Clinical effects of phospholipase D2 in attenuating acute pancreatitis

BACKGROUND The objective of the current study was to elucidate the clinical mechanism through which phospholipase D2(PLD2)exerted a regulatory effect on neutrophil migra-tion,thereby alleviating the progression of acute pancreatitis.AIM To elucidate the clinical mechanism through which PLD2 exerted a regulatory effect on neutrophil migration,thereby alleviating the progression of acute pan-creatitis.METHODS The study involved 90 patients diagnosed with acute pancreatitis,admitted to our hospital between March 2020 and November 2022.A retrospective analysis was conducted,categorizing patients based on Ranson score severity into mild(n=25),moderate(n=30),and severe(n=35)groups.Relevant data was collected for each group.Western blot analysis assessed PLD2 protein expression in patient serum.Real-time reverse transcription polymerase chain reaction was used to evaluate the mRNA expression of chemokine receptors associated with neutrophil migration.Serum levels of inflammatory factors in patients were detected using enzyme-linked immunosorbent assay.Transwell migration tests were conducted to compare migration of neutrophils across groups and analyze the influence of PLD2 on neutrophil migration.RESULTS Overall data analysis did not find significant differences between patient groups(P>0.05).The expression of PLD2 protein in the severe group was lower than that in the moderate and mild groups(P<0.05).The expression level of PLD2 in the moderate group was also lower than that in the mild group(P<0.05).The severity of acute pancreatitis is negatively correlated with PLD2 expression(r=-0.75,P=0.002).The mRNA levels of C-X-C chemokine receptor type 1,C-X-C chemokine receptor type 2,C-C chemokine receptor type 2,and C-C chemokine receptor type 5 in the severe group are significantly higher than those in the moderate and mild groups(P<0.05),and the expression levels in the moderate group are also higher than those in the mild group(P<0.05).The levels of C-reactive protein,tumor necrosis factor-α,interleukin-1β,and interleukin-6 in the severe group were higher than those in the moderate and mild groups(P<0.05),and the levels in the moderate group were also higher than those in the mild group(P<0.05).The number of migrating neutrophils in the severe group was higher than that in the moderate and mild groups(P<0.05),and the moderate group was also higher than the mild group(P<0.05).In addition,the number of migrating neutrophils in the mild group combined with PLD2 inhibitor was higher than that in the mild group(P<0.05),and the number of migrating neutrophils in the moderate group combined with PLD2 inhibitor was higher than that in the moderate group(P<0.05).The number of migrating neutrophils in the severe group+PLD2 inhibitor group was significantly higher than that in the severe group(P<0.05),indicating that PLD2 inhibitors significantly stimulated neutrophil migration.CONCLUSION PLD2 exerted a crucial regulatory role in the pathological progression of acute pancreatitis.Its protein expression varied among patients based on the severity of the disease,and a negative correlation existed between PLD2 expression and disease severity.Additionally,PLD2 appeared to impede acute pancreatitis progression by limiting neutrophil migration.

Cytosolic phospholipase A2α modulates cell-matrix adhesion via the FAK/paxillin pathway in hepatocellular carcinoma

