Diabetes and hyperlipidemia induce dysfunction of VSMCs: contribution of the metabolic inflammation/miRNA pathway.

Vascular endothelial cell injury is considered to be the major factor inducing vascular complications in metabolic diseases and plays an important role in other organ damage. With diabetic and hyperlipidemic rats and cultured VSMCs, the present study was aimed at investigating whether the early damage of VSMCs during metabolic diseases plays a critical role in vascular dysfunction and the underlying mechanisms and would be a promising treatment target. With diabetic and hyperlipidemic rats and cultured VSMCs, the changes and relationships of vascular relaxation and contractile function to the vital organ damage and the underlying mechanisms were investigated; meanwhile, the protective and preventive effects of lowering blood lipid and glucose and inhibition of diabetes and hyperlipidemia-induced vascular hyperreactivity were observed. Diabetic and hyperlipidemic rats presented hyperreactivity in vascular contractile response in the early stages. Hyperglycemia and hyperlipidemia directly affected the contractile function of VSMCs. Early application of fasudil, a specific antagonist of Rho kinase, significantly alleviated diabetes and hyperlipidemia-induced organ damage by inhibiting vascular hyperreactivity. Diabetes and hyperlipidemia-induced inflammatory response could upregulate the expression of connexins and Rho kinase by selective downregulation of the expression of miR-10a, miR-139b, miR-206, and miR-222. These findings suggest that hyperglucose and lipid may directly impair VSMCs and induce vascular hyperreactivity in the early stages. Metabolic inflammation-induced changes in the miRNA-connexin/Rho kinase regulatory pathway are the main mechanism for vascular hyperreactivity and organ damage. Measures inhibiting vascular hyperreactivity are promising for the prevention of organ damage induced by metabolic diseases.

Protective effects of HBOC on pulmonary vascular leakage after haemorrhagic shock and the underlying mechanisms.

Volume resuscitation is an important early treatment for haemorrhagic shock. Haemoglobin-based oxygen carrier (HBOC) can expand the volume and provide oxygen for tissues. Vascular leakage is common complication in the process of haemorrhagic shock and resuscitation. The aim of this study was to observe the effects of HBOC (a bovine-derived, cross-linked tetramer haemoglobin oxygen-carrying solution, 0.5 g/L) on vascular leakage in rats after haemorrhagic shock. A haemorrhagic shock rat model and hypoxic vascular endothelial cells (VECs) were used. The role of intercellular junctions and endothelial glycocalyx in the protective effects of HBOC and the relationship with mitochondrial function were analysed. After haemorrhagic shock, the pulmonary vascular permeability to FITC-BSA, Evans Blue was increased, endothelial glycocalyx was destroyed and the expression of intercellular junction proteins was decreased. After haemorrhagic shock, a small volume of HBOC solution (6 ml/kg) protected pulmonary vascular permeability, increased structural thickness of endothelial glycocalyx, the levels of its components and increased expression levels of the intercellular junction proteins ZO-1, VE-cadherin and occludin. Moreover, HBOC significantly increased oxygen delivery and consumption in rats, improved VEC mitochondrial function and structure. In conclusion, HBOC mitigates endothelial leakage by protecting endothelial glycocalyx and intercellular junctions through improving mitochondrial function and tissue oxygen delivery.

Study on the blood-borne virus co-infection and T lymphocyte subset among intravenous drug users.

