Role of nitric oxide in the inhibition of lipopolysaccharide-induced thoracic aortic hyporeactivity by cholecystokinin in vitro

AIM:To investigate the effect of cholecystokinin octapeptide(CCK-8) on the L-arginine-nitric oxide(NO) pathway in rabbit thoracic aortae treated with lipopolysaccharide(LPS).METHODS:The isolated thoracic aortic rings(TARs) from rabbits were incubated with LPS, LPS+CCK or vehicle for 14 h. Then the contractility to phenylephrine(PE) by TARs and the response to L-arginine(L-Arg) by pre-contractile TARs were measured. In addition, we added NO synthase(NOS) inhibitors aminoguanidine(AG)and N^ω-nitro-L-arginine(L-NNA) into organ baths to observe the changes of vascular contractility to PE. NOS activity in isolated TARs were also detected. RESULTS:Incubation of TARs with LPS for 14 h resulted in an increase of NOS activity and a reduction of contractility to PE. Treatment with CCK-8 significantly inhibited the increased NOS activity in thoracic aortae and improved the hypocontractility of TARs to the same degree as AG.CONCLUSION:CCK-8 may improve the hypocontractility of TARs induced by LPS by inhibiting the activity of NOS.     

Inhibitory effect of cholecystokinin octapeptide on expression of CD14 on rat pulmonary interstitial macrophages induced by lipopolysaccharide in vitro

AIM:To study the effect of cholecystokinin octapeptide(CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophage(IM)in vitro.METHODS:Pulmonary IM were isolated and cultured in the presence of LPS, CCK-8, proglumide(the antagonist of CCK receptors) and vehicle alone or together. The expression of mCD14 protein was assayed by flow cytometry, and sCD14 in the supernatant was analyzed semi-quantitatively by Western blot, and TNF-α in the supernatant was detected with ELISA.RESULTS:CCK-8, at concentrations from 10^-7mol/L to 10^-6mol/L inhibited significantly the expression of mCD14, the release of sCD14 and TNF-α to the supernatant up-regulated by LPS(1mg/L). The effect of CCK-8 was inhibited by proglumide. CONCLUSION:CCK-8 modulated negatively several functions of LPS-stimulated pulmonary IM through CCK receptors, which may be one of the mechanisms for CCK-8 to alleviate the inflammation in lung tissues during endotoxemia.     

Effects of cholecystokinin octapeptide on expression of IL-1β, IL-6, IL-4 and IL-10 in lipopolysaccharide-attacked mice

AIM:To observe the effects of cholecystokinin octapeptide (CCK-8) on the expression of proinflammatory cytokines IL-1β, IL-6 and anti-inflammatory cytokines IL-10, IL-4 in LPS- attacked mice. METHODS:Kunming mice were randomly assigned and injected intraperitoneally with LPS alone or/and CCK-8 at different time points. The expression of IL-1β, IL-6, IL-10 and IL-4 in the serum and lung tissues were assayed by ELISA and RT-PCR. RESULTS:The expression of IL-1β, IL-6, IL-10 and IL-4 were upregulated in LPS-attacked mice. Pre-treatment of CCK-8 decreased both IL-1β and IL-6 expression and augmented IL-10 and IL-4 expression in LPS-attacked mice. CONCLUSIONS:CCK-8 exerts an anti-inflammatory effect by inhibiting the expression of IL-1β, IL-6 and increasing the expression of IL-10, IL-4 in LPS-attacked mice, which could alleviate the inflammatory response in lung tissue.     

Reversion of the decline of cardiac function in endotoxin shock rats by cholecystokinin octapeptide

AIM: To verify the effect of cholecystokinin octapeptide(CCK-8) on cardiac function in endotoxin shock (ES) rats. METHODS: The rats were divided into four groups:control,lipopolysaccharide(LPS),CCK-8 and CCK-8+LPS. The left ventricle pressure(LVP),the maximal/minimum rate of LVP,heart rate (HR) and mean arterial pressure (MAP) were measured. The activity of superoxide dismutase (SOD),the contents of malondialdehyde (MDA) and nitric oxide (NO) in both serum and myocardium were also measured,respectively. RESULTS: CCK-8 (40 μg·kg^-1, iv) elicited bradycardia in short time and gently increase MAP,LVP and ±LVdp/dt_max. Lipopolysaccharide(LPS, 8 mg·kg^-1, iv) caused a variation in heart rate (HR)(a bradycardia following a tachycardia) and rapid decreases in MAP,LVP and ±LVdp/dt_max. The rapid variation of HR and the decline of MAP,LVP and ±LVdp/dt_max were reversed by pretreatment with CCK-8 in ES rats, but didn't restore to normal. The activity of SOD was increased and the contents of MDA and NO were decreased by pretreatment with CCK-8 in ES rats. CONCLUSION: The decline of cardiac function in ES rats could be reversed by pre-administration of CCK-8 and the decrease in NO production may be one of the mechanisms.     