Objective: To explore the effect of cytosolic phospholipase A2α(cPLA2α) on hepatocellular carcinoma(HCC) cell adhesion and the underlying mechanisms.Methods: Cell adhesion, detachment, and hanging-drop assays were utilized to examine the effect of cPLA2α on the cell-matrix and cell-cell adhesion. Downstream substrates and effectors of cPLA2α were screened via a phospho-antibody microarray.Associated signaling pathways were identified by the functional annotation tool DAVID. Candidate proteins were verified using Western blot and colocalization was investigated via immunofluorescence. Western blot and immunohistochemistry were used to detect protein expression in HCC tissues. Prognosis evaluation was conducted using Kaplan-Meier and Cox-proportional hazards regression analyses.Results: Our findings showed that cPLA2α knockdown decreases cell-matrix adhesion but increases cell-cell adhesion in HepG2 cells. Microarray analysis revealed that phosphorylation of multiple proteins at specific sites were regulated by cPLA2α. These phosphorylated proteins were involved in various biological processes. In addition, our results indicated that the focal adhesion pathway was highly enriched in the cPLA2α-relevant signaling pathway. Furthermore, cPLA2α was found to elevate phosphorylation levels of FAK and paxillin, two crucial components of focal adhesion. Moreover, localization of p-FAK to focal adhesions in the plasma membrane was significantly reduced with the downregulation of cPLA2α. Clinically, cPLA2α expression was positively correlated with p-FAK levels. Additionally, high expression of both cPLA2α and p-FAK predicted the worst prognoses for HCC patients.Conclusions: Our study indicated that cPLA2α may promote cell-matrix adhesion via the FAK/paxillin pathway, which partly explains the malignant cPLA2α phenotype seen in HCC.

Functions and mechanisms of cytosolic phospholipase A_(2)in central nervous system trauma

Central nervous system(CNS)trauma,including traumatic brain injury and spinal cord injury,has a high rate of disability and mortality,and effective treatment is currently lacking.Previous studies have revealed that neural inflammation plays a vital role in CNS trauma.As the initial enzyme in neuroinflammation,cytosolic phospholipase A_(2)(cPLA2)can hydrolyze membranous phosphatides at the sn-2 position in a preferential way to release lysophospholipids andω3-polyunsaturated fatty acid dominated by arachidonic acid,thereby inducing secondary injuries.Although there is substantial fresh knowledge pertaining to cPLA2,in-depth comprehension of how cPLA2 participates in CNS trauma and the potential methods to amelio rate the clinical res ults after CNS trauma are still insufficient.The present review summarizes the latest understanding of how cPLA2 participates in CNS trauma,highlighting novel findings pertaining to how cPLA2 activation initiates the potential mechanisms specifically,neuroinflammation,lysosome membrane functions,and autophagy activity,that damage the CNS after trauma.Moreover,we focused on testing a variety of drugs capable of inhibiting cPLA2 or the upstream pathway,and we explored how those agents might be utilized as treatments to improve the results following CNS trauma.This review aimed to effectively understand the mechanism of cPLA2 activation and its role in the pathophysiological processes of CNS trauma and provide clarification and a new referential framework for future research.

Cytosolic Phospholipase A2 and Its Role in Cancer

Cytosolic phospholipase A2α （cPLA2α） catalyzes the release of arachidonic acid （AA） and lysophosphoglyceride from membrane phospholipids. Although the roles of AA and eicosanoids in cellular viability, the processes of inflammation and cancer cell development have been extensively studied, the function of cPLA2α in the processes of inflammation and cancer cell development is not clear. This review summarizes published evidences for the biochemical properties and regulatory mechanisms of cPLA2α. The potential for use of cPLA2α as a novel diagnostic target and predictive biomarker for tumors is also discussed.

Time-dependent changes of glial fibriliary acidic protein and cytosolic phospholipase A2 in hippocampal area of focal cerebral ischemia/reperfusion in rats

