Effect of autologous bone marrow mononuclear cell local transplantation on peripheral neovascularization

AIM: To investigate the effect of autologous bone marrow mononuclear cells (BM-MNCs) local transplantation (BM-LT) on a rabbit model of hindlimb ischemia. METHODS: After unilateral femoral artery and its branches were surgically ligated and excised in rabbits (n=14) 1 hour or so, autologous BM-MNCs were injected into the ischemic skeletal muscles in 7 rabbits, and phosphate buffered saline for another 7 ones as control. Hemodynamics parameters were measured at day 0, day 7 and day 28, respectively. On day 28, the animals were sacrificed in groups for pathological analysis. RESULTS: Compared with control group, color Doppler flow image in BM-LT group was recovered significantly, and its' systolic peak velocity and end-diastolic velocity were also improved (P<0.05). Resistance index and flow volume were significantly improved on day 28 (P<0.05). A higher capillary density was detected in BM-LT group than that in control group (P<0.01). Ratio of the number of capillary and the number of myocyte fiber were significant difference (P＜0.05). CONCLUSION: BM-LT improves peripheral neovascularization. It might provide a new therapeutic method for ischemic peripheral vascular disease.     

Intravenous transfusion of endothelial progenitor cells reduces neointima formation

AIM: To investigate the feasibility of transplanting endothelial progenitor cells (EPCs) obtained from spleen in vascular endothelium repairmen after vascular injury. METHODS: EPCs were isolated by using a Ficoll density gradient centrifugation, and were cultured in plate. The endothelial characteristics of EPCs were identified by immunochemical staining and fluorescent labeling. Dil-Ac-LDL labeled spleen-derived EPCs were transplanted into the rats by intravenous injection directly after induction of arterial injury and again 24 hours later. Rats received FITC-labeled lectin intravenously before euthanasia. The distribution of fluorescent labeled EPCs was traced. The morphology of arterial intima and media was studied by optical microscopy and image analysing system. RESULTS: The adherent cells were considered EPCs that showed spindle shape and form blood-siland-like structures during development. The adherent cells had many endothelial characteristics. Fluorescent labeling showed that the intravenously injected EPCs specifically restricted to the vascular injury site, and lectin binding confirmed the endothelial phenotype. The ratio of neointimal/media area in EPCs transplantation group was obviously reduced than that in injury group and M199 group (0.82±0.09 vs 1.52±0.21, 1.48±0.19, P<0.01). The PCNA positive expression cells were evidently decreased compared with injury group and M199 group (19.25±3.96 vs 31.42±5.23, 29.37±3.16, P<0.05). CONCLUSION: EPCs incorporate into the process of injured carotid reendothelialization. EPCs transplantation induces an increase in the circulating EPCs, accelerates the process of endothelial repairmen and reduces neointima formation.     

Effects of pentoxifylline on ventricular remodeling and cardiac function of dilated cardiomyopathy rats

AIM: To explore the effects of pentoxifylline (PTX) on ventricular remodeling and cardiac function in dilated cardiomyopathy (DCM) rats.METHODS: Lewis rats were randomly allocated to a myocin-induced dilated cardiomyopathy (DCM) group receiving saline (n=10), a DCM group receiving PTX (PTX group; 25 mg·kg^-1·d^-1, ip, for 30 days, n=10) or healthy control group (n=10). The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10 in the blood plasma were analyzed by ELISA. The extent of fibrosis was estimated using Massons staining and immunohistochemistry analyses. Cardiac structure and function were measured by echocardiography.RESULTS: PTX decreased plasma levels of TNF-α and IL-6, and increased IL-10 level in DCM animals compared with DCM group ［TNF-α: (7.21±0.24) μg/L vs (19.30±1.31) μg/L, P<0.01; IL-6: (119.60±36.58) ng/L vs (189.50±13.25) ng/L, P<0.05; IL-10: (41.26±3.27) μg/L vs (32.45±4.32) μg/L, P<0.05］. Collagen volume fraction (CVF), perivascular collagen area (PVCA) and collagen Ⅰ/Ⅲ ratio were lower in PTX group than those in DCM group ［CVF: (16.45±3.01)% vs (23.33±4.43)%, P<0.05; PVCA: 4.58±2.10 vs 13.74±4.29, P<0.05; Ⅰ/Ⅲ ratio: 2.84±0.67 vs 4.22±0.54, P<0.01］. Left ventricular end-diastolic dimension reduced ［(6.11±0.51) mm vs (6.46±0.28) mm, P<0.05］ and left ventricular ejection fraction elevated ［(77.29±5.20)% vs (62.73±10.11)%, P<0.01］ by PTX compared with DCM.CONCLUSION: PTX modulates plasma levels of inflammatory cytokines, delays the ventricle remodeling and improves the heart function in DCM rats.     