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.     

Role of heme oxygenase in cholecystokinin octopeptide ameliorating acute lung injury induced by lipopolysaccharide

AIM: To study the role of heme oxygenase (HO)-1 in the mechanism of cholecystokinin-octapeptide (CCK-8) for attenuation of acute lung injury (ALI) induced by lipopolysaccharide (LPS).METHODS: Adult male rats were randomly divided into five groups: control group,LPS group,CCK-8+LPS group,LPS+ Hm (hemin,HO-1 donor) group and LPS+ZnPP (zinc protoporphyrin,specific inhibitor of HO-1) group.PMN number in bronchoalveolar lavage fluid (BALF),the structure of the lung,MDA content,HO-1 activity,the expressions of HO-1 mRNA and protein in the lung were detected respectively.RESULTS: The lung injury in LPS group was observed,at the same time the numbers of PMN,the content of MDA,the activity and the expression of HO-1 were all higher than those in control group (all P<0.05).The degree of lung injury,PMN numbers and MDA content were lower,while the activity and the expression of HO-1 in CCK-8+LPS and LPS+Hm group were higher than those in LPS group (all P<0.05).However,the degree of lung injury,PMN numbers and MDA content were higher,the activity and the expression of HO-1 were lower in LPS+ZnPP than those in LPS group respectively (all P<0.05).CONCLUSION: CCK-8 attenuates the LPS-induced ALI by means of anti-oxidation and inhibits PMN aggregation,which are both mediated by HO-1 partly.     

Protective role of endogenous hydrogen sulfide in cholecystokinin octapeptid against acute lung injury induced by lipopolysaccharide

AIM: To explore the role of endogenous hydrogen sulfide (H₂S) in the mechanism of cholecystokinin octapeptide (CCK-8) to alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: Eighty-four Sprague-Dawley rats were randomly divided into seven groups: control, LPS (instilled intratracheally to reproduce the model of ALI), NaHS (H₂S donor) +LPS, propargylglycine ［inhibitor of cysathionine-γ-lyase (CSE), PPG］+LPS, CCK-8+LPS, PPG+CCK-8+LPS and CCK-8 group. Animals were sacrificed at 4 h and 8 h after agent instillation. The wet and dry ratio (W/D) of the lung weight was measured and calculated. Morphological changes of lung tissues were observed. H₂S concentration in plasma, malondialdehyde (MDA) content, myeloperoxidase (MPO) and CSE activities in the lung were determined. Furthermore, the level of P-selectin of lung tissue was measured by radioimmunoassay, the CSE mRNA expression in the lung was detected by RT-PCR, and the protein content in bronchoalveolar lavage fluid (BALF) was detected. RESULTS: Compared with control, severe injury of lung tissues and increase in W/D, protein content in BALF, MDA content, MPO activity and P-selectin level in the lung were observed in rats treated with LPS. LPS also lead to a drop in plasma H₂S concentration, lung CSE activity and CSE mRNA expression. Administration of NaHS before LPS could attenuate the changes induced by LPS, while H₂S concentration, CSE activity and CSE mRNA expression were higher than those in LPS group. However, pre-treatment with PPG exacerbated the lung injury induced by LPS, H₂S concentration, CSE activity and CSE mRNA expression were lower than those in LPS and CCK-8 +LPS group, respectively. CONCLUSION: CCK-8 attenuates LPS-induced acute lung injury by means of anti-oxidation and inhibition of PMN adhesion and aggregation, both of which are mediated by endogenous H₂S.     

Inhibiting role of polydatin to expression of intercellular adhesion molecule-1 induced by lipopolysaccharide on vascular endothelial cells

AIM and METHODS:To investigate expression of intercellular adhesion molecule-1(ICAM-1) on human umbilical vein endothelial cells (HUVEC) induced by lipopolysaccharide (LPS) , and inhibiting role of polydatin by cellular immune fluorescent staining and laser confocal microscope scanning technology. RESULTS: Compared with basic expression of ICAM-1 on HUVEC, the ICAM-1 expression was enhanced significantly after stimulated by LPS from 8 h to 36 h, dose-dependent relation appeared between expression of ICAM-1 and LPS. ICAM-1 expression on endothelial cells treated only by polydatin had no abvious change,but inducing role of LPS to expression of ICAM-1 was inhibited significantly by polydatin pretreating endothelial cells. CONCLUSION:The expression of ICAM-1 on endothelial cells can be promoted by LPS , and polydatin can inhibit LPS-induced ICAM-1 expression.     

