Changes of pulmonary apoptosis and NOS mRNA in the lipopolysaccharide-induced acute lung injury

AIM: To observe the chronological changes of pulmonary apoptosis and the expression of iNOS mRNA,nNOS mRNA and eNOS mRNA in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to investigate the mechanisms of ALI.METHODS: Rats were randomly divided into 2 groups: control group and LPS treated group.The rats were injected with either saline or LPS and killed at 1,3,6,9 and 12 h after LPS injection.The expressions of iNOS mRNA,nNOS mRNA and eNOS mRNA in the lung tissue were respectively measured with RT-PCR methods.Apoptosis and expressions of Bcl-2 and Bax were respectively determined by flow cytometry (FCM) and immunohistochemistry (IHC).The pathological changes of lung tissue were observed under light and electron microscope.RESULTS: Compared with that in control group,the expression of iNOS mRNA was significantly increased at 3,6,9 and 12 h after administration of LPS (P<0.05).The eNOS mRNA was significantly decreased at 3,6,9 and 12 h after administration of LPS (P<0.05).The nNOS mRNA had no significant change during the 12 h in LPS group.Degree of ALI was gradually worsened after administration of LPS.Apoptosis of pulmonary cells was significantly increased,and reached the top level at 9 h after administration of LPS (P<0.01).The expression of Bcl-2 was markedly decreased and the expression of Bax was significantly enhanced in alveolar and airway epithelial cells in LPS treated group.CONCLUSION: The expressions of iNOS mRNA,eNOS mRNA and nNOS mRNA are not identical in LPS-induced acute lung injury.NOS regulates the apoptosis of pulmonary cells through affecting the balance of Bcl-2 and Bax.     

Effects of NG-nitro-L-arginine on LPS-induced lung injury in rats

AIM: To investigate the effects of nitric oxide (NO) and NO synthase (NOS) inhibitor N^G-nitro-L arginine (L-NA) on LPS induced-lung injury in rats. METHODS: Forty healthy male SD rats, weighing 300±20 g, were used. The animals were anesthetized with 20% urethane 1 g·kg^-1. Common carotid artery (CAA) and jugular vein were exposed through a median incision in the neck. Mean arterial pressure (MAP) was measured through a pressure transducer connected with intubation of CAA. The animals were randomly divided into five groups: group 1: control; group 2: LPS (5 mg·kg^-1, iv); group 3: high dose L-NA (20 mg·kg^-1 intraperitoneal injection, ip); gropu 4: middle dose L-NA (10 mg·kg^-1, ip); group 5: low dose L-NA (5 mg·kg^-1, ip). Group1 : 0.9% saline solution was given and the animals were killed 6 h after the saline solution. Gruop 2: saline solution was given 3 h after LPS and the animals were killed 3 h after administration. Group 3, 4 and 5: L-NA was given 3 h after LPS iv and the animals were killed 3 h after administration, respectively. The pulmonary was removed immediately. The pulmonary coefficient and water content in pulmonary tissue were calculated (%). The NO₂^-/NO₃^- content in plasma, MDA content and NOS, SOD activity in the pulmonary tissue were measured. RESULTS: L-NA significantly decreased pulmonary coefficient and water content in pulmonary tissue and ameliorated LPS induced lung injury. The effect in high dose group was better than that in low dose group. L-NA significantly decreased NO₂^-/NO₃^- content in plasm, decreased MDA content and inhibited NOS activity and enhanced SOD activity in the pulmonary tissue. CONCLUSION: It may be concluded that L-NA has a beneficial effect on lung injury induced by LPS.     

Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system in acute lung injury induced by LPS in rats

