Establishment of a transgenic heterozygous mouse model of ApcMin/+ precancerosis of colorectal cancer with p110δ mutation

AIM：To establish a transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation in the C57BL/6J background for serving the studies on colorectal cancer research mediated by p110δ. METHODS：The transgenic heterozygous mice were generated by crossing in p110δD910A/D910A mouse and ApcMin/+ mouse, and the genotype was detected by PCR. Compared with ApcMin/+ mice, transgenic heterozygous mice (ApcMin/+; p110δD910A/D910A)were counted, and the number and size of intestine polyps were analyzed after methylene blue staining. The intestinal tissue structure was assessed by HE staining. RESULTS：The transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation was established. The number and size of polyps in the transgenic heterozygous mice were declined. CONCLUSION：A transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation was successfully established. The initial phenotype of intestinal tumors in transgenic mice was observed. This model will greatly contribute to the related research of colorectal cancer in mice.     

Expression of MMP-2 and NF-κB in myocardium of mice with viral myocarditis

AIM: To observe the effects of TNF-α/nuclear factor-κB(NF-κB)/matrix metalloproteinase-2(MMP-2) pathway on the expression of MMP-2 in the mice with viral myocarditis. METHODS: Six-week-old inbred male mice were randomly assigned to control and myocarditis group. The mice in myocarditis group and control group were intraperitoneally inoculated with 0.1 mL 10^-5.69 TCID50/mL coxsackievirus B3 and vehicle (PBS), respectively. Ten mice were sacrificed at the 4th and 10th days after injection. The blood and heart specimens were harvested. The serum content of TNF-α was measured by ELISA. The myocardial levels of MMP-2, NF-κB p65 and IκBα were determined by Western blot. RESULTS: Compared with control group, the protein expression of MMP-2 and NF-κB p65 in the myocardium and the serum content of TNF-α were significantly increased in myocarditis group (P<0.05). The protein expression of IκBα was lower in myocarditis group than that in control group (P<0.05).CONCLUSION: TNF-α, NF-κB p65 and MMP-2 were higher in the mice with acute viral myocarditis. The increased expression of them might be involved in the pathogenesis of viral myocarditis.     

Effects of IL-32γ on proliferation and cell cycle of rat vascular smooth muscle cells

AIM: To observe the effects of interleukin-32γ (IL-32γ)on the proliferation and cell cycle of rat vascular smooth muscle cells (VSMCs). METHODS: The VSMCs were isolated from the thoracic aorta of SD rats by the method of tissue-piece inoculation. The cells were cultured and treated with different concentrations of IL-32γ. The proliferation of the cells was examined by MTT assay. The cell cycles were analyzed by flow cytometry. The protein levels of NF-κB p65 and cyclin D1 were detected by Western blotting. The expression of proliferating cell nuclear antigen (PCNA)was examined by immunocytochemical staining. RESULTS: Administration of IL-32γ at the concentrations of 10~50 μg/L for 24~48 h significantly promoted the proliferation of VSMCs in a dose- and time-dependent manner. After stimulation with IL-32γ at the concentration of 50 μg/L for 24 h, the cell cycle transition from G₁ phase to S/G₂ phase was accelerated and the expression levels of NF-κB p65, cyclin D1 and PCNA increased as compared with those in control group. CONCLUSION: IL-32γ promotes the proliferation of rat VSMCs and accelerates the cell cycle transition via upregulating the expression of NF-κB p65 and cyclin D1.     

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.     

Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing expression of MCL-1

AIM: To investigate the synergistic effect of toosendanin on regulating the cytotoxicity of γδ T cells to colorectal cancer cells. METHODS: γδ T cells amplified in vitro were identified by flow cytometry. Lactate dehydrogenase (LDH) release was detected to evaluate the cytotoxicity of γδ T cells and toosendanin to SW480 cells. The role of toosendanin in regulating the protein expression of Bcl-xL, Bcl-2 and MCL-1 was determined by Western blot. The effect of toosendanin on regulating the secretion of TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) by γδ T cells was evaluated by ELISA. The mitochondrial membrane potential and apoptosis in SW480 cells treated with γδ T cells and toosendanin were analyzed by flow cytometry. The activation of caspase-9 and caspase-3 were determined by Western blot. RESULTS: CD3 and γδ T-cell receptor (TCR) were highly expressed in the γδ T cells amplified in vitro. Combination with toosendanin significantly enhanced the cytotoxicity of γδ T cells to SW480 cells. Toosendanin did not influence the secretion of TRAIL and FasL secreted by γδ T cells. Toosendanin did not regulate the expression of Bcl-xL and Bcl-2 but suppressed the expression of MCL-1 in SW480 cells. In addition, enforced expression of MCL-1 obviously suppressed the synergistic effect of toosendanin on γδ T cell-induced cell death in SW480 cells. Meanwhile, co-treatment with toosendanin was able to enhance the γδ T cell-induced apoptosis and decrease of mitochondrial membrane potential. γδ T cell-dependent activation of caspase-9 and caspase-3 was significantly enhanced by toosendanin co-treatment in SW480 cells. CONCLUSION: Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing the expression of MCL-1.     

Comparison of vascular remodeling between small artery and aorta in spontaneous hypertensive rats

AIM: To examine the difference of vascular remodeling between aorta and small artery in sponta-neous hypertensive rats (SHR) and control rats.METHODS: Male SHR (20-week-old) were used as experiment group, and age matched male Wistar-Kyoto (WKY) rats were used as control group. The systolic blood pressure and body weight were measured once a week. At 43 weeks old, the rats were anaesthetized, blood samples were collected, and thoracic aorta and mesenteric small artery tissue were harvested. The morphological changes of the arterial tissue were observed with HE staining. The collagen and elastine fibers were detected by the Sirius red-Victoria blue staining. The protein expression of type I and Ⅲ collagens were analyzed by confocal laser-scanning microscopy and Western blot. The changes of the vascular ultrastructure were imaged by transmission electron microscopy. The expression of proliferating cell nuclear antigen (PCNA) and the cell apoptosis in the arterial wall were examined by immunohistochemical method and TdT-mediated dUTP nick and labeling (TUNEL) detection.RESULTS: The inner diameter (ID) and luminal cross-sectional area (LCSA) of mesenteric small artery were decreased, whereas ratio of wall thickness (WT) to ID (WT/ID) and ratio of wall cross-sectional area (WCSA) to LCSA (WCSA/LCSA) were increased. Meanwhile, adventitia fibroblast migrated to the media, with overload collagens, especially collagen Ⅲ. Proliferation index (PI) and apoptotic index (AI) of the mesenteric small artery wall cells were increased. The ID, LCSA, WT/ID and WCSA/LCSA of the aorta were increased. Moreover, the vascular smooth muscle cells (VSMCs) showed hypertrophy and hyperplasia, with overload collagens. The PI and AI of the aortic wall cells were increased.CONCLUSION: The difference of vascular remodeling between the aorta and small artery is significant. The small artery mainly appears hyperplasia of matrix, especially the adventitial collagen Ⅲ. Meanwhile, the cell apoptosis in the small artery wall is increased. The aorta mainly appears hyperplasia and hypertrophy of media VSMCs.     

Expression profiles of CaMKⅡδ at different stages of osteoclast differentiation

AIM: To study the expression profiles and the role of Ca²⁺/calmodulin-dependent kinase Ⅱ delta (CaMKⅡδ) during osteoclast differentiation.METHODS: Mouse RAW264.7 cells were induced by receptor activator of nuclear factor κB ligand (RANKL) at 50 μg/L for osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption lacunae examination were performed to verify osteoclast formation. The expression of CaMKⅡδ at mRNA and protein levels was also determined by immunofluorescent cytochemistry, RT-qPCR and Western blot at days 0, 1, 3 and 5.RESULTS: TRAP positive multinuclear cells with bone resorption function were formed after 5 d of induction. The mRNA levels of CaMKⅡδ detected by RT-qPCR were 1.028±0.041, 2.478±0.087, 10.524±1.284 and 42.914±2.667 at days 0, 1, 3 and 5, respectively, while the protein levels of CaMKⅡδ detected by Western blot were 0.762, 0.963, 1.802 and 3.136, respectively. The changes of protein level were also verified by immunofluorescence cytochemistry, in which the fluorescence intensity increased in a time-dependent manner (P<0.05).CONCLUSION: The expression of CaMKⅡδ increases with the differentiation of osteoclasts. CaMKⅡδ may play a key role in the osteoclastogenesis.     