Effect of mycophenolic acid on T lymphocyte proliferation and cytokine expression in vitro

AIM: To investigate the effect of mycophenolic acid (MPA) alone or in combination with anti B7-1 mAb on proliferation of T lymphocytes and the possible mechanisms. METHODS: The proliferation of T lymphocytes was detected by BrdU incorporation method. The expressions of IL-2, IFN-γ and IL-10 in mRNA and protein levels were detected by RT-PCR and ELISA, respectively. RESULTS: (1) MPA markedly inhibited the T lymphocyte proliferation as compared with control (P<0.01). (2) MPA significantly inhibited the levels of IL-2 and IFN-γ (42.73 ng/L±14.64 ng/L vs 99.70 ng/L±9.15 ng/L, P<0.01; 7.87 ng/L±4.22 ng/L vs 82.42 ng/L±25.55 ng/L, P<0.05), and significantly increased content of IL-10 compared with control (770.95 ng/L±126.85 ng/L vs 545.71 ng/L±22.45 ng/L, P<0.05). MPA in combination with anti B7-1 mAb obviously enhanced the content of IL-10 compared with MPA alone (941.90 ng/L±56.61 ng/L vs 770.95 ng/L±126.85 ng/L, P<0.05). (3) The expression levels of IL-2 and IFN-γ mRNA in the MPA group were obviously lower than those in control (0.74±0.10 vs 1.17±0.15, 0.52±0.05 vs 0.75±0.12, P<0.01). MPA in combination with anti-B7-1 mAb showed a statistically significant increase in IL-10 mRNA expression (1.28±0.06 vs 0.84±0.09, P<0.01) as compared with MPA alone. CONCLUSION: MPA induces the changes of cytokine expressive spectrum and the Th1 and Th2 shift might be involved in the immunosuppressive effect. The combination of MPA with anti B7-1 mAb might have a synergic effect.     

Effects of spermine on myocardial ischemia/ reperfusion injury in rats

AIM: To observe the effect of exogenous spermine (low concentration) on myocardial ischemia/reperfusion injury in rats.METHODS: 40 Wistar rats were randomly divided into 4 groups: sham- operation group (Sham), ischemic reperfusion group (I/R), spermine group (Sp) and natural saline group (NS). The model of ischemic/reperfusion injury was established by ligating rat coronary artery. In Sp group, spermine (0.5 mmol/L, 2 mL/kg) was injected slowly into rat vein. During the process, we recorded the electrocardiogram and the LV functional parameters, assayed the levels of SOD, LDH, NO and MDA in serum, and examined the ultrastructure of the myocardium. RESULTS: In I/R group, the incidence of arrhythmia was 90%, myocardial ultrastructure was injured seriously, values of LVSP and ±dp/dtmax decreased, levels of LDH, NO and MDA increased while SOD activity decreased (P<0.05 or P<0.01, compared with Sham group). Compared with I/R and NS group, all those indexes in Sp group changed significantly (P<0.05 or P<0.01). CONCLUSION: Exogenous spermine alleviates myocardial ischemia/ reperfusion injury in rats. The mechanism may be related to its antioxidant effect and relieving the injury caused by oxygen free radical.     

Effects of metformin on nuclear factor-κB expression and serum high sensitivity C-reactive protein level in atherosclerosis model of rabbit