AIM: To observe the changes of endogenous hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) system while acute lung injury induced by LPS in rats. METHODS: Eighty rats were randomly divided into six groups (n=8): Ⅰ, control group;Ⅱ, LPS 1 h group; Ⅲ, LPS 3 h group; Ⅳ, LPS 6 h group; Ⅴ, LPS 9 h group; Ⅵ, LPS 12 h group. The ALI model of rats was prepared with LPS. The rats were respectively killed at 1, 3, 6, 9 or 12 h after administration of LPS. The morphological changes of lung tissues were observed by light and electron microscope. The lung coefficient and the wet-to-dry weight ratio were measured. The contents of IL-1β and IL-10 in serum, the H2S level in plasma and the CSE activity in lung tissue were respectively detected. RESULTS: ⑴ In LPS 1 h group, the morphology, the lung coefficient, the wet-to-dry weight ratio, the H2S level and the CSE activity showed no changes compared with the control group. The contents of IL-1β and IL-10 were increased compared with the control group (IL-1β, P<0.05;IL-10, P<0.01). ⑵ In LPS 3 h, 6 h, 9 h and 12 h groups, compared with the control group, the lung tissues were significantly damaged, the lung coefficient and the wet-to-dry weight ratio were significantly increased respectively (LPS 3 h, P<0.05; LPS 6 h, 9 h, 12 h, P<0.01). The contents of IL-1β and IL-10 in serum were markedly increased (P<0.01). The H2S level in plasma and the CSE activity in lung tissue were significantly decreased (P<0.01).CONCLUSION: The changes of inflammatory cytokines may be the pathological foundation of the ALI induced by LPS and the endogenous hydrogen sulfide/cystathionine-γ-lyase system is possibly involved in the formation of the ALI.     

Effect of rhSOD on pulmonary nuclear factor-kappa B and MIP-1α expression in meconium-induced acute lung injury

AIM: To evaluate the role and mechanisms of recombinant human superoxide dismutase (rhSOD) in meconium-induced acute lung injury (ALI) by evaluating pulmonary MIP-1α and NF-κB expression. METHODS: 24 health male Sprage-Dawley rats were randomized to 3 groups (8, each group), followed by intratracheal (IT) administration with (1) saline at 1 mL/kg (control group); (2) 20% human newborn meconium suspension at 1 mL/kg, followed by saline at 1 mL/kg (Mec/saline group); (3) 20% human newborn meconium suspension at 1mL/kg, followed by rhSOD at 20 mg/kg (Mec/rhSOD group). The animal was killed 24 h after treatment. The measurements included the bronchoalveolar lavage (BAL) cell count, RT-PCR analysis of pulmonary MIP-1α mRNA expression, Western blotting analysis of pulmonary NF-κB expression. RESULTS: Meconium-induced ALI was characterized by increased BAL cell count, increased expressions of pulmonary MIP-1α mRNA and NF-κB protein ［(4.68±1.40)×109 cells/L vs (0.53±0.19)×109 cells/L, 3.60±0.75 vs 1.56±0.33, 0.72±0.31 vs 0.23±0.12, respectively in control rats, all P<0.01］. IT administration of rhSOD early in the ALI rat significantly decreased meconium-induced BAL cell count ［(3.13±0.77)×109 cells/L vs (4.68±1.40)×109 cells/L in Mec/saline rats, P<0.01］, inhibited the expression of pulmonary MIP-1α mRNA (2.20±0.39 vs 3.60±0.75, in Mec/saline rats, P<0.01) and NF-κB protein (0.44±0.21 vs 0.72±0.31 in Mec/saline rats, P<0.05). CONCLUSION: The early IT administration of rhSOD in ALI rat following meconium aspiration protects lung from inflammatory injury through inhibiting meconium-induced pulmonary MIP-1α mRNA and NF-κB protein expression.     

中甘21号

AIM: To observe the pathological role of 3-nitrotynosine (3-NT) on Escherichia coli LPS-induced vascular hyporeactivity in rats and the therapeutic effect of antioxidants. METHODS: Forty male SD rats weighting from 200 g to 250 g were randomly divided into four groups: the control group (n=10); LPS shock group (n=10); uric acid-treated group (n=10); melatonin-treated group (n=10). 6 h after LPS shock, phenylephrine (0.5-2.5 μg·kg-1) was applied intravenously to all groups and the percentage increase in MAP was detected, respectively. The concentration-response curve of aorta rings from all groups rats were obtained by cumulative addition of phenylephrine (PE), and PE Emax, EC50 were calculated. The concentrations of plasma malondialdehyde (MDA), nitrate/nitrite and 3-NT were assayed in all groups 6 h after LPS shock. RESULTS: The MAP level induced by PE significantly decreased to 54.60% in LPS shock rats compared with the control (P<0.05). However, PE induced MAP level increased 37.70% and 43.05% in uric acid and melatonin treated rats, respectively, compared with the LPS shock rats (P<0.05). The maximum response and EC50 to PE were significant reduced in LPS shock rats ［Emax, 35.30%±9.80%; EC50, (15.70±4.50)nmol/L］ compared with control group ［Emax, 100%; EC50, (4.71±2.04)nmol/L, P<0.05］; but the reactivity of aorta to PE was improved obviously in uric acid and melatonin treated groups (P<0.05). The plasma concentration of MDA, nitrate/nitrite and 3-NT were much lower in uric acid and melatonin groups than those in LPS shock group (P<0.05). CONCLUSIONS: 3-NT is an important pathological factor on vascular hyporeactivity in LPS shock. Antioxidants effectively improve α-adrenergic receptor-mediated vascular reactivity in LPS shock rats partially by removing lipid peroxidative production, reducing nitric oxide and 3-NT biosynthesis in LPS shock. These results suggest that antioxidants have potential beneficial therapeutic effect for septic shock patients.     