Role of vascular remodeling for urotensin Ⅱ in rat thoracic aorta after balloon injury

AIM: To explore the possible effect of UII in the process of remodeling after vascular injury. METHODS: The rat model of balloon injury in thoracic aorta was established. Male rats were randomized to 4 groups (n=5), including sham injury group, injury group, UII group (UII pumped into the rats after thoracic aorta balloon injury at 1.0 nmol·kg-1·h-1) and urantide group (urantide pumped into the rats after thoracic aorta balloon injury at 10 nmol·kg-1·h-1). At 21 days, the thoracic aortas were taken out to measure the changes of pathology, the expression of UII, the proliferation of VSMC and the expression of collagen. RESULTS: (1) At the 21 days after operations, the systolic blood pressure was higher in UII group than that in injury group ［(140.0±10.0) mmHg vs (132.0±3.4) mmHg, P>0.05］. The systolic blood pressure was also obviously higher than that in urantide group ［(140±10.0) mmHg vs (128.0±2.4) mmHg, P<0.05］. (2) Urotensin Ⅱ was expressed strongly in the injured area after thoracic aorta injury. (3) In contrast to injury group, the intimal thicken in urotensin Ⅱ group enhanced, the decrease in lumen area was marked (0.13±0.05 vs 0.07±0.02, P<0.05), the cell proliferation index was markedly increased (0.74±0.16 vs 0.40±0.11, P<0.01), and the expression of collagen was also markedly increased (counted as IOD, 318±127 vs 78±26, P<0.01). (4) In contrast to injury group, the decrease in lumen area was not abolished (0.09±0.03 vs 0.07±0.02, P>0.05) after chronic infusion of urotensin Ⅱ receptor antagonist urantide, the cell proliferation index was markedly increased (0.73±0.15 vs 0.40±0.11, P<0.01) and the expression of collagen was not statistically increased (counted as IOD, 200±79 vs 78±26, P>0.05). CONCLUSION: Urotensin Ⅱ expresses strongly in the myointimal cells after thoracic aorta injury in rat. The extra UII enhances the proliferation of VSMC and expression of collagen in the myointimal, increases the stenosis of injured vasculature, indicating that UII might take part in the process of repairing after vessel injury.     

Effects of short-chain acyl-CoA dehydrogenase on hypertensive vascular remodeling

AIM: To investigate the changes of short-chain acyl-CoA dehydrogenase (SCAD) in hypertensive vascular remodeling and to explore the relationship between SCAD and vascular remodeling in hypertension.METHODS: The spontaneously hypertensive rats (SHR; 24 weeks old) and Wistar rats (24 weeks old) were used as experimental control groups. The SHR and Wistar rats of 16 weeks old were trained by swimming as experimental groups. The systolic pressure was measured periodically. The thickness of vascular wall and the diameter of the vascular lumen were measured. The contents of ROS and ATP, the enzyme activity of SCAD, and the expression of SCAD at mRNA and protein levels in the aorta were determined. The free fatty acid in the serum and aorta was also measured.RESULTS: Compared with Wistar group, the diameter of vascular lumen decreased in SHR group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR group were increased significantly (P<0.05). Compared with SHR group, the diameter of vascular lumen increased in SHR+swim group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR+swim group were decreased significantly. Compared with control group, the expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, and the content of ATP were decreased in SHR group. However, the free fatty acid in the serum and aorta, and the content of ROS in the aorta were increased in SHR group. The expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, the content of ATP were increased in Wistar+swim group and SHR+swim group. However, the free fatty acid in serum and aorta, and the content of ROS in the aorta were decreased in Wistar+swim group and SHR+swim group.CONCLUSION: Decrease in SCAD expression may be associated with hypertensive vascular remodeling. Swimming training can reverse hypertensive vascular remodeling by increasing the expression of SCAD in the aorta.     

Changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mice in early stage

AIM:To investigate the changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mouse in the early stage of diabetes mellitus.METHODS:The effects of 60 mmol/L KCl, phenylephrine (PE), sodium nitroprusside (SNP) were measured and concentration-response curves to SNP were determined in the presence and in the absence of the inhibitors of potassium channels on the thoracic aortic rings of diabetic and age-matched control mice in vitro. RESULTS:STZ-diabetic mice showed a significant increase in the maximum contractile response and sensitivity of thoracic aorta to 60 mmol/L KCl and PE. The endothelium-independent relaxation response to SNP was increased by diabetes and were decreased significantly by pretreatment of the vessels with 1 mmol/L tetraethylammonium (TEA), 1 mmol/L 4-aminopyridine (4-AP) and 10 μmol/L glibenclamide in diabetes thoracic aorta. Only 4-AP decreased relaxation response to SNP in age-matched control mice. The -logIC₅₀ difference of TEA in thoracic aorta rings of diabetes was significantly higher than age-matched control mice.CONCLUSION:In early stage of diabetes mellitus, the opening or expression of K_Ca channels is significantly enhanced.The opening of K_ATP channels is also enhanced in this stage.     

Expression and significance of TLR4/NF-κB in pulmonary vascular remodeling in smoking rats

ATM: To observe the expression of Toll-like receptor 4 (TLR4), nuclear factor-κB subunit P65 protein (NF-κB P65) and proliferating cell nuclear antigen (PCNA) in the pulmonary vascular tissues of the rats exposed to smoke, and to explore the possible mechanism of TLR4/NF-κB signaling pathway in pulmonary vascular remodeling. METHODS: SPF male healthy rats (n=48) were randomly divided into control group, smoke exposure for 4 weeks group (S4 group), smoke exposure for 8 weeks group (S8 group) and smoke exposure for 12 weeks group (S12 group), with 12 rats in each group. HE staining was used to observe the morphological changes of pulmonary vessels, and then the pulmonary vascular wall area/total vascular area (WA%) and vascular wall thickness/vascular external diameter (WT%) were measured by the medical image analysis system. The expression of TLR4, NF-κB P65 and PCNA in the pulmonary vascular tissues was detected by immunohistochemical staining. The protein content was expressed by the average integral absorbance. The mRNA expression of TLR4 in the pulmonary vessels was detected by RT-qPCR. The relationships between WA%, WT%,TLR4 protein, TLR4 mRNA, P65 protein, PCNA protein and pulmonary vascular remodeling, and another relationships between WA%, WT%, P65 protein, PCNA protein and TLR4 protein were analyzed.RESULTS: The WA% and WT% in smoke exposure groups significantly increased compared with control group, and the ratio was proportional to the time of smoke exposure. The protein expression of TLR4, p65 and PCNA, and the mRNA expression of TLR4 in smoke exposure groups also increased significantly compared with control group. CONCLUSION: The extent of pulmonary vascular remodeling in the rats increases when the protein expression of TLR4 is up-regulated. There is a positive correlation between pulmonary vascular remodeling and the protein expression of TLR4 and NF-κB P65. Pulmonary vascular remodeling may be related to the activation of TLR4/NF-κB signaling pathway.     

Aerobic exercise protects cardiac function of T2DM mice by activation of PI3K (p110α)/Akt signaling pathway

AIM: To study the protective effect of aerobic exercise on cardiac dysfunction in mice and its mechanism, and to provide theoretical and practical basis for the exercise therapy of diabetic cardiac dysfunction.METHODS: The mice were divided into normal control non-exercise (NNC) group, normal control exercise (ENC) group, diabetic non-exercise (NDM) group and diabetic exercise (EDM) group. At the end of the experiment, the cardiac function was evaluated by echocardiography. The pathological changes of the myocardial tissues and the development of fibrosis were observed. The mRNA expression of ANP, and the protein levels of PI3K (p110α) and Akt were determined. RESULTS: The decrease in cardiac function of diabetic mice was observed, and the cardiac function recovered after exercise intervention (P<0.05). Under light microscope with HE and Masson staining, the myocardial structure in NDM group was in extreme disorder, cell arrangement was not neat, and the degree of fibrosis increased, but the myocardial damage was improved in ENC group. Compared with NNC group, the mRNA expression of ANP in the myocardium of diabetic mice was up-regulated (P<0.05). The protein levels of PI3K (p110α) and Akt were decreased (P<0.05), and the cascade was inactivated. Compared with NDM group, the mRNA expression of ANP was down-regulated and the protein levels of PI3K (p110α) and Akt were up-regulated in EDM group (P<0.05). CONCLUSION: Diabetes results in myocardial damage in mice, and reduces cardiac function. Exercise intervention alleviates the heart dysfunction induced by high glucose via activating PI3K(p110α)/Akt signaling pathway to protect the structure and function of the myocardium.     

