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Objective: To study the effect of Leptin on neuron apoptosis in mice with cerebral ischemia injury and its mechanism., Methods: Seventy-five mice were randomly divided into three groups. Focal cerebral ischemia/reperfusion injury model in mice was reproduced by middle cerebral artery occlusion for 2 hours followed by reperfusion. In Leptin intervention group mice were given Leptin 1 μg/g during cerebral ischemia by intraperitoneal injection. Mice in the model group were given equal amount of phosphate buffer saline. After reperfusion for 24 hours, the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The mRNA and protein expression of apoptosis relative gene caspase-3 and bcl-2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immuno histochemistry., Results: Most of neuron necrosis was observed in cerebral ischemia center in model group. Compared with sham-operation group, neuron apoptosis rate, mRNA and protein expression of caspase-3 and bcl-2 in model group increased significantly [apoptosis rate: (68.65 ± 0.79)% vs. (4.40 ± 0.00)%, caspase-3 mRNA: 2.563 ± 0.250 vs. 0.153 ± 0.020, bcl-2 mRNA: 0.337 ± 0.100 vs. 0.125 ± 0.030, caspase-3 protein (absorbance value, A value): 0.57 ± 0.05 vs. 0.37 ± 0.03, bcl-2 protein (A value): 0.51 ± 0.04 vs. 0.35 ± 0.01, all P<0.01]. The apoptosis rate of penumbra neurons was reduced in Leptin intervention group significantly compared with model group [(42.30 ± 8.45)% vs. (68.65 ± 0.79)%, P<0.01]. Compared with model group, the mRNA and protein expression of caspase-3 in Leptin intervention group were reduced significantly [caspase-3 mRNA: 2.267 ± 0.040 vs. 2.563 ± 0.250, caspase-3 protein (A value): 0.45 ± 0.04 vs. 0.57 ± 0.05, P>0.05 and P<0.01], and the mRNA and protein expression of bcl-2 in Leptin intervention group upregulated significantly [bcl-2 mRNA: 0.662 ± 0.040 vs. 0.337 ± 0.100, bcl-2 protein (A value): 0.76 ± 0.09 vs. 0.51 ± 0.04, both P<0.01]., Conclusion: Leptin could reduce apoptosis of neurons through down-regulation of the expression of caspase-3 and up-regulation of the expression of bcl-2. The results suggest that Leptin plays a neuroprotective role in cerebral ischemia injury.

Objective: To determine Leptin levels in supernatant fluid of culture of human umbilical vein endothelial cells (ECV-304) after being challenged by lipopolysaccharide (LPS) and calcium ion vector A23187, and to explore the possible relation between Leptin release and cytosolic phospholipase A(2) (cPLA(2)) activity in an inflammatory cell model., Methods: ECV-304 cells were cultured in vitro. Experiment 1: the cells were divided into seven groups: blank control group, LPS 5, 10, 20 μg/ml stimulation groups, A23187 0.1, 1.0, 10.0 μmol/L stimulation groups. The supernatants were collected at 6, 12 and 24 hours.Experiment 2: according to the results of experiment 1, the cells were divided into eight groups: blank control group, LPS 20 μg/ml stimulation group, the inhibitor of cPLA2 AACOCF3 0.1, 1.0, 10.0 μmol/L plus LPS stimulation groups, the inhibitor of mitogen-activated protein/extracellular signal-regulated protein kinase kinase 1/2 (MEK1/2) U0126 0.1, 1.0, 5.0 μmol/L plus LPS stimulation groups, with AACOCF3 or U0126 added 1 hour before the addition of LPS, and the supernatants were collected 24 hours after the addition of LPS. Leptin level was determined by radioimmunoassay., Results: Experiment 1: with increase in LPS concentration and prolongation of time, Leptin release was decreased gradually. After 24 hours of interaction the concentration of Leptin (ng/ml) in LPS 20 μg/ml group was decreased significantly compared with the blank control group (0.540±0.109 vs. 0.823±0.048,P<0.05). However, A23187 had no significant effect on Leptin release. Experiment 2: LPS rendered cells to release less Leptin (ng/ml: 0.558±0.069 vs. 0.825±0.067,P<0.05); by adding AACOCF3 or U0126 in different concentration before adding LPS rendered the cells to release more Leptin (ng/ml), and it showed concentration-dependent (the AACOCF3 0.1, 1.0, 10.0 μmol/L groups were 0.673±0.135, 0.723±0.055, 0.797±0.062, respectively; the U0126 0.1, 1.0, 5.0 μmol/L groups were 0.698±0.112, 0.862±0.184, 0.935±0.145, respectively). The release of Leptin in AACOCF3 1.0 μmol/L, 10.0 μmol/L and U0126 1.0 μmol/L, 5.0 μmol/L groups was significantly higher than LPS 20 μg/ml stimulation group (all P<0.05)., Conclusion: There is a possible relation between Leptin release and cPLA 2 activity in inflammatory cells induced by LPS.

