High-cholesterol diet with or without corn oil for establishing atherosclerotic model in rabbits

AIM：To compare the reliability and plaque area between using high-cholesterol diet and high-cholesterol diet with corn oil to establish a rabbit atherosclerotic model. METHODS：Eighteen New Zealand rabbits were randomly divided into 3 groups (6 rabbits each): normal diet group (group C), high-cholesterol diet group (group H1) and high-cholesterol diet containing 6% corn oil group (group H2). All rabbits were fed for 12 weeks, and their body mea-sured was weighed at the end of every weeks. The serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and triglyceride (TG) were measured by automatic chemistry analyzer at 0 week and 12 weeks. At the end of 12 weeks, the thoracic aorta of 8-cm length since aortic root was isolated from the rabbit after anesthesia, and stained with Sudan IV or oil red O to verify the existence of plaque. The percentage of plaque area (PA/IA) in the intima area was further calculated by ImageJ2x software. RESULTS：At the end of 12-week feeding, the serum levels of HDL-C, LDL-C and TC in both group H1 and group H2 were significantly higher than those in group C, and serum TG in group H2 was significantly higher than that in group C. Serum HDL-C in group H2 was significantly higher than that in group H1, but no significant difference of serum LDL-C, TC and TG between group H1 and group H2 was found. There was no plaque in the intima in group C, and plaques were observed in the intima of all rabbits in group H1 and group H2. Rabbit atherosclerotic models in both group H1 and group H2 were established with a success rate of 100%. The values of PA/IA in group H1 ［(49.74±18.78)%］ and group H2 ［(56.95±26.74)%］ were both significantly higher than that in group C (0%), and no significant difference of PA/IA between group H1 and group H2 was observed. CONCLUSION：High-cholesterol diet with or without corn oil can establish a rabbit atherosclerotic model with a success rate of 100% after 12-week feeding, and the percentage of plaque area in the total aortic intimal area is not different in the 2 feeding methods.     

Roles of PI3K/Akt/mTOR signaling pathway in macrophage autophagy and atherosclerotic plaque instability

AIM：To investigate whether selective inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.METHODS：In in vitro study, casodex (20 μmol/L), rapamycin (10 nmol/L) or mTOR-siRNA (30 nmol/L) was used to treat mouse macrophage cell line RAW 264.7. Inflammation-related cytokines secreted by macrophages were measured by means of ELISA. Ultrastructural changes of the macrophages were examined by transmission electron microscopy. The mRNA and protein expression levels of Akt, mTOR and autophage-related protein Beclin 1 were assayed by real-time fluorescence quantitative RT-PCR and Western blotting. The expression of autophagy-related indicator LC3-II was detected by immunofluorescence and Western blotting. In in vivo study, 24 New Zealand white rabbits underwent balloon-induced abdominal aortic wall injury and were fed with a diet of 1% cholesterol for 8 weeks. The rabbits were randomly divided into control group (n=8), casodex group (1.0 mg·kg-1·d-1, n=8) and rapamycin group (0.5 mg·kg-1·d-1, n=8). Four weeks after drug administration, intravascular ultrasound (IVUS) was carried out to observe the plaque imaging. Ultrastructural changes of the macrophages and the protein expression of Akt, mTOR and LC3-II in the macrophages were also measured.RESULTS：In in vitro study, more typical autophagosomes were detected in casodex-, rapamycin- or mTOR-siRNA-treated cells. The expression level of LC3-II increased, but Beclin 1,p-Akt and p-mTOR significantly decreased in the 3 treatment groups. The concentration of IL-10 decreased while IFN-γ significantly increased in the treatment groups. In in vivo study, IVUS found that external elastic membrane area (EEMA),plaque area(PA) and plaque burden (PB) significantly decreased in casodex and rapamycin treatment groups. Expression of LC3-II increased significantly in the 2 treatment groups. The staining of RAM-11 and p-mTOR in the macrophages was significantly reduced as compared with control group.CONCLUSION：Selective inhibition of PI3K/Akt/mTOR signaling pathway reduces the infiltration of macrophages and stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.     