AIM: To investigate the features of various blood-borne virus infections and co-infection in intravenous drug users (IDUs), and to examine the correlation of T lymphocyte subsets with virus co-infection. METHODS: Four hundred and six IDUs without any clinical manifestation of hepatitis and 102 healthy persons were enrolled in this study. HBV-DNA and HCV-RNA were detected by fluorescence quantitative PCR. HBsAg, HBeAg, anti-HBc, anti-HCV, HDV-Ag, anti-HGV, anti-HIV, and HCMV-IgM were assayed by enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests. The levels of Th1 and Th2 cytokines were measured by ELISA and radioactive immune assay (RIA). The T lymphocyte subpopulation was detected by using fluorescence immunoassay. The similar indices taken from the healthy persons served as controls. RESULTS: The viral infection rate among IDUs was 36.45% for HBV, 69.7% for HCV, 47.3% for HIV, 2.22% for HDV, 1.97% for HGV, and 3.45% for HCMV. The co-infection rate of blood-borne virus was detected in 255 of 406 (62.81%) IDUs. More than 80% (161/192) of subjects infected with HIV were co-infected with the other viruses, such as HBV, HCV. In contrast, among the controls, the infection rate was 17.65% for HBV and 0% for the other viruses. Our investigation showed that there was a profound decrease in the proportion of CD4/CD8 and the percentage of CD3 and CD4, but not in the percentage of CD8. The levels of PHA-induced cytokines (IFN-gamma and IL-4) and serum IL-2 were obviously decreased in IDUs. On the other hand, the level of serum IL-4 was increased. The level of IFN-gamma and the percentage of CD4 were continuously decreased when the IDUs were infected with HIV or HIV co-infection. IDUs with HIV and HBV co-infection was 15.1% (29/192). Of those 29 IDU with HIV and HBV co-infection, 51.72% (15/29) and 37.93% (11/29) were HBV-DNA-positive and HBeAg-positive, respectively. But, among IDUs without HIV infection, only 1.68% (2/119) of cases were HBV-DNA-positive. CONCLUSION: HCV, HBV and HIV infections are common in this population of IDU, leading to a high incidence of impaired Th1 cytokine levels and CD4 lymphocyte. IDUs with HIV and HBV/HCV co-infection have lower expression of Th1 cytokine with enhancement of the Th2 response. HIV may be causing HBV replication by decreasing Th1 function.

[Two-dimensional gel electrophoresis of subcellular fractions of hepatoma cells].

OBJECTIVES: To seek a better profiling of proteins of hepatoma cells. METHODS: The homogenate of hepatoma cells QGY-7703 was fractionated into four parts by differential centrifugation: the nuclei, the pellet by 20,000 x g, the pellet by 100,000 x g and the cytosolic supernatant. The four fractions were submitted to two-dimensional gel electrophoresis and their electrophoretic patterns were analyzed. RESULTS: In comparison with the protein pattern of hepatoma cells not fractionated, the patterns of the four fractions display many more protein spots, and a large number of proteins present in the nuclei and cytosolic supernatant were not shown in the not-fractionated samples. CONCLUSION: Preparation of subcellular fractions before electrophoretic procedures proves to be very useful; not only can it improve the results of two-dimensional gel electrophoresis, but also can lead to research into the subcellular level.

[Changes in systemic and local vascular reactivity in hemorrhagic shock and the therapeutic effect of delta opioid receptor antagonist ICI174,864].

OBJECTIVE: To investigate the changes in systemic and local vascular reactivity following hemorrhagic shock of different severity and the therapeutic effect of alpha opioid receptor antagonist ICI174,864. METHODS: Fifty-six Wistar rats were used in two experiments. In experiment I, 32 rats were equally divided into sham operation group, 1 hour, 2 hours and 3 hours hypotension groups. In the latter groups, rats were bled to a mean arterial blood pressure (MAP) of 40 mm Hg (1 mm Hg=0.133 kPa) and maintained at this level for 1, 2, 3 hours, respectively. The pressor response of blood pressure and the contractile response of superior mesenteric artery (SMA) to norepinephrine(NE, 3 ug/kg) were observed after shed blood was reinfused. In experiment II, 24 rats were divided into shock control, ICI174,864 0.5 mg/kg and 1.0 mg/kg groups. The response of blood pressure and SMA contractility to NE (3 microg/kg) were observed at 1, 2, and 4 hours after ICI174,864 administration. RESULTS: Following hemorrhagic shock, the systemic and local (SMA) vascular responsiveness was significantly decreased significantly and it was time dependent. Shed blood reinfusion alone did not restore the decreased vascular reactivity. ICI174,864 improved the decreased vascular reactivity in dose-dependent manner. CONCLUSION: Hemorrhagic shock can induce systemic and local vascular hyporeactivity. The decreased vascular reactivity is closely associated with the severity and duration of shock. Loss of systemic vascular reactivity parallels to that of the regional vessel. delta opioid receptor antagonist ICI174,864 has some beneficial effect in improving vascular hyporeactivity.

Effect of initial fluid resuscitation on subsequent treatment in uncontrolled hemorrhagic shock in rats.

