Osteoarthritis(OA) and intervertebral disc degeneration(IVDD) are degenerative musculoskeletal disorders characterized by degeneration of cartilaginous tissues and inflammation. While inflammation is implicated in the...Osteoarthritis(OA) and intervertebral disc degeneration(IVDD) are degenerative musculoskeletal disorders characterized by degeneration of cartilaginous tissues and inflammation. While inflammation is implicated in the pathogenesis of OA and IVDD, and cytosolic phospholipase A2(cPLA2) is a key mediator of inflammation, direct evidence linking cPLA2 to chondrocyte homeostasis and cartilage degeneration is lacking. This study aims to investigate the role of cPLA2 in chondrocytes and its contribution to the development of cartilage degenerative conditions such as OA and IVDD. Here, single-cell RNA sequencing was used to examine cPLA2 expression in chondrocytes. To explore its importance in chondrocytes and OA/IVDD, various cell-based assays and genetically modified mouse models with age-related and surgically induced OA/IVDD were employed. Furthermore, the therapeutic potential of fexofenadine, an over-the-counter drug recently identified as a cPLA2 inhibitor, was explored in these models. cPLA2 is predominantly expressed in prehypertrophic chondrocytes, characterized by elevated levels of cartilage degeneration markers and senescence-related genes. Genetic deletion and pharmacological inhibition of cPLA2 reduced inflammation induced catabolic activity and senescence in chondrocytes, as well as cartilage degeneration in various OA and IVDD models. This study identifies cPLA2 as a pivotal driver of cartilage degeneration and senescence in OA and IVDD, highlighting its potential as a dual-action therapeutic target that suppresses both inflammation and senescence to preserve cartilage integrity. These findings position cPLA2as a promising candidate for developing disease-modifying therapies for cartilage degenerative conditions such as OA and IVDD.展开更多
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 random...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.展开更多
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 parti...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.展开更多
The phosphatidylserine-specific phospholipase A1(PLA1A)is an essential host factor in hepatitis C virus(HCV)assembly.In this study,we mapped the E2,NS2 and NS5A involved in PLA1A interaction to their lumenal domains a...The phosphatidylserine-specific phospholipase A1(PLA1A)is an essential host factor in hepatitis C virus(HCV)assembly.In this study,we mapped the E2,NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts,through which they form oligomeric protein complexes to participate in HCV assembly.Multiple regions of PLA1A were involved in their interaction and complex formation.Furthermore,the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A,and strongly suggest PLA1 A-E2,s physical interaction in cells.Meanwhile,we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1.Interestingly,these amino acids in the sequence are also essential for viral RNA replication.Further experiments revealed that these four proteins interact with each other.Moreover,PLA1A expression levels were elevated in livers from HCV-infected patients.In conclusion,we exposed the structural determinants of PLA1A,E2,NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.展开更多
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 un...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.展开更多
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...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.展开更多
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 ne...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.展开更多
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 is...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）.展开更多
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 proce...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.展开更多
Bee venom phospholipase A2(BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids.In this work,a new BvPLA2(AccPLA2...Bee venom phospholipase A2(BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids.In this work,a new BvPLA2(AccPLA2) gene from the Chinese honeybee(Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector.Tn-5B-4(Tn) cells were transfected with the recombinant bacmid DNA for expression.Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa.Products of hexahistidine AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins.The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2(BvPLA2 of the European honeybee,Apis mellifera) polyclonal serum.The reaction resulted in a double glycosylation band,which agrees with the band generated by the native AmPLA2 in Western blot analysis.The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg).In summary,the recombinant AccPLA2 protein,a native BvPLA2-like structure with corresponding biological activities,can be glycosylated in Tn cells.These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry.展开更多
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)...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.展开更多