Effects of Astragalus polysacharin on fibroblast proliferation and adhesion between HUVECs and white cells

AIM: To investigate the effects of Astragalus polysacharin(APS) on human fibroblast and human umbilical vein endothelia cell (HUVEC) proliferation, as well as its acts on adhesion between white cells and HUVECs. METHODS: Human fibroblasts from distal and proximal skin away the ulcer were cultured as normal fibroblasts(NF) and wounded fibroblasts(WF). MTT assay was used for detecting cell proliferation, Rose Bengal staining and fluorescence immunohistology assay were used for examining the adhesion of human polymorpho-nuclear cell(PMN) and TPH-1 to HUVECs. RESULTS: 2.44-156 mg/L APS promoted WF proliferation, and 2.44-39 mg/L APS also promoted NF proliferation, but it did not show any proliferating effect on HUVECs. APS inhibited the adhesion of PMN or TPH-1 to HUVECs induced by tumor necrosis factor(TNF). At 25-100 mg/L, it also inhibited both VCAM-1 and ICAM-1 expression in HUVECs induced by TNF. Treatment with APS for 12 h also inhibited CD44 expression in HUVECs. CONCLUSION: APS shows mitogenic activity on both human normal and wounded fibroblasts. It also exerts anti-inflammation effects by inhibiting adhesion molecule expression and adhesion of white cells to HUVECs.     

Effect of lipopolysaccharide on viability and secretion function of human umbilical vein endothelial cells

AIM: To observe the direct effect of lipopolysaccharide (LPS) on secretion of endothelin-1 (ET-1) and nitric oxide by human umbilical vein endothelial cell and cell viability of the secretor. METHODS: The third passage of human umbilical vein endothelial cells were incubated with different concentrations of LPS (1 g/L, 100 mg/L, 10 mg/L, 1 mg/L, 100 μg/L, 10 μg/L, 1 μg/L) for 6 hours, and the culture supernatants were collected. The concentrations of ET-1 were determined by radioimmunoassay, the concentrations of nitric oxide were determined using Greiss's method. The viabilities of cells were measured by MTT method. RESULTS: The concentration of ET-1 (pg/L) of normal control group was 251.64±10.90. The concentrations of ET-1 (pg/L) of LPS treated groups were 220.85±19.14, 278.67±15.45, 306.40±11.60, 312.87±33.50, 324.38±17.02, 291.49±14.30, 282.11±13.38, respectively (each group compared with normal control group, P<0.05 or P<0.01). The concentration of NO_x (μmol/L) of normal control group was 629.46±13.36. The concentrations of NO_x (μmol/L) of LPS treated groups were 732.58±23.21, 669.87±9.32, 661.24±16.80, 650.33±13.24, 606.59±12.94, 626.75±9.83, 627.61±5.61, respectively (each group compared with normal control group, P<0.05 or P<0.01). The viabilities of endothelial cells of LPS treated groups were 74%, 81%, 86%, 88%,91%, 93%, 93%, respectively. CONCLUSION: LPS of lower concentrations had no significantly lethal effect on human umbilical vein endothelial cells, but enhanced secretion of ET-1 and inhibited NO production. LPS in higher concentrations showed significant lethal effect on human umbilical vein endothelial cells, inhibited secretion of ET-1 and enhanced NO production.     

Effect of cGMP-dependent protein kinase on endothelial cytoskeleton changes

AIM: To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of burn shock. METHODS: Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with 10% burn serum or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 were used to pretreat the endothelial cells before the administration of burn serum or PKG activator 8-Br-cGMP. RESULTS: Exposures to burn serum and 8-Br-cGMP led to a rapid time-dependent increase in endothelial PKG activity and the polar distribution of intracellular filamentous actin, and preincubation with KT5823 abolished those effects. CONCLUSIONS: The results suggest that burn serum induces PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably contributes to the endothelial hyperpermeability after burn shock.     

Effects of pueraria lobata isoflavones on myocardiac cell hypertrophy induced by Neuropeptide-Y in vitro

AIM: To investigate the effects of pueraria lobata isoflavones (PLIs) on neuropeptide-Y(NPY)-induced myocardiac cell hypertrophy in vitro. METHODS: Rat cardiomyocytes cultured in vitro were randomized to control, NPY, NPY+PLIs and PLIs group. The protein synthesis in cardiomyocytes was assessed by [³H-Leu] uptake, C-jun mRNA by RT-PCR and CaN activity by histochemistry. RESULTS: The rate of [³H-Leu] uptake, the C-jun mRNA expression and the CaN activity of myocardiac cells in 100 nmol/L NPY group were higher than those in control (P<0.01,P<0.05). On the other hand, the effects of NPY on myocardiac cells in vitro were inhibited by PLIs at concentrations of 1-3 mg/L.CONCLUSION: NPY could induce the myocardiac cell hypertrophy in vitro, which was inhibited by PLIs. The underlying mechanism may be attributed to the inhibition of CaN activity and blocking of Ca²⁺/CaM-dependent pathway.     