SUMMARY: Using a modified uncontrolled hemorrhage shock model with massive splenic and vascular injury, we evaluated outcome and tissue oxidation injury with different resuscitation interventions during prehospital and hospital phases. The aim of our study was to explore the effect of initial fluid resuscitation on subsequent treatment of uncontrolled hemorrhagic shock in rats. Uncontrolled hemorrhagic shock was produced in 114 Wistar rat by sharp transection both of the splenic parenchyma at one location between the major branches of the splenic artery into the spleen and of one of the major branches of the splenic artery. Experimental design consisted of three phases: a "prehospital phase" (resuscitation with balanced saline to a mean arterial pressure (MAP) of 40, 50, 60, 80, and 100 mmHg, respectively, when MAP reached 30 mmHg), followed by a "hospital phase" (120 min, including control of hemorrhage and resuscitation with balanced saline and whole blood (2:1) or balanced saline alone to a MAP >80 mmHg), and a 240-min observation phase. Blood loss, infused volume, hematocrit, and survival rate were recorded. At the end of the experiment, survivors were sacrificed, and the lung, kidney, and distal ileum were harvested for determination of malondialdehyde (MDA) content and total antioxidative capacity (T-AOC). All rats that were resuscitated to a MAP >80 mmHg in the prehospital phase and received balanced saline alone in the hospital phase died, whereas those that had been resuscitated to a MAP of 40 or 50 mmHg during the prehospital phase and then resuscitated with balanced saline and whole blood in the hospital phase survived throughout the experiment. The animals whose MAP was kept higher than 80 mmHg had significantly higher MDA content and lower T-AOC than those whose MAP was maintained 40, 50, or 60 mmHg during the prehospital phase. In the hospital phase, resuscitation with balanced saline and whole blood not only relieved tissue damage but also improved the survival, as indicated by 44.4% survival rate in the rats that resuscitated to a MAP of 80 or 100 mmHg in the prehospital phase. These results suggested that in our uncontrolled hemorrhagic shock model, limited resuscitation in the prehospital phase had benefit for subsequent treatment in the hospital phase in terms of alleviated tissue damage and improved survivorship.

PMN apoptosis and its relationship with the lung injury after chest impact trauma.

BACKGROUND: Polymorphonuclear neutrophil (PMN), one of the most important inflammatory cells, functions throughout the initiation, progression and resolution of inflammation. This study aimed at investigating the relationship between PMN apoptosis and the lung injury after chest impact trauma. METHODS: PMNs were purified from rabbits subjected to the chest impact trauma and their apoptosis, necrosis, survival and respiratory burst were detected by flow cytometry. Meanwhile, lactate dehydrogenase and (LDH) [Ca2+]i were measured. RESULTS: The delayed apoptosis of PMNs in bronchoalveolar lavage fluid was observed from 2 hours to 12 hours after trauma, and viable cells increased. Respiratory burst of PMNs in bronchoalveolar lavage fluid was increased significantly from 2 hours with the peak at 8 hours. Meanwhile, lactate dehydrogenase in bronchoalveolar lavage fluid was higher than that in control (P < 0.05) from 4 hours to 24 hours, and intracellular free Ca2+ in PMN was increased temporarily. CONCLUSIONS: Retention of PMN in tissues and the abnormality in apoptotic pathway inevitably generate persistent activation of PMN and excessive release of toxic substances, resulting in tissue injury. The temporary increase of intracellular free Ca2+ may be responsible for the delayed apoptosis of PMN.

[Mechanism of better result of limited resuscitation in a model of uncontrolled hemorrhagic shock].

OBJECTIVE: To investigate the mechanism of better result of limited resuscitation in a model of uncontrolled hemorrhagic shock. METHODS: Uncontrolled hemorrhagic shock was produced in 54 rats by a standardized massive splenic injury with transection of the middle branch of splenic artery (MSIA). The rats were randomly assigned to six groups (n=6) by maintenance of the level of mean arterial pressure (MAP): sham-operated group (SS), 40 (RS40), 50 (RS50), 60 (RS60), 80 (RS80) and 100 mm Hg (RS100, 1 mm Hg=0.133 kPa). When the MAP reached 40 mm Hg, resuscitation was begun. Ringer's solution was continued as needed to maintain the following desired endpoints for 45 minutes (T45 point): MAP of 40, 50, 60, 80 and 100 mm Hg. After the bleeding was controlled, resuscitation was continued with Ringer's solution and whole blood (2:1) to raise the MAP to 100 mm Hg for 120 minutes (T165 point), followed by a 240 minutes observation period (T405 point). All animals were observed for 240 minutes or till death. The blood samples were withdrawn from artery for hematocrit (Hct), blood lactate (BL), base excess(BE) at T0, T45, T165, T405 points. At the end of the experiment, a small amount of hepatic tissue was collected for measuring tissue blood perfusion, total antioxidative capacity (T-AOC), Na(+)-K(+)-ATPase, and malondialdehyde (MDA). RESULTS: At T45, T405 points, Hct in SS, RS50 and RS60 were significantly higher than in RS80 and RS100(P<0.05). At T405 point, BL and BE levels in RS80 and RS100 were significantly higher than that of the other groups (P<0.05). The contents of MDA in SS, RS40, and RS50 were significantly lower than in RS80 and RS100(P<0.05). T-AOC level, Na(+)-K(+)-ATPase were significantly lower in RS80 and RS100 than that in the other groups. Blood perfusion was significantly lower in RS80 and RS100 than that in SS, RS40, and RS50. CONCLUSION: In the setting of uncontrolled hemorrhagic shock, limited resuscitation could balance well the needs of organ perfusion and decrease lactate level. It might also exert a protective effect against ischemia/reperfusion injury to liver tissue.