Effect of mesenteric lymph duct ligation on actue lung injury in rats

AIM：To explore the effect of mesenteric lymph duct ligation against actue lung injury (ALI) in rats.METHODS：45 Wistar rats were divided into three groups：the ligation group,the non-ligation group and sham operated group,and the two-hit model was established by hemorrhage and LPS injection.Mesenteric lymph was diverted by ligating mesenteric lymph duct in ligation group.All rats facilitated blood withdrawal for blood sample to arterial gas analysis after 24 hours.Then the WBC,NO,NOS,MDA,SOD and lung permeability index (LPI) were determined in bronchoalveolar lavage fluid (BALF),the MPO and ATPase activity were determined in lung homogenate.The ultrastructure was also observed.RESULTS：After two-hit,the PaCO2,the total cells and PMN,the NO2-/NO3-,NOS and MDA content in BALF and MPO activity in lung homogenate and LPI in non-ligation group were significantly increased than those in sham operated group.PaO2 and pH in arterial blood,SOD in BALF and the ATPase in lung homogenate were significantly lower (P<0.01 or P<0.05).The total cells and PMN,MDA,NO2-/NO3- in BALF,LPI in ligation group were significantly increased than those in sham operated group,and SOD in BALF was significantly lower (P<0.01 or P<0.05).The pH and PaO2 in arterial blood,the ATPase in lung homogenate in ligation group were significantly increased than those in non-ligation group,and the PaCO2,the total cells,PMN,NO2-/NO3-,NOS,MDA in BALF,LPI,and MPO in lung homogenate in ligation group were significantly lower than those in non-ligation group (P<0.01 or P<0.05).The injury of pulmonary vascular endothelium in ligation group was lighter than that in non-ligation group.CONCLUSION：The ligation of mesenteric lymph duct attenuates the ALI of rats.Mesenteric lymph might play an important role in the pathogenesis of ALI.     

Meconium induces formation of nitrotyrosine and expression of inducible nitric oxide synthase in the rat lung

AIM: To evaluate the contribution of inducible nitric oxide synthase (iNOS) and nitrotyrosine to acute lung injury (ALI) in rats with meconium aspiration. METHODS: 16 health male Sprage-Dawley rats were randomized to control group and meconium group, followed by intratracheally administration of 1 mL/kg saline or 1 mL/kg 20% human newborn meconium suspension. The animals were killed after 24 h of treatment. The measurements included bronchoalveolar lavage fluid (BALF) cell count, pulmonary myoloperoxidase (MPO) activity and nitric oxide (NO) level. Western bloting was used to determine the expression of pulmonary nitrotyrosine-a specific “footprint” of peroxynitrite and iNOS. RESULTS: Compared to control group, the rats in the meconium group had increased BALF cell counts ［(4.04±1.01)×109cells/L vs （0.53±0.19)×109cells/L］, pulmonary MPO activity ［(1.49±0.22）U/g wet lung tissue vs (0.62±0.16) U/g wet lung tissue］, NO level ［(12.77±5.00） mmol/g protein vs （4.89±1.32) mmol/g protein］, increased expression of nitrotyrosine and iNOS (0.46±0.19 and 1.49±0.60 vs 0.15±0.04 and 0.09±0.04, respectively), all P<0.01. CONCLUSIONS: Meconium results in an increase in expression of pulmonary iNOS, leading to over production of NO and nitrotyrosine, which may be of pathogenic importance in the ALI with meconium aspiration.     