Objective: To explore the effect of rat myocardial ischemia/reperfusion (I/R) injury on serum Leptin, endothelin (ET), C-reactive protein (CRP) and myocardial Leptin expression, and discuss the role of Leptin in myocardial I/R injury., Methods: Fifty Sprague-Dawley (SD) rats were randomly divided into sham-operation, ischemia and I/R 1, 2, 3 hours groups, with 10 rats in each group. Anterior descending artery of the left coronary artery was ligated for 45 minutes and released for 1, 2 and 3 hours to establish myocardial I/R model, and the said artery of the rats in sham-operation group was not ligated. Blood from left femoral artery was collected at different time points, and serum Leptin, ET and CRP contents were detected. Myocardial tissue was harvested, and stained with hematoxylin-eosin (HE) and immunohistochemistry for its observation of the myocardial pathological changes and Leptin protein expression., Results: Serum Leptin content (μg/L) of ischemia group was significantly lower than that of sham-operation group (4.69±1.67 vs. 6.48±2.02, P<0.05); as the reperfusion time was prolonged, serum Leptin level increased gradually, and the level of I/R 3-hour group recovered to that before injury [(6.59±2.58) μg/L]. ET content (ng/L) of ischemia group was significantly higher than that of sham-operation group (110.58±37.86 vs. 80.74±34.43, P<0.05), the levels of ET in I/R 1, 2 and 3 hours groups were significantly lower than those of ischemia group (35.87±13.56, 31.98±10.88, 34.56±14.37 vs. 110.58±37.86, all P<0.05). CRP content (mg/L) of ischemia group was significantly higher than that of sham-operation group (13.12±4.82 vs. 3.24±1.72, P<0.01); as the reperfusion time was prolonged, serum CRP level increased gradually, and the levels of I/R 1, 2 and 3 hours groups were significantly higher than those of ischemia group (18.37±6.48, 24.30±9.51, 27.08±8.32 vs. 13.12±4.82, all P<0.05). Pathological examination showed that there was necrosis of ischemic myocardial cells in ischemia group, with mild congestion and edema in interstitial spaces. After I/R injury, the myocardial cells showed coagulative necrosis, and there was severe congestion of myocardial interstitial. Immunohistochemistry results showed that there was a tendency of decrease in Leptin protein expression in the early phase but increase in the late phase after the injury., Conclusion: Leptin content in the serum and myocardial tissue decreases significantly in the early phase after myocardial I/R but increases gradually in the rehabilitative phase, suggesting that Leptin maybe a stress protective factor against I/R-induced myocardial injury. There is a possible association between Leptin and the early increase followed by a delayed decrease of ET as well as the increase of CRP.

