Effects of ambroxol combined with low-dose heparin on production of ICAM-1 and E,P-selectin in acute lung injury

AIM: To study the production of intercellular adhesion molecule-1(ICAM-1), E-selectin and P-selectin in serum, lung tissues and bronchoalveolar lavage fluid(BALF)of acute lung injury(ALI) model and to observe the effects of ambroxol combined with low-dose heparin on the changes of the 3 factors above.METHODS: Twenty-four healthy rabbits were randomly divided into 3 groups: normal saline control group (NC), oleic acid injury group (OA), ambroxol+ heparin treatment group (AH). The rabbit ALI model was induced by oleic acid injection through auricular vein. Partial pressure of O₂ in artery(PaO₂) was analyzed.The concentrations of ICAM-1 and E-selectin were detected by ELISA.The apoptosis index(AI) was measured by TUNEL method.The expression of P-selectin was determined by immunohistochemical method.The ultrastructural changes of the lung tissues were observed under electron microscope, and the lung wet/dry ratio(W/D) was calculated.RESULTS: PaO₂ in AH group and OA group was significantly lower (P<0.01) than that in NC group, and PaO₂ in AH group was significantly higher than that in OA group (P<0.01). The concentrations of ICAM-1 and E-selectin in serum, lung tissues and BALF, and AI and W/D in lung tissues in AH group were higher (P<0.05 or P<0.01) than those in NC group, and was lower than those in OA group (P<0.05 or P<0.01). In NC group, no significant change of the above parameters at all time points was observed (P>0.05). In OA group, PaO₂ was significantly decreased (P<0.01) with the pathological process developed, and the concentrations of ICAM-1 and E-selectin were significantly increased. In AH group, PaO₂ was decreased (P<0.05),and the concentrations of ICAM-1 and E-selectin were increased with the process of ALI developed. The P-selectin expression in lung tissues of OA group was distributed mainly in inflammatory cells, capillary endothelial cells and plasma. From low to high levels, the order was NC group < AH group < OA group in the expression of P-selectin. The most obvious apoptosis was observed in OA group. No apoptosis or occasional positive cells were found in NC group. The apoptotic rate in AH group was significantly reduced compared with that in OA group.CONCLUSION: In ALI induced by OA, ICAM-1, E-selectin and P-selectin are significantly increased and are involved in the occurrence and development of ALI. Ambroxol combined with low-dose heparin reduces the levels of ICAM-1, E-selectin and P-selectin, the pulmonary edema and the lung injury, improves pulmonary functions, and plays an important role in the prevention and treatment of acute lung injury.     

Agkistrodon halys venom PCA improves hemorheology in rats with coronary artery microthrombosis

AIM: To explore the effect of protein C activator (PCA) from Wannan Agkistrodon halys venom on hemorheology in the rats with coronary artery microthrombosis(CAM). METHODS: Fifty SD rats were randomly divided into sham operation group, CAM group, low dose (0.5 mg/kg), medium dose (2 mg/kg ) and high dose (8 mg/kg) of PCA treatment groups. The model of CAM was induced by injecting sodium laurate at dose of 1.0 mg/kg with the concentration of 10 g/L from aortic root to left ventricle. Thrombelastogram was obtained by thromboelastography instrument system. The content of endothelin-1(ET-1) and P-selectin, and the activity of creatine kinase-MB (CK-MB),lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in plasma were determined by ELISA. The protein expression of myocardial P-selectin and TNF-α was detected by Western blotting. The structural changes of the myocardial cells and interstitial tissues were observed under microscope. RESULTS: Compared with CAM group, clotting time and clot formation time significantly prolonged in high-dose PCA group. The alpha angle, maximum amplitude, coagulation index, and the levels of ET-1, P-selectin, CK-MB, LDH and AST in plasma were decreased. The protein expression levels of P-selectin and TNF-α in myocardial tissues were down-regulated (P<0.05). Cardiac histological observation showed that coronary microthrombi in medium-dose and high-dose PCA groups were not found. CONCLUSION: Agkistrodon halys venom PCA inhibits the formation of coronary microthrombi, down-regulates the expression of P-selectin and TNF-α, improves the abnormal hemorheology after microthrombi, and effectively protects myocardial cells.     

