[Hydrogen sulfide reduces lipopolysaccharide-induced acute lung injury and inhibits expression of phosphorylated p38 MAPK in rats].

To investigate the influence of hydrogen sulfide (H2S) on p38 MAPK signaling pathway during acute lung injury (ALI) caused by lipopolysaccharide (LPS), the rats were randomly divided into six groups: control group, LPS group, LPS + NaHS group, LPS + PPG (cystathionine-γ-lyase inhibitor) group, NaHS group and PPG group. The rats were sacrificed 6 h after injection and lung tissues were obtained. The structure of lung tissues and the number of polymorphonuclear leucocyte (PMN) was observed under optical microscope; the lung myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were tested; intercellular adhesion molecule-1 (ICAM-1) protein expression changes were detected by immunohistochemical staining; phosphorylated p38 MAPK (p-p38 MAPK) protein expression was detected by Western blotting. The results showed that the lung injury in LPS group was observed, at the same time the MPO activity, the content of MDA, ICAM-1 and p-p38 MAPK protein expressions, the number of PMN were all higher than those in control group (all P < 0.05). Pre-injection of NaHS alleviated the changes induced by LPS, while pre-injection of PPG aggravated those alterations (all P < 0.05). ICAM-1 and p-p38 MAPK protein expressions in lung tissue were positively correlated (r = 0.923, P < 0.01). The results suggest that H2S may reduce LPS-induced ALI through inhibiting the conjugation of p38 MAPK and reducing the expression of ICAM-1.

[Administration of hydrogen sulfide intraperitoneally reverses the hyporesponsiveness of rat pulmonary artery induced by lipopolysaccharide and its relationship with carbon monoxide].

OBJECTIVE: To explore the effect of hydrogen sulfide (H(2)S) on abnormal pulmonary artery reactivity induced by lipopolysaccharide (LPS) and its relationship with carbon monoxide (CO). METHODS: Forty eight rats were divided into four groups randomly according to table of random number: control group (normal saline, NS), LPS group, a donor of H(2)S sodium hydrosulfide (NaHS)+LPS group, and NaHS+NS group (n=12 in each group). Rats were given LPS by intratracheal instillation (0.8 ml/kg). 0.5 ml of NaHS (28 µmol/kg) was injected intraperitoneally 10 minutes before LPS or NS instillation and 2 hours after LPS or NS instillation in NaHS+LPS and NaHS+NS groups. Twelve hours after instillation of LPS, 6 rats from each group were sacrificed. The pulmonary artery rings (PARs) were prepared and the changes in cumulative relaxation response of PARs to NaHS were detected before and after incubation with an inhibitor of heme oxygenase-1 (HO-1) zinc protoporphyrinIX (ZnPPIX) using isolated vascular ring tension detecting technique. Twelve hours after LPS instillation, the remaining 6 rats in each group were sacrificed, and the contents of carboxyhemoglobin (COHb) in efferent pulmonary blood (EPB) and afferent pulmonary blood (APB) were measured, and the difference between the contents of COHb in EPB and that of APB was calculated to represent content of CO from pulmonary circulation. RESULTS: In the present study, compared with control group, after the instillation of LPS the percentage of relaxation response of PARs to NaHS was significantly declined [(75.72±7.22)% vs. (96.40±4.40)%, P<0.01]. After being incubated with ZnPPIX, the decreased relaxation response of PARs to NaHS induced by LPS was further depressed [(62.91±8.22)% vs. (75.72±7.22)%, P<0.01]. Administration of NaHS intraperitoneally reversed the hyporesponsiveness of PARs to NaHS, the percentage of relaxation response of PARs to NaHS was significantly increased [(94.65±8.45)% vs. (75.72±7.22)%, P<0.01]. However ZnPPIX also attenuated the effect [(83.75±9.76)% vs. (94.65±8.45)%, P<0.01]. NO significant changes were observed between NaHS+NS group and control group, also between the results before and after ZnPPIX incubation . Compared with control group, the difference between the contents of COHb in EPB and that of APB increased after instillation of LPS [(3.12±0.48)% vs. (2.12±0.32)%, P<0.05], which further increased after intraperitoneal administration of NaHS [(4.03±0.56)%, P<0.01]. CONCLUSION: The results suggested that intraperitoneal administration of H(2)S could reverse hyporesponsiveness of PARs to H(2)S induced by LPS, and the result might be related to an intensification of HO-1/CO system in pulmonary artery tissue.

[Effect of hydrogen sulfide on rat pulmonary artery reactivity and injury induced by lipopolysaccharide].