BACKGROUND： Interaction between astrocyte and neuron may two-dimensionally influence on ischemic injury; however, glial fibriliary acidic protein （GFAP） and cytosolic phospholipase A2 （cPLA2） are both important markers to reflect changes of astrocyte and neuron after cerebral ischemia, respectively. OBJECTIVE： To observe the changes of GFAP and positive cPLA2 cells in hippocampal area of model rats with focal cerebral ischemia in various phases of cerebral ischemia/reperfusion. DESIGN ： Randomized contrast observation SETTING： Department of Basic Medical Science of Human Anatomy and Histology ＆ Embryology, Medical College of Wuhan Polytechnic University; Faculty Medical College of Wuhan University. MATERIALS： The experiment was carried out in the Department of Basic Medical Science, Medical College of Wuhan Industry College from May to June 2004. A total of 28 healthy SD rats of either gender and weighing 200-250 g were provided by Animal Department of Medical College of Jianghan University. METHODS： All 28 rats were randomly divided into 7 groups, including sham operation group, 2-, 6-, 12-, 24- and 48-reperfusion groups, and triphenyltetrazolium chloride （TTC） group, with 4 in each group. Two hours after ischemia, ischemia/reperfusion models were established in left middle cerebral artery （MCA）; common carotid artery was ligated and line cork was inserted into it with the depth of （1.8±0.5） cm. Rats in sham operation group were inserted with the depth of 1.0 cm, and other operations were as the same as those in 2-hour ischemia/reperfusion groups. Models in TTC group were established as the same as those in 2-hour ischemia/24-hour reperfusion group, and they were used to evaluate the therapeutic effect. Changes of GFAP and cPLA2 in hippocampal area in various phases were detected with immunohisto- chemical method. MAIN OUTCOME MEASURES ： Changes of GFAP and positive cPLA2 cells in hippocampal area of rats with focal cerebral ischemia in various phases of ischemia/reperfusion. RESULTS： All 28 rats were involved in the final analysis without any loss. （1） Animal models successfully showed the effect of focal cerebral ischemia. （2） Changes of GFAP and cPLA2 in hippocampal area in various phases： Two hours after ischemia/reperfusion, changes of GFAP and cPLA2 were increased gradually, reached at peak at 24 hours, and decreased gradually. CONCLUSION ： Courses of GFAP and cPLA2 are changed at the onset of focal cerebral ischemia, and this suggests that both of them participate in injury or protection of brain tissue of focal cerebral ischemia.

Plasma Lysophosphatidylcholine and Phospholipase A2 Activity in Chagas Disease Patients: A Comparative Analysis

Chagas disease (CD) affects 21 countries in the Americas and is caused by the parasite Trypanosoma cruzi. A key molecule involved in CD is lysophosphatidylcholine (LPC), which has been studied in various contexts: in the saliva of insect vectors, during the establishment of infection in the vertebrate host, and for the parasite itself. This lipid can be produced by the action of phospholipases A2 (PLA2), enzymes that catalyze the hydrolysis of phospholipids releasing fatty acids and lysophospholipids, such as LPC. This study investigates LPC levels and PLA2 activities in the plasma of CD patients and compares these levels with those in healthy individuals and patients with idiopathic dilated cardiomyopathy (IDCM). Plasma from 64 CD patients, 54 healthy individuals, and 16 IDCM patients were analyzed. LPC levels and the activity of two types of phospholipase A2: secreted (sPLA2) and lipoprotein-associated (Lp-PLA2) were measured. LPC levels and sPLA2 activity were similar between CD patients and the control groups. However, there were notable differences in LPC levels and sPLA2 activity between subgroups of CD patients and IDCM patients. This study is the first to identify LPC in patients with CD across various stages of the disease. It also offers new insights into the biochemical changes observed in the plasma of patients with IDCM.

Changes of gastric and intestinal blood flow, serum phospholipase A_2 and interleukin-1β in rats with acute necrotizing pancreatitis