Experimental study on induction of atherosclerotic plaque instability in rabbits after transfer of wild-type p53 gene

AIM: To study the apoptotic role of wild-type p53 in induction of plaque instability in atherosclerotic rabbits. METHODS: Fifty-four New Zealand White rabbits underwent balloon-induced abdominal aortic wall injury and then were fed on a diet of 1% cholesterol. At the end of the eighth week, the rabbits were randomly divided into two groups: group A and group B. Recombinant adenovirus carrying p53 and β-galactosidase (LacZ) genes were injected in group A and B, respectively. Two weeks later, 10 rabbits each in group A and B was killed and the remaining rabbits all underwent pharmacological triggering with injection of Chinese Russell's viper venom and histamine. RESULTS: Compared with group B, p53 gene over-expression in group A resulted in a marked increase in number of positive apoptotic cells (2.5%±0.8% vs 1.0%±0.3%, P<0.05) and a significant decrease in vascular smooth muscle cells (47.5%±6.8% vs 80.4%±10.6%, P<0.01), the thickness of the fibrous cap ［(132.9±56.7）μm vs （181.8±59.7） μm, P<0.05］ and the cap/intima-media ratio (0.20±0.18 vs 0.21±0.11, P<0.05). CONCLUSION: Transfer of human wild-type p53 genes effectively promotes apoptosis of VSMCs in atherosclerotic plaques, which makes the plaques vulnerable to rupture.     

Overexpression of SHP-1 mediated by lentivirus restrains atherosclerotic progression in mice

AIM: To investigate the role of SH2-domain-containing protein-tyrosine phosphatase-1 (SHP-1) lentivirus in atherosclerotic mice. METHODS: ApoE knock-out mice were randomly assigned to 3 groups: control group, GFP transfection group and SHP-1 transfection group. All mice were placed with carotid collars on the right common carotid arteries near its bifurcation, following feeding with high-fat diet for 8 weeks and then transfected with GFP blank vector or SHP-1 lentivirus (SHP-1-LV). The fluorescence density of the plaques, body weight, the levels of plasma total cholesterol (TC) and triglyceride (TG) were determined at 1st, 2nd, and 6th week after lentivirus transfection. Furthermore, the mRNA and protein expression of SHP-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-2 and MMP-9 were analyzed by real-time PCR and Western blot. Additionally, pathological analysis of the plaques was also performed by HE and oil red O staining. RESULTS: The fluorescence of the plaques was observed at 1st, 2nd, and 6th week after lentivirus transfection, with a highest density at 2nd week. The body weight and the levels of TC and TG in the mice were not influenced by lentivirus transfection. Moreover, SHP-1-LV transfection significantly upregulated the expression of SHP-1 at mRNA and protein levels, but inhibited the expression of IL-6, TNF-α, MMP-2 and MMP-9. In addition, SHP-1-LV transfection also decreased the plaque size ratio and lipid content in right common carotid arteries. CONCLUSION: SHP-1 overexpression accelerates the regression of atherosclerotic plaque, thus emerging SHP-1 as a target for prevention and treatment of atherosclerosis.     

3,4-Dihydroxyacetophenone reduces TG content in L02 cells and hepatic tissue via AMPK pathway

AIM: To investigate the mechanism of 3,4-dihydroxyacetophenone (3,4-DHAP) regulating lipid metabolism in human hepatic cells (L02 cells) and rabbit hepatic tissue. METHODS: L02 cells were divided into normal control group, model group, 3,4-DHAP group and simvastatin group. The cells were collected after treated with drugs for 8 h. Triglyceride (TG) content in the cells was detected by TG kit. RT-qPCR was applied to detect the mRNA expression level of AMP-activated protein kinase (AMPK). The protein levels of AMPK, phosphorylated AMPK (p-AMPK), phosphorylated sterol regulatory element binding protein-1c (p-SREBP-1c) and phosphorylated acetyl-CoA carboxylase (p-ACC) were detected by Western blot. Male New Zealand white rabbits (n=32) were randomized into normal control group, model group, 3,4-DHAP group and simvastatin group. The rabbits were treated with the drugs from week 2 to week 12. At the end of week 12, all rabbits were sacrificed. The liver lipids were measured by oil red O staining, and TG content was analyzed by TG kit. The protein levels of AMPK, p-AMPK, p-SREBP-1c and p-ACC in hepatic tissue were detected by Western blot. RESULTS: In L02 cells, compared with model group, TG content in 3,4-DHAP group was significantly decreased, and the expression of AMPK at mRNA and protein levels and the protein levels of p-AMPK, p-SREBP-1c and p-ACC were significantly increased. In rabbits of 3,4-DHAP group, the TG content was significantly decreased compared with model group, and the protein levels of AMPK, p-SREBP-1c and p-ACC were significantly increased. CONCLUSION: The AMPK signaling pathway may be involved in the mechanism of 3,4-DHAP to reduce TG content in L02 cells and rabbit hepatic tissue.     