OBJECTIVE: To explore the effects of hydrogen sulfide (H2S) on abnormal pulmonary artery reactivity and injury induced by lipopolysaccharide (LPS). METHODS: Seventy-two rats were divided into four groups randomly according to table of random number: control group, LPS group, sodium hydrosulfide (NaHS) as a donor of H2S+LPS group and NaHS+normal saline (NS) group (n=18 in each group). Rats were challenged with 0.8 ml/kg LPS (200 microg/200 microl) by intratracheal instillation. NaHS (28 micromol/kg, 0.5 ml) was injected intraperitoneally 10 minutes before LPS instillation and 2 hours after LPS instillation. Twelve hours later, 6 rats from each group were sacrificed. Blood from carotid artery was collected to detect H2S content in serum. After that, pulmonary artery rings (PARs) were prepared carefully, then the contraction response of PARs to phenylephrine (PE, 10(-6) mol/L) and the endothelium-dependent relaxation response to acetylcholine (ACh, 10(-6) mol/L) were measured using isolated vascular ring tension detecting technique. Six rats from each group were sacrificed for determination of malondialdehyde (MDA) content of pulmonary artery, and the remaining 6 rats from each group were sacrificed for observation of morphological changes in pulmonary artery tissue. RESULTS: Compared with control group, after LPS instillation, the contraction response (g/mg) of PARs to PE increased greatly (0.86+/-0.20 vs. 0.56+/-0.13), the relaxation response to ACh significantly decreased [(65.18+/-7.05)% vs. (84.13+/-8.84)%]. MDA content (mmol/L) in pulmonary artery tissues increased (32.03+/-7.81 vs. 5.82+/-0.92), and H2S (micromol/L) content in serum decreased (175.23+/-27.36 vs. 238.12+/-16.38). Changes of all results were significant (P<0.05 or P<0.01). The pulmonary artery tissue and endothelium were injured. However, these changes were reversed by administration of NaHS intraperitoneally, the contraction response of PARs to PE decreased [(0.61+/-0.17) g/mg], the relaxation response to ACh increased [(82.92+/-9.71)%], MDA content in pulmonary artery tissue decreased [(16.88+/-3.54) mmol/L] and H2S content in serum increased [(242.70+/-38.80) micromol/L]. There was significant difference in all results (P<0.05 or P<0.01). The injury to the tissue induced by LPS were alleviated significantly. There was no statistical difference in above indexes between NaHS+NS group and control group, except for the level of H2S. CONCLUSION: Exogenous H2S could not only reverse abnormal vascular reactivity of PARs induced by LPS but also alleviate the injury to pulmonary artery tissue induced by LPS.

[Role of hydrogen sulfide/cystathionine-gamma-lyase system in acute lung injury induced by lipopolysaccharide in rats].

OBJECTIVE: To explore the role of hydrogen sulfide/cystathionine-gamma-lyase (H(2)S/CSE) system in lipopolysaccharide (LPS)- induced acute lung injury (ALI) in rats and the underlying mechanisms. METHODS: Sixty-four Sprague-Dawley (SD) rats were randomly divided into four groups: control, LPS (instilled intratracheally to induce ALI), sodium hydrosulfide (NaHS), propargylglycine (PPG). Animals were sacrificed at 4 and 8 hours (n=8) after administration of the above agents. Morphological changes in lung tissues were determined, H(2)S, nitrogen monoxide (NO) and carbon monoxide (CO) concentration in plasma were determined. Malondialdehyde (MDA) content, and myeloperoxidase (MPO), CSE, inducible nitric oxide synthase (iNOS), heme oxygenase (HO) activity of the lung were also determined. The level of P-selectin of lung tissue was measured by radioimmunoassay. Immunohistochemistry technique was performed to examine the expression of iNOS and HO-1 protein in lung tissues. RESULTS: Severe injuries of lung tissues and raised MDA content, MPO activity and P-selectin level were observed in rats treated with LPS. LPS also led to a drop in plasma H(2)S concentration and lung CSE activity. The enzyme activity of iNOS and HO, and their protein expression, plasma NO, and CO levels increased after LPS instillation (P<0.05 or P<0.01). Pre-administration of NaHS before LPS could attenuate the changes induced by LPS. Pre-administration of PPG exacerbated the injuries induced by LPS, with increased MDA content, MPO activity, P-selectin level, the plasma NO level, lung iNOS activity and its protein expression, but there was no prominent variation in CO level, HO activity and HO-1 protein expression compared with those of LPS group. CONCLUSION: Downregulation of H(2)S/CSE is involved in the pathogenesis of ALI induced by LPS. Endogenous and exogenous H(2)S provide protection against ALI, which may be explained by its anti-oxidative effects, attenuation of inflammatory over-reaction in lung induced by polymorphonuclear neutrophils, downregulation of NO/iNOS system and the upregulation of CO/HO-1 system.

[The expression of cholecystokinin-octapeptide receptor in vascular endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the expression of cholecystokinin-octapeptide receptor (CCK-R) mRNA, and observe the effect of lipopolysaccharide (LPS) on CCK-AR mRNA and CCK-BR mRNA expression in ECV-304. METHODS: The human umbilical vein endothelial cell line ECV-304 was cultured and treated with LPS in dosage of 0.01, 0.1, 1, 10 mg/L for 2 hours, or treated with LPS in dosage of 1 mg/L for 0.5, 2, 6, 12 hours. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the expression of CCK-AR mRNA and CCK-BR mRNA in ECV-304, and to analyse the sequences of the amplification products. RESULTS: Compared with control group, the expression of CCK-AR mRNA and CCK-BR mRNA was significantly upregulated in a dose-dependence manner after incubated with 0.01, 0.1 and 1 mg/L LPS for 2 hours (all P<0.05). However, the expression of CCK-AR mRNA showed no significant increase,while that of CCK-BR mRNA was increased, after being incubated with 0.01 mg/L LPS. The expressions of CCK-AR mRNA and CCK-BR mRNA in the 10 mg/L LPS group showed no significant difference compared with 1 mg/L LPS group (both P>0.05). The expression of CCK-AR mRNA and CCK-BR mRNA was significantly upregulated in a time-dependence manner after incubated with 1 mg/L LPS from 0.5 hour to 2 hours compared with control group (all P<0.05). After incubated with 1 mg/L LPS for 6 hours, the expression of CCK-AR mRNA and CCK-BR mRNA was significantly decreased compared with 2-hour group, but was still higher than that of control group (both P<0.05). Its expression was decreased further after being incubated with 1 mg/L LPS for 12 hours compared with the 6 hours group (both P<0.05), but showed no significant difference compared with the control group (both P>0.05). CONCLUSION: Both CCK-AR mRNA and CCK-BR mRNA are expressed in ECV-304. LPS can up-regulate the expression of CCK-AR mRNA and CCK-BR mRNA.