Elevated levels of serum IL-6, ICAM-1 and P-selectin in stable survivors with liver transplantation

AIM: To study the profile of serum IL-6， ICAM-1 and P-selectin in stable survivors with clinical liver transplantation (LTx). METHODS: Flow cytometric analysis was used to determine the phenotype of T cell subsets in peripheral blood mononuclear cells （PBMCs） from stable survivors with liver transplantation (n=22), and healthy volunteers (n=12). Serum levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF-α) and interleukin (IL-6), intercellular adhesion molecules (ICAM-1) and P-selectin in stable survivors with liver transplantation and healthy volunteers were assessed by enzyme-linked immunoabsordent assay (ELISA). Recently performed 6 cases of liver transplantation were also dynamically observed in this study. RESULTS: Percentage of CD4+ T cells， CD8+ T cells and CD3+ T cells, as well as ratio of CD4 to CD8 were no difference between two groups (P>0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group (P<0.05). Significantly increased concentrations of IL-6, ICAM-1 and P-selectin were found in stable liver transplantation group as compared to healthy group (P<0.05). A high TNF-α level was detected in stable liver transplantation group while no significant difference was found as compared to healthy volunteers group (P>0.05). There was not found no regular change of serum cytokines (IL-6, TNF-α) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patients during post-operation from day 1 to day 30, indicating that was associated with the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involved in the repair of the injury induced by TNF-α in allo-transplanted livers.     

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.     

Effect of metronomic cyclophosphamide combined with recombinant human endostatin on a nude mouse xenograft model of non-small-cell lung cancer

AIM：To investigate the effects of combination of metronomic (MET) cyclophosphamide (CPA) with recombinant human endostatin (Endostar) as maintenance therapy on treating non-small-cell lung cancer (NSCLC). METHODS：The lung adenocarcinoma model of BALB/c nude mice was established by subcutaneous inoculation of A549 cells. Following 4 circles of CPA chemotherapy at maximum tolerated dose (MTD), the tumor-bearing mice were randomly divided into 4 groups：the mice in control group were treated with saline, the mice in MET CPA group were treated with CPA, the mice in Endo group were treated with Endostar, and the mice in MET CPA+Endo group were treated with CPA+Endostar. The volume of xenograft tumors and the survival rate of the mice were recorded. The circulating endothelial cells (CECs) and viable CECs in peripheral blood of tumor-bearing mice were detected by flow cytometry. The microvessel density (MVD) and pericyte coverage were observed by confocal microscopy. RESULTS： At the 6th week of maintenance therapy, the tumor volume in both MET CPA group and Endo group was significantly smaller than that in control group, and the highest inhibitory effect was observed in MET CPA+Endo group. The survival time of the mice in both MET CPA group and Endo group was significantly longer than that in control group, and the mice in MET CPA+Endo group showed the longest survival time. Compared with control group, MET CPA or Endostar significantly reduced the total number and viable CECs in peripheral blood of tumor-bearing mice and the MVD in the xenograft tumors. Endostar also considerably reduced pericyte coverage in xenograft tumors. MET CPA combined with Endostar even more greatly inhibited the angiogenesis-related indicators mentioned above. CONCLUSION：Combination of MET CPA with Endostar shows effective anti-tumor activity and leads to improved survival in a xenograft model of lung adenocarcinoma, which might be partly attributed to the enhanced anti-angiogenic effect of the combination therapy.     

Effects of Xijiao Dihuang and Yinqiao San decoction on ICAM-1 and VCAM-1 expression in mouse lung tissues and rat pulmonary microvascular endothelial cells infected with influenza virus