[Beneficial effect of thyrotropin-releasing hormone in combination with HSD on hemorrhagic shock with pulmonary edema at high altitude in the rat].

OBJECTIVE: To study the effects of thyrotropin-releasing hormone (TRH) in combination with hypertonic saline/dextran (7.5% NaCl + 6% Dextran 40, HSD ) on hemorrhagic shock with pulmonary edema in the rats which were recently brought to high altitude. METHODS: Forty-nine SD rats, transported to Lasa, Tibet, which was 3,760 meters above the sea level, were anesthetized one week later with sodium pentobarbital (30 mg/kg, intraperitoneal). Hemorrhagic shock with pulmonary edema was induced by hemorrhage (50 mm Hg maintained for 1 hour,1 mm Hg=0.133 kPa) plus intravenous injection of oleic acid (50 microl/kg). They were equally divided into seven groups (n=7): normal control, hemorrhagic shock, hemorrhagic shock with pulmonary edema (HSPE), HSPE plus TRH (5 mg/kg), HSPE plus HSD (4 ml/kg), and HEPE plus TRH and HSD in combination. Hemodynamic parameters including mean arterial blood pressure(MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (+/- dp/dt max) were observed at 15, 30, 60 and 120 minutes, blood gases were analyzed at 30 and 120 minutes, and the water content of lung and brain was determined at 120 minutes after drug administration. RESULTS: TRH or HSD used alone or in combination significantly increased MAP, LVSP and +/- dp/dt max (P<0.05 or P<0.01 ), ameliorated acid-base imbalance, and decreased the water content of lung and brain. The effect of the two in combination was superior to either drug used alone. CONCLUSION: TRH in combination with HSD can be used in the treatment of hemorrhagic shock with pulmonary edema at high altitude.

[Effect of different volumes of fluid resuscitation on hemorrhagic shock with pulmonary edema at high altitude in the unacclimated rat].

OBJECTIVE: To study the effects of different volumes of fluid resuscitation on hemorrhagic shock with pulmonary edema at high altitude in the unacclimated rat. METHODS: One hundred and twenty-six SD rats transported to Lasa, Tibet, 3 760 meters above the sea level, were anesthetized one week later with sodium pentobarbital (30 mg/kg, intraperitoneal). Hemorrhagic shock with pulmonary edema model was induced by hemorrhage (50 mm Hg for 1 hour, 1 mmHg=0.133 kPa) plus intravenous injection of oleic acid (50 microl/kg). Experiments were then conducted in two parts. Sixty-three rats in part I were equally divided into nine groups (n=7): normal control, hemorrhagic shock control, hemorrhagic shock with pulmonary edema (HSPE) without fluid infusion, HSPE plus infusing lactated Ringer's solution (LR) with 0.5-, 1-, 1.5-, 2- or 3- fold volume shed blood, and 1 volume of LR plus mannitol (10 ml/kg). Hemodynamic parameters including mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (+/- dp/dt max) were observed at 15, 30, 60 and 120 minutes after infusion, blood gases were measured at 30 and 120 minutes after infusion and the water content of lung and brain was determined at 120 minutes after infusion. In part II, additional 63 rats were used to observe the effect of different volumes of fluid resuscitation on survival time of HSPE rats. RESULTS: 0.5 volume of LR infusion significantly improved MAP, LVSP and +/- dp/dt max, prolonged the survival time of HSPE animals (all P<0.01), while it did not increase the water content of lung and brain and had no marked influence on blood gases. One volume of LR infusion slightly improved hemodynamic parameters, prolonged the survival time and increased the water content of lung. More than 1 volume of LR infusion including 1.5-, 2- and 3- fold volume LR deteriorated the hemodynamic parameters and decreased the survival time of shocked animal, meanwhile they apparently increased the water content of lung. One volume of LR plus mannitol (10 ml/kg) infusion did not improve the hemodynamic parameters and blood gases; also it did not decrease the water content of lung. CONCLUSION: The tolerance to fluid infusion for the unacclimated animal subjected to hemorrhagic shock with pulmonary edema at high altitude is significantly decreased. 0.5-1 volume of LR infusion appears to be beneficial effect on resuscitation at high altitude, while over 1 volume of LR infusion would aggravate pulmonary edema and exacerbate fluid resuscitation effect.