Mechanisms of hepatocytic mitochondria damage following septic shock

AIM: To study mechanism of hepatocytic mitochondria damage following septic shock. METHODS: 30 SD rats were randomly divided into three groups: sham operation group, 12 h cecal ligation and puncture (CLP) group and 16 h CLP group. The model of septic shock was made by cecal ligation and puncture. The liver mitochondria respiratory control rate (RCR), phosphate/oxygen (P/O) and ATPase activities were assayed. RESULTS: In 12 h CLP group mean artery pressure (MAP) [(9.54±1.26)kPa] was significantly lower than sham operation group [(14.58±1.32)kPa,P<0.05]. However, mortality was obviously higher than sham operation group (P<0.05), the liver mitochondria respiratory control rate (1.27±0.25), phosphate/oxygen (1.67±0.34) and Na⁺-K⁺-ATPase (40.80±3.45), Ca²⁺-ATPase (58.00±2.43), Mg²⁺-ATPase (78.30±4.16), Ca²⁺-Mg²⁺-ATPase(2.70±2.25) activities decreased strikingly. The difference between 12 h CLP group and sham operation group was significant (P<0.05), 16 h CLP groups was more lower than 12 h CLP group. As RCR, P/O and ATPase activities were significantly reduced, mortality significantly increased. Futhermore, obvious positive correlation was showed between them (r=0.892,P<0.01;r=0.834,P<0.01). CONCLUSION: Liver mitochondria function of ingestion-oxygen and phosphorus-acidification are decreased and membrane fluxion is weaken. Energy metabolism is blocked and Ca²⁺-Mg²⁺ shows imbalanced. All of them cause hepatocytic mitochondria injury following septic shock.     

Expression of intercellular adhesion molecule in lung tissues of experimental acute lung injury and the effect of rhubarb

AIM:To approach the relationship between the expression of intercellular adhesion (ICAM-1 mRNA) and acute lung injury (ALI) as well as the mechanisms of rhubarb in the prevention and treatment of the lung injury. METHODS:ALI animal model was performed by Lipopolysaccharide (LPS). The rats were divided into 4 groups: LPS group, control group, rhubarb+LPS group and dexamethasone+LPS group. Histopathological examination and biological markers were measured for the lung specimens. Molecular hybridization method was used to determine the expression of ICAM-1 mRNA. RESULTS:The ICAM-1 mRNA expression in the lung tissues of LPS group significantly increased compared with control group (P＜0.01), rhubarb and dexamethasone had the action of decreasing the ICAM-1 mRNA expression (P＜0.05, P＜0.01); pathologic changes and the biological markers of ALI significantly decreased or ameliorated. CONCLUSION:The increase in the expression of ICAM-1 mRNA in the lung tissues of ALI is involved in the formation of ALI. Rhubarb and dexamethasone can ameliorate the lung damage, mechanism of which may be related to the inhibition of ICAM-1 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.     

Effect of heat shock protein 70 expression induced by glutamine on vascular hyporeactivity in rats caused by LPS

AIM: To observe the effect of heat shock protein 70(HSP70) expression induced by glutamine on Escherichia coli lipopolysaccharides(LPS)-induced vascular hyporeactivity in rats.METHODS: Twenty four healthy male Sprague-Dawley rats were randomly divided into: the control group (n=8);LPS shock group (n=8);glutamine(Gln) treated group (Gln 0.75 g·kg-1 iv,n=8).6 h after LPS shock,phenylephrine (PE,0.5-2.5 μg·kg-1 ) was applied intravenously to all groups and the percentage increase in mean arterial pressure(MAP) was detected,respectively.The concentration-response curves of aorta rings were obtained by cumulative addition of phenylephrine (PE),and PE Emax,EC50 were calculated.The blood concentration of malondialdehyde (MDA),TNF-α and IL-6 were assayed in all groups 30 min and 360 min after LPS shock,respectively.The expressions of HSP70 from heart and aorta were also assayed after 6 h LPS shock.RESULTS: The MAP level induced by PE significantly decreased by 51.4% in LPS shock group compared with the control (P<0.05).However,PE induced MAP level increased by 17.5% in Gln group compared with LPS shock group (P<0.05).Emax and EC50 to PE were significant reduced in LPS shock group compared with control group (P<0.05),but significantly improved in Gln group (P<0.05).The expressions of HSP70 from heart and aorta were much higher in Gln group than those in LPS shock group (P<0.05).The blood concentrations of TNF-α,IL-6 and MDA were much lower in Gln group than those in LPS shock group.CONCLUSION: Glutamine effectively improves α-adrenergic receptor-mediated vascular reactivity through inducing the expression of HSP 70,reducing inflammatory cytokine release and peroxide biosynthesis in LPS shock.These results suggest that glutamine have potential beneficial therapeutic effect for septic shock patients.     