Effects of phytosterol ester on aortic aging and expression of related genes in rats

AIM: To explore the protective effect of phytosterol ester (PSE) on aortic aging in rats. METHODS: The female SD rats (12 months old, n=42) were randomly divided into control group, model group and PSE group. During the experiment, the rats in control group, model group and PSE group were treated with basic feed, high-fat diet (HFD) and HFD with 2% PSE (W/W) for 6 months, respectively. The morphological changes of the aorta were observed by HE staining and Masson staining, and the absolute area of smooth muscle cells and collagen fiber in the vascular wall were measured by image analysis. The levels of advanced glycosylation end products (AGEs), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the plasma were detected. The expression of silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor γ (PPARγ) at mRNA and protein levels in the vascular tissue was determined by real time PCR and Western blot, respectively. RESULTS: PSE significantly lowered plasma TC and LDL-C, and increased plasma HDL-C level (P<0.05), but had no effect on plasma TG level. PSE significantly attenuated the thickening of intima and media of aging aortic, and decreased the migration of vascular smooth muscle cells (VSMC) and the amount of VSMC and collagen fiber in the aorta (P<0.05). PSE significantly reduced the contents of AGEs and MDA (P<0.05), but had no effect on the activity of SOD and CAT in the plasma. PSE also down-regulated the expression of PPARγ and up-regulated the expression of SIRT1 (P<0.05). CONCLUSION: PSE is able to attenuate the senescence process in the aorta by reducing the production of reactive oxygen species in plasma, and activating SIRT1, or inhibiting the expression of PPARγ in vascular tissues.     

Role of angiotensin Ⅱ on typeⅠcollagen synthesis and its mRNA expression in vascular adventitial fibroblasts

AIM:Effects of angiotensin Ⅱ on typeⅠcollagen synthesis and its mRNA expression in cultured vascular adventitial fibroblasts. METHODS:Vascular adventitial fibroblasts (VAF)were isolated, cultured from rat thoracic aorta by explant method. ELISA was used to study typeⅠcollagen synthesis and competitive RT-PCR was employed to detect its mRNA expression after angiotensin Ⅱ administration. RESULTS: Angiontensin Ⅱ caused a dose dependent increase of typeⅠcollagen synthesis and its mRNA expression in VAF. CONCLUSION:The results support that angiotensin Ⅱ is an important factor controlling collagen metabolism of VAF and VAF may play an important role in vascular remodelling of hypertension.     

Effect of N-acetyl-L-cystein on blood pressure and endothelial function in aorta of rats exposed to chronic intermittent hypoxia

AIM: To investigate the effect of N-acetyl-L-cystein (NAC) on blood pressure and endothelial function in the aorta of the rats exposed to chronic intermittent hypoxia (CIH). METHODS: Thirty healthy male SD rats were randomly divided into 3 groups: control group, CIH group and CIH+NAC group. The systolic blood pressure (SBP) was measured with tail-cuff me-thod. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of endothelial nitric oxide synthase (eNOS） and endothelin-1 (ET-1) in the thoracic aorta. The protein expression of eNOS in the thoracic aorta was examined by Western blotting. The levels of ET-1 in the thoracic aorta and serum were detected by radioimmunoassay. The serum nitric oxide was determined by nitric acid reduction method.The superoxide dismutase (SOD) activity in peripheral blood plasma was detected by xanthine oxidase method.The serum malondialdehyde content was detected by thiobarbituric acid method, and superoxide anion (O-·2) in thoracic aorta was determined by chemical colorimetric method. RESULTS: Compared with the control animals, CIH exposure was associated with decreased SOD level, and NAC-treated CIH animals showed recovery in SOD level. NAC treatment prevented CIH-induced hypertension as well as CIH-induced increase in MDA. The aorta eNOS mRNA and protein, and serum NO levels in CIH group were lower than those in control group, and those in NAC treatment group were higher than those in CIH group. The increases in ET-1 mRNA,ET-1 protein and O-·2 levels in the aorta, and the elevated circulating ET-1 level were also observed in CIH-exposed animals. Treatment with NAC significantly decreased the mRNA and protein levels of ET-1, the O-·2 content, and the circulating ET-1 level in CIH-exposed animals. CONCLUSION: NAC protects endothelial function and alleviates hypertension by suppressing the oxidant stress in the aorta tissues, indicating that oxidant stress may be involved in the mechanism of endothelial disorder of CIH-induced hypertension.     