[The role of hydrogen sulfide in acute lung injury during endotoxic shock and its relationship with nitric oxide and carbon monoxide].

OBJECTIVE: To explore the role of hydrogen sulfide (H2S) in acute lung injury (ALI) during endotoxic shock (ES) and its relationship with nitric oxide (NO) and carbon monoxide (CO). METHODS: Sixty-four adult male SD rats were randomly divided into 4 equal groups: control group injected with normal saline via the caudal vein, lipopolysaccharide (LPS)-treated group injected with LPS to establish ES model, LPS + NaHS group injected with LPS and sodium hydrosulfide (NaHS, an exogenous H2S donor], and LPS + PPG group injected with LPS and polypropylene glycol (PPG, a H2S synthase inhibitor). The mean artery pressure (MAP) was measured via a polyethylene catheter in the right common carotid artery for 6 h. Then the rats were sacrificed with their lungs taken out to determine the lung water content, lung tissue malonyldialdehyde (MDA), NO, and CO contents, as well as lung tissue cystathionine-gamma-lyase (CSE), myeloperoxidase (MPO), nitric oxide synthase (NOS), and heme oxygenase (HO) activities. The H2S content in blood plasma was detected also. Morphological changes of the lung tissues were observed under light microscope and the index of quantitative assessment (IQA) of lung injury was calculated. Immunohistochemistry and Western blotting were used to detect the lung tissue inducible NOS (iNOS) and HO-1 protein expression. RESULTS: Compared with the control group, the MAP of the LPS group was significantly lower, the pathological changes in lung tissue was more obvious, and the IQA, lung water content, lung MDA content, lung MPO and CSE activities as well as plasma H2S content were all significantly higher (P < 0.05 or P < 0.01). Compared to the LPS group, the plasma H2S and lung CSE activity of the LPS + NaHS group were higher, the lung injury was more severe, and the MAP was lower. And compared to the LPS group, the MAP of the LPS + PPG group was higher, and the lung injury was milder (both P < 0.05). The eNOS activity in the lung tissue of the LPS group was (5.26 +/- 0.25) Uxmg(-1)xprot(-1), significantly lower than that of the control group [(6.45 +/- 0.42) Uxmg(-1)xprot(-1)]; and the iNOS activity and NO content of the LPS group were (12.6 +/- 0.6) Uxmg(-1)xprot(-1) and (144 +/- 25) micromol/L respectively, both higher than those of the control group [(10.5 +/- 0.7) Uxmg(-1)xprot(-1) and (68 +/- 5) micromol/L respectively] (P < 0.05 or P < 0.01). Compared with the LPS group, the lung tissue eNOS activity of the LPS + PPG group was significantly higher, and the iNOS activity [(10.2 +/- 0.4) Uxmg(-1)xprot(-1)], iNOS protein expression, and NO content [(74 +/- 5) micromol/L]were all significantly lower (P < 0.05 or P < 0.01). Compared with the LPS group, the lung tissue eNOS activity of the LPS + NaHS group [(4.81 +/- 0.23) Uxmg(-1)xprot(-1)] was significantly lower, and the iNOS activity [(14.6 +/- 0.4) Uxmg(-1)xprot(-1)], iNOS protein expression, and NO content [(217 +/- 18) micromol/L] were significantly higher (P < 0.05 or P < 0.01). The lung tissue HO activity [(173 +/- 31) pkat/g], HO protein expression, and CO content [(3.63 +/- 0.24)%] of the LPS group were all significantly higher than those of the control group [(125 +/- 22) pkat/g, (2.48 +/- 0.33)%, both P < 0.05], and the LPS + PPG group [(88 +/- 17) pkat/g, (2.98 +/- 0.23)%, both P < 0.05]. Compared to the LPS group, the lung tissue HO activity [(263 +/- 37) pkat/g], HO protein expression, and CO content [(4.35 +/- 0.32)%] of the LPS + NaHS group were all significantly higher (all P < 0.05). CONCLUSION: The increase of H(2)S generation participates in the lung tissue injury during ES and this event is related to eNOS activity decrease, iNOS activity increase that causes the production of large amount of NO. H2S up-regulates the HO-1/CO system in the lung tissues during ES, which may be the endogenous compensatory response against the injury.

[Inhibitory effect of melatonin on the expression of nuclear factor-kappaB during acute lung injury in rats].

OBJECTIVE: To investigate the protective effect of melatonin (MT) on lung tissue during acute lung injury (ALI) in rats and its possible mechanism. METHODS: Ninety-six Sprague-Dawley (SD) rats were randomly divided into four groups: control group, lipopolysaccharide (LPS) group, dexamethasone (DEX) and MT treatment group, with 24 rats in each group. Rat model of ALI was established by instilling LPS intratracheally, and DEX and MT were injected intraperitoneally. All rats in each group were sacrificed at 3, 6 and 12 hours after intratracheal instillation of LPS, and lung tissue samples were harvested. Myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents in lung tissue samples were detected in each group. In addition, the expression of nuclear factor-kappaB (NF-kappaB) was assessed with immunohistochemistry staining in lung tissues. RESULTS: Compared with control group, SOD activity in LPS group decreased at different time points significantly (P<0.05 or P<0.01), but MPO activity, MDA content and the expression of NF-kappaB increased obviously (P<0.05 or P<0.01); the administration of MT and DEX could mitigate above values significantly (P<0.05 or P<0.01). The changes in above each indexes were most obvious at 6 hours, either reaching the peak or the trough, respectively. CONCLUSION: MT possesses protective effect on lung tissues during ALI through scavenging free radicals and inhibiting the activation of NF-kappaB.