Effect of ghrelin on iNOS expression in alveolar macrophages and lung tissues in rats with sepsis-associated lung injury

AIM: To investigate the effect of ghrelin on inducible nitric oxide synthase (iNOS) expression in alveolar macrophages and lung tissues in sepsis-induced acute lung injury (ALI) rats. METHODS: The septic rat model was established by cecal ligation and puncture (CLP). Male SD rats were divided into sham group, CLP group and CLP+ghrelin group. The rats in the former 2 groups were further divided into 3 subgroups, which were 6 h, 12 h and 20 h post-operation groups. Ghrelin was administered by intraperitoneal injection at 3 h and 15 h after operation in ghrelin group. The samples were harvested 20 h after operation. The mRNA expression of iNOS in alveolar macrophages collected from bronchoalveolar lavage was detected by RT-PCR. The protein levels of lung iNOS were measured by Western blotting. The lung pathological examination was performed 20 h after operation. RESULTS: In CLP group, the mRNA expression levels of iNOS in the alveolar macrophages were 1.33±0.05, 1.44±0.08, 1.57±0.11 at 6 h, 12 h and 20 h after CLP, respectively, which were higher than that in sham group, but did not show time correlation. However, it was lower in CLP group than that in CLP+ghrelin group at 20 h after CLP (2.27±0.37, P<0.05). At 20 h after CLP, the protein level of lung iNOS was decreased in CLP+ghrelin group (0.87± 0.03) as compared with CLP group (1.08±0.05). Compared with sham group, the histopathological score was increased in both CLP group and CLP+ghrelin group, but it was lower in CLP+ghrelin group (5.83±0.477) than that in CLP group (7.83±0.75). CONCLUSION: Ghrelin treatment improves the degree of ALI. During 6 h to 20 h after CLP, the mRNA expression of iNOS in alveolar macrophages was elevated, but the difference was not seen as the time went on. Ghrelin up-regulates the mRNA expression of iNOS in alveolar macrophages and inhibits iNOS expression in lungs of septic rats.     

Effect of endogenous H2S on pulmonary hypertension during acute lung injury induced by LPS and its interaction with NO

AIM: To investigate the effect of H2S on pulmonary artery hypertension during acute lung injury induced by LPS and the interaction between the systems of hydrogen sulfide (H2S)/cystathionine-β-lyase (CSE) and nitric oxide (NO)/nitric oxide synthase (NOS) in this process. METHODS: Seventy-two adult male rats were randomly divided into four groups: control group, LPS group, LPS+L-NAME group and LPS+propargylglycine (PPG) group. Mean pulmonary artery pressure (mPAP) of each rat was examined at 2 h, 4 h, 6 h and 8 h after treatment. H2S and NO contents in plasma, NO content, iNOS, cNOS and CSE activity in lung were measured at 4 h or 8 h after treatment, respectively. Expression of iNOS in lung tissue was also detected by immunohistochemistry technique, and the injury of lung was evaluated with morphological changes under microscope. RESULTS: LPS could induce severe lung injury, and mPAP, NO content, iNOS activity and its protein expression in LPS group significantly increased, but cNOS activity, H2S content and CSE activity decreased compared with those of control group. Administration of L-NAME before LPS could attenuate the changes induced by LPS. Pre-administration of PPG, a CSE inhibitor, exacerbated the injury by LPS, but there was no prominent variation in cNOS activity. CONCLUSION: Reduced endogenous H2S could increase pulmonary artery hypertension during acute lung injury induced by LPS. There is a negative effect between H2S/CSE system and NO/NOS system in this process.     