Effect of 17β-estradiol on the levels of plasma oxidized low density lipoprotein in ovariectomized rabbits

AIM: To observe the changes of plasma oxidized low density lipoprotein(oxLDL) levels of ovariectomized cholesterol-fed rabbits with or without 17β-estradiol(E₂) replacement therapy.METHODS: All rabbits were ovariectomized and fed standard chow supplemented with 1.5% cholesterol for 14 weeks. Two weeks after operation, the rabbits were randomly assigned to four groups. Three groups were treated with E₂ 1 mg, 2 mg, 4 mg respectively, the other group served as control. Serum superoxide dismutase (SOD) levels and plasma oxLDL levels were measured at 0, 3, 8, 12 weeks after hormone replacement therapy. The aortic atherosclerotic lesion areas were determined by computer. RESULTS: We found that there were striking increase of serum SOD levels ( P<0.05 ) and significant decrease in both the plasma oxLDL levels and the aortic atherosclerotic lesion areas ( P<0.01 respectively). Moreover, a positive correlation was found between plasma oxLDL levels and the areas of atherosclerotic plaque in all rabbits. CONCLUSIONS: Estrogen attenuates atherogenesis in cholesterol-fed ovariectomized rabbits. And this beneficial effect of E₂ may be duo to its lowering of plasma oxLDL level.     

Effects of oxidized high density lipoprotein on tissue factor expression in ECV304 cell line

AIM: To investigate the expression of tissue factor (TF) induced by oxidized high density lipoprotein (oxHDL) in human umbilical vein cell line, ECV304, and the related mechanisms. METHODS: Four main groups were designed: the negative, the positive (ECV304 with histamine), the HDL group and the oxHDL group. Quantitative real-time polymerase chain reaction (RQ-PCR) and Western blotting were used to detect the expression level of TF. The specific inhibitors of MAPKs, SP600125 (c-jun terminal NH2 kinase, JNK), SB203580 (p38 MAP kinase, p38 MAPK), PD98059 (extracellular signal-regulated kinase, ERK1/2) were used to investigate the underlying mechanisms. RESULTS: The TF expression in normal ECV304 cell line was not detected. Histamine administration resulted in a significant expression of TF in ECV304 cell line, with strongest effect after 1 h co-incubation at concentration of 1×10-5 mol/L histamine (about 4.8-fold higher expression of TF compared with that of 1×10-9 mol/L histamine). Expression level of TF was detected after stimulated with oxHDL in dose- and time- dependent manners. The highest expression of TF mRNA was found at 20 mg/L oxHDL and 6 h co-incubation, with 1.8-fold and 5.3-fold increase in TF expression, respectively, compared with that at 10 mg/L oxHDL and 2 h co-incubation. 20 mg/L oxHDL also caused an apparent augmentation of TF protein expression, about 1.5-fold higher compared with that stimulated by 40 mg/L oxHDL. HDL co-incubation did not cause a detectable expression of TF protein. The mRNA levels of TF in ECV304 cell line induced by oxHDL were decreased by 95.0%, 81.0%, 87.0%, respectively (all P<0.05) after application of inhibitors against p38MAPK, JNK and ERK1/2. CONCLUSION: The results suggest that oxHDL stimulates TF expression in ECV304 cell line in both dose- and time- dependent manners, in which MAPKs signal transduction may play an important role.     

Expression of ROCK I and TGF-β1 in pulmonary arterioles of rat with chronic thromboembolism pulmonary hypertension