Objective: To explore the effect of ethyl pyruvate (EP) and alkaline phosphatase (ALP) on injuries of sepsis and the mechanism involved., Methods: A murine sepsis model of cecal ligation and puncture was reproduced, and 90 male Kunming mice were divided randomly into sham-operation, model and EP-intervention groups. 75 mg/kg EP was intraperitoneally injected in EP groups 1 hour after establishment of model, and the mice in model group were given a same volume of Ringer's solution. The eyeballs were removed in the latter two groups, and mice were sacrificed at 15 minutes and 1, 3 and 6 hours in subgroups of 10 mice each. ALP, uric acid (UA) and ratio of lactic acid and pyruvic acid were determined in serum and homogenized lung tissue by autonomous biochemical analyzer, and pathological changes in intestine were observed by hematoxylin-eosin (HE) staining., Results: Compared with sham-operation group, serum ALP in model groups and EP groups decreased significantly (P<0.05 or P<0.01), and ALP level of EP group was significantly lower than model group at 6 hours after injury (P<0.05). Compared with sham-operation group, serum UA in model group increased significantly at 1 hour, and reached the highest level at 3 hours (both P<0.05) but decreased significantly later. UA in EP group was significantly lower than that in model group at 1 hour and 3 hours (both P<0.05). Lactic acid/pyruvic acid ratio in lung homogenate of EP group was significantly lower than that of the model group at all the time points (all P<0.05). Intestinal structural damages were distinctly improved in EP group compared with model group at 3 hours and 6 hours (both P<0.05 )., Conclusion: EP promotes the utilization of serum ALP, decreases serum UA, ameliorates acidosis and intestinal damages, thus exerting a protective effect on sepsis-induced organ injuries.

Objective: To explore the underlying mechanism of lipopolysaccharide (LPS)-induced interleukin-1 beta (IL-1 beta) and IL-6 release via p38 mitogen-activated protein kinase (MAPK) pathway in HeLa cells for further identification of involved down-stream message factors., Methods: HeLa cells were challenged with LPS to reproduce inflammatory cell model. The activity or expression of p38 MAPK, cytosolic phospholipase A(2) (cPLA(2)) and COX-2, was inhibited with pretreatment of inflammatory HeLa cells with the inhibitors (SB203580, AACOCF(3), NS-398) or transfected with the cPLA(2) antisense oligonucleotide (SK7111), then the activities and/or expression of p38 MAPK, cPLA(2), COX-2, and relationship with levels of IL-1 beta and IL-6 supernatants were determined in each group., Results: SB203580 obviously down-regulated the activities of p38 and cPLA(2), as well as the release of IL-1 beta and IL-6. AACOCF(3) and SK7111 blocked dose-dependently the activity or expression of cPLA(2), IL-1 beta and IL-6 production. However, the expression of COX-2 could hardly be detected in HeLa cells, even after LPS treatment. At the same time, pre-treatment with NS-398 had no effect on IL-1 beta, IL-6 production., Conclusion: p38 MAPK/cPLA(2) pathway mediates the expression of IL-1 beta and IL-6 resulting from LPS treatment of HeLa cells, while COX-2, as a down-stream enzyme of cPLA(2) has no effect in this process.

Objective: To detect the effect of sepsis on renal function and corresponding enzymes in mice, and to explore the role of leptin in acute inflammation., Methods: Sepsis was reproduced by cecum ligation and puncture in mice. Serum uric acid (UA) and four enzymes related with synthesis of free radicals in kidney homogenized fluids, myeloperoxidase (MPO), glutathione-S-transferase (GST), xanthine oxidase (XOD) and superoxide dismutase (SOD) were determined with spectrophotometry, and leptin level in kidney was detected by radioimmunoassay. Histopathologic changes in kidney were observed with hematoxylin-eosin staining., Results: Twelve hours after leptin (0.08 mg/kg, i.p.) and indomethacin (8 mg/kg, i.p.) injection, serum UA was significantly decreased [(295.79+/-80.86) micromol/L and (281.78+/-46.35) micromol/L, respectively, vs. sepsis group (474.03+/-75.22) micromol/L]. At the same time, renal leptin levels in leptin injection group [(196.00+/-134.30) microg/g] 12 hours after sepsis and in indomethacin injection group [(169.30+/-132.00) microg/g] 6 hours after sepsis were also significantly higher than sepsis group [(61.65+/-27.29) microg/g]. Six and 12 hours after leptin and indomethacin injection, renal MPO, GST, XOD and SOD activities were affected to certain extent, as the results were not completely inhibited or enhanced. Nevertheless, leptin and indomethacin could promote scavenge and deactivation of free radicals., Conclusion: Low dose leptin can ameliorate sepsis-induced renal injury, which may be related with scavenge and deactivation of free radicals in renal cells, and this mechanism is similar with that of indomethacin.

