Effects of burn sera on IκBα degradation and NF-κB activation in monocytes in vitro

AIM: To investigate the effects of burn sera on IκBα degradation, NF-κB activation in peripheral blood monocytes (PBMCs) in order to explore the role of burn sera on activation of monocytes. METHODS: PBMCs isolated from healthy volunteers were stimulated by sera from healthy volunteers and burn patients and by burn sera together with PDTC (pyrrolidine dithioncarbamate). Activation of monocytic NF-κB was tested by electrophoretic mobility shift assay (EMSA) and the degradation of monocytic IκBα was determined by Western blotting. RESULTS: When compared to that in control group, cytosolic IκBα degradation occurred within 30 min after PBMCs stimulated by burn sera, and peaked at 60 min. But IκBα gradually recovered in the cytoplasm after 2 h of stimulation. Meanwhile, activity of monocytic NF-κB was markedly increased, reached the peak at 30 min to 60 min after stimulation, and gradually decreased after 2 h of stimulation. PDTC (an antioxidants) effectively inhibited the monocytic IκBα degradation and activation of NF-κB induced by burn sera. CONCLUSION: Burn sera might induce the degradation of IκBα, then activate NF-κB, which ultimately lead to the secretion of cytokines from the monocytes.     

Effects of lipopolysaccharide,IL-6 and TNFα on the tissue factor expression of astrocytes

AIM:To study the effects of lipopolysaccharide(LPS), interleukin-6(IL-6)and tumor necrosis factor α (TNFα) on tissue factor(TF) expression of astrocytes. METHODS:Astrocytes were identified with anti-glial fibrillary acidic protein antibody. The TF activity of cell lysate was measured with one stage clotting assay. RESULTS:TF activity of astrocytes of LPS,IL-6,TNFα groups were obviously higher than that of the control group(P <0.05); While LPS,IL-6 and TNFα were combined with trifluoperazine or H₇, their inductive effects were inhibited. CONCLUSION:LPS,IL-6 and TNFα promoted the TF expression of astrocytes and its mechanisms may connected with Calcium/Camodulin and protein kinase C pathway.     

Antagonistic effect of captopril on activation and injury of vascular endothelial cells induced by lipopolysaccharide

AIM: To investigate the antagonistic action of Captopril (Cap) on the activation and injury of human umbilical vascular endothelial cells (HUVECs) induced by lipopolysaccharide(LPS) and the possible mechanisms. METHODS: After 18 h exposure of the cultured HUVECs to LPS (1 mg/L), or LPS (1 mg/L) plus Cap at the concentration of 10^-7mol/L, 10^-5mol/L and 10^-3mol/L, the expression of vWF protein in the conditioned media was tested by enzyme-linked immunosorbent assay (ELISA), the expression of ICAM-1 protein in HUVECs was determined by indirect immunofluorescence technique with flow cytometry as well. In addition, the expression of TNFα mRNA was determined by in situ hybridization. RESULTS: The results of ELISA and indirect immunofluorescence technique showed that exposure to LPS at a concentration of 1 mg/L led to a significant increase in the vWF and ICAM-1 expression in HUVECs as compared to the control (P＜0. 05), whereas they were somewhat decreased when exposed to Cap at three increasing concentrations mentioned above, especially in the Cap (10^-3mol/L) plus LPS group (P＜0.05). Cap inhibited vWF secretion and ICAM-1 expression of HUVECs caused by LPS in a concentration-dependent manner. In situ hybridization revealed that the expression of TNFα mRNA was inhibited by Cap both in a concentration of 10^-3mol/L, and in a lower concentration of 10^-5mol/L. CONCLUSION: Cap antagonizes the activation and injury of HUVECs induced by LPS, which may be related to the decrease in TNFα mRNA expression.     

Effect of tissue factor on intravascular migration of tumor cells

AIM: To evaluate the effect of tissue factor on intravascular migration of tumor cells.METHODS: Expression of tissue factor in tumor cells (HT1080) was analyzed by flow cytometry and immunofluorescence staining. An in vitro model was used to observe intravascular migration of tumor cells. RESULTS: High expression of tissue factor was observed in tumor cells (HT1080). The antibody for tissue factor inhibited intravascular migration of tumor cells. CONCLUSIONS: Tissue factor stimulated tumor metastasis through promoting intravascular migration of tumor cells.     