AIM: To investigate the effects of metformin on nuclear factor-κB (NF-κB),its inhibitor IκB,and the level of serum high sensitivity C-reactive protein (hs-CRP) in rabbits.METHODS: 24 New Zealand male rabbits were randomly divided into control group,atherosclerosis (AS) group and metformin (Met) group.AS group and Met group were made as models by cholesterolenriched diets feeding and vascular intimal immunologic injury.The AS model was confirmed by high frequency ultrasound.Met group were given metformin 150 mg·kg-1·d-1 for 8 weeks.At the end of experiment,serum hs-CRP and serum lipids in all three groups were detected.Immunohistochemistry and Western blotting technique were applied to detect the expression of nucleus NF-κB p65 and cytoplasma IκBα in aorta in all three groups.RESULTS: Compared to normal control group,the level of serum hs-CRP was elevated (1.27±0.43 vs 3.96±0.63,P<0.01),the expression of nucleus NF-κB p65 increased significantly (P<0.01) while the expression of IκBα reduced significantly (P<0.01).Compared to AS group,metformin significantly reduced the level of serum hs-CRP (2.79±0.40 vs 3.96±0.63,P<0.05) and the expression of nucleus NF-κB p65 (P<0.01),and increased the expression of IκBα (P<0.05).CONCLUSION: Metformin inhibits the activation of NF-κB p65 and the degradation of IκBα,and decreases the levels of serum hs-CRP in AS rabbits.These results suggest that metformin exerts direct vascular anti-inflammatory effects.It may be one important mechanism of metformins antiatherogenic properties.     

Comparative analysis of cultured endothelial progenitor cells in vitro from PBMCs and enriched CD133+ cells

AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+ cells in CD133+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133+ group on 7 d (P<0.01). Compared to CD133+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that its not a preferential way to obtain EPCs for clinic therapy.     

Effects of homocysteine on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether homocysteine (Hcy) has influences on endothelial progenitor cell (EPCs) number and activity from peripheral blood. METHODS: Total mononuclear cells (MNCs) were plated on fibronectin-coated culture dishes and cultured for 7 days, and then attached cells were stimulated with Hcy or vehicle control for 6 h, 12 h, 24 h and 48 h. The adhesion, proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed, respectively. RESULTS: Incubation of isolated human MNCs with Hcy dose and time-dependently decreased the number of EPCs with maximum at 200 μmol/L for 24 hours (35.7±6.7 vs 62.5±10.6， P＜0.01). In addition, Hcy impaired EPC proliferative (0.531±0.061 vs 0.328±0.055， P＜0.05), migratory (26.3±6.4 vs 6.4±3.7， P＜0.01), adhesive (33.1±8.1 vs 17.4±7.5， P＜0.01) and vasculogenesis capacity (25.4±9.1 vs 10.4±4.7， P＜0.01) in a dose and time-dependent manner. CONCLUSION: It is suggested that Hcy may result in the reduction of EPCs and decrease EPC functional activity.     

Hepatocyte protection of ethyl pyruvate in septic mice

AIM:To study the protective effect of ethyl pyruvate (EP) on hepatocytes in septic mice. METHODS:The cecal ligation-perforation was made in mice as septic model. Ringers ethyl pyruvate solution (REPS) and Ringers lactic solution (RLS) were used to resuscitate septic mice. Anti-oxidative capacity of hepatic tissue and liver function were detected in different groups. RESULTS:Anti-oxidative capacity in septic mice was significantly lower than that in sham group (P<0.01). EP promoted the anti-oxidative capacity of hepatic tissue in septic mice. Malondialdehyde level was lower in REPS group than that in RLS group ［(48.18±5.98) μmol·g-1 protein vs (78.34±11.16) μmol·g-1 protein］, superoxide dismutase ［(5.19±1.41)103 U/g protein vs (3.20±1.08)103 U/g protein］ and total anti-oxidative capacity ［(7.02±1.79)103 U/g protein vs (4.77±1.35)103 U/g protein］ level were higher in REPS group than those in RLS group (P<0.01). Alanine aminotransferase in REPS group were lower than that in RLS group ［(210.06±23.36) U vs (458.86±51.55) U, P<0.01］. CONCLUSION:Ethyl pyruvate is an effective anti-oxidant in septic mice, which significantly increases the anti-oxidative capacity in hepatic tissue and ameliorates liver function.     

Premature response to luteinizing hormone of granulosa cells from women with polycystic ovary syndrome