[The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells].

OBJECTIVE: To investigate the peroxynitrite damage to the lens epithelial cells (LEC) and the prevention of this damage by puerarin in vitro. METHODS: This paper was experimental study. Rabbit LEC were isolated and cultured and the third or forth passage LEC were used in this experiment The experiment groups included: (1) CONTROL GROUP: Heat-pathogen free saline (NS) 200 microl was added to the medium; (2) ONOO- group: ONOO- 200 microl was added to obtain the terminal concentration at 0. 5 mmol/L; (3) Puerarin group: 5 microg/ml ONOO- and 10 microg/ml puerarin were added simultaneously. Then, the cells were cultured and collected after 6,12 or 24 hours. The nitrotyrosine (NT), a symbol of the ONOO-, was tested with immunofluorescence technique. The expression of NT protein was examined with Western blot method. The cell morphology was observed with light microscope. Cell apoptosis was examined via DNA ladder, flow cytometry and Fas/FasL immunohistochemical staining. These datas were analyzed by one-way-ANOVA and q test. RESULTS: During the 6 to 24 hours of experiment period, green color could be observed in the cell nucleus and cytoplasm of control group. Staining ranged from yellow to brown-yellow, then to brown color were observed in STZ group. Staining ranged from faint green to yellow green or faint green color were observed in puerarin group. Slight expression of nitrotyrosine (NT) could be seen in the control group. A moderate to strong expression of NT was observed at different stages in the STZ group (A = 77.22 +/- 2.44, 145.00 +/- 3.94, 235. 8 +/- 5.97). At 6 hours, a slight expression of NT could be seen in the control group (A = 72.78 +/- 2.64), this increased at 12 hours (A =89. 94 +/- 3.01) and decreased at 24 hours (A = 74. 44 +/- 3.00). With computer photo-analysis, there were significant differences between the control, STZ and puerarin groups at different period during the experiment (q = 78.12, 82.76, 69.98, P <0. 01). In the control group, cell morphology and gene DNA ladder were normal, minor apoptosis could be observed but no expression of Fas/FasL in the membrane and cytoplasm of the cells. Distinctive cell morphology changes and the typical "ladder bands" as well as the expression of Fas/FasL could be observed in STZ group. All of these aspects were comparatively normal in puerarin group. CONCLUSIONS: The LEC apoptosis induced by ONOO- in vitro could be alleviated by puerarin. Fas/FasL cell signal transduction pathway may affect and strengthen the apoptosis process mediated by ONOO-.

[Relationship between hydrogen sulfide and myocardial damage in endotoxemic rats].

To investigate the changes and role of hydrogen sulfide (H2S) in myocardial damage in endotoxemic rats, a rat model of endotoxemia induced by injection of lipopolysaccharide (LPS) was developed. Male Wistar rats were divided into four groups: control group, LPS group, LPS + propargylglycine (PPG, a metabolic enzyme inhibitor of H2S) group and LPS + NaHS (H2S donor) group. The mean arterial pressure (MAP) of rats within 4 h was observed, TNF-alpha and H2S contents in plasma, TNF-alpha and H2S contents, lactate dehydrogenase (LDH) and myeloperoxidase (MPO) activity in cardiac muscles were determined. The morphological structure of cardiac muscle was observed. Administration of LPS caused a sustained fall in MAP within 4 h, and significant increases in TNF-alpha and H2S contents in plasma (P<0.05). Plasmic H2S content was negatively correlated with MAP (r = -0.936, -0.913 and -0.908 at 1, 2 and 4 h, respectively, P<0.05). LPS also induced increases in TNF-alpha and H2S contents, LDH and MPO activity in cardiac muscles and myocardial damage. Treatment with PPG reduced the increases in TNF-alpha and H2S contents in plasma, TNF-alpha and H2S contents, LDH and MPO activity in cardiac muscles, ameliorated the hypotensive effect and myocardial damage caused by LPS administration (P<0.05). However, treatment with NaHS increased TNF-alpha and H2S contents in plasma, TNF-alpha and H2S contents, LDH and MPO activity in cardiac muscles, and aggravated the hypotensive action and tissue injuries caused by LPS administration (P<0.05). It is suggested that hypotension and myocardial damage in endotoxemic rats are partly induced by increase in H2S content.

[Protective effect of endogenous carbon monoxide on organs during septic shock in rat and its mechanisms].

OBJECTIVE: To study the protective effect of endogenous carbon monoxide (CO) on lung and liver during septic shock in rat and its mechanism. METHODS: Septic shock model was replicated by cecal ligation and puncture (CLP). Ninety-six rats were randomly divided into sham operation group, CLP group, CLP+ hemin (Hm) group and CLP+zinc protoporphyrin (ZnPP) group. The carboxyhemoglobin (COHb) levels of in-flowing pulmonary blood (IPB) and out-going pulmonary blood (OPB) were determined at 2, 4 and 6 hours after treatments. Malondialdehyde (MDA) contents and superoxide dismutase (SOD) activities in the lung, liver and blood were also determined. Pathological changes in lung and liver were examined with light microscope, and immunohistochemical technique was used for analysis of heme oxygenase-1 (HO-1) protein expression and distribution in lung and liver. RESULTS: Compared with sham operation group, the COHb level in OPB and IPB as well as MDA contents of lung, liver and blood significantly increased in CLP group, while the SOD activities significantly decreased at different time points (P<0.05 or P<0.01), and the pathological changes and expressions of HO-1 in two tissues were more marked. However, in CLP+Hm group the results of MDA, SOD activities and pathological changes were reversed. The content of COHb increased compared to those of CLP group. Immunohistochemical studies showed that there were more HO-1 positive deposits in CLP+Hm group than those in CLP group. CONCLUSION: Increase in endogenous CO may play a protective role in lung and liver during septic shock.