Objective: To explore the effect of operative trauma induced stress responses on serum leptin levels., Methods: Serum samples of patients who had undergone resection of hepatic tumors or cholecystectomy were collected, and highly sensitive radioimmunoassay and enzyme-linked immunoadsorbent assay (ELISA) were used to determine serum levels of leptin, granulocyte-clone stimulating factor (G-CSF), C-reactive protein (CRP) and adrenocorticotropin hormone (ACTH) in the blood of these patients., Results: Compared with self-control before operation, serum leptin levels decreased slightly right after an abdominal operation (T0), it reached the highest level 1 day after operation (T1), and began to decrease from 2 days (T2) to 4 days after operation (T4), but the level was still higher than that before operation. Serum leptin levels of patients undergoing laparoscopic operation showed no significant difference when compared with that of laparotomy patients. G-SF levels decreased significantly after operation in both groups, and didn't recover to the levels before operation from T1 to T4. CRP levels slightly decreased in both groups at T0, but increased significantly higher than the levels before operation from T1 to T4. ACTH levels of decreased significantly in laparotomy patients from T0 to T1, and began to recover on T2, while that of laparoscopic operation patients showed no significant difference before and after operation., Conclusion: Serum leptin levels of patients increase significantly and constantly subsequent to operative trauma induced stress responses, but this change has no correlation with that of CRP, G-SF and ACTH.

Objective: To investigate the effect of long tubular bone fracture (LTBF) on serum levels of leptin, acute phase proteins and biochemical markers for organ functions, and to look for the role of leptin in traumatic inflammatory responses., Methods: Serum samples of LTBF patients and normal controls were collected, and immunoassays were used to determine serum levels of leptin and three acute phase proteins, including C-reactive protein (CRP), interleukin-1 (IL-1) and IL-2, and 21 biochemical markers for organ and metabolic functions were measured simultaneously with automatic biochemical analyzer. Correlation between leptin and all the markers was then analyzed., Results: Compared with normal control, serum levels of leptin, CRP, IL-1 and IL-2 increased significantly (all P<0.05), with various degrees of changes in the markers for hepatic, cardiac, renal and metabolic functions. Leptin was independent to all the markers investigated, and it seemed to exert its unique roles., Conclusion: Leptin increases significantly in LTBF-induced acute traumatic inflammatory response, showing a comparatively strong responsiveness to the stimulation, and it may play a role as an anti-inflammatory cytokine.

Objective: To determine serum leptin levels in patients with acute myocardial infarction (AMI) and coronary atherosclerosis (CS), and to analyze its correlation with C reactive protein (CRP), troponin T (TnT) and endothelin (ET)., Methods: Serum samples from confirmed AMI and CS patients were collected. Leptin and ET were assayed with high sensitive radioimmunoassay, TnT was determined with automatic biochemical analyser, and CRP was determined with enzyme-linked immunosorbant assay (ELISA)., Results: Compared with normal control group, serum leptin, TnT, CRP and ET levels increased significantly (all P<0.01) in AMI patients. Serum levels of other cytokines, except TnT in CS patients, increased significantly compared with normal control group (all P<0.01). Correlation analysis showed that all the changes were not correlated with each other, each being an independent factor. Only serum TnT levels of AMI and CS patients showed a significant difference (P<0.01)., Conclusion: Serum leptin levels of both AMI and CS patients increase significantly without a significant difference between each other, and there is no correlation for leptin with CRP, TnT and ET.