Effects of the Bushen Ningxin Chinese herb-containing serum on the adherence of human monocytes to endothelial cells

AIM: To study effect of the Bushen Ningxin decoction, a Chinese medicine, on the adherence of monocytes to endothelial cells and its mechanism. METHODS: Using cultured human umbilical vein endothelial cells (HUVECs) as target cells, oxidized low density lipoprotein (ox-LDL) was added to the endothelial cell culture to prepare the model of human endothelial cell injury. The serum of rabbits having been treated with Bushen Ningxin decoction was added to trial architecture, the adherence of monocyte-like cell line U937 to HUVECs was analyzed using Rose Bengal staining. In addition, the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1) and E-selectin in HUVECs was measured by flow cytometry. RESULTS: Treatment of HUVEC with ox-LDL (100 mg/L) for 24 hours significantly increased adhesion of U937 to HUVECs. If serum of the animal treated with Bushen Ningxin decoction was added to trial architecture, the adhesion decreased significantly. The flow cytometry analysis showed that ox-LDL could induce the expression of ICAM-1, VCAM-1 and E-selectin in HUVECs. Serum of the animal treated with Bushen Ningxin decoction significantly decreased the expression of ICAM-1, VCAM-1 and E-selectin in HUVECs. CONCLUSION: The Bushen Ningxin Chinese herb-containing serum has an inhibitory effect on the adherence of monocytes to endothelial cells, probably by way of down-regulating the expression of ICAM-1, VCAM-1 and E -selectin in endothelial cells.     

Effects of pretreatment with captopril on the infarct size and myocardial cell apoptosis in experimental rabbits

AIM:To investigate the effects of pretreatment of captopril on the infarct size and myocardial cell apoptosis in rabbits. METHODS:Rabbits were randomly divided into sham-operated control group (SO), acute infarct group (AI) and captopril pretreatment group (CP). The rabbits of CP group were treated with captopril (25 mg·kg^-1.d^-1) for 1 week before harvest. The left circumflex branch of coronary (LCX) was ligated to develop acute ischemic model. The systolic and diastolic function of left ventricle(LV) was measured before and at 15, 30, 60 min after ligating LCX, and the blood viscosity and hematocrit before and at 60 min after ligating LCX were measured also. 6 hours later LCX ligation, the hearts were harvested for determining the infarction size, which was expressed as the ratio of infarct area to the total ischemic area, and evaluating apoptosis index expressed as the percentage of myocardial cells with TUNEL positive staining. RESULTS:1.Compared with AI group, captopril pretreatment significantly reduced the infarction size (16.60%±0.94% vs 36.24%±1.94%, P＜0.05), and improved the LV function and viscosity of blood. 2. Apoptosis of myocardial cell was found in the myocardium surrounding to the infarction area, however, the apoptosis index of CP group was significantly lower than that of AI group (26.30%±0.71% vs 42.44%±2.32%,P＜0.05).CONCLUSION:Apoptosis of myocardial cell exists in the area surrounding the infarction. Captopril pretreatment can reduce infarction size and myocardial apoptosis index, and improve the LV function as well as blood viscosity in this acute ischemic model.     

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

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

Effects of monocyte-endothelium interaction on the expression of CD36 in monocytes

AIM: To examine the effects of monocyte-endothelium interaction on the expression of CD36 in monocytes and observe the functions of cytokines in this process. METHODS: The monocytes and endothelial cells were cultured alone or cocultured together to form different cell culture conditions. The level of M-CSF in culture medium was determined by enzyme linked immune sandwich assay(ELISA) technique, and the expression of CD36 in monocytes was determined by flow cytometry. RESULTS: The expression of CD36 in monocytes was low in monocytes cultured alone but increased signiFcantly when monocytes and endothelial cells were cocultumd(P<0.05).M-CSF and PDGF -BB played certain roles in increasing expression of CD36 in monocytes, but they didn't seize the principal positions. CONCLUSION: The monocyte-endothelium interaction increased the expression of CD36 in monocytes through various mechanisms and may participate in the pathogenesis of atherosclerosis.     