AIM:To explore the relationship between gastric and intestinal microcirculatory impairment and inflammatory mediators released in rats with acute necrotizing pancreatitis (ANP). METHODS: A total of 64 rats were randomized into control group and ANP group. ANP model was induced by injection of 5% sodium taurocholate under the pancreatic membrane. Radioactive biomicrosphere technique was used to measure the gastric and intestinal tissue blood flow at 2 and 12 h after the induction of ANP, meanwhile serum phospholipase A2 (PLA2) activities and interleukin-1β levels were determined. Pathologic changes in pancreas, gastric and intestinal mucosae were studied. RESULTS: The gastric blood flow in ANP group (0.62±0.06 and 0.35±0.05) mL/(min·g) was significantly lower than that in control group (0.86±0.11 and 0.85±0.06) mL/(min·g) (P<0.01) at 2 and 12 h after induction of ANP. The intestinal blood flow in ANP group (0.80±0.07 and 0.50±0.06) mlV(min·g) was significantly lower than that in control group (1.56±0.18 and 1.61±0.11) mL/(min·g) (P<0.01). Serum PLA2 activities (94.29±9.96 and 103.71± 14.40) U/L and IL-1β levels (0.78±0.13 and 0.83±0.20)μg/L in ANP group were higher than those in control group (65.27±10.52 and 66.63±9.81) U/L, (0.32±0.06 and 0.33±0.07)μg/L (P<0.01). At 2 and 12 h after introduction of the model, typical pathologic changes were found in ANP. Compared with control group, the gastric and intestinal mucosal pathologic changes were aggravated significantly (P<0.01) at 12 h after induction of ANP. Gastric and intestinal mucosal necrosis, multiple ulcer and hemorrhage occurred. CONCLUSION: Decrease of gastric and intestinal blood flow and increase of inflammatory mediators occur simultaneously early in ANP, both of them are important pathogenic factors for gastric and intestinal mucosal injury in ANP.

Bromophenacyl bromide,a phospholipase A_2 inhibitor attenuates chemically induced gastroduodenal ulcers in rats

AIM: To study the effect of bromophenacyl bromide (BPB), a phospholipase A2 inhibitor on gastric secretion and to protect chemically induced gastric and duodenal ulcers in rats. METHODS: Acid secretion studies were undertaken in pylorus-ligated rats with BPB treatment (0, 5, 15 and 45 mg/kg). Gastric and duodenal lesions in the rats were induced by ethanol and cysteamine respectively. The levels of gastric wall mucus, nonprotein sulfhydryls (NP- SH) and myeloperoxidase (MPO) were also measured in the glandular stomach of rats following ethanol induced gastric lesions. RESULTS: BPB produced a dose-dependent inhibition of gastric acid secretion and acidity in rats. Pretreatment with BPB significantly attenuated the formation of etha- nol induced gastric lesion. BPB also protected intestinal mucosa against cysteamine-induced duodenal ulcers. The antiulcer activity of BPB was associated with signifi- cant inhibition of ethanol-induced depletion of gastric wall mucus, NP-SH and MPO. These findings pointed towards the mediation of sulfhydryls in BPB induced gas- trointestinal cytoprotection. CONCLUSION: BPB possesses significant antiulcer and cytoprotective activity against experimentally induced gastroduodenal lesions.

Oxidized phospholipids and lipoprotein-associated phospholipase A_2 as important determinants of Lp(a) functionality and pathophysiological role

Lipoprotein（a） [Lp（a）] is composed of a low density lipoprotein（LDL）-like particle to which apolipoprotein（a）[apo（a）] is linked by a single disulfide bridge. Lp（a） is considered a causal risk factor for ischemic cardiovascular disease（CVD） and calcific aortic valve stenosis（CAVS）. The evidence for a causal role of Lp（a） in CVD and CAVS is based on data from large epidemiological databases, mendelian randomization studies, and genome-wide association studies. Despite the well-established role of Lp（a） as a causal risk factor for CVD and CAVS, the underlying mechanisms are not well understood. A key role in the Lp（a） functionality may be played by its oxidized phospholipids（OxPL） content. Importantly, most of circulating OxPL are associated with Lp（a）; however, the underlying mechanisms leading to this preferential sequestration of OxPL on Lp（a） over the other lipoproteins,are mostly unknown. Several studies support the hypothesis that the risk of Lp（a） is primarily driven by its OxPL content.An important role in Lp（a） functionality may be played by the lipoprotein-associated phospholipase A_2（Lp-PLA_2）,an enzyme that catalyzes the degradation of OxPL and is bound to plasma lipoproteins including Lp（a）. The present review article discusses new data on the pathophysiological role of Lp（a） and particularly focuses on the functional role of OxPL and Lp-PLA_2 associated with Lp（a）.