Cardioprotective effect of sevoflurane preconditioning on ROS production in rat heart with ischemia-reperfusion

AIM: To investigate the effects of sevoflurane preconditioning on the production of reactive oxygen species (ROS) and nitric oxide (NO), the activity of superoxide dismutase(SOD), glutathione peroxide(GPx) and catalase(CAT) in myocardial ischemia-reperfusion injury(IRI). METHODS: Sixty rats were randomly allocated to 8 groups. Following 2% sevoflurane preconditioning for 30 min, the left anterior descending artery was ligated for 30 min and then reperfused for 120 min in vivo. The infarction size of the hearts was measured with the staining of 2,3,5-triphenyltetrazoliumchloride. The myocardial apoptotic index was measured by the method of TUNEL. The ROS fluorescent probe dihydroethidium was used for the measurement of ROS. The myocardium was homogenized for the measurement of NO, SOD, GPx and CAT. To evaluate the effects of ROS and NO on the cardioprotection of sevoflurane preconditioning, ROS scavenger N-(2-mercaptopropionyl) glycine (2-MPG) or NOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) were employed to block their actions. RESULTS: Compared with control group, the production of ROS was induced by sevoflurane preconditioning before ischemia-reperfusion injury (12.0±0.8 vs 2.6±0.5, P<0.05) and decreased after ischemia-reperfusion injury (16.2 ±0.9 vs 24.9±1.3, P<0.05). 2-MPG decreased the elevation of ROS caused by sevoflurane preconditioning before ischemia-reperfusion injury (5.1±0.7 vs 12.0±0.8, P<0.05). No difference of ROS production between treating with 2-MPG+Sevo+IRI and with IRI (24.9±1.4 vs 24.9±1.3, P>0.05) was observed. Compared with control group, sevoflurane preconditioning also induced the generation of NO (34.5±3.2 vs 15.9±1.4, P<0.05) and the activity of SOD(1.5±0.5 vs 0.6±0.2, P<0.05), GPx(22.8±2.5 vs 12.7±2.2, P<0.05) and CAT(15.5±1.8 vs 11.2±1.4, P<0.05). 2-MPG blocked the increase in NO production and inhibited the activity of SOD,GPx,CAT. L-NAME also attenuated the activity of SOD,GPx,CAT. CONCLUSION: Sevoflurane preconditioning protects the rat heart against ischemia-reperfusion injury by reducing the infarction size and apoptosis. Production of ROS at sub-injury dose induced by sevoflurane preconditioning stimulates the myocardium to create SOD,GPx,CAT and NO, thus inhibiting the further formation of ROS and protecting the heart under the condition of ischemia-reperfusion.     

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.     

Changes of NO- and H2O2-dependent soluble guanylate cyclase pathway in the hypoxic hypercapnic pulmonary hypertension rats

AIM: To study the effect of hypoxia and hypercapnia on nitric oxide (NO) in plasma and superoxide dismutase (SOD), catalase (CAT), soluble guanylate cyclase (sGC), cyclic guanosine monophospholate (cGMP) in lung tissue in rats, and to explore the effect of NO- and H2O2-sGC pathway on the development of the pulmonary hypertension. METHODS: The model of hypoxic and hypercapnic 1, 2, 4-week group (HH 1 week, HH 2 weeks, HH 4 weeks) and control group was set up. NO content in plasma, CAT and SOD in rat lung were determined by spectrophotometry. The sGC activity in lung tissue was detected by enzyme kinetic analysis. cGMP content in lung tissue was examined with ［125I］-radioimmunoassay. RESULTS: The mean pulmonary artery pressure (mPAP) showed significantly higher in HH 1 week, HH 2 weeks and HH 4 weeks groups compared with control group (all P<0.05). NO concentration in plasma, CAT, SOD, basal or nitroprusside-or H2O2- stimulated sGC activity and cGMP concentration in lung homogenates were significantly lower (P<0.05, P<0.01, P<0.01, respectively) in HH 1 week, HH 2 weeks and HH 4 weeks groups compared with control group. CONCLUSION: The inhibition of NO- and H2O2-sGC pathway by hypoxia and hypercapnia plays an important role in the development of pulmonary hypertension.     