Objective: To determine serum levels of leptin and some related cytokines in severely ill patients, including severe pulmonary infection-induced multiple organ dysfunction syndrome (MODS), acute myocardial infarction (AMI) and arrhythmia (AR), and to explore the possible role of leptin in the pathogenesis and diagnosis of MODS., Methods: Radioimmunoassay was used to determine leptin, fatty acid binding protein (FABP), transferrin (Ferr) and interleukin-1beta (IL-1beta), and enzyme-linked immuno adsorbent assay (ELISA) was used to assess C reactive protein (CRP)., Results: Compared with normal individuals, leptin levels in MODS, AMI and AR patients increased significantly (all P<0.01). CRP and IL-1beta levels also increased significantly in MODS, AMI and AR patients, but the changes were more marked (all P<0.05) in MODS patients than in the patients of other two diseases (both P<0.05). Though FABP and Ferr levels of patients in all the three groups of patients showed a trend toward increase, especially in MODS patients, there was no significant difference between them and normal individuals., Conclusion: Serum leptin level increases significantly in pulmonary infection-induced MODS patients with a simultaneous increase of CRP and IL-1beta levels, and the result suggests that leptin plays a possible role in the pathogenesis and prognosis of MODS.

Objective: To explore the distribution of Orexin-A mRNA expression in different organs, and its variation in acute inflammation, and to evaluate the role of Orexin-A in acute inflammatory responses., Methods: Hypothalamus, adipose tissue, lung, stomach, duodenum, liver, spleen and testis of male Sprague-Dawley rats (including normal and those of intestinal ischemia/reperfusion injury model) were harvested, and reverse transcription-polymerase chain reaction(RT-PCR) was used to assess Orexin-A mRNA expression in different tissues of normal rats, and that in the hypothalamus of ischemia/reperfusion injured rats., Results: All tissues expressed Orexin-A mRNA, and it was especially high in the stomach, lung and kidney. After the acute stimulus of ischemia/reperfusion injury, Orexin-A mRNA expression showed a trend of fluctuant declination in the hypothalamus., Conclusion: Orexin-A mRNA is widely expressed in the major organs, and it has a time-dependent response to acute inflammatory stimuli. The results suggest that its expression variation may be used as a novel marker for acute inflammation.

Objective: To investigate the effect of intestinal ischemia/reperfusion injury on Leptin levels in serum and adipose tissue, and evaluate the potential role of Leptin in acute inflammatory response., Methods: An intestinal ischemia/reperfusion injury model of rats was reproduced. Serum and adipose tissue Leptin concentrations and Leptin mRNA expression in adipose tissue were determined by a highly-sensitive murine Leptin radioimmunoassay and reverse transcription-polymerase chain reaction (RT-PCR), respectively., Results: Compared with animals prior to injury, serum Leptin level decreased significantly in ischemia for 60 minutes followed by reperfusion for 30 minutes (I60R30), but it tended to increase in I60R150 and significantly increased in I60R360. Compared with sham group, serum Leptin level tended to increase in I60R240 and elevated significantly in I60R360; Leptin levels in adipose tissue significantly decreased in I60R30 and I60R90, while they increased significantly in I60R360. Compared with sham group, Leptin mRNA levels elevated significantly in I60R30, I60R240 and I60R360, but lowered significantly in I60R150., Conclusion: Leptin has a time-dependent response to acute inflammatory stimuli such as intestinal ischemia/reperfusion injury, and it migh play a role as an inflammatory cytokine.

Objective: To explore the effect of intestinal ischemia/reperfusion injury on Leptin concentrations in serum and adipose tissue, and to find out the role of Leptin in acute inflammatory responses., Methods: An intestinal ischemia-reperfusion injury model of rats was established, and used a highly-sensitive murine Leptin radioimmunoassay to check the change of Leptin concentrations in serum and adipose tissue., Results: Serum Leptin level (10.82+/-0.83) microg/L significantly decreased after an injury of 60-minute ischemia and 30-minute reperfusion versus pre-experimental serum levels (16.46+/-3.21) microg/L; Leptin level in serum was higher than that in adipose tissue (4.466+/-2.63) mg/100g, and they both showed a similar changeable trend to increase step by step as reperfusion time extended (P=0.047)., Conclusion: Leptin may be an inflammatory cytokine and may play a role in inflammatory responses such as intestinal ischemia/reperfusion.