Phospholipase A_2 and Its Relationship with Acute Lung Injury in Acute Pancreatitis in Dogs

Acute fulminant pancreatitis was produced in dogs by injection of autobile into the main pancreatic duct.After injection the phospholipase A_2(PLA_2)activities in serum,lung lymph and bronchoalveolar lavage fluid(BAL)were elevated significantly,lung lymph flow and pulmonary transvascular potein clearance increased progressively,protein content and cell numbers in BAL in the experimental animals were significantly higher than those in the control animals.Furthermore the lung index,wet to dry lung weight ratio,extravascular lung water to bloodless dry lung weight ra- tio,extravascuar lung water to bloodless dry lung weight ratio increased significantly as compared to control animals.Pretreatment with PLA_2 inhibitor,chloroquine,blocked the changes mentioned above.This experiment suggests:1.PLA_2 activity in lung lymph fluid as well as in serum and BAL is elevated in acute hemorrhagic pancreatitis.2.Elevated PLA_2 activity may increase the pulmonary vascular permeability.3.PLA_2 is the major factor leading to pulmonary edema in acute hemorrhagic pancreatitis.4.Phagocytes contribute to the lung injury induced by PLA_2 to some ex- tent.

Regulatory effects of phospholipase A_2 inhibitors on platelet activating factor in endotoxic shock in rabbits

The regulatory effects of phospholipase A2(PLA2) inhibitors, chloroquine and dexamethasone, on the activity of blood PLA2 and its related lipid mediators during endotoxic shock were observed in rabbits. The rabbits were randomized into 4 groups as follows : The normal control (NC) group consisted of 12 rabbits with sham injection . the endotoxic shork (ES) group of 31 rabbits, the chloquine pretreated (CQ) group of 16 rabbits receiving 3 mg/kg of chlorqouine and the dexamethasone-pretreated (DM) group of 10 rabbits receiving 5 mg/kg of dexamethasone. Blood was sampled before and 5 and 30 min, 1 ,3, 5 and 8 h after the administration of endotoxin for the determination of PLA2, platelet activating factor (PAF) , TXB2 and 6-keto-PGF1α. In addrtion, changes of mean arterial pressure (MAP) and respiratory rate (RR) were also carefully recorded. It was found that the activities of PLA2 and PAF and the levels of TXB2 and 6-keto-PGF1α. were significantly increased after the infusion of endotoxin. CQ and DM markedly suppressed the activities of PLA2 and PAF. The inhibition of CQ on TXB2 and 6-keto-PGF1α was greater than that of DM. Besides, CQ and DM could increase the survival rate of the animals from 48% to 75% (CQ group) and 70% (DM group). These findings suggest that PLA2 inhibitors such as CQ and DM can significantly attenuate the formation of shock mediators such as PLA2, PAF, TXB2 and 6-keto-PGF1α, and so improve the prognosis of the victims of endotoxic shock.

CONTROL OF ANGIOGENESIS BY INHIBITOR OF PHOSPHOLIPASE A_2

Objective To investigate the potential effects of angiogenic process by secretory phospholipase A2 (sPLA2)inhibitor-HyPE(linking N-derivatized phosphatidyl-ethanolamine to hyaluronic acid)on human bone marrow endothelial cell line(HBME-1). Methods In order to examine the suppressing effects of HyPE on HBME-1 proliferation, migration, and capillary-like tube formation, HBME-1 were activated by angiogenic factor, specifically by basic fibroblast growth factor(b-FGF), vascular endothelial growth factor(VEGF),and oncostatin M(OSM)(at a final concentration of 25, 20, and 2.5 ng/mL, respectively), then HBME-1 proliferation, migration, and tube forma-tion were studied in the absence or presence of HyPE. HBME-1 tube formation was specially analyzed in fibrin gel. Results HyPE effectively inhibited HBME-1 proliferation and migration as a dose-dependent manner, whatever HBME-1 were grown in the control culture medium or stimulated with b-FGF, VEGF, or OSM. In fibrin, the formations of HBME-1 derived tube-like structures were enhanced by all angiogenic factors, but these were strongly suppressed by HyPE. Conclusions The results support the involvement of sPLA2 in angiogenesis. It is proposed that sPLA2 inhibitor introduces a novel approach in the control of cancer development.