The antagonism of cholecystokinin octapeptide-8 to the peroxynitrite oxidation on a diabetic cataractal rat model.

BACKGROUND: Cataracts is considered be formed because of an abnormal glucose metabolic pathway or oxidative stress. We explored the damaging role of ONOO- and antagonism of cholecystokinin octapeptide-8 (CCK-8) in diabetic cataractal rat lenses. METHODS: A diabetic cataractal animal model was established by peritoneal injection of streptozotocine (STZ). Thirty-six normal SD rats were taken as control group; seventy-two were given STZ (45 mg/kg) and then divided into STZ group and CCK-8 group (peritoneal injection CCK-8). STZ induced diabetic rats were treated with CCK-8 for 60 days. Lenses were examined with slit lamp at 20, 40 and 60 days. Immunofluorescent staining and Western blot analysis were used for determining nitrotyrosine (NT, a marker for ONOO-). PT-PCR and gene array analysis were used for determining the expression of inducible nitric oxide synthetase mRNA (iNOS mRNA) in lens epithelium (LEC). RESULTS: STZ group rats developed lens opacity by 20 days that reached a high level by 60 days after STZ injection. CCK-8 group rats delayed the cataract formation. CCK-8 group rats delayed the cataract formation. There was no distinct expression of NT and iNOS mRNA in control group. In STZ group, there were distinct expression of NT and upregulation of iNOS mRNA; however, CCK-8 group showed weak expression of NT and downregulation of iNOS mRNA. CONCLUSIONS: NT, which may be a new form of oxidative stress, was expressed in diabetic rat LEC although CCK-8 could reverse NT damage in LEC. The results suggested that CCK-8 might be a useful therapeutic agent against diabetic cataract. The antagonizing mechanism of CCK-8 may be related to direct antagonism of ONOO- as well as its inhibition of the expression of iNOS mRNA for production of NO and therefore decrease in the formation of ONOO-.

Puerarin decreases lens epithelium cell apoptosis induced partly by peroxynitrite in diabetic rats.

The present study was designed to observe if puerarin decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO(-)). One hundred and eight rats were randomly divided into control group (n=36), streptozotocin (STZ) group (n=36) and STZ + puerarin group (n=36). The rats in the control group intraperitoneally (i.p.) received 0.5 ml of saline. The rats in STZ group and STZ + puerarin group received intraperitoneal injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin (140 mg/kg per day, i.p.). On days 20, 40 and 60 of the experiment, morphologic changes of lenses were observed with slit lamp. Then the animals were sacrificed for further analysis. The amount and percentage of apoptotic LECs were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO(-)) was examined by immunohistochemistry. Apoptosis-related genes (iNOS, etc.) were analyzed by gene array. The results showed that in the control group, all the lenses were clear. In STZ group, gradually severe opacity of the lens was observed on days 20, 40 and 60. But in STZ + puerarin group, mild opacity of the lens was observed on day 20 and more severe on day 40, but markedly decreased on day 60. In the control group, mild apoptosis of LECs was observed. In STZ group, time-dependent increase in apoptosis of LECs was observed. In STZ + puerarin group, mild apoptosis of LECs was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of NT in the lens in the control group, but an increased expression of NT in STZ group. In STZ + puerarin group, mild expression of NT was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of iNOS in the lens in the control group, but continuous up-regulation of iNOS expression in STZ group. In STZ + puerarin group, mild expression of iNOS was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. Except the changes of iNOS related to NO production, the other apoptosis-related genes, including BCL-2 and SOD were down-regulated, while NF-kappaB and TNFR1-FADD-caspase signal transduction way were up-regulated in STZ group. The results were opposite in STZ + puerarin group and the control group. These findings show that NT is expressed in diabetic rat lens, which proves that LEC apoptosis in diabetic lens is partly induced by ONOO(-) which may be a new oxidative damage way to form cataract. Puerarin partly decreases LEC apoptosis induced by ONOO(-) and is a potential medicine for therapy of diabetic cataract. The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO(-) in diabetic rats.

[Inhibitory effect of cholecystokinin octapeptide on lipopolysaccharide-elicited inducible nitric oxide synthase expression in vascular endothelial cells].

OBJECTIVE: To investigate the effect of cholecystokinin octapeptide (CCK-8) on lipopolysaccharide (LPS)-elicited inducible nitric oxide synthase (iNOS) expression in vascular endothelial cells. METHODS: Human umbilical vein endothelial cell line (ECV-304 cells) was stimulated with vehicle (normal saline) or LPS in the presence (0.01, 0.1, 1 mg/L) or absence (0.1 mg/L) of CCK-8 (10(-6)-10(-8)mol/L). Nitric oxide (NO) level and cellular nitric oxide synthase (NOS) activity were determined with spectrophotometrically. The iNOS expression was detected with immunocytochemical technique and Western blot. RESULTS: Compared with normal saline, LPS significantly induced the upregulation of iNOS protein expression in the cultured ECV-304 cells, and NOS activity in ECV-304 cells and NO level in cultured media were increased. CCK-8 obviously inhibited above-mentioned effect of LPS in a dose-dependent manner. Whereas CCK-8 alone did not showed effect on iNOS protein expression, NO level and cellular NOS activity as compared with those values when vehicle was used. CONCLUSION: CCK-8 inhibited LPS-elicited iNOS expression and NO production in ECV-304 cells.