AIM：To investigate the effects of Xijiao Dihuang and Yinqiao San decoction (XDY) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in mouse lung tissues and rat pulmonary microvascular endothelial cells (RPMVECs) infected with influenza virus, and to explore its mechanism for treatment of viral pneumonia. METHODS：Fifty-four male BALB/c mice were randomly divided into normal group, model group and XDY group (n=18 in each group). The viral pneumonia model was established by intranasally dripping influenza A (H1N1) virus into the mice. The mice in XDY group were treated with XDY 1 h after dripping the virus. The expression of ICAM-1 and VCAM-1 in lung tissues was examined by immunohistochemical staining 2, 4 and 6 d after infection. On the other hand, RPMVECs were obtained from male Wistar rats and primarily cultured. The cells were randomly divided into control group, virus group, virus+XDY group, tumor necrosis factor α (TNF-α) group and TNF-α+XDY group. The mRNA and protein expression of ICAM-1 and VCAM-1 was evaluated by real-time PCR and flow cytometry 24 h after infection. RESULTS：Virus exposure increased ICAM-1 and VCAM-1 expression in mouse lung tissues (P<0.01), and XDY treatment attenuated this effect (P<0.01). Virus and TNF-α both led to the increases in mRNA and protein expression of ICAM-1 and VCAM-1 in RPMVECs (P<0.01), which were also reduced by treatment with XDY (P<0.01). CONCLUSION：Treatment with XDY decreases virus-induced ICAM-1 and VCAM-1 expression, suggesting an important role of XDY in treatment of viral pneumonia.     

Effect of mininally modified low density lipoprotein on the adhesive function of vascular endothelial cell and its mechanism

AIM: To observe the effect of MM-LDL (minimally modified LDL) on the interaction between human umbilical vein endothelial cell (HUVEC) and U937 monocyte-like cell line and the exporession of vascular cell adhesion-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), P-selectin.METHODS:The adhesive percentage between HUVEC treated with MM-LDL and U937 was determined by counting and the expression of VCAM-1, ICAM-1, P-selectin were examined by ELISA. RESULTS: Treatment of HUVEC with MM-LDL (75 mg/L) for 4 hours significantly increased adhesion of U937 to HUVEC ( P <0.01) and did not induce the surface expression of VCAM-1, ICAM-1, P-selectin . Recombination tumor necrosis factor-alpha (rTNF_α) 5.0 μg/L, as a positive control, induced the expression of these adhesion molecules ( P <0.05). Prolonged (18 h) exposure to MM-LDL resulted in the expression of P-selectin, but not VCAM-1.CONCLUSION: the adhesion of monocytes to endothelial cells induced by MM-LDL is not mediated by VCAM-1, ICAM-1. P-selectin induction may be partly involved in the process.     

Transwell contact co-culture promotes growth and differentiation of single-dissociated iPSCs

AIM：To investigate the promoting role of Transwell contact co-culture system in the growth and differentiation of single-dissociated induced pluripotent stem cells (iPSCs). METHODS：Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃ and 5% CO2 for 8 h. Accutase digestion and 40 μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs, and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks. The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction (qPCR), immunofluorescence staining, live & dead cell staining and alkaline phosphatase (ALP) staining. The group of iPSCs cultured in conventional medium was used as control group 1. The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2. RESULTS：The bovine CECs showed typical hexagonal cobblestone shape. iPSCs showed colony-like growth, while became single-dissociated cells after Transwell contact co-culture with bovine CECs for 3 d. The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4. The mRNA expression levels of Nanog, Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05). The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01). After 14 d of induced differentiation co-culture, the single-dissociated iPSCs showed rather uniform polygonal morphology, increased dimension and no obvious colony existence. Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed. The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased, and had statistically significant difference compared with control group 1 (P<0.01). CONCLUSION：When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs. Transwell contact co-culture system not only enhances the growth of single-dissociated iPSCs, but also promotes their differentiation.     

Effect of N,N-dimethylsphingosine on the expression of adhesion molecules in human umbilical vein endothelial cell line EA.hy926

AIM: To investigate whether and how N, N-dimethylsphingosine (DMS) plays a role in modulating the adhesion of monocytes to vascular endothelial cells, and identify whether human umbilical vein endothelial cell line EA.hy926 take place of the vascular endothelial cells.METHODS: Adhesion ratio was measured by flow cytometry, and immunohistochemistry was used to detect the expression of ICAM-1 and P-selectin in HUVEC: EA.hy926 cells after the effect of DMS. RESULTS: DMS inhibited the adhesion of monocytes to HUVEC: EA.hy926 cells in a time-dependent and concentration-dependent manner by reducing the expression of ICAM-1 and P-selectin. CONCLUSIONS: DMS reduced adhesion molecule expression in vascular endothelial cells. DMS may be an important contributor to reduce adhesion ratio, suggesting that DMS plays a negative role in proinflammatory and immune functions of the modified vascular endothelial cells during atherosclerosis and restenosis.     