AIM:To demonstrate the relationship between hormones in follicular fluid and the expression of LH receptor in granulosa cells (GC) in anovulatory women with polycystic ovary syndrome (PCOS). METHODS:Follicles were obtained from 12 women with PCOS and 15 women with normal menstrual period through surgery at time between day 7 and day 10 of menstrual cycle. The accumulations of estrogen (E2), progesterone (P), luteinizing hormone (LH), follicle stimulating hormone (FSH) and insulin in follicular fluid were determined by a automatism chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination. The accumulation of androstenediol (A) was determined by ELISA. The amounts of the mRNA expressions of LH receptors from GC and theca cells (TC) respectively were measured by RT-PCR using β-actin as intra-control simultaneously. RESULTS:The levels of LH ［(3.8±2.1 vs 1.7±0.8)IU/L, P<0.01］, A ［(600.0±373.4 vs 212.4±205.4)μg/L, P<0.05］ and expressions of LH receptor mRNA of GC (0.29±0.16 vs 0.12±0.13, P<0.01) and TC (0.46±0.14 vs 0.34±0.09，P<0.05) in the women of PCOS group were statically higher than those in control group. The expression of LH receptor mRNA was not detected by RT-PCR in control group when the diameter of an follicle was less than 7 mm, while it was detected in women with PCOS when it remained as small as 4 mm. Expression of LH receptor mRNA in granulose cells was positive related to the concentration of LH (r=0.67, P<0.01) and insulin (r=0.51, P<0.05) in follicular fluid, and that in theca cells (r=0.60, P<0.01). CONCLUSION:The high level of LH in follicular fluid occurs and GC responds to LH prematurely and more intensively in anovulatory women with PCOS. Larger amount of A and P was produced as a result. All of above may contribute to the mechanism of anovulatory.     

Development and evaluation for volume overload-induced and pressure overload-induced cardiac hypertrophy in rats

AIM: To compare the evaluations for the structure and function of the hypertrophic hearts induced by volume overload or pressure overload in rats. METHODS: Volume overload-induced cardiac hypertrophy was established by abdominal aortacaval fistula (ACF) and pressure overload-induced cardiac hypertrophy was developed by constriction of aorta (CA). The cardiac structure and function were analyzed by echocardiography, hemodynamic determination, heart weight measurement and histological examination. RESULTS: Heart weight of rats in all the operated groups was increased compared to the sham-operated groups. In 1-week ACF group, the internal diameter ［(0.67±0.03)cm vs (0.60±0.02)cm, P<0.01］ and volume of left ventricle increased ［(0.69±0.10)mL vs (0.50±0.04)mL, P<0.01］，relative wall thickness (RWT) decreased (0.46±0.05 vs 0.55±0.05, P<0.01), compared with the sham-operated group. In 1-week CA group, interventricular septal thickness ［(0.20±0.03)cm vs (0.16±0.02)cm, P<0.05］, left ventricular posterior wall thickness ［(0.20±0.03)cm vs (0.16±0.02)cm, P<0.01］, RWT (0.71±0.17 vs 0.56±0.12, P<0.05) and +dp/dtmax (4 886±1 304 vs 3 674±325, P<0.05) were all increased compared with the sham-operated group. In 2-week-groups, these parameters changed more significiantly. CONCLUSION: Cardiac structure and function could be evaluated by echocardiography and hemodynamic determination. RWT is a sensitive index for the cardiac hypertrophy induced by both volume overload and pressure overload.     

Effect of granulocyte-colony stimulating factor on the reendothelialization and intima hyperplasia of ballon-injured rat carotid artery

AIM:To study the effect of mobilization of stem cells by exogenous recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the repairing process of reendothelialization and neointima hyperplasy on ballon injured rat carotid arteries.METHODS:Male Wistar rats were randomly divided into rhG-CSF group and NS+injury group.The animals were injected daily with 30 μg/kg rhG-CSF or 0.9% NaCl for 7 days,then underwent balloon angioplasty of the common carotid arteries which were harvested and processed for scanning electron microscopy (SEM),Evans blue staining,morphometric analysis of endothelialization and neointimal formation at 1 h,3 d,5 d,7 d,14 d after injury.Immunohistochemistry for proliferation cell nuclear antigen (PCNA) and RT-PCR for eNOS mRNA were also conducted for evaluating the proliferation of cells of the vessel wall and the possible mechanism of the repairing.RESULTS:SEM and Evan’s blue staining showed increased reendothelialization of the denuded vessels in rhG-CSF-treated animals compared with that NS+injury animals ［(60.6±7.3)% vs (41.6±3.3)%，P<0.01］.Neointima thickness was reduced by 37.3% in rhG-CSF group compared with NS+injury group 2 weeks after injury.Immunohistochemical staining for PCNA positive cells was less in rhG-CSF group compared with that in NS+injury group (42.6% vs 72.8%,P<0.01).CONCLUSION:rhG-CSF has beneficial effects on the reendothelialization and neointima thickness of the ballon-injured arteries.Mobilization of EPCs by exogenous granulocyte colony stimulating factor may be a potential therapeutic strategy for prevention of restenosis after percutenous coronary artery intervention.     