[Differential expression of mitochondrial proteins in hepatoma cells analyzed using two-dimensional electrophoresis].

BACKGROUND & OBJECTIVE: Mitochondria play a key role in cell apoptosis. Proteomic analysis of mitochondria will contribute to the discovery of tumorigenic mechanism, early detection of cancer, and findings of anticarcinogens. This study was to identify the differentially expressed mitochondrial proteins in hepatoma cells using two-dimensional gel electrophoresis (2-DE). METHODS: Mitochondria were isolated from normal hepatocytes L02, and hepatoma QGY-7703 cells before and after treatment of epirubicin by density gradient centrifugation; the proteins of mitochondrial samples were analyzed with 2-DE. RESULTS: After density gradient centrifugation, the mitochondrial marker enzyme activities were increased by 11.8, 13.8, and 10.2 times, respectively, for L02 cells, untreated QGY-7703 cells, and epirubicin-treated QGY-7703 cells, and the 2-DE profiles identified 206, 217, and 214 protein spots, respectively, from the 3 samples. Compared L02 cells and QGY-7703 cells, 49 differentially expressed protein spots were identified; compared QGY-7703 cells before and after treatment of epirubicin, 29 differentially expressed protein spots were identified. CONCLUSION: A set of differentially expressed protein spots are detected in mitochondria of QGY-7703 cells with density gradient centrifugation and 2-DE.

[The inflammatory mediators induced endothelial cell injury through delay neutrophils apoptosis].

This study aimed at elucidating the relationship between NTP apoptosis and endothelial cell injury. After isolation and purification, activated NTPs were incubated with endothelial cells directly or indirectly contact in a ratio of 5:1 and 10:1, meanwhile 10ng/ml IL-6 and 10% (v/v) burned serum added or not. Apoptosis and necrosis of endothelial cells were detected in situ. Activated NTP alone exerted no obviously detrimental effects on endothelial cells. However, when IL-6-or serum from burn patients was added, NTP apoptosis was delayed and endothelial cells were injured in the form of necrosis. Inflammatory mediators can delay NTP apoptosis, further research demonstrated that direct contact between the two types of cells was essential for NTP to impact injury on endothelial cells.

Death style and respiratory burst of neutrophils in peripheral blood and pulmonary alveolus under endotoxemia in rats.

To study the difference of changes on apoptosis, necrosis and respiratory burst of the polymorphonuclear neutrophils (PMN) in endotoxemia rat model. LPS (O(55)B(5), 5 mg/kg) was injected into abdominal cavity of 20 random normal Wistar rat. 2, 4, 8 and 12 hours after injection, the changes of apoptosis, necrosis and respiratory burst of the rats between PMN from the peripheral blood and from the bronchoalveolar lavage fluid were observed using the flow cytometer. At the same time, 5 uninjected rats were taken as control. The results demonstrated that the quantity proportions of apoptosis of PMN between the peripheral blood PMN and the bronchoalveolar lavage fluid PMN in rat's endotoxemia were similar. However, comparison with the uninjected LPS rat, the necrosis of peripheral blood PMN obviously increased and the respiratory burst capacity was clearly inhibited. Contrarily, the necrosis of bronchoalveolar lavage fluid PMN obviously decreased and the respiratory burst obviously increased in the injecting LPS rat. It was concluded that the necrosis and apoptosis displayed differently between the pulmonary and peripheral blood PMNs in endotoxemia. Under state of inflammation, the surviving PMN in tissue increased and kept the activated state due to tissue injury.