AIM:To investigate the dynamic expression of Rho kinase (ROCK I) and transforming growth factor β₁ (TGF-β₁) in pulmonary arterioles of rat with chronic thromboembolic pulmonary hypertension. METHODS: Sixty-four male Wister rats were randomly divided into eight groups: beginning control group, embolism for 3 d, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks groups and end control group. The pulmonary thromboembolism (PTE) model was established by injecting thrombin into jugular vein two times in two weeks and each rat underwent peritoneal injection with tranexamic acid one time a day during experiment to prevent thrombolysis. The mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), relative medial thickness of small pulmonary arteries (PAMT) and vessel wall area/total area (WA/TA) were measured. The levels of ROCK I mRNA and TGF-β₁ protein in rat pulmonary artery were determined by in situ hybridization, immunohistochemistry and image analysis, respectively. RESULTS: mPAP, PAMT and WA/TA were higher respectively in embolism from 4 weeks group to 12 weeks group than those in beginning control group (mPAP: all P<0.01, PAMT and WA/TA: 4 weeks group P<0.05, 8 weeks group and 12 weeks group P<0.01). RVHI was elevated in 8 weeks group P<0.05, in 12 weeks group P<0.01. ROCK I mRNA and TGF-β₁ protein in pulmonary arterioles got the enhanced positive signals of in situ hybridization or immunohistochemistry staining with prolonging the time of rats with pulmonary thromboembolism. ROCKⅠ mRNA: embolism from 3 d group to 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01, TGF-β₁ protein: 1 week group and 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01. Linear correlation analysis showed that ROCK I mRNA and TGF-β₁ protein were positively correlated with mPAP, RVHI and vessel remodeling index (all P<0.01), ROCK I mRNA were positively correlated with TGF-β₁ protein (P<0.01). CONCLUSION:ROCK I and TGF-β₁ play a role in the pathogenesis of chronic thromboembolic pulmonary hypertension and pulmonary vessel remodeling. TGF-β₁ produces biological effect by active ROCK signal pathway.     

Effects of pravastatin combined with warfarin on steroid-induced femoral head necrosis in rabbits

AIM: To investigate the role of pravastatin combined with warfarin in treating steroid-induced osteonecrosis of femoral head (SIONF) in rabbits. METHODS: Forty-eight Japan healthy adult female rabbits were randomly divided into 3 groups: control group (CTR group, 12 rabbits), experimental group (SIONF group, 18 rabbits) and pravastatin+warfarin treatment group (PW group, 18 rabbits). The rabbits in SIONF group and PW group were injected with methylprednisolone (20 mg/kg) intramuscularly to establish the animal model of SIONF. The rabbits in CTR group were only injected with normal saline intramuscularly. The rabbits in PW group were also received pravastatin (2.5 mg·kg^-1·d^-1) and warfarin(1.5 mg·kg^-1·d^-1)orally. The blood samples were collected from 2 rabbits in each group at time point of pre-experiment and 2, 4, 6, 8, 10, 12 weeks post-experiment for measuring the contents of total cholesterol (TC) and triglyceride (TG), prothrombin time (PT) and activated partial thromboplastin time (APTT). In the latter 4 time points, the X-ray examination of bilateral hip joints were taken, and the animals were then killed to collect the bone tissues for ultrastructural observation under transmission electron microscope. The SIONF incidence rate was also determined. RESULTS: From the 6th week to the end of experiment, the level of TC in PW group was higher than that in CTR group (P<0.05), but lower than that in SIONF group (P<0.05). The level of TC in SIONF group was higher than that in CTR group (P<0.01). The levels of TG showed the similar changes as TC. From the 2nd week of experiment, significant prolonged PT was observed in PW group as compared with CTR group and SIONF group (P<0.01). On the contrary, significant shortened PT in SIONF group was found (P<0.01). The changes of APTT were similar to the changes of PT. Compared with SIONF group, the bone tissues in PW group showed significant decrease in the conversion rate of intramedullary fat in femoral head and the rate of bone recess. No thrombus formation was observed, and bone karyopyknosis and autolysis were also significantly reduced. In PW group, the SIONF incidence rate was 31 % (5/16), significantly lower than 63% (10/16) in SIONF group (P<0.05). CONCLUSION: Combination of pravastatin with warfarin reduces the incidence rate of SIONF in rabbits, indicating a relation with the improvement of hyperlipid and hypercoagulant state.     