[Receptor mechanisms underlying the modulation of lipopolysaccharide-induced nuclear factor-kappaB expression in vascular endothelial cells by cholecystokinin octapeptide].

OBJECTIVE: To elucidate the receptor mechanisms underlying the modulation of lipopolysaccharide (LPS)-induced nuclear factor-kappaB (NF-kappaB) expression in human umbilical vein endothelial cell line ECV-304 cells by cholecystokinin octapeptide (CCK-8). METHODS: Human umbilical vein endothelial cell line ECV-304 cells were stimulated with vehicle, LPS, CCK-8 (10(-9)-10(-7) mol/L), CCK receptor non-specific antagonist proglumide, CCK-A receptor (CCK-AR) specific antagonist CR-1409 or CCK-B receptor (CCK-BR) specific antagonist CR-2945 singularly or in combination. The NF-kappaB p65 protein level was determined by Western blot and immunocytochemistry technique. RESULTS: LPS resulted in an increase in the up-regulatory expression and nuclear translocation of NF-kappaB p65 protein in ECV-304 compared with vehicle stimulation. CCK-8 obviously inhibited LPS-induced the changes in NF-kappaB p65 protein in a dose-dependent manner. The inhibitory effects of CCK-8 on NF-kappaB p65 protein expression were attenuated by proglumide>CR-2945>CR-1409. CONCLUSION: CCK-AR and CCK-BR are involved in the mediation of CCK-8 inhibitive regulation for LPS-induced NF-kappaB protein expression in ECV-04 cells, whereas the effect of CCK-BR are more than that of CCK-R.

Cholecystokinin octapeptide improves cardiac function by activating cholecystokinin octapeptide receptor in endotoxic shock rats.

AIM: To explore the effect of sulfated cholecystokinin octapeptide (sCCK-8) on cardiac functions and its receptor mechanism in endotoxic shock (ES) rats. METHODS: The changes of the mean arterial pressure (MAP), heart rate (HR), the left ventricular pressure (LVP) and the maximal/minimum rate of LVP (+/-LVdp/dt(max))) were measured by using physiological record instrument in eight groups of rats. The expression of cholecystokinin-A receptor (CCK-AR) and cholecystokinin-B receptor (CCK-BR) mRNA of myocardium in ES rats was examined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: (1) Low doses of sCCK-8 (0.4 microg/kg) caused tachycardia (441+/-27, normal control 391+/-22 s/min) and slight increase in MAP, LVP and +/-LVdp/dt(max) (16.96+/-1.79, 18.21+/-1.69 and +768.85+/-31.28/-565.04+/-27.71 kPa, respectively, all P<0.01), while medium doses (4.0 microg/kg) and high doses of sCCK-8 (40 microg/kg) elicited bradycardia and marked increase in MAP, LVP and +/-LVdp/dt(max) (17.29+/-1.63, 19.46+/-2.57 and +831.46+/-22.57/-606.08 +/-31.32; 17.46+/-1.08, 19.83+/-2.91 and +914.52+/-35.95/-639.15+/-30.23 kPa, respectively, all P<0.01). Proglumide (1.0 mg/kg), a nonselective antagonist of CCK-receptor (CCK-R), significantly inhibited the pressor effects of sCCK-8 (15.96+/-1.38, 17.36+/-0.66 and +748.18+/-19.29/-512.12+/-14.39 kPa, respectively, all P<0.01), whilst reversing the bradycardiac responses. (2) High doses of LPS (8 mg/kg) elicited marked decrease in MAP, LVP and +/-LVdp/dt(max). (7.16+/-0.59, 7.6+/-0.68 and +298.01+/-25.52/-166.96+/-19.25 kPa, respectively, all P<0.01). Pretreatment with sCCK-8 (40 microg/kg) could reverse the decline of cardiac functions (10.71+/-0.45, 11.7+/-1.26 and +446.04+/-67.18/-347.90+/-36.98 kPa, respectively, all P<0.01), while proglumide could cause further decline of cardiac function in ES rats (4.71+/-0.67, 5.58+/-1.25 and +226.48+/-15.84/-142.83+/-20.23 kPa, respectively, all P<0.01). (3) CCK-A/BR mRNAs were expressed in myocardium of control rats. Gene expression of CCK-AR and CCK-BR significantly increased in myocardium of ES rats. The increase of CCK-AR mRNA induced by LPS began at 0.5 h, peaked at 2 h, kept a high level at 6 h and declined at 12 h, respectively. Similar to CCK-AR mRNA, the expression of CCK-BR mRNA peaked at 2 h and kept a high level at 6 h, but it did not change at the first 0.5 h and was stable at a high level at 12 h. CONCLUSION: The above results indicate that endogenous and exogenous sCCK-8 may significantly improve cardiac function and intractable hypotension of ES rats, which was likely related to high expression of CCK-A/BR in myocardium induced by LPS.

Multiple factors contributing to lipopolysaccharide-induced reactivity changes in rabbit pulmonary artery.