Calcium-independent phospholipase A<sub>2</sub> (iPLA<sub>2</sub>) belongs to the group VI family of phospholipase superfamily (PLA<sub>2</sub>) that catalyses the hydrolysis of gly...Calcium-independent phospholipase A<sub>2</sub> (iPLA<sub>2</sub>) belongs to the group VI family of phospholipase superfamily (PLA<sub>2</sub>) that catalyses the hydrolysis of glycerophospholipids at the sn-2 ester bond, producing unesterified fatty acids and 2-lysophospholipids. Research interests on iPLA<sub>2</sub> have not been as significant as those on secretary PLA<sub>2</sub> and cytosolic PLA<sub>2</sub>. However, more efforts have been made recently on understanding the expression, regulation and biological function of iPLA<sub>2</sub>. iPLA<sub>2</sub> plays important roles in several biological processes, including signal transduction, phospholipid remodelling, eicosanoid formation, cell proliferation, cell differentiation and apoptosis. Modulation of iPLA<sub>2</sub> activity can have prominent effects on cellular metabolism, central nervous system and cardiovascular functions. Thus, dysregulation iPLA<sub>2</sub> can play a vital role in the pathogenesis of several diseases. The aim of this review is to provide the current understanding of the structure, function and regulation of group VI iPLA<sub>2</sub> and highlight its potential mechanisms of action in mediating several neurological disorders and cancer.展开更多
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 endothel...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.展开更多
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 m...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.展开更多
A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary...A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary development of the as say. Various combinations of human, sheep, rat, and mouse red blood cells (RBC) with human,rat,and mouse sera were tested on their effectiveness to detect PLA2.Complete hemolysis (b hemolysis) was evident in the plate with rat RBC mixed with mouse serum.No hemolysis was detected in plates containing human RBC and human serum. Human RBC mixed with mouse serum proved to be ideal, even though this combination displayed incomplete hemolysis(a hemolysis).Susceptible RBC, in conjunction with rat or mouse serum, are excellent indicators for the presence of PLA2.Mixtures of RBC and serum in combination with BB4(E. coli) cells,λbacteriophage,and IPTG on LB agar plates provide an excellent detection system for cDNA clones that express venonl PLA2. Hemolysis surrounding a plaque is identified as positive for PLA2.展开更多
Objective: To imastigate the effects of anisodamine on pulmonary α1- adrenergic receptor andphospholipase A, in acute lung injurg. Methods: Change of α1--adrenergic receptor (al AR ) in lung tissllesduring endotoxin...Objective: To imastigate the effects of anisodamine on pulmonary α1- adrenergic receptor andphospholipase A, in acute lung injurg. Methods: Change of α1--adrenergic receptor (al AR ) in lung tissllesduring endotoxin--induced rat acute lung injury was measured with radioligand biding assay. The effects ofanisodamine on pulmonary α1--AR and phospholipase A2 (PLA2 ) were observed. Results: 1. 4 h after theendotoxin injection, there was a significant decrease in the maximal binding capacity of α1--AR by 34% ascompared with the control group. meanwhile elevated activity of PLA2 in rat lung and reduction of thephospholipids content of cell membrane was found. 2. Anisodamine could attenuate endotoxin--induced acutelung injury in rats. Conclusion: This effect might be related to anisodamine’s blockage of α1--AR andsuppression of PLA2, prevention of membranous phospholipids from degradation. and the reduction ofarachidonic acid release.展开更多
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...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.展开更多
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 ...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.展开更多
A fraction of cobra （Naja naja atra） venom has been discovered to have protective effect on rat cultural cerebellar granule neurons （CGNs） from apoptosis induced by removing serum and reducing the extracellular po...A fraction of cobra （Naja naja atra） venom has been discovered to have protective effect on rat cultural cerebellar granule neurons （CGNs） from apoptosis induced by removing serum and reducing the extracellular potassium concentration from 25 to 5 mM. This component has been purified and identified as secreted phospholipase A2 （cobra sPLA2）. In order to study the relationship between the protection on CGNs and enzymatic activity of phospholipase A2, CGNs stained by Hoechst 33258 were quantified to determine survival rate under the fluorescence microscopy; the protective potencies on apoptosis of cultural CGNs were compared among cobra sPLA2, the cobra sPLA2 modified in carboxylate groups with water soluble carbodiimide and semicarbazide, and the heated cobra sPLA2 at 80℃ for 30 rain. The results showed that the CGN survival rate was unaffected significantly both in modified cobra sPLAz whose enzymatic activity of PLA2 had decreased by 80%, and in cobra sPLA2 adding 7, 7-Dimethyleicosadienoic acid, an inhibitor of sPLA2 at concentration of 10-fold IC50; contrary, the neuronal survival rate fell about 60% in heated cobra sPLA2, although its PLA2 activity only decreased by 10%. The protection on CGNs were also found in some of sPLA2s derived from venoms of bee, Naja naja mossambica, Crotalus atroxalso and Vipera Ammodytes Ammodytes but could not be found in other sPLA2s from bovine pancreas and Streptomyces violaceoruber. Above results suggest that the protection on CGNs of cobra sPLA2 is independent of its enzymatic activity.展开更多