Cardiac protective effect of granulocyte colony-stimulating factor combined with ischemic postconditioning on acute myocardial infarction in rabbits

AIM: To investigate the protective effect of granulocyte colony-stimulating factor (G-CSF) combined with ischemic postconditioning (IP) on acute myocardial infarction (AMI). METHODS: Male New Zealand rabbits were randomly divided into 4 groups (n=15) after 30 min of left ventricular artery (LVA) occlusion: the rabbits in ischemia-reperfusion (IR) group were directly given reperfusion|the rabbits in G-CSF group were subsequently treated with G-CSF (10 μg·kg^-1·d^-1) by subcutaneous injection after direct reperfusion|the rabbits in IP group received 4 episodes of 30 s reperfusion and 30 s occlusion before total reperfusion|the rabbits in IP combined with G-CSF (IP+G-CSF) group were treated with both IP and G-CSF. Electrocardiogram (ECG) monitoring was performed during the operation. Blood was drawn to evaluate white blood cell count (WBC) and cardiac troponin I (cTnI) before operation and 7 d later. Ultrasound cardiography was performed to evaluate left ventricular remodeling and functions 4 weeks after operation. The sizes of infarcted myocardium were determined by triphenyltetrazolium chloride (TTC) staining. Apoptosis of cardiomyocytes was measured by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: ST-segment resolutions were significantly decreased in IP group and IP+G-CSF group compared with direct reperfusion groups (P<0.05). WBC significantly increased in the groups treated with G-CSF for 1 week. The values of cTnI after operation were significantly lowered in G-CSF group, IP group and IP+G-CSF group as compared with IR group (P<0.05). Left ventricular ejection fraction, the size of infarcted myocardium and apoptosis of cardiomyocytes were better in IP group, G-CSF group and IP+G-CSF group than those in IR group. CONCLUSION: G-CSF combined with IP is a promising strategy against cardiac reperfusion injury and accelerates cardiac repair in AMI.     

Effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia

AIM:To investigate the effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia. METHODS:The rabbit model of atherosclerosis was produced by feeding high lipid forages. RAW264.7 foam cell and HepG2 injured cell models were established by incubation with oxidized low density lipoprotein (ox-LDL). The atherosclerotic plaque area was measured, and serum lipids were detected. The cellular lipid accumulation was examined by oil red O staining. The cellular contents of total cholesterol and cholesterol ester were quantified by high performance liquid chromatography. The hepatic lipase (HL) mRNA expression was determined by RT-PCR. RESULTS:(1) Compared with hyperlipid group, the aorta atherosclerosis plaque area and the serum triglyceride level were significantly decreased in sodium ferulate-treated rabbits, but the serum cholesterol level showed little change. (2) Compared with ox-LDL group, the HL mRNA expression in HepG2 cells was enhanced significantly in sodium ferulate-treated group, but the cellular contents of total cholesterol and cholesterol ester in RAW264.7 foam cells showed little change. CONCLUSION:Sodium ferulate inhibits the formation of atherosclerotic plaque in high-cholesterol-fed rabbits aorta. This antiatherosclerotic function may reduce serum triglyceride level through enhancing the expression of HL mRNA without influencing serum cholesterol level and foam cell formation.     

Effects of pituitary adenylate cyclase-activating polypeptide on cardiac remodeling in coronary atherosclerotic rabbits

AIM:To study the intervention effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cardiac remodeling during the development of rabbit coronary atherosclerosis. METHODS:60 male New Zealand rabbits were equally divided into 3 groups randomly: control group (C group), atherosclerosis model group (A group) and PACAP intervention group (P group). At the 4th, 8th and 12th week, 5-6 cases of rabbits in each group were sacrificed, cardiac tissue with coronary arteries were harvested to make paraffin sections. The sections were stained with hematoxylin-eosin and van Gieson separately. The qualitative observation and/or quantitative analysis were made by light microscope. RESULTS:（1）There was no lesion in C group. For A group and P group, there were plaques in large epicardial coronary arteries and small coronary arteries; an impressive accumulation of collagen was also observed in myocardium. In P group, the lesions of small coronary arteries were less serious, and the degrees of perivascular and myocardial fibrosis also appeared to be less.（2）For A group, the wall-to-lumen ratios in small coronary arteries were significantly greater at the 12th week (2.58±1.54) than C group (1.34±0.58) and P group (1.39±0.48) （P<0.05）； and the width of cardiomyocyte (13.85 μm±2.27 μm) was already remarkably narrower than C group (14.68 μm±2.40 μm) at the 8th week (P<0.05) and narrower significantly than C group and P group at the 12th week. （3）There were not difference significantly between the above-related parameters of P group and C group (P>0.05). CONCLUSION:Structure changes exist in coronary arteries and myocardium during the development of rabbit coronary atherosclerosis, PACAP can inhibit the cardiac remodeling.     