Effects of dehydroepiandrosterone on expression of ICAM-1 in rabbit aorta and HUVECs induced by high lipid levels

AIM: To investigate the effects of dehydroepiandrosterone (DHEA) on the expression of intercellular adhesion molecule-1 (ICAM-1) induced by high lipid levels in rabbit aorta and human umbilical venous endothelial cells (HUVECs), and the effects of all-trans retinoic acid (ATRA) in this process.METHODS: For in vitro experiments, the cultured HUVEC were divided into control group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+DHEA group, ox-LDL+DHEA+ATRA group and DHEA group. The HUVECs in all groups were treated with the corresponding reagents for 24 h. The expression of ICAM-1 at mRNA and protein levels in all groups were determined by RT-PCR and ELISA, respectively. For in vivo experiments, the rabbits were divided into control group, high lipid group, high lipid+DHEA group, high lipid+DHEA+ATRA group and DHEA group. The rabbits in all groups were fed with the corresponding diets for 10 weeks. The expression of ICAM-1 in the rabbit aorta at mRNA and protein levels was determined by RT-PCR and immunohistochemistry. RESULTS: The expression of ICAM-1 in the HUVECs in ox-LDL group was significantly increased compared with control group (P<0.05). Compared with ox-LDL group, the expression of ICAM-1 in ox-LDL+DHEA group was obviously decreased (P<0.05). The expression of ICAM-1 was similar in both control group and DHEA group (P>0.05). The expression of ICAM-1 was similar in both ox-LDL+DHEA group and ox-LDL+DHEA+ATRA group (P>0.05). The expression of ICAM-1 in the rabbit aorta in high lipid group was significantly increased compared with control group (P<0.05). Compared with high lipid group, the expression of ICAM-1 in high lipid+DHEA group was obviously decreased (P<0.05). No remarkable difference in the expression of ICAM-1 between control group and DHEA group was observed (P>0.05), so did between high lipid+DHEA group and high lipid+DHEA+ATRA group (P>0.05). CONCLUSION: DHEA inhibits high lipid-induced ICAM-1 expression in rabbit aorta and HUVECs. That may be one of the mechanisms of antiatherosclerotic effect of DHEA. ATRA seems no positive effect on DHEA function.     

Alteration of circulating endothelial cells from acute promyelocytic leukemia patients before and after treatment and its influential factors

AIM: To determine the biological feature of circulating endothelial cells (CECs) in acute promyelocytic leukemia (APL) patients before and after treatment, and to analyze the relationship between CECs and the clinical characteristics. METHODS: The CECs were sorted from peripheral blood by magnetic-activated cell sorting and then counted by 3-color flow cytometry. The cells were identified by immunofluorescence staining for the expression of CD146, CD31, CD144, VEGFR-2, CD45 and CD133. The CECs were cultured in vitro, and the tube formation and colony-forming rate were determined. RESULTS: Increased quantity of CECs was observed in CD34 positive group and group with WBC>10×10⁹/L (P<0.05). The quantity of CECs had a significant difference among low risk, medium risk and high risk groups (P<0.05). The positive rate of CD133 and quantity of CECs significantly reduced in 32 APL patients when they gain complete remission after treatment (P<0.05). The amount of tube formation and colony-forming rate were significantly reduced after treatment (P<0.05). The ratio of CECs quantity from APL patients after treatment to that before treatment had a negative correlation with arsenic concentration in urine on day 7 during As₂O₃ treatment (P<0.05). CONCLUSION: Accurately counting CECs may be helpful for evaluating prognosis and designing treatment strategy.     