Overexpression of tissue factor levels in plasma and tissue of hepato-cellular carcinoma (HCC) patients and its clinical significance

AIM:To detect tissue factor (TF) level both in plasma and in tissue of hepatocellular carcinoma (HCC) patients and to elucidate their association with clinical features.METHODS:Plasma TF levels of 50 cases of HCC patients and 30 cases of control were detected by ELISA.27 HCC tissue samples with their adjacent tissue samples and 27 normal liver tissues were detected by RT-PCR.RESULTS:① Plasma TF levels were increased significantly in HCC group when compared with control (P<0.05).TF levels were higher in poor differentiation,large size and cirrhosis subgroup in HCC patients (P<0.05).Plasma TF levels were also significantly increased in extra-hepatic metastasis,lymphatic metastasis and portal venous tumor thrombus subgroups (P<0.05).② The mRNA expression rate of TF in HCC tissue was 62.96% (17/27) and the relative mRNA expression intensity of TF was 0.567±0.268.Those were significantly higher than that in their adjacent tissue samples or 27 normal liver tissue samples (P<0.05).While the relative expressive intension of TF were also significantly higher in larger size and several invasive and metastatic subgroups (P<0.05).CONCLUSION:TF might play an important role in hepatic carcinogenesis,invasiveness and metastasis.     

Effects of captopril on expression of calcineurin and NF-κB p65 in the heart of hypertensive rats

AIM: To study the effect of captopril on calcineurin and NF-κB p65 in the signal transduction pathway of the cardiovascular remodeling in hypertensive rats. METHODS: Using a animal model of hypertension induced by abdominal aortic banding, the rats were treated with captopril for 10 weeks. The blood pressure was observed with a tail cuff method. The heart weight and heart weight/body weight were measured. The expression of calcineurin and NF-κB p65 were studied by using immunohistochemistry. RESULTS: After treated with captopril, the blood pressure of the model rats was decreased (P<0.01), the heart weight or heart weight/body weight were also decreased (P<0.01). The calcineurin and NF-κB p65 protein overexpression was down-regulated, NF-κB-positive area and area percentage were reduced in the heart of hypertensive rats (P<0.01，P<0.01). CONCLUSION: Captopril reverses the cardiovascular remodeling by affecting the overexpression of calcineurin and NF-κB p65 involved in the cardiovascular remodeling in hypertensive rats.     

Effect of captopril and betaloc on the changes in expression of MHC of left ventricle in hypertensive rats

AIM:To investigate myosin heavy chain(MHC)gene expression and the effects of captopril and betaloc at an early stage of hypertension. METHODS:Model of hypertension was made by partly narrowing two bilateral renal arteries(2K2C). The rats were divided into four groups at random. (1) control group; (2)2K2C group;(3)captopril group;(4)betaloc group.Levels of α-MHC and β-MHC mRNA were determined by dot-blot. RESULTS:α-MHC mRNA expression were gradual1y reduced in 2K2C group, while β-MHC mRNA expression were increased, and the marked changes were observed at 72h postoperation. Captopril could inhibit the changes in MHC gene expression; but betaloc could not. CONCLUSION: The expression of MHC gene has changed at an early stage of renal hypertensive rat, and renin-angiotensin system may play an important role in this change.     

Effects of sodium ferulate on function of macrophages in the colonic tissue of colitis rats

AIM: To explore the effects of sodium ferulate (SF) on function of macrophages in colonic tissue of the colitis rats in vivo. METHODS: The immunological colitis model of rats was produced. SF was used intracolonically for 21 days. The contents of malondialdehyde (MDA), nitric oxide (NO), prostaglandin E₂ (PGE₂) and the activity of superoxide dismutase (SOD), interleukin-1 (IL-1), TNF-α, myelopexoxidase (MPO), and the expression level of NF-κB p65 in colonic tissue of the rats were detected. RESULTS: SF (200,400,800 mg/kg) decreased the elevated contents of MDA, NO, PGE₂, the activity of IL-1, TNF-α, MPO, and the expression level of NF-κB p65, while increased the reduced activity of SOD in colonic tissue of the colitis rats in a dose-depended manner. CONCLUSION: SF restrained the activity of activated colonic macrophages and relieved the colonic inflammation reaction in vivo in colitis rats, which may be related to the suppression of NF-κB activation.     