Phospholipase A1(PLA1)is a kind of specific phospholipid hydrolase widely used in food,medical,textile.However,limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-...Phospholipase A1(PLA1)is a kind of specific phospholipid hydrolase widely used in food,medical,textile.However,limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS.In this study,we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation.Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells,resulting in improved cell growth and increased protein solubility of the protein.The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS(ΔN27 PlaS),highlighting their binding capabilities.Furthermore,in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 andΔN27 PlaS,exhibiting high affinity.Molecular docking analysis provides insights into the hydrogen bond interactions betweenΔN27 PlaS and PlaA1,identifying key amino acid residues crucial for their binding.Finally,the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test.Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1,offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.展开更多
基金supported partly by NIH research grants R01AR078035, R01AR062207,R01AR076900 and R01NS070328。
文摘Osteoarthritis(OA) and intervertebral disc degeneration(IVDD) are degenerative musculoskeletal disorders characterized by degeneration of cartilaginous tissues and inflammation. While inflammation is implicated in the pathogenesis of OA and IVDD, and cytosolic phospholipase A2(cPLA2) is a key mediator of inflammation, direct evidence linking cPLA2 to chondrocyte homeostasis and cartilage degeneration is lacking. This study aims to investigate the role of cPLA2 in chondrocytes and its contribution to the development of cartilage degenerative conditions such as OA and IVDD. Here, single-cell RNA sequencing was used to examine cPLA2 expression in chondrocytes. To explore its importance in chondrocytes and OA/IVDD, various cell-based assays and genetically modified mouse models with age-related and surgically induced OA/IVDD were employed. Furthermore, the therapeutic potential of fexofenadine, an over-the-counter drug recently identified as a cPLA2 inhibitor, was explored in these models. cPLA2 is predominantly expressed in prehypertrophic chondrocytes, characterized by elevated levels of cartilage degeneration markers and senescence-related genes. Genetic deletion and pharmacological inhibition of cPLA2 reduced inflammation induced catabolic activity and senescence in chondrocytes, as well as cartilage degeneration in various OA and IVDD models. This study identifies cPLA2 as a pivotal driver of cartilage degeneration and senescence in OA and IVDD, highlighting its potential as a dual-action therapeutic target that suppresses both inflammation and senescence to preserve cartilage integrity. These findings position cPLA2as a promising candidate for developing disease-modifying therapies for cartilage degenerative conditions such as OA and IVDD.
基金Supported by the Traditional Chinese Medicine Administration Bureau Foundation of Jiangsu Province,No.9965the Applied Basic Research Program of Science and Technology Commission Foundation of Jiangsu Province,No.BJ2000327
文摘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.
基金supported by the National Natural Science Foundation of China [Grant No. 81973112 and Grant No.9204930002]
文摘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.
基金supported by grants of the National Basic Research Priorities Program of China (2015CB554304)
文摘The phosphatidylserine-specific phospholipase A1(PLA1A)is an essential host factor in hepatitis C virus(HCV)assembly.In this study,we mapped the E2,NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts,through which they form oligomeric protein complexes to participate in HCV assembly.Multiple regions of PLA1A were involved in their interaction and complex formation.Furthermore,the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A,and strongly suggest PLA1 A-E2,s physical interaction in cells.Meanwhile,we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1.Interestingly,these amino acids in the sequence are also essential for viral RNA replication.Further experiments revealed that these four proteins interact with each other.Moreover,PLA1A expression levels were elevated in livers from HCV-infected patients.In conclusion,we exposed the structural determinants of PLA1A,E2,NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.