Effect of Shenshuguanxin granula on coronary circulation in rats with myocardial infarction

AIM：To explore the effect of traditional Chinese medicine Shenshuguanxin granula on coronary circulation in a rat model of myocardial infarction (MI). METHODS：SD rats (n=50, SPF grade) were randomly divided into 5 groups (n=10):sham group, MI group, and high-dose, middle-dose and low-dose Shenshuguanxin granula treatment groups. The rat MI model was established by ligation of the coronary artery. The cardiac markers, small and medium-sized blood vessels ［microvessel count (MVC) value］ in the infarct zone, and platelet endothelial cell adhesion mo-lecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) expression in the infarct border zone were measured. RESULTS：After 4 weeks of coronary artery ligation, the significant increases in MVC in the infarct zone, and the expression of PECAM-1 and VEGF in the infarct border zone were detected compared with sham group (P<0.05). The differences of cardiac markers between MI group and other groups were insignificant (P>0.05). CONCLUSION:Shenshuguanxin granula improves coronary circulation in the rats with myocardial infarction by increasing the expression of PECAM-1 and VEGF, and promoting small and medium-sized angiogenesis.     

Inhibitory effects of local transfection of vascular endothelial growth factor 165 gene on intimal hyperplasia of artery in rabbits after operation injury

AIM: To observe the effects of local transfection of vascular endothelial growth factor 165 (VEGF165) gene on inhibiting intimal hyperplasia and restenosis of artery in rabbits after operation injury, and its possible mechanisms. METHODS: Microsurgery injury was used to establish the intimal injury model of right external iliac artery in rabbits. 105 male New Zealand rabbits were randomly divided into 3 groups (35 rabbits in each group). Group A was physiological saline control group, group B was pBudCE4.1-transfected group, group C was pBudCE4.1/VEGF165-transfected group. The physiological saline, pBudCE4.1 and pBudCE4.1/VEGF165 transfection solutions were injected into injured vessel walls of above-mentioned groups. The injured vascular specimen was harvested for pathologic examination, electric microscope observation, RT-PCR examining and immunohistochemical staining. RESULTS: Rabbit intimal thickness and area of vessel walls in group C at every time point after operation were significantly less than those in group A and group B (P<0.01). The stenosis ratio of vessels in group C at 28 days after operation decreased by 51.6% and 49.8%, respectively, as compared with groups A and B. The expression of VEGF165 mRNA and VEGF165 positive cells in Group C were increased significantly than those in group A and B at every time point after operation (P<0.01). CONCLUSION: Local transfection of VEGF165 gene restrains intimal hyperplasia and restenosis of vessels, which lays a foundation for future gene therapy of vascular intimal hyperplasia.     

Irbesartan suppresses expression of VCAM-1 and ICAM-1 in myocardial ischemia-reperfusion areas in rabbits

AIM: To investigate the effects of irbesartan on acute myocardial ischemia-reperfusion injury and on expression of ICAM-1 and VCAM-1 in myocardial ischemia-reperfusion areas in rabbits. METHODS: Anterior descending branch of left coronary artery (LAD) was ligated in ischemia-reperfusion (IR) group but not in the sham group. Rabbits of IR group were subjected to 60 min of LAD occlusion, and 360 min of reperfusion. In the treated group, the rabbits were given irbesartan until the trial ended. Then, all rabbits were killed and tissue samples were removed from IR areas. After disposed with routine histological methods and stained with hematoxylin and eosin (HE), these samples were examined by light microscopy. The expression of ICAM-1 and VCAM-1 were also investigated by employing immunohistochemical SP method in the tissue samples. RESULTS: Degree of damage in cardiocytes and of neutrophil infiltration were more serious in IR group than those in sham group (P<0.01). Expression of ICAM-1 and VCAM-1 in IR areas of the treated group was much less up-regulation than that in IR group (P<0.01). CONCLUSION: The expression of ICAM-1 and VCAM-1 in I/R areas is up-regulated in the IR group. Irbesartan effectively alleviates myocardial IR injury in rabbits.     