The effects of FTY720 on lymphocytes and monocytes in mouse intestinal transplantation

AIM：To test the effects of FTY720 on mouse intestinal allografts.METHODS：C3H mice（H-2k）were used as donor and C57BL/6 mice (H-2b) as recipients.FTY720 group,allogeneic control group and isogeneic control group were set up.6 and 14 days after transplantation,murine intestinal grafts were harvested for histologic assessment.Lymphocytes were collected from mesenteric lymph nodes (MLN),Peyer’s patch (PP),lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) in the graft,then were analyzed by cytometry.RESULTS：Rejection was inhibited in FTY720 group at the 6th post-transplant day,although not at the 14th day.Recipient CD4+ and CD8+ T cells,CD19+ B cells,as well as γδ TCR lymphocytes,were greatly reduced by FTY720 therapy.The similar action of FTY720 was also revealed in Gr1+CD11b+ monocytes.CONCLUSION：FTY720 is efficient on alleviating allo-immune response by reducing the infiltration of both lymphocytes and monocytes into the graft in a mouse intestinal transplantation model.     

Effects of changing PKC activity on proliferation and telomerase expression in human hepatocellular carcinoma cells

AIM: To investigate whether protein kinase C (PKC) is involved in the proliferation and the telomerase expression in human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (BEL-7402) were treated with exogenous phorbol-12-myristate-13-acetate (PMA, PKC activator) and staurosporine (SP, PKC inhibitor) for 48 hours. The techniques of cell culture and the telomeric repeat amplification protocol silver staining in combination with computer image scanning system in vitro were used to observe the variations of the growth and the telomerase expression. RESULTS: The proliferative potential of BEL-7402 cells was decreased by the action of PMA as well as SP, and the telomerase expression was also inhibited by PMA and SP. CONCLUSION: Our findings suggest that the proliferation of human hepatocellular carcinoma cells and the telomerase expression may be related to PKC.     

The effect of vasoactive substances on synthesis and release of C-type natriuretic peptide in cultured human endothelial cells

AIM:To investigate the effect of endothelin (ET), angiotensin II (AngII) and homocysteine (Hcy) on C-type natriuretic peptide (CNP) synthesis and release. METHODS: Human endothelial cell was cultured; CNP was measured by radioimmunoassay method. RESULTS: ET and AngII could augment CNP synthesis in human endothelial cells. Compared with control group, 10^-9,10^-8,10^-7 mol/L ET and Ang II increased CNP content of endothelial cells by 1%(P＞0.05), 49%(P＜0.05),117%(P＜0.01) and 137% (P＜0.01),165%(P＜0.01),201%(P＜0.01),respectively. A great dose of ET and Ang II also stimulated CNP release from cultured human endothelial cells. Hcy had no effect on CNP synthesis, but 10^-9,10^-8,10^-7 mol/L Hcy enhanced CNP release from cultured human endothelial cells by 17%(P＞0.05),84%(P＜0.01) and 555%(P＜0.01), respectively. CONCLUSION: ET, AngII and Hcy might be involved in the synthesis and release of human endothelial cell CNP.Fig 1 Time-course of CNP syntheis and release in cultured human endothelial cell ( ±s,n=6)     

Effects of oxidized HDL on the levels of MCP-1, ICAM-1 and free calcium in cultured human umbilical venous endothelial cells