磷脂酶A_2氨基末端衍生肽的合成及其抗细菌活性的研究

目的 探讨磷脂酶A_(2)(PLA_(2))氨基末端衍生肽的合成及抗菌活性。方法 根据PLA_(2)氨基末端氨基酸残基的顺序合成为多肽PLA_(2)N_(11)、PLA_(2)N_(15)、PLA_(2)N_(20),将其分别与2种细菌(大肠埃希菌、枯草杆菌)在特定条件下培养,并以生理盐水作为对照,分析抗菌活性。结果 与对照比较,多肽不同作用浓度时PLA_(2)N_(11)、PLA_(2)N_(15)、PLA_(2)N_(20)对革兰氏阳性枯草杆菌杀菌活性更强,各多肽间比较,差异有统计学意义(P<0.05);当多肽作用浓度为500μg/ml时,PLA_(2)N_(15)对革兰氏阳性枯草杆菌杀菌活性可达99.8%,高于PLA_(2)N_(11)、PLA_(2)N_(20),及对照(P<0.05)。与对照比较,各多肽不同作用浓度时对大肠埃希菌杀菌活性比较,差异有统计学意义(P<0.05);多肽作用浓度为7.81、125.00、500.00μg/ml时,PLA_(2)N_(15)的杀菌活性均高于PLA_(2)N_(11)和PLA_(2)N_(20),及对照(P<0.05);多肽作用浓度为31.25μg/ml时,PLA_(2)N_(15)的杀菌活性低于PLA_(2)N_(11)的杀菌活性,且高于PLA_(2)N_(20)的杀菌活性(P<0.05)。结论 多肽PLA_(2)N_(11)、PLA_(2)N_(15)、PLA_(2)N_(20)对枯草杆菌、大肠埃希菌有很强的杀菌作用。

Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications

Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2(Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the LpPLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed.

The Use of Lipoprotein-Associated Phospholipase A2 in a Chinese Population to Predict Cardiovascular Events

Objective To explore associations between lipoprotein-associated phospholipase A2(Lp-PLA2)and the risk of cardiovascular events in a Chinese population,with a long-term follow-up.Methods A random sample of 2,031 participants(73.6%males,mean age=60.4 years)was derived from the Asymptomatic Polyvascular Abnormalities Community study(APAC)from 2010 to 2011.Serum Lp-PLA2 levels were determined by enzyme-linked immunosorbent assay(ELISA).The composite endpoint was a combination of first-ever stroke,myocardial infarction(MI)or all-cause death.Lp-PLA2 associations with outcomes were assessed using Cox models.Results The median Lp-PLA2 level was 141.0 ng/m L.Over a median follow-up of 9.1 years,we identified 389 events(19.2%),including 137 stroke incidents,43 MIs,and 244 all-cause deaths.Using multivariate Cox regression,when compared with the lowest Lp-PLA2 quartile,the hazard ratios with95%confidence intervals for developing composite endpoints,stroke,major adverse cardiovascular events,and all-cause death were 1.77(1.24–2.54),1.92(1.03–3.60),1.69(1.003–2.84),and 1.94(1.18–3.18)in the highest quartile,respectively.Composite endpoints in 145(28.6%)patients occurred in the highest quartile where Lp-PLA2(159.0 ng/m L)was much lower than the American Association of Clinical Endocrinologists recommended cut-off point,200 ng/m L.Conclusion Higher Lp-PLA2 levels were associated with an increased risk of cardiovascular event/death in a middle-aged Chinese population.The Lp-PLA2 cut-off point may be lower in the Chinese population when predicting cardiovascular events.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa

Aim： To determine the cellular distribution of secretory phospholipase A2 （sPLA2） in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods： Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA2 and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Results： Although sPLA2 was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA2 was associated with enhanced binding of annexin V-fluoroscein isothiocyanate （FITC） to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA2, disturbance of acrosome structure, and loss of vitality. Conclusion： The ability of spermatozoa to release secretory phospholipase A2 is related to the acrosomal state. Premature destabi- lization of the acrosome and loss of sPLA2 can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA2 in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Effects ofβ_2-Adrenergic Antagonist on Cytosolic Ca^(2+) in Ventricular Myocytes from Infarcted Rat Heart

Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 ＋ （[Ca^2＋ ]i） in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending coronary artery of rat hearts. Rats in the control group were sham-operated. Cardiomyocytes were dissociated at two, four, eight weeks after myocardial infarction （MI） and [Ca^2＋]i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol in presence or absence of betal-adrenergic antagonist atenolol, beta2-adrenergic antagonist ICI118, 551 or non-selective β1, 2- adrenergic antagonists propranolol was examined. Results The followings were found that ICI 118, 551 had no significant effects on the rise of [Ca^2＋]i induced by isoproterenol in normal ventricular myocytes （P 〉 0.05）, ICI118, 551 only significantly attenuated the rise of [Ca^2＋]i induced by isoproterenol at four weeks and eight weeks after MI （24.5%±5.7% vs 57.8% ± 13.2%, P〈 0.01; 12.2%±7.9% vs 44.6%±11.3%, P〈 0.01）. Atenolol had suppressive effects only in the control group and the post-MI group of two weeks （P 〈 0.05）, and propranolol had suppressive effects in the control and all the three post-MI groups （P 〈 0.01）. Conclusions Beta2-adrenergic antagonist ICI118, 551 may exert negative effects on Ca^2＋ overload initiated by sympathetic stimulation after MI.

磷脂酶CG2在溃疡性结肠炎小鼠模型中的表达

目的:探讨磷脂酶CG2(phospholipase C Gamma 2,PLCG2)在溃疡性结肠炎(ulcerative colitis,UC)小鼠模型中的表达及意义。方法:选择8~12周龄C57BL/6野生型(WT)雄性小鼠10只,其中5只分离不同肠段,另5只分离肠上皮细胞及固有层淋巴细胞,分别提取各个肠段和细胞总RNA,采用逆转录PCR(RT-PCR)和实时荧光定量PCR(qRT-PCR)检测PLCG2 mRNA表达。另选择8~12周龄WT雄性小鼠15只,随机分成3组,分别为对照组、急性发病期组和恢复期组,每组5只;急性发病期组和恢复期组用含2.5%葡聚糖硫酸钠饮用水饲养5 d,随后改为常规饮用水饲养,对照组予以常规饮用水饲养,在UC发展的不同阶段处理小鼠并提取其结肠固有层淋巴细胞;另选择8~12周龄WT雄性小鼠15只,以同样方式造模,提取小鼠结肠上皮细胞;RT-PCR和qRT-PCR检测结肠固有层淋巴细胞和肠上皮细胞中PLCG2 mRNA表达。结果:PLCG2 mRNA在WT小鼠结肠中表达与十二指肠、空肠、回肠、盲肠相比无明显差异(P>0.05)。PLCG2 mRNA在WT小鼠结肠固有层淋巴细胞中表达明显高于肠上皮细胞(P<0.05)。在小鼠结肠固有层淋巴细胞中,与对照组相比,急性发病期组(第3天)和恢复期组(第9天)PLCG2 mRNA相对表达均明显降低(P<0.05);在小鼠肠上皮细胞中,PLCG2 mRNA在对照组、急性发病期组(第5天)和恢复期组(第9天)中相对表达无明显差异(P>0.05)。结论:PLCG2 mRNA在UC小鼠结肠固有层淋巴细胞中呈低表达,其可能在UC的发生发展中起一定作用。