Effects of grape seed procyanidin on vascular remodeling in renovascular hypertensive rats

AIM: To study the effect of grape seed procyanidin (GSP) on vascular remodeling in renovascular hypertensive (RH) rats. METHODS: The RH rat model was established by two-kidney one-clip method. Two weeks after operation, 28 rats were selected according to the increased tail systolic pressure above 130 mmHg and randomly divided into 4 groups (n=7): RH model group, low GSP treatment group (50 mg·kg^-1·d^-1),high GSP treatment group (200 mg·kg^-1·d^-1) and captopril treatment group (30 mg·kg^-1·d^-1). Meanwhile, 7 rats with sham operation served as controls. Tail systolic pressure, medial thickness (MT), luminal diameter (LD), and the ratio of MT to LD in thoracic aortic wall were determined 6 weeks after treatment. Masson staining and ELISA were used to detect the content of collagen and angiotensin II (Ang II) in aortic tissues. The protein expression of tumor necrosis factor α (TNF-α) in abdominal aortic tissues was determined by Western blotting. RESULTS: Compared with control group, the tail systolic pressure, MT, MT/LD, the content of collagen and the protein expression of TNF-α in aorta were significantly increased in RH model group, but LD decreased. Treatment with GSP and captopril reduced the raised parameters, and increased the LD in RH model rats. These effects were more notable in high GSP treatment group, and equal to captopril treatment group. CONCLUSION: GSP treatment significantly decreases tail systolic pressure in RH rats, and effectively attenuates arterial vascular remodeling by decreasing the content of AngII and reducing the protein expression of TNF-α in aorta.     

Protective effect of C1q/tumor necrosis factor-related protein 3 on vascular endothelium in rats with hyperuricemia

AIM To study whether C1q/tumor necrosis factor （TNF）-related protein 3 （CTRP3）protect vascular endothelium in rats with hyperuricemia and its potential mechanisms. METHODS An animal model of hyperuricemia was established by using male SD rats drinking 10% fructose water （n=10）. The rats drinking normal water served as normal controls （n=10）. After 12 weeks, the rats were given a single injection with Ad-CTRP3 or Ad-GFP. The experiment was ended at 14th day after transfection.The serum levels of uric acid and nitric oxide （NO） were evaluated. The serum contents of TNF-α and interleukin-6 （IL-6） were measured by ELISA. HE staining and TUNEL assay were used to assess the morphological changes of intima and apoptosis of endothelial cells in thoracic aorta, respectively. The mRNA levels of endothelial nitric oxide synthase （eNOS）, TNF-α and IL-6 were detected by RT-qPCR. The protein levels of CTRP3 and Toll-like receptor 4 （TLR4） were determined by Western blot. RESULTS Compared with normal control group, the rats with hyperuricemia showed lower CTRP3 and higher TLR4 protein levels in the thoracic aorta （P<0.05）. Hyperuricemic rats had higher serum contents of uric acid, TNF-α and IL-6 （P<0.05）. Also, the intima structure disturbance of thoracic aorta, increased apoptotic rate, higher mRNA levels of TNF-α and IL-6 as well as lower mRNA levels of eNOS were observed （P<0.05）. By contrast, CTRP3 over-expression decreased TLR4 protein levels, reduced inflammatory cytokines, and obviously improved the morphology and function of thoracic aorta in the rats with hyperuricemia. CONCLUSION CTRP3 protect vascular endothelium in rats with hyperuricemia maybe via down-regulation of TLR4- mediated inflammatory signaling pathway.     