Effects of ERK1/2 on pressure overload-induced cardiomyopathy and heart failure in mice

AIM: To explore the changes in extracellular regulated protein kinase (ERK1/2) in the hypertrophic myocardium induced by pressure overload at the different time courses and to determine the molecular mechanism in the myocardium from hypertrophy to heart failure. METHODS: C57/BL mice, aged 12 week old, were subjected to sham-operation (SH) or transversing aortic constriction (TAC) to establish left ventricular hypertrophy. Echocardiographic assessments, hemodynamic determination, organ weight measurement, morphological and histological examination were performed at 1, 4, 8, 12 and 16 weeks after surgery. Meanwhile mRNA levels of atrial natriuretic peptide (ANP), α-myosin heavy chain (α-MHC), bcl-2 and bax were measured by RT-PCR, and ERK1/2 levels were detected by Western blotting. The animals in SH group were performed the same tests then sacrificed at 16 weeks. RESULTS: (1) Compared to SH group, LVESd, LVEDd, Awsth, Awdth, Pwsth and Pwdth progressively increased after TAC. Meanwhile, ejection fraction (EF%) significantly decreased at 16th week (P<0.05). LVSP, dp/dtmax and dp/dtmin in TAC group were progressively increased after 4 weeks. From 8-12 weeks these parameters maintained stable and then sharply decreased at 16th week (all P<0.05). However, LVEDP was statistically increased at 8th week. These echocardiographic and hemodynamic changes indicated a development of LVH and eventually progressing towards to heart failure. (2) Histologically, cardiac collagen measured by percentage of Sirius red positive stained area and apoptosis index showed progressive increases from 4 to 16 weeks. (3) Compared to SH group, mRNA levels of ANP was time-dependently increased while α-MHC and Bcl-2 were time-dependently decreased. The ratio of Bcl-2 /Bax was decreased. Phosphorylation of ERK1/2 was increased at 4th week, then decreased with age of TAC (all P<0.05). CONCLUSION: Pressure-overload induced by TAC results in a development of LVH from early concentric hypertrophy to late eccentric hypertrophy, and eventually toward cardiac dysfunction or heart failure. Those changes are associated with increase in cell size and cardiac fibrosis. ERK1/2 signaling pathway may involve in the regulation of myocardial cell apoptosis in hypertrophic and failure heart.     

Impact of hydrogen sulfide donor on endothelin-1 and connective tissue growth factor expression in rats with pulmonary hypertension induced by high pulmonary blood flow

AIM: To explore the possible impact of hydrogen sulfide (H2S) donor-sodium hydrosulfide (NaHS) on endothelin-1 (ET-1) and connective tissue growth factor (CTGF) expressions in rats with pulmonary hypertension induced by high pulmonary blood flow. METHODS: Thirty-two male SD rats were randomly divided into 4 groups: shunt group, shunt+NaHS group, sham group and sham+NaHS group. Rats in shunt group and shunt+NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. After 11 weeks of experiment, rat systolic pulmonary artery pressure (SPAP), lung tissue H2S, plasma ET-1 concentration and lung tissue ET-1mRNA expression, as well as pulmonary artery CTGF protein expression were detected.RESULTS: After 11 weeks of experiment, SPAP, lung tissue ET-1mRNA, plasma ET-1 as well as pulmonary artery CTGF expressions were increased markedly, respectively, whereas H2S in lung tissue decreased significantly in rats of shunt group as compared with that in sham group (all P<0.05). After administration of NaHS for 11 weeks, H2S in lung tissue increased significantly, whereas SPAP, plasma ET-1 and lung tissue ET-1 mRNA expression as well as pulmonary artery CTGF protein expression decreased significantly, respectively, in rats of shunt+NaHS group as compared with that in shunt group (all P<0.05).CONCLUSION: NaHS might be involved in the development of pulmonary hypertension induced by high pulmonary blood flow by down-regulating vasoactive peptides ET-1 and CTGF expressions in lung tissues of rats.     