Procainamide inhibits A23187－induced human platelet aggregation and increase in cytosolic free－Ca2＋ in the cells

Effects of procainamide (PA) on human platelet aggregation and the cytosolic free-Ca2+ concentration ([Ca2+]) were investigated in vitro. PA at doses of 8.5 , 34 and 136 μmol·L-1 could inhibit the human platelet aggregation induced by 0. 5 μmol · L-1 A23187 with a good concentration -effect relationship. One min and maximal aggregation rates were also inhibited ( P<0. 01 ,vs control). The [Ca2+], was decreased in the presence of PA ,and the changes of [Ca2+], showed a significant linear correlation with 1 min or maximal aggregation rate (P<0.05). These results suggest that the mechanisms of PA inhibiting platelet aggregation relate to the decrease of [Ca2+].

Relation of phospholipase A2-Ⅴ and indoxam to hippocampal neuronal death

BACKGROUND: Ⅴ secretory phospholipase A2 (sPLA2-Ⅴ) is abundant in many mammal tissues. However, it remains unknown whether sPLA2-Ⅴ causes biological or pathological response in central nervous system. OBJECTIVE: To observe the effect of phospholipase A2-Ⅴ (PLA2-Ⅴ) and its inhibitor (indoxam) on hippocampal neuron survival. DESIGN: A repetitive measurement. SETTING: The Animal Center of South Carolina University. MATERIALS: Sprague-Dawley pregnancy day-7, 14, 21 female rats were selected; Reagents: sPLA2- Ⅴ and indoxam were obtained from the Dennis Research Laboratories METHODS: The experiment was finished at the animal center in South Carolina University from January to December, 2004. 0, 12.5, 25, 50 and 100 μg/L sPLA2-Ⅴ were added to neuron with none-MgCl2 Eagle’s medium at 37 ℃, then changed to normal neuron culture medium after 3 hours. 1, 2.5, 5 and 10 μmol/L indoxam was added at 6 hours after 100 μg/L sPLA2-Ⅴwas put to Day-21 SD rat hippocampal embryonic neurons with none-MgCl2 Eagle’s medium at 37 ℃. After 3 hours in the inhibition experiment, it was changed to normal neuron culture medium. The embryonic hippocampal neurons were primarily cultured, and the neuron survival ratio was detected with morphological method. MAIN OUTCOME MEASURES: Survival ratio of hippocampal neurons. RESULTS: ① Effects of sPLA2-Ⅴon neuron survival: When sPLA2-Ⅴ was 0, 12.5, 25, 50 and 100 μg/L, the neuron survival ratios in embryonic neurons of day-7 SD rats were (95.3±1.1)%, (81.4±3.1)%, (74.2±2.2)%, (62.4±1.7)% and (48.9±1.6)%, those in embryonic neurons of day-14 rats were (93.2±1.4)%, (74.3±1.9)%, (68.1±1.7)%, (56.1±1.4)% and (42.5±1.1)%, and those in embryonic neurons of day-21 rats were (91.2±1.2)%, (69.4±2.1)%, (60.3±2.2)%, (49.1±1.2)% and (35.5±1.9)%. There were significant differences among different concentrations (P < 0.05). ② Effects of indoxam on neuron survival: In case of sPLA2-Ⅴ 100 μg/L, the neuron survival ratios were (58.65±1.4)%, (69.34±1.1)%, (82.11±1.2)% and (95.28±0.9)% when indoxam was 1, 2.5, 5 and 10 μmol/L, respectively. There were significant differences among different concentrations (P < 0.05). CONCLUSION: ① The of neuronal death ratio is in a concentration-dependent manner with sPLA2-Ⅴ, and increases as the embryonic aging. ② Indoxam inhibits the proapoptotic effect of sPLA2-Ⅴ.