57BL/6J mice and the underlying mechanism. METHODS: RAW246.7 mononuclear macrophage in vitro were divided into control group, DMSO group, BaP group, low dose (1 μmol/L) amfostine treated group, middle dose (5 μmol/L) amfostine treated group and high dose (25μmol/L) amfostine treated group. The influence of BaP on the expression of matrix metalloproteinase (MMP)-9, MMP-12, TNF-α, NF-κB in the RAW246.7 mononuclear macrophages in vitro was determined by Western blot. Male C57BL/6J mice (8 months old) were divided into control group, model group (AngII+BaP group), low dose (50 mg/kg) amfostine treated group and high dose (100 mg/kg) amfostine treated group. After 6 weeks, the abdominal aorta were isolated. The aortic tissues were subjected to HE and Masson staining. The vascular wall structure, infiltration of macrophage, the expression of MMP-9, MMP-12, TNF-α, NF-κB were evaluated by Western blot and immunochemistry staining. RESULTS: Amifostine attenuated BaP-induced expression of TNF-α, MMP-9, MMP-12, NF-κB in the RAW246.7 mononuclear macrophages (P<0.05). The results of animal experiments showed that the incidence of AAA in high dose amifostine treated group were significantly lower than that in low dose amifostine treated group and model group (P<0.05). Immunohistochemistry staining observation showed that amifostine inhibited the aortic macrophage infiltration more obviously in high amifostine treated group compared with model group and low dose amifostine treated group (P<0.05). Compared with model group and low dose amifostine treated group, the MMP-9, MMP-12, TNF-α and NF-κB expression of abdominal aorta in high amifostine treated group was reduced significantly (P<0.05). CONCLUSION: Amifostine inhibits BaP-induced activation of macrophages, and also prevents the formation of abdominal aortic aneurysm in C57BL/6J mice induced by BaP by inhibition of the NF-κB pathway, macrophage infiltration and the expression of TNF-α and MMPs.     

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

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

Effect of polygonum cuspidatum and hawthorn on anti-atherosclerosis unstable plaque

AIM: To investigate the effect of detoxifying herbs polygonum cuspidatum, and hawthorn, herb of promoting blood flow, on pathologic morphology and inflammatory factors in apolipoprotein E gene knockout mice, in order to approach the possible regulatory mechanism of polygonum cuspidatum and hawthorn for treating artherosclerosis (AS) unstable plaque. METHODS: The animals were divided into 7 groups (12 mice in every group). The ApoE (-/-) mice fed with high fat diet were divided into polygonum cuspidatum group, hawthorn group, polygonum cuspidatum + hawthorn group, Xuezhikang group and high fat diet model group. Moreover, ApoE (-/-) mice fed with normal diet (normal diet group) and C57BL/6J mice fed with normal diet (normal control group) were set up. After intragastric administration for 17 weeks, serum hs-CRP was detected, aorta structure was observed under light microscope and NF-κB protein expression was determined by immunohistochemistry. RESULTS: The pathological change of AS in aorta in all groups fed with high fat diet and normal diet group were observed with different degree. The changes of aortic lesion in all treatment groups were reduced. The levels of NF-κB and hs-CRP in high fat diet group were significant higher than those in normal control group and normal diet group. Serum NF-κB and hs-CRP levels decreased in every treatment group, which were significant different from those in high fat diet model group (P<0.01). Among them, the changes in polygonum cuspidatum and hawthorn groups were the best. CONCLUSION: Chinese herbs of polygonum cuspidatum and hawthorn reduce inflammatory factors NF-κB and hs-CRP expression and play a role in anti-AS formation.     

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.     

Expression of scavenger receptor B1 (SR-B1) in the liver of high fat and sugar diet-induced diabetic mice

AIM: To investigate serum lipid and the expression of SR-B1 in the livers of diabetic mice. METHODS: Ten normal diet, female C57BL/6J mice, fifteen high fat and sugar diet female C57BL/6J mice, five fed 8 weeks and ten fed 16 weeks were used in the experiment. Serum total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), insulin (INS) and the expression of SR-B1 in the livers were measured. RESULTS: 1. In the high fat and sugar diet mice, serum TC and FBG at 16 weeks were significantly higher than that in normal diet mice (P<0.05). 2. The expression of SR-B1 protein in the liver of high fat and sugar diet mice was the higher than that in normal mice, and the SR-B1 expression in the liver of the mouse fed 16 weeks was also higher than that fed 8 weeks. CONCLUSION: The expression of SR-B1 protein in the liver of type 2 diabetes mice is higher than that in normal mice, perhaps it is related to the decrease in serum HDL-C.     

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

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

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.     

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

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

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

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

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

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

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 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.