Interventional effects of tongxinluo combined with atorvastatin and aspirin on adventitial inflammation in early stage of atherosclerosis

AIM: To investigate the effect of a treatment proposal, which consisted of tongxinluo, atorvastatin and aspirin, on adventitial inflammation of early atherosclerosis in rabbit carotid artery. METHODS: The atherosclerotic model was established in the rabbits with silicone collar, which was positioned around the carotid arterial adventitia+high-cholesterol diet. New Zealand rabbits (n=72) were randomly divided into 6 groups (n=12): control group, model group, tongxinluo group, atorvastatin group, aspirin group, and three-drug combination group. The rabbits in control group were fed with common foodstuffs, and the rabbits in all the other groups were fixed the right carotid arteries with the silicone tube, and were fed with fatty foodstuffs. The rabbits in tongxinluo group, atorvastatin group and aspirin group were given the suspension of tongxinluo supermicropowder (0.3 g·kg^-1·d^-1), atorvastatin (2.5 mg·kg^-1·d^-1) and aspirin (12 mg·kg^-1·d^-1) respectively,and the rabbits in three-drug combination group were given the suspension of tongxinluo supermicropowder (0.3 g·kg^-1·d^-1), atorvastatin (2.5 mg·kg^-1·d^-1) and aspirin (12 mg·kg^-1·d^-1) together. The rabbits in each group were fed with the corresponding medicines for 4 weeks. The tissue slices of carotid artery were observed under light microscope with HE staining. The change of blood lipid was detected by biochemical assay. The protein levels of MCP-1, IL-1β and IL-10 in the carotid arterial adventitia were detected by ELISA. The immunohistochemical staining was used to detect the protein expression of IL-8 around the carotid arterial adventitia.RESULTS: Compared with control group, the contents of TC, TG and LDL-C were significantly increased, and the content of IL-10 was significantly decreased in model group. The levels of TC, TG and LDL-C were significantly decreased in tongxinluo group and atorvastatin group compared with model group, no significant difference between tongxinluo group and atorvastatin group was observed. In the three-drug combination group, the levels of TC, TG and LDL-C were lower than those in atorvastatin group and tongxinluo group. Compared with control group, the contents of MCP-1 and IL-1β were significantly increased, and the content of IL-10 was significantly decreased in model group. Compared with model group, the contents of MCP-1 and IL-1β were decreased in tongxinluo group, atorvastatin group and aspirin group, no significant difference between the 3 groups was observed. The content of IL-10 was decreased in three-drug combination group, and the contents of TC, TG and LDL-C were lower than those in tongxinluo group, atorvastatin group and aspirin group. The content of IL-8 was decreased in tongxinluo group, atorvastatin group, aspirin group and three-drug combination group.CONCLUSION: The strategy of three-drug combination enhances the effect of regulating the lipid metabolism and inhibiting the adventitia inflammation. It plays an important role to intervene in the process of atherosclerosis.     

Effects of macrophage polarization during development of liver cirrhosis in rats

AIM: To explore the state of macrophage polarization and its relation with intestinal endotoxemia-endoplasmic reticulum stress in the development of liver cirrhosis induced by multiple pathogenic factors in rats. METHODS: The male SD rats (n=36) were randomly divided into normal control group and liver cirrhosis model group, and sacrificed at the end of the 4th, 6th and 8th weeks. The rat model of liver cirrhosis was induced by multiple pathogenic factors. The levels of alanine aminotransferase (ALT), endotoxin, homocysteine (Hcy) in the plasma, and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), arginase-1 (Arg-1) and interleukin-10 (IL-10) in the liver tissues were detected by ELISA. Histopathological change of the liver was observed under microscope with the staining of hematoxylin and eosin (HE) and van Gieson (VG). The expression of glucose-regulated protein 78 (Grp78), nuclear factor-kappa B (NF-κB), interferon-regulatory factor 5 (IRF5), CD86, CD206 and transforming growth factor-β1 (TGF-β1) at mRNA levels in the liver tissues were detected by the method of real-time fluorescence quantitative PCR.RESULTS: Compared with the corresponding normal control group, the levels of ALT, endotoxin, Hcy in the plasma and Grp78 mRNA in the liver tissues in liver cirrhosis model group were significantly and gradually increased (P<0.05). The mRNA expression of NF-κB, IRF5 and CD86, and the protein levels of iNOS, TNF-α and IL-6 in the liver tissues were significantly increased (P<0.05), and they successively increased from the 4th week to the 6th week and decreased reversely at the 8th week. The mRNA expression of CD206, TGF-β1, Arg-1 and IL-10 in the liver tissues were significantly increased from the 6th week to the 8th week (P<0.05), and no significant difference at the 4th week was observed. The level of endotoxin in the plasma was correlated with the mRNA expression of Grp78 in the liver tissues (P<0.01). Both endotoxin in the plasma and Grp78 mRNA in the liver tissues were correlated with the mRNA expression of CD86 and CD206 in the liver tissues (P<0.01).CONCLUSION: The pathway of liver damage-intestinal endotoxemia-endoplasmic reticulum stress-macrophage polarization may be critical in the pathogenesis of liver cirrhosis induced by multiple pathogenic factors.     