Characteristics in the secretion of liver TNF-α induced by infusion of LPS into veins in goats

AIM: To study the role of liver in immune regulation in experimental endotoxemia. METHODS: 17 castrated male goats were subjected to simultaneously installing catheters in jugular, hepatic and portal veins by surgery. Four days later, lipopolysaccharide (LPS) was infused in term of three groups as followings: In group ①, LPS of 20 EU (endotoxin unit, EU)·kg^-1 was infused into portal vein; In group ②, LPS of 20 EU·kg^-1 was infused into jugular vein and LPS of 1 500 EU·kg^-1 infused into jugular vein in group ③. Before and after infusion, blood samples were collected from the three veins through the catheters for 8 h.The plasma levels of TNF-α were measured by RIA. RESULTS: In group ①, the plasma TNF-α levels of hepatic and portal vein rose to peak value at 5 h, but that of the jugular vein did not changed. In group ②, the plasma TNF-α levels in hepatic vein rose to peak value at 3 h. The TNF-α levels of jugular vein rose to peak value at 1 h and the one in portal vein enhanced continuously between 0-8 h. In group ③, the plasma TNF-α levels in jugular, hepatic and portal vein rose to significant peaks at 1 h simultaneously. CONCLUSION: During experimental endotoxemia,liver showed different dynamic characteristics in TNF-α secretion according to the pathway and doses of LPS delivery.     

Effects of amlodipine on apoptosis of vascular endothelial cells induced by oxyeterols

AIM:To investigate the apoptosis of endothelial cells (EC) induced by oxysterols and to examine the effect of amlodipine on the apoptosis induced by oxysterols.METHODS:Light microscope, electron microscope, DNA agarose gel electrophoresis and flow cytometry were used to detect the apoptosis of EC.RESULTS:The characteristic morphological features of apoptosis were observed under light and electron microscope; DNA electrophoresis showed"DNA Ladder"; Flow cytometry showed the sub-G1 peak, apoptotic rate is 32.25% and 23.04% in Triol-treated and 25-OH-treated groups, respectively. While treated EC with amlodipine at the same time, the apoptotic rate decreased significantly.CONCLUSION:Both Triol and 25-OH can induce apoptosis of EC, which can be inhibited by amlodipine.     

Effect of lipopolysaccharide on apoptosis of human umbilical vein endothelial cells

AIM: To explore the mechanism of lipopolysaccharide (LPS) on vascular endothelial cell(VEC) damage. METHODS: By using cytometry techniques, we studied the effects of LPS on apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: LPS was able to induce apoptosis of HUVECs in a time-dose-dependent fashion.CONCLUSION:Apoptosis might play a role in LPS-induced damage of vascular endothelial cells.     

Effect of atrial natriuretic peptide on acute lung injury induced by lipopolysaccharide

AIM: To investigate effect of atrial natriuretic peptide (ANP) on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rat. METHODS: Mean arterial blood pressure (MAP) was recorded with model 6280 physiology intelligentialize grapher, nitric oxide (NO) and endothelin (ET) concentrations in plasma were measured after lipopolysaccharide (LPS) or following LPS ,ANP was injected into vein in rats. After experiment,lung water as well as pulmonary histopathological changes was measured and observed, respectively. RESULTS: Administration of LPS elicited a persistence decrease in MAP (8.1 kPa±2.6 kPa,at 4 h,P<0.01 vs control); NO and ET concentration in plasma was evident higher than that in control group, respectively (P<0.01); Wet-dry ratio of lung was higher than that in control group (5.15±0.43,at 4 h) (P<0.05); Alveolus detelectasis was observed and pulmonary mesenchyme was thicker than that in control group. No erythrocyte and leukocytes in alveolus,which show an interstitial pulmonary edema, was observed in LPS+ANP group, ANP maintained MAP at higher levels (13.35 kPa±2.93 kPa, at 4 h, P<0.05 vs LPS) after an transient decline when LPS was injected; NO and ET concentration of plasma had all significantly decrease, respectively (P<0.05 vs LPS, at 4 h); Wet-dry ratio of lung was lower than LPS group (4.57±0.35, P<0.05). Compared with control group the ratio was not evident difference (P>0.05); The histopathological of lung displayed markedly improved. CONCLUSION: ANP attenuates ALI induced by LPS in the rat. The effect of ANP may be via decreasing secretion of ET,NO and regulation arterial blood pressure.