文摘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.
文摘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.
基金supported by the National Natural Science Foundation of China,No.82072192(to KLZ)Public Welfare Technology Research Project of Zhejiang Province,No.LGF20H150003(to KLZ)+1 种基金the Natural Science Foundation of Zhejiang Province,Nos.LY17H060009 and Y21H060050(both to WFN)Wenzhou Science and Technology Bureau Foundation,No.Y20210438(to KLZ)。
文摘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.
文摘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）.
文摘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.
基金Project (No 2007AA10Z324) supported by the National High-Tech Research and Development Program (863) of China
文摘Bee venom phospholipase A2(BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids.In this work,a new BvPLA2(AccPLA2) gene from the Chinese honeybee(Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector.Tn-5B-4(Tn) cells were transfected with the recombinant bacmid DNA for expression.Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa.Products of hexahistidine AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins.The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2(BvPLA2 of the European honeybee,Apis mellifera) polyclonal serum.The reaction resulted in a double glycosylation band,which agrees with the band generated by the native AmPLA2 in Western blot analysis.The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg).In summary,the recombinant AccPLA2 protein,a native BvPLA2-like structure with corresponding biological activities,can be glycosylated in Tn cells.These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry.
文摘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.
文摘Calcium-independent phospholipase A<sub>2</sub> (iPLA<sub>2</sub>) belongs to the group VI family of phospholipase superfamily (PLA<sub>2</sub>) that catalyses the hydrolysis of glycerophospholipids at the sn-2 ester bond, producing unesterified fatty acids and 2-lysophospholipids. Research interests on iPLA<sub>2</sub> have not been as significant as those on secretary PLA<sub>2</sub> and cytosolic PLA<sub>2</sub>. However, more efforts have been made recently on understanding the expression, regulation and biological function of iPLA<sub>2</sub>. iPLA<sub>2</sub> plays important roles in several biological processes, including signal transduction, phospholipid remodelling, eicosanoid formation, cell proliferation, cell differentiation and apoptosis. Modulation of iPLA<sub>2</sub> activity can have prominent effects on cellular metabolism, central nervous system and cardiovascular functions. Thus, dysregulation iPLA<sub>2</sub> can play a vital role in the pathogenesis of several diseases. The aim of this review is to provide the current understanding of the structure, function and regulation of group VI iPLA<sub>2</sub> and highlight its potential mechanisms of action in mediating several neurological disorders and cancer.
文摘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.
文摘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.
文摘A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary development of the as say. Various combinations of human, sheep, rat, and mouse red blood cells (RBC) with human,rat,and mouse sera were tested on their effectiveness to detect PLA2.Complete hemolysis (b hemolysis) was evident in the plate with rat RBC mixed with mouse serum.No hemolysis was detected in plates containing human RBC and human serum. Human RBC mixed with mouse serum proved to be ideal, even though this combination displayed incomplete hemolysis(a hemolysis).Susceptible RBC, in conjunction with rat or mouse serum, are excellent indicators for the presence of PLA2.Mixtures of RBC and serum in combination with BB4(E. coli) cells,λbacteriophage,and IPTG on LB agar plates provide an excellent detection system for cDNA clones that express venonl PLA2. Hemolysis surrounding a plaque is identified as positive for PLA2.
文摘Objective: To imastigate the effects of anisodamine on pulmonary α1- adrenergic receptor andphospholipase A, in acute lung injurg. Methods: Change of α1--adrenergic receptor (al AR ) in lung tissllesduring endotoxin--induced rat acute lung injury was measured with radioligand biding assay. The effects ofanisodamine on pulmonary α1--AR and phospholipase A2 (PLA2 ) were observed. Results: 1. 4 h after theendotoxin injection, there was a significant decrease in the maximal binding capacity of α1--AR by 34% ascompared with the control group. meanwhile elevated activity of PLA2 in rat lung and reduction of thephospholipids content of cell membrane was found. 2. Anisodamine could attenuate endotoxin--induced