To explore the underlying mechanism(s) of pulmonary arterial hypertension in endotoxic shock, the roles of N-acetylcysteine (NAC), nitric oxide (NO) and carbon monoxide (CO) were investigated. Pulmonary arterial rings (3-mm width) were prepared from 24 rabbits. Lipopolysaccharide (LPS), after 7-hour incubation, decreased the endothelium-dependent relaxation response of the arterial ring (pre-contracted with phenylephrine) to acetylcholine (1 mumol/L), but did not affect the endothelium-independent relaxation response to sodium nitroprusside. The LPS effects were reduced by a concomitant incubation with the free radical scavenger (NAC), NO donor (L-arginine), and CO donor (hemin), respectively. On the other hand, the LPS effects were enhanced by applying heme oxygenase-1 (HO-1) inhibitor (zinc protoporphyrin) to block CO production. The response to acetylcholine changed from relaxation to contraction, however, the contractile response to phenylephrine increased significantly after pre-incubation with nitric oxide synthase (NOS) inhibitor (L-NAME) to block NO production, confirming the importance of CO and NO. These results show that LPS impairs endothelium-dependent relaxation of the pulmonary artery, which can be greatly reduced by the antioxidant, or by supplying with NO and CO. Thus, multiple factors are involved in this model of endotoxin-induced pulmonary hypertension.

[Melatonin improves vascular reactivity of endotoxemia rats].

The purpose of the present study was to investigate the effect of melatonin (MT) on the abnormal reactivity of thoracic aorta and pulmonary artery induced by lipopolysaccharide (LPS) in rats. Sprague-Dawley rats were divided into four groups randomly: (1) Vehicle group; (2) LPS group: LPS (4 mg/kg, i.p.); (3) LPS+MT group: MT (5 mg/ml, i.p.) was given 30 min before LPS and 60 min after LPS (4 mg/kg ,i.p); (4) MT group: received two doses of MT, 90 min after the first injection of MT another dose of MT was given. Six hours after LPS injection,the rats were killed and both thoracic aortic rings (TARs) and pulmonary artery rings (PARs)were prepared. The reactivity of TARs and PARs in the four subgroups was tested separately. The contraction response to phenylephrine (PE) and the endothelium-dependent relaxation response (EDRR) to ACh were observed with the isolated artery ring technique. Concentration-response curves were generated with ACh or PE (1 x 10(-8) - 1 x 10(-5) mol/L). Superoxide dismutes (SOD) activity and the content of malondialhyde (MDA) in artery tissues were detected. For TARs, LPS significantly reduced the contraction response to PE compared with the vehicle group (P<0.01) and the curve of cumulative dose responses to PE in the LPS group shifted downward. Although EDRR to ACh in the LPS group had the tendency to decrease but still showed no significant difference compared with the vehicle group (P>0.05). For PARs, EDRR to ACh was depressed significantly in the LPS group (P<0.01), while no effect on contraction response to PE in the LPS group was observed, compared with the vehicle group (P> 0.05). Compared with the LPS group, TARs in the LPS+MT group exhibited an increased contraction response to PE, but were still lower than that in the vehicle group. Similarly, EDRR to ACh of PARs in the LPS+MT group was improved significantly and there was no difference between the LPS+MT group and the vehicle group. The vascular reactivity was unaffected in MT group compared with the vehicle group in both TARs and PARs. SOD activity in the LPS +MT group increased significantly and the content of MDA decreased markedly compared with the LPS group. These results suggest that MT may improve the vascular reactivity in endotoxemia rats due to its antioxidant properties.

[Effect of exogenous carbon monoxide on sequestration of polymorphonuclear neutrophils in the lung following limb ischemia-reperfusion: an experimental study].

OBJECTIVE: To investigate the effect of exogenous carbon monoxide (CO) in inhibiting the sequestration of polymorphonuclear neutrophils (PMNs) in the lung following limb ischemia-reperfusion (IR) and the mechanism thereof. METHODS: PMNs of peripheral blood were isolated from the venous blood of a healthy volunteer. Serum was collected from a patient undergoing bilateral knee joint replacement as IR serum. Human pulmonary microvascular endothelial cells (PMVECs) were cultured and divided into 4 groups: control group (cultured under the condition of room air containing 5% CO2 for 5 h and cultured in normal human serum instead of medium during the last 4 hours of experiment), IR group (cultured under the condition of air containing 5% CO2 for 5h and cultured in the serum of IR patient during the last 4 hours), IR + CO group (cultured under the condition of air containing 0.025% CO and 5% CO2 for 5 hours and cultured in IR serum during the last 4 hours), and control + CO group (cultured under the condition of air containing and 0.025% CO and 5% CO2 for 5 hours and cultured in normal human serum during the last 4 hours). Immunofluorescence flow cytometry was used to detect the expression of intercellular adhesion molecule (ICAM)-1 and integrin CD11b in the PMVECs. Human PMVECs were put into the wells of a 96-well plate and added with PMNs to calculate the PMVEC-PMN adhesion rate. Tourniquettes were bound at the bilateral hind thighs of 32 healthy male SD rats for 4 hours so as to establish a rate IR model. The rats were randomly divided into 4 equal groups: control group (undergoing the same operation without causing limb ischemia and exposed to room air), IR group (undergoing bilateral hind limb ischemia for 4 h and reperfusion for 4 h and exposed to room air), IR + CO group (exposed to the containing 0.025% CO one hour before reperfusion till 4 hours after reperfusion), and control + CO group (exposed to air containing 0.025% CO at the corresponding time point as that of the IR + CO group). Then the rats were killed and their middle pulmonary lobes were taken out for microscopy and calculation of the number of PMNs in alveolar septum. Western blotting was used to examine the ICAM-1 protein expression in the lung. RESULTS: The ICAM-1 expression and integrin CD11b expression of the IR group PMVECs were significantly stronger than those of the IR + CO group PMVECs (both P < 0.05) and there were no significant differences in the ICAM-1 expression and CD11b expression between the control + CO and control groups (both P > 0.05). The PMN-PMVEC adhesion rate of the IR group PMVECs was 30 +/- 2.9%, significantly higher than those of the IR + CO group and control group PMVECs (19.8 +/- 1.5% and 13.4 +/- 1.1% respectively, both P < 0.05) and there was no significant difference in the PMN-PMVEC adhesion rate between the CO + control group and control group (P > 0.05). The lung tissues of the IR group rats showed edema and hemorrhage. The number of PMNs in the alveolar septum was 60.6 +/- 1.7/10 high power fields, significantly higher than those of the IR + CO group and control group (36.4 +/- 1.6 and 22.5 +/- 1.6 respectively, both P < 0.05) and there was no significant difference between the latter 2 groups (P > 0.05). The ICAM-1 protein expression in the lung of the IR group was the strongest, followed by the IR + CO group, control + CO group, and control group. CONCLUSION: Exogenous CO inhibits the limb/IR-induced PMN sequestration in the lung, probably by the mechanism of down-regulation of the expression of adhesion molecules and suppression of the PMN\PMVEC adhesion following IR.