Effects of splenectomy on CD4+ , CD8+ and spleen function in cirrhotic rats with portal hypertension

AIM:To investigate the effect of subtotal splenectomy on the expression of CD4+、CD8+ and tuftsin in cirrhosic rats with portal hypertension (PHT) . METHODS:Rats liver cirrhosis was induced by subcutaneous injection of 40% CCl4. Fifty rats were randomly divided into five groups (n=10). Group A:control rats;group B:PHT rats;group C:normal rats with total splenectomy;group D:PHT with total splenectomy and group E:PHT with subtotal splenectimy. The hepatic function, the expression of CD4+, CD8+, the ratio of CD4+ to CD8+ and tuftsin were analyzed at the fourth week after treatment. RESULTS:The expression of tuftsin ,the ratio of CD4+ to CD8+ was significantly decreased in PHT rats with total splenectomy compared with PHT rats ［（171±21） ng/L vs （433±44）ng/L,P<0.01;(2.01±0.22 vs 1.12±0.12),P<0.01］. In the group of PHT rats with subtotal splenectomy, the expression of tuftsin, the ratio of CD4+ to CD8+ was higher than those in the PHT rats with total splenectomy ［（434±42） ng/L vs （171±21） ng/L,P<0.01;（1.97±0.18 vs 1.12±0.12,P<0.01］, however, the hepatic function was not show difference（P>0.05）. CONCLUSION:Spleen and immune function is significantly improved in PHT rats after subtotal splenectomy, but the hepatic function is not changed significantly.     

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.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

Effects of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children

AIM: To investigate the effect of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children. METHODS: Glucocorticoid inhalator was inhaled by 70 children with attack asthma. The levels of CD4+CD25+Tr in peripheral blood of asthmatic children were tested by flow cytometry (FCM). RESULTS: The CD4+CD25+Tr levels in peripheral blood of asthmatic children were (5.62%±1.29%) and (7.05%±1.61%) before and after of regulated glucocorticoid inhalation, respectively (P<0.01). The Tr levels were (7.56%±1.88%), (7.09%±1.23%) and (6.11%±1.96%) in the complete control group, part control group and poor control group, respectively (P<0.05). The Tr level in formal treatment group (7.05%±1.61%) was higher than that in irregular treatment group (5.91%±1.76%), P<0.01. CONCLUSION: The level of CD4+CD25+Tr is remarkable increased by regulated glucocorticoid inhalation, and the level of Tr can reflect the effects of glucocorticoid inhalation.     

Correlation between intracellular magnesium and expression of beta 2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice

AIM: To explore the effect of intracellular magnesium on expression of beta 2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice. METHODS: Ninety-six healthy, 4-6 weeks old and female C57BL/6 mice, weighting (12±2) g, were randomly divided into the following A, B, C, D groups with 24 mice in each group. The mice were sensitized and challenged by ovalbumin to establish the asthmatic model. A and B groups were fed with magnesium deficient diet. C and D groups were fed with normal magnesium level diet. B and D groups were injected intraperitoneally with 0.2 mL sulphate salbutamol solution after OVA provocation. A and C groups were injected intraperitoneally with 0.2 mL saline as control. Eight mice in each group were randomly taken out at 1 d, 21 d, 34 d to detect plasma Mg2+, intracellular Mg2+, the beta 2-AR mRNA and protein in lung tissue. RESULTS: No significant difference in plasma Mg2+, intracellular Mg2+, the beta 2-AR mRNA and protein in lung tissue among all groups at 1st d was observed (P>0.05, respectively). Plasma Mg2+, intracellular Mg2+, the beta 2-AR mRNA and protein in lung tissue in group C at 21st d and 34th d were significantly higher than those in group A at 21st d and 34th d ［21st d:(0.84±0.09)mmol/L vs 0.57±0.10)mmol/L, (2.39±0.14)mmol/L vs (2.11±0.08) mmol/L,(0.75±0.09)pmol/g vs (0.59±0.06)pmol/g, (88.50±8.50)pmol/g vs (60.10±7.70)pmol/g, P<0.05, respectively; 34th d:(0.67±0.10)mmol/L vs (0.51±0.09)mmol/L, (2.17±0.08)mmol/L vs (2.05±0.09)mmol/L,(0.61±0.05)pmol/g vs (0.53±0.06)pmol/g, (76.60±7.10)pmol/g vs (58.00±7.60)pmol/g, P<0.05, respectively］. Also, plasma Mg2+, intracellular Mg2+, the beta 2-AR, mRNA and protein of lung tissue in group D at 21st d and 34th d were significantly higher than those in group B at 21st d and 34th d ［21st d:(0.95±0.33)mmol/L vs (0.46±0.09)mmol/L,(2.32±0.18)mmol/L vs (1.87±0.14)mmol/L,(0.73±0.10)pmol/g vs (0.43±0.07)pmol/g, (96.90±8.00)pmol/g vs (47.90±4.90)pmol/g, P<0.05, respectively; 34th d:(0.71±0.10)mmol/L vs (0.31±0.08)mmol/L, (1.66±0.13)mmol/L vs (1.45±0.16)mmol/L,(0.40±0.07)pmol/g vs (0.33±0.05)pmol/g, (61.50±3.20)pmol/g vs (35.30±7.10)pmol/g, P<0.05, respectively］.CONCLUSION: The expression of β2-adrenergic receptor mRNA in the lung of C57BL/6 asthmatic mice with deficient intracellular magnesium is suppressed and C57BL/6 asthmatic mice with deficient intracellular magnesium are even easier to induce downregulation of β2-adrenergic receptor when β2-AR agonist is administered.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.     