AIM:To study the effects of oxidized high-density lipoprotein (oxHDL) on the expression of monocyte chemoattractant protein-1(MCP-1) and intercellular adhesion molecule-1(ICAM-1) and intracellular free calcium concentration ([Ca²⁺]i) level in cultured human umbilical venous endothelial cells(HUVECs). METHODS:The MCP-1 protein content in the medium of conditioned HUVEC was measured by ELISA, and the ICAM-1 on HUVECs was detected by indirect immunofluorescence, and [Ca²⁺]i was determined by Fluo-3/AM, the injury of cells was observed by scanning electron microscopy (SEM).RESULTS:oxHDL could induce the expression of MCP-1 and ICAM-1 in HUVECs. In oxHDL group (HUVECs were incubated with 100 mg protein/L oxHDL for 24 h), the levels of MCP-1, ICAM-1 and [Ca²⁺]i increased by 160%, 60% and 70% respectively compared with the control group (P＜0.01). When HUVECs were incubated with 300 mg protein/L oxHDL for 24 h, cells were injured obviously. CONCLUSION:By inducing the expression of ICAM-1 and MCP-1 in endothelial cells, oxHDL may promote monocyte-endothelium adhesion and monocyte migration to intima, it may promote atherosclerosis as oxidized low-density lipoprotein (oxLDL).     

Influence of captopril on intracellular free calcium concentration and involved ion channels mechanisms in cardiac myocytes of the neonatal rat undergone anoxia-reoxygenation injury

AIM:To observe the influence of captopril on intracellular free calcium concentration (［Ca2+］ i) and the involved ion channels mechanisms in cardiac myocytes of the neonatal rat undergone anoxia-reoxygenation injury.METHODS:The anoxia-reoxygenation model in cultured neonatal rat ventricular myocytes was established.Groups were divided into ① normal;② anoxia-reoxygenation;③anoxia-preconditioning (5 min anoxia+5 min reoxygenation);④ captopril preconditioning.Flou-3 /AM loading and flow cytometry technique were used to observe the ［Ca2+］i,and whole-cell patch clamp technique was used to record the L-type calcium current and Na+/Ca2+ exchange current.RESULTS:① Compared to normal group,［Ca2+］i in anoxia -reoxygenation group was increased significantly (789.42±9.05 vs 414.08±37.40,P<0.01),L-type calcium current density was decreased (P<0.01),the current-voltage curve was moved up,the inactivation curve was moved left and Na+/Ca2+ exchange current was increased in anoxia-deoxygenating.② Compared to anoxia-reoxygenation group,anoxia and captopril preconditioning resulted in a significant decrease in ［Ca2+］i (593.84±5.06,507.08±31.89 vs 789.42±9.05,P<0.01),and a significant increase in L-type calcium current density (P<0.01),the current-voltage curve was moved down,the inactivation curve was moved right and Na+/Ca2+ exchange current was decreased ③ Compared to normal oxygen condition,the anoxia and captopril precondition resulted in a lightly increase in ［Ca2+］i (507.08±31.89 vs 414.08±37.40,P<0.05) and Na+/Ca2+ exchange current.④ Compared to anoxia-preconditioning group,captopril-preconditioning resulted in no significant difference in all the markers mentioned above.CONCLUSIONS:The anoxia-reoxygenation injury in cardiac myocytes results in ［Ca2+］i abnormal increase and calcium overload by increasing Na+/Ca2+ exchange current.Late preconditioning in cardiac myocytes is triggered by transient and repeated anoxia and captopril,which slightly increases Na+/Ca2+ exchange current and ［Ca2+］i and restraines the abnormal increasing of Na+/Ca2+ exchange current and calcium overload induced by subsequenced anoxia-reoxygenation injury,so it plays an delayed protective role in cardiac myocytes.L-typed calcium passage is not involved in calcium overloaded and late preconditioning of calcium in myocytes during reperfusion.     

Effect of fibrinogen,fibrin and fibrin degradation products on the proliferation and migration of human vascular smooth muscle cells

AIM: To investigate the effect of fibrinogen (Fg), fibrin (Fb) and fibrin degradation products (FDPs) on the proliferation and migration of human vascular smooth muscle cells (VSMC).METHODS: The effects of Fg, Fb and FDPs on the proliferation of VSMC were observed by means of cell counting and MTT test, migration assays were performed using the wounding model and the transwell cell culture apparatus.RESULTS: Fg itself did not stimulate the proliferation of VSMC, but stimulated VSMC migration. Fb and FDPs both stimulated the proliferation and migration of VSMC, meanwhile the effect of Fb was in a dose-dependent manner.CONCLUSION: Fb, in particular FDPs, may play an important role by stimulating the proliferation and migration of VSMC in restenosis and atherogenesis.