CCK-8 inhibits expression of TNF-alpha in the spleen of endotoxic shock rats and signal transduction mechanism of p38 MAPK.

AIM: To study the effect of sulfated cholecystokinin-octapeptide (CCK-8) on systemic hypotension, gene and protein expression of TNF-alpha in the spleen of lipopolysaccharide (LPS)-induced endotoxic shock (ES) rats, and further investigate the signal transduction mechanism of p38 mitogen-activated protein kinase (MAPK). METHODS: The changes of blood pressure were observed using physiological record instrument in the four groups of rats: LPS (8 mg x kg(-1), iv), CCK-8 (40 microg x kg(-1), iv) pretreatment 10 min before LPS (8 mg x kg(-1)), CCK-8 (40 microg x kg(-1), iv) or normal saline (control) group. The content of TNF-alpha in the spleen was assayed 2 h after LPS administration using ELISA kit and the expression of TNF-alpha mRNA was examined 30 min, 2 h and 6 h after LPS administration by reverse transcribed polymerase chain reaction (RT-PCR). Activation of p38 MAPK was detected with Western blot 30 min after LPS administration. RESULTS: CCK-8 reversed LPS-induced decrease of mean arterial pressure (MAP) in rats. The content of TNF-alpha in the spleen was (282+/-30) ng x L(-1) in control group, while it increased to (941+/-149) ng x L(-1) in LPS group, P<0.01. CCK-8 significantly inhibited the LPS-induced increase of TNF-alpha content in spleen. It decreased to (462 +/-87) ng x L(-1) in CCK-8+LPS group, P<0.01. The expression of TNF-alpha mRNA 30 min and 2 h after treatment was stronger in LPS group, while it was lowered after CCK-8 pretreatment. The p38 MAPK expression increased significantly in LPS group (5.84 times of control) and CCK-8 increased the activation of p38 MAPK in ES rats (10.74 times of control). CONCLUSION: CCK-8 reverses the decrease of MAP in ES rats and has inhibitory effect on the gene and protein expression of TNF-alpha in spleen, and p38 MAPK may be involved in its signal transduction mechanisms.

Anti-inflammatory effect of cholecystokinin and its signal transduction mechanism in endotoxic shock rat.

AIM: To study the anti-inflammatory effects of cholecystokinin-octapeptide (CCK-8) on lipopolysaccharide (LPS)-induced endotoxic shock (ES) and further investigate its signal transduction pathways involving p38 mitogen-activated protein kinase (MAPK) and IkappaB-alpha. METHODS: Eighty-four rats were divided randomly into four groups: LPS (8 mg.kg(-1), iv) induced ES; CCK-8 (40 microg.kg(-1), iv) pretreatment 10 min before LPS (8 mg.kg(-1)); CCK-8 (40 microg.kg(-1), iv) or normal saline (control) groups. The inflammatory changes of lung and spleen, phagocytic function of alveolar macrophage, quantification of inflammatory cells in bronchoalveolar lavage (BAL) were investigated in rats by using hematoxylin and eosin (HE) staining, phagocytosis of Candida albicans and differential cell counting. Nitric oxide (NO) production in serum, lung and spleen was measured with the Griess reaction. The mechanism involving p38 MAPK and IkappaB-alpha signal pathways was investigated by Western blot. RESULTS: Inflammatory changes of lung and spleen induced by LPS were alleviated by CCK-8, the increase of NO induced by LPS in serum, lung and spleen was significantly inhibited and the neutrophil infiltration in BAL was significantly reduced by CCK-8. The number of neutrophils was (52+/-10)X10(6) cells. (-1) in LPS group, while it decreased to (18+/-4)X10(6) cells. (-1) in CCK-8+LPS (P<0.01). The phagocytic rate of CCK-8 group increased to (62.49+/-9.49) %, compared with control group (48.16+/-14.20) %, P<0.05. The phagocytosis rate was (85.14+/-4.64) % in LPS group, which reduced to (59.33+/-3.14) % in CCK-8+LPS group (P<0.01). The results of phagocytosis indexes showed similar changes. CCK-8 may play an important role in increasing the expression of p38 MAPK and decreasing the degradation of IkappaB-alpha in lung and spleen of ES rats. CONCLUSION: CCK-8 can result in anti-inflammatory effects, which may be related to activation of p38 MAPK and inhibition on the degradation of IkappaB-alpha.