Hindlimb ischemia and granulocyte colony-stimulating factor accelerates mobilization of endothelial progenitor cells in mice peripheral blood

AIM: To evaluate the mobilization of endothelial progenitor cells (EPCs) in mice peripheral blood during hindlimb ischemia alone or in combination with granulocyte colony stimulating factor (G-CSF). METHODS: Hindlimb ischemia was established in mice by surgical excision of both femoral arteries. Fluorescence-activated cell sorting (FACS) was used to detect the expression of cell-surface CD34 and vascular endothelial growth factor recepter-2 (VEGFR2) antigens. The ratio of double-positive cells for CD34 and VEGFR2 was regarded as the level of EPCs in peripheral blood. In G-CSF administration in combination with hindlimb ischemia group, the percentage of double-positive cells was also detected. RESULTS: As compared with control group, hindlimb ischemia increased the percentage of EPCs in mice peripheral blood. The hindlimb ischemia combined with G-CSF administration significantly enhanced the percentage of EPCs. CONCLUSION: Ischemia increases the number of EPC in peripheral blood. It may induce the migration of EPC from barrow to peripheral blood. By mobilizing barrow, G-CSF enhances this effect.     

Effects of botulinum toxin-A in lower esophageal sphincter in cats

AIM: To investigate the effect of botulinum toxin-A (BTA) on lower esophageal sphincter (LES) after BTA injection into the LES of cats, and then LES pressure, content of Ach and AchE activity were determined to provide scientific proof to treat achalasia with BTA. METHODS: LES pressure was tested on 20 cats. BTA was endoscopically injected circumferentially into the LES in 10 cats, and saline injection served as control (10 cats). One week later, LESP was measured again. Then in vitro, LES samples were gathered. Contents of acetylcholine and cholinesterase activity were determined. By means of Karnovsky-Roots technique, ultrastructure of nerve terminal and vesicles containing Ach were studied under light and electronic transmission microscopes after LES was cut into ultraslices. RESULTS: (1) The LES pressure (LESP) in BTA-treated cats was obviously weakened ［(28.17±3.55) mmHg vs (9.93±1.06) mmHg, P<0.01］ and LESP in control group didnt change ［(28.60±2.79) mmHg vs (26.93±2.05) mmHg, P>0.01］. (2) Contents of LES acetylcholine in BTA-treated cats remarkably decreased, compared with control group ［(75.48±4.67) mg/g vs (93.03±4.65) mg/g, P<0.01］. Consequently, LES acetylcholinesterase activity in BTA-treated cats also apparently decreased, compared with control group ［(38.20±2.17) 103 U/g vs(69.88±6.73) 103 U/g, P<0.01］. (3) The colours of motor end plate fainted away and the number of which became small under light microscopy. The vesicles containing Ach and AchE-positive reactant in the nerve terminal reduced distinctly under the electronic microscopy. CONCLUSION: When BTA is injected into LES, LESP decreases significantly. The reason may be that BTA destroys or inhibits the vesicles of the cholinergic nerve terminal, and as a result, the contents of Ach and AchE activity are evidently reduced.