Construction of a New Zealand rabbit model of diabetic nephropathy

AIM: To establish a method to produce animal model of diabetic nephropathy (DN) in New Zealand rabbits using alloxan (ALX). METHODS: Twenty male New Zealand rabbits were randomly divided into 2 groups: 8 rabbits in control group were fed with conventional feed such as buffer solution;12 in diabetes milleuts (DM) group were fed with high-sugar and high-fat diet (conventional feed plus 5% sucrose, 5% pork fat and 1% cholesterol) for 2 weeks, then intravenous injection of ALX at 50 mg/kg was given, and another dosage of 100 mg/kg ALX was intravenously injected after 48 h. The levels of blood glucose, blood cholesterol, triglyceride, urine microalbumin, and serum creatinine were detected before and 12 weeks after the processes of modeling. After 12 weeks, the rabbits were sacrificed,and the kidneys were taken for pathological examination. RESULTS: The successful model of DN in the rabbits had the characteristics of high blood glucose, abundant urine microalbumin (P<0.01), and corresponding morphological and functional changes of the kidneys. CONCLUSION: Rabbits with high-sugar and high-fat diet plus twice intravenous injection of ALX at a total dose of 150 mg/kg (50 mg/kg and 100 mg/kg, respectively) shows low mortality and stable diabetes mellitus state. This method induces a typical DN model in 12 weeks.     

Role of homocysteine in tissue factor expression in human vascular smooth muscle cells

AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.     

Effects of Tongxinluo on activation of platelet in rabbit model of atherosclerosis

AIM: To study the effects of Tongxinluo on the activation of platelets in a rabbit model of atherosclerosis. METHODS: New Zealand rabbits were randomly divided into 7 groups: normal group, model group, the groups treated with high, medium and low doses of Tongxinluo micropowder (0.15, 0.3 and 0.6 g·kg^-1·d^-1), atorvastatin group (2.5 mg·kg^-1·d^-1), and aspirin group (12.5 mg·kg^-1·d^-1). The rabbits in normal group was fed with common diet for 12 weeks, and the rabbits in model group were fed with high-fat diet for 12 weeks to establish atherosclerosis model. The rabbits in the rest groups were treated with the corresponding drugs, at the same time to give high-fat diet. Fasting for 12 h after the last treatment, whole blood was collected to perform the blood routine test, and to measure serum and plasma levels of lipids, platelet factor 4 (PF4) and soluble CD62P (sCD62P). Flow cytometry was used to analyze platelet calcium ion concentration. Electron microscopy was used for platelet superfine observations, and light microscopy for observing the pathological changes. RESULTS: Compared with normal group, the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), platelet counts, and mean platelet volume in model group were significantly elevated, and the levels of PF4, sCD62P and calcium were also significantly increased (P < 0.05). Compared with model group, except aspirin group, the levels of TG, TC and LDL-C in high, medium and low doses of Tongxinluo groups and atorvastatin group were effectively decreased. The platelet counts and mean platelet volume in all treatment groups were markedly decreased, and the serum levels of PF4, sCD62P and Ca²⁺ in platelet (P < 0.05) were reduced. In electron microscopic observation, the shape of platelet was regular and organelles distributed uniform in normal group. However, in model group, the shape of platelet was irregular, pseudopodia forming was obviously observed, and α particles and dense granules decreased, indicating that the platelet was activated. To a different extent, the platelet shape, increase in the number of α particles and dense granules were improved in treatment groups and the damage of the cytoplasm was attenuated. Through histopathological observation, the intimal was smooth and complete in normal group. In the model group, the intimal thickness markedly increased, foam cell aggregated, and plaque was formed. Compared with model group, the intimal thickening and the number of foam cells were significantly decreased, and plaque formation was not obvious in atorvastatin group and high dose of Tongxinluo group. The pathological damages in the other treatment groups were alleviated in different degrees. CONCLUSION: Tongxinluo significantly inhibits the activation of platelets in the process of atherosclerosis, and has important clinical value to delay the atherosclerotic thrombosis.