Effect of exogenous HGF gene transfection on pulmonary perfusion and pressure in pulmonary artery hypertension in rabbits

AIM: To investigate the feasibility of exogenous hepatocyte growth factor (HGF) gene transfection to promote pulmonary collateral angiogenesis, improve pulmonary perfusion and reduce pulmonary artery pressure in the rabbit model of pulmonary artery hypertension (PAH). METHODS: The model rabbits of PAH were randomly divided into control group, empty vector group and HGF gene transfection group. The rabbits in HGF gene transfection group were transfected with Ad-HGF via intratracheal instillation. Pulmonary hemodynamic indicators were monitored in the 4th week after HGF gene transfection. Density of pulmonary vessels was examined with double-labeling immunofluorescence (endothelial cells were labeled with anti-FⅧ and vascular smooth muscle cells were marked with anti-α-SMA). Double-labeling immunofluorescence of FITC-lectin and anti-α-SMA was also performed to evaluate the pulmonary blood perfusion. RESULTS: Four weeks after transfection, the density of pulmonary arterioles of the rabbits in HGF gene transfection group was higher than that in control group and empty vector group (P<0.05), which was confirmed by double-labeling immunofluorescence. Pulmonary blood perfusion in HGF group was significantly increased compared with that in the other two groups, in which pulmonary arterial stenosis and occlusion were observed. The mean pulmonary artery pressure in HGF transfection group was much lower than that in control group and empty vector group (P<0.05). CONCLUSION: Four weeks after intratracheal adenoviral-mediated HGF gene transfection, pulmonary collateral vessels and pulmonary perfusion increase, and the pulmonary artery pressure is effectively reduced.     

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.     

Effects of perindopril at different doses on cardiac function and ACE2/Ang-(1-9)/Ang-(1-7) axis of ischemic cardiac dysfunction rabbits

AIM: To investigate the different dose of perindopril on cardiac function in the rabbits with ischemic cardiac dysfunction. METHODS: Male rabbits weighing 2.5~3.0 kg(n=30) were randomly divided into 3 groups(n=10):high dose perindopril group(HD group), low dose perindopril group(LD group) and cardiac dysfunction group(CD group). The Left anterior descending coronary artery of the rabbits was ligatured for model preparation. In HD group, the rabbits were treated with perindopril split normal saline solution(1 g/L)2 mL·kg^-1·d^-1. In LD group, the rabbits were treated with perindopril split normal saline solution(0.33 g/L)2 mL·kg^-1·d^-1. In CD group, the rabbits were treated with normal saline solution 2 mL·kg^-1·d^-1. Four weeks after treatment, the cardiac function was measured via echocardiography, the mRNA expression of angiotensin-converting enzyme 2(ACE2) and angiotensin type 2 receptor(AT2R) was analyzed by real-time PCR, serum angiotensin(Ang)-(1-9) and Ang-(1-7) levels were detected by ELISA. RESULTS: Compared with CD group, the cardiac function of the 2 groups treated with perindopril was significantly improved(P<0.01), and more improvement in HD group was observed than LD group(P<0.05). The serum angiotensin(Ang)-(1-9) and Ang-(1-7) level and the mRNA expression of ACE2 and AT2R in the 2 groups treated with perindopril were significantly improved(P<0.01). Compared with LD group, the mRNA expression of ACE2 and AT2R and the serum levels of Ang-(1-9) in HD group were significant improved(P<0.05), while no difference of serum Ang-(1-7) level was observed. Correlation analysis revealed that the improvement of the cardiac function was associated with serum Ang-(1-9) level, mRNA expression of ACE2 and AT2R(P<0.01), but has no significant correlation with serum Ang-(1-7) level. CONCLUSION: High dose of perindopril may improve more cardiac function in ischemic cardiac dysfunction model in rabbits. The mechanism may relate to increasing serum Ang-(1-7) level to activate AT2R.     

Prevention and treatment of restenosis after PTA operation in rabbit artery by 3,3-diindolylmethane