Atorvastatin inhibits atherogenesis by RXRα-mediated depressing oxidative stress in STZ-induced diabetic ApoE-/- mice with fat-rich diet

AIM: To explore the effects of atorvastatin (Atorv) on atherosclerosis in streptozotocin (STZ)-induced diabetic apolipoprotein E knockout (ApoE-/-) mice with fat-rich diet and the possible mechanism. METHODS：C57 mice served as control. ApoE-/- mice (n=34) fed with high-fat diet were randomly divided into ApoE-/- group, STZ-ApoE-/- group and STZ-ApoE-/-+Atorv group. Intraperitoneal injection of streptozotocin was performed to create diabetic animal model. Blood glucose was determined by glucose oxidase method. Blood lipid levels were detected by enzymic method or selective homogeneous method. The plaque area in the thoracic aorta was measured by HE staining. The protein level of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox in the thoracic aorta was determined by Western blotting. The levels of reactive oxygen species (ROS) in blood and thoracic aorta homogenates were detected by Fenton reaction and Griess reagent. Human umbilical vein endothelial cells (HUVECs) were isolated from healthy umbilical cords by collagenase I and cultured. ROS production was detected by flow cytometry. NADPH oxidase activity was measured using lucigenin assay.Effects of retinoid X receptor α (RXRα) on inhibition of oxidative stress by atorvastatin were evaluated by RNA interference and plasmid transfection. RESULTS：(1) Compared with C57 group, the plaque areas of the thoracic aorta in ApoE-/- group were increased. No difference of the fasting glucose between the 2 groups was observed. The levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in ApoE-/- group than those in C57 group. (2) Compared with ApoE-/- group, the plaque areas of the thoracic aorta in STZ-ApoE-/- group were further enlarged ［(314.13±35.72) μm2 vs (215.88±34.19) μm2, P<0.05］. The levels of blood glucose, TG, TC and LDL-C, thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in STZ-ApoE-/- group than those in ApoE-/- group (P<0.05). (3) Compared with STZ-ApoE-/- group, the plaque areas of the thoracic aorta in STZ-ApoE-/- +Atorv group were reduced ［(217.47±24.56） μm2 vs （314.13±35.72） μm2, P<0.05］. The levels of blood glucose, LDL-C, TC, HDL-C and TG showed no significant difference between the 2 groups. Thoracic aorta gp91phox protein level and ROS production in blood and thoracic aorta homogenates were lower in STZ-ApoE-/- +Atorv group than those in STZ-ApoE-/- group (P<0.05). (4) High glucose-induced increases in NADPH oxidase activity and gp91phox expression were significantly inhibited by atorvastatin (10-6 mol/L) in HUVECs. The inhibitory effects of atorvastatin on high glucose-induced ROS production and NADPH oxidase activation were largely impaired when the cells were transfected with RXRα siRNA. However, the effect of atorvastatin was significantly strengthened when RXRα was over-expressed in the HUVECs transfected with RXRα plasmid. CONCLUSION：Atorvastatin inhibits atherogenesis by depressing high glucose-induced oxidative stress in diabetic ApoE-/- mice with fat-rich diet. The anti-oxidative stress effect of atorvastatin is mediated by RXRα.     

Role of estrogen on expression of TNF-α and MMP-9 in intracranial arterial vascular wall of spontaneous hypertensive rats

AIM: To study the effects of estrogen on the inflammatory response and vascular remodeling of intracranial artery in rats.METHODS: Thirty-two female spontaneous hypertensive rats (SHR) were randomized into 4 groups: spontaneous hypertensive group(sham-operated), ovariectomized group, ovariectomized+17 beta-estradial group and ovariectomized+vehicle group (8 rats in each group).On day 14, estradiol was detected by radioimmunoassay.The pathological changes were observed under light microscope.The protein expression of tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9) in vascular wall of Willis circle was detected by Western blotting.RESULTS: The estrogen level was lower in ovariectomized group than that in sham-operated group (P<0.01).The estrogen level was higher in ovariectomized rats treated with 17 beta-estradial than that in ovariectomized rats treated with vehicle (P<0.01).Advanced aneurysm was not found in all groups.Early aneurysmal change was not found in sham-operated group.Early aneurysmal changes in some rats were observed in ovariectomized group (2 rats), ovariectomized+vehicle group (3 rats) and ovariectomized+17 beta-estradial group (1 rat).The protein levels of TNF-α and MMP-9 in the vascular wall of Willis circle in sham-operated group were lower than that in ovariectomized group (P<0.01).Additionally, the protein levels of TNF-α and MMP-9 in the vascular wall of Willis circle of ovariectomized rats treated with 17 beta-estradial were lower than those of ovariectomized rats treated with the vehicle (P<0.01).CONCLUSION: Estrogen can influence the vascular remodeling of intracranial artery by inhibiting the inflammatory response and degradating MMP-9 in the vascular wall.