Protective effect of ambroxol against the lung damage in chronically hypoxic rats

AIM: To investigate the effect of ambroxol on pulmonary and vascular injury in chronically hypoxic rats. METHODS: 36 male Wistar rats were randomly divided into 3 groups: normal control,chronically intermittent hypoxia(CIH) and ambroxol precaution group(AP).The CIH and AP groups were made into the chronically hypoxic models.The mean pulmonary artery pressure(PAPM) and the levels of plasma superoxide dismutase(SOD) and plasma nitric oxide(NO),lipid peroxide(LPO) were determined. The levels of the lung homogenates SOD, LPO, NO and the changes in pulmonary vascular structure were also examined. RESULTS: The levels of plasma and lung homogenates SOD,NO in CIH group were respectively significantly lower than that of normal control and AP group( P <0.01),but the levels of plasma and lung homogenates LPO were significantly higher( P< 0.01). PAPM in AP group is significantly lower than that of CIH group( P< 0.01);The damage of pulmonary artery smooth muscle cells and extra cell matrix of AP group is much slighter than that of CIH group. CONCLUSION: Ambroxol might be an effective protector in chronically hypoxic rats.     

Antagonistic effect of bilirulin on acute lung injury induced by LPS in rats

AIM: To investigate the effect of bilirubin on acute lung injury (ALI) and the mechanism. METHODS: 30 male Wistar rats were divided into normal group, ALI group and bilirubin treatment group. Lung specimens were examined by histopathological technique. Lung index (LI) and lung permeability index (LPI) were measured. Moreover, white blood cell (WBC) count, neutrophil percentage (PMN%) and the content of protein (Pr) in the bronchoalveolar lavage fluid (BALF), as well as the contents of superoxide dismutase (SOD), malonaldehyde (MDA) and glutathione peroxidase (GSH-Px) in the lung homogenate were determined. RESULTS: (1) In ALI group: LI, WBC count, PMN%, Pr and LPI increased significantly compared with normal group (P<0.01). In bilirubin treatment group, all the values determined decreased compared with ALI group (P<0.01; P<0.05). No notable discrepancy between bilirubin treatment group and normal group (P>0.05) was observed. (2) In ALI group, the content of MDA was significantly higher (P<0.01), but the contents of SOD and GSH-Px were significantly lower than those in normal group (P<0.01). In bilirubin treatment group, the content of MDA decreased significantly (P<0.01) but the contents of SOD and GSH-Px increased significantly (P<0.01; P<0.05) compared with ALI group. No notable discrepancy between bilirubin treatment group and normal group was observed (P>0.05). CONCLUSION: Bilirubin relieves ALI induced by LPS in rats via antioxidation.     

Effects of human urotensin II on ischemia/reperfusion injury in isolated perfused rat hearts

AIM: To investigate the effects of human urotensin II (hUII) on ischemia/reperfusion (I/R) injury in isolated rat hearts. METHODS: In the ischemia/reperfusion (I/R) model of isolated perfused rat hearts, the effects of hUII pretreatment on cardiac function was monitored with cardiac function software of MFL Lab200. ATP, total calcium, and malondialdehyde (MDA) content in myocardium were detected. The coronary perfusion flow (CPF) and lactate dehydrogenase (LDH) activity in coronary effluent were measured during reperfusion. RESULTS: In the hUII pretreated group, the release of LDH from myocardium was lower [(78.3±18.1)U/L] than I/R group [(109.3±23.9) U/L, P< 0.05], with decreased contents of MDA and calcium in myocardium (decreased by 24% and 27%, respectively, P< 0.05) and an increased myocardial ATP content [(3.8±0.4)μmol/g dw vs (2.2±0.4)μmol/g dw, P< 0.05)]. At the same time, hUII pretreatment increased CPF [(5.4±0.7) mL/min vs (3.8±0.8) mL/min in I/R group, P< 0.05], reduced left ventricular end-diastolic pressure (LVEDP) by 20% ( P< 0.05) with increased±d p /d t max [(217±38) kPa/s and (119±18) kPa/s vs (173±29) kPa/s and (82±25) kPa/s in I/R groups, respectively, P< 0.05]. hUII pretreatment also increased natrite/natrate (NO₂^-/NO₃^-) content in coronary effluent [(52.2±12.0)μmol/L vs (32.1±10.2)μmol/L in I/R group, P< 0.05)]. CONCLUSION: hUII pretreatment attenuated I/R injury in isolated perfused rat hearts. The protective mechanism might be associated with NO-mediated coronary vasodilation.