AIM: To evaluate the safety and efficacy of 3,3-diindolylmethane (DIM) on neointimal proliferation of rabbit artery after balloon angioplasty. METHODS: Restenosis models of carotid artery after balloon injury was established in rabbits. 30 rabbits were randomly divided into 4 groups: sham-operated group, model group, low-dose DIM group and high-dose DIM group. DIM was given to the rabbits in low-dose treatment group (2 mg·kg-1·d-1) and high-dose treatment group (8 mg·kg-1·d-1) once a day from 3 d before operation to 4 weeks after operation. The two treatment groups were administered with intraperitoneal injection of emulsified DIM, while the other two groups with saline at the same volume. All rabbits were killed after 28 d and carotid arteries were removed. With HE staining, automatic image analysis and immunohistochemistry, artery morphology was observed and the thickness of arterial intima and media was measured. Platelet derived growth factor (PDGF) and transfer growth factor β1 (TGF-β1) were examined. The expression of 〖STBX〗bcl-2〖STBZ〗 gene was detected by in situ hybridization (ISH) with computer-assisted picture analysis system. RESULTS: No significant difference was found between low-dose DIM group and model group in intimal thickness, media thickness, luminal area and the expression of PDGF and TGF-β1 in low-dose DIM group (P>0.05). The intimal thickness was decreased in high-dose DIM group compared with model group and low-dose DIM group (P<0.01). The luminal area was significantly larger in high-dose DIM group than that in model group and low-dose DIM group (P<0.01). The expression of PDGF and TGF-β1 in low-dose DIM group was significantly reduced compared with model group and low-dose DIM group (P<0.01). CONCLUSION: The inhibition and treatment of DIM on arterial restenosis are safe and effective. The effect might be achieved by preventing neointimal proliferation and the expression of PDGF and TGF, enhancing apoptosis in the vascular smooth muscle cells.     

Pathophysiologic changes of local ischemic coronary artery and cardiac muscle after ligating canine LAD

AIM: To evaluate the reliability of making a research model of coronary artery stenosis and local myocardial infarction reproduced in dog by ligating canine LAD. METHODS: We disparted 30 aged healthy cross-breed dogs ［(18.5±6.7) kg］ into three groups. The near part of the LAD through left minimal thoracic incision was ligated to interdict 25% (group A), 50% (group B), 75% (group C) of the flux, respectively. The changes of plasma endothelium-derived factors NO, ET-1, sP-selectin and CTnT were measured before ligation and at different time points after ligation. The expression of P-selectin gene in cardiac muscle was detected by Western blotting. The segments of distal parts of the ligated LAD were cut and pathological changes of the patches of topical cardiac muscle were observed by electronic microscope. RESULTS: After ligation, NO/ET-1, P-selectin and CTnT had significant changes in group B (P<0.05) and group C (P<0.01). The expression of P-selectin of cardiac muscle was highly up-regulated after ligating in B (50%) and C (75%) group, In C group animals, a typical far more intense expression pattern was found. Under electronic microscope, the endothelium and other structures of the LAD wall and ultrastructure of myocardial cells had obvious changes in later two groups, especially in group C. There were a typical stenosis of LAD and myocardial infarction. CONCLUSIONS: Ligating the LAD 75% severely damages the endothelial cell and cardiac muscle cells of local ischemic vessel and cardiac muscle, thus forms the typical local stenosis of coronary artery and myocardial infarction, such method is a safe and reasonable way for making a disease model for studying CABG in surgery.     

Effect of puerarin on expression of Fas/FasL mRNA in lung tissue with pulmonary injury induced by ischemia-reperfusion in rabbits

AIM：To investigate the effect of puerarin (Pur) on expression of Fas/FasL mRNA in lung tissue during pulmonary ischemia and reperfusion injury (PIRI) in rabbits.METHODS：Single lung ischemia and reperfusion animal model was used.The rabbits were randomly divided into three groups,sham operated group (sham,n=10),PIR group (I-R,n=30) and PIR+ Pur group (Pur,n=30).Changes of several parameters included apoptotic index (AI),wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured at 60,180 and 300 minutes after reperfusion in lung tissue.Meanwhile,the location and expression of Fas/FasL mRNA were observed.Lung tissue was prepared for light microscopic and electron microscopic observation at 60,180,300 minutes after reperfusion.RESULTS：As compared with group I-R,Fas/FasL mRNA slightly expressed in intima and extima of small pulmonary artery,alveoli,and bronchiole epithelia in group Pur.The values of AI,W/D and IQA showed significantly lower than that in group I-R at 60,180,300 minutes after reperfusion in lung tissue (P<0.01 and P<0.05).Meanwhile,abnormal changes of the lung tissue in morphologically were lessen markedly in group Pur.CONCLUSION：Puerarin produces a notable protective effects on PIRI in rabbits by inhibiting Fas/FasL mRNA expression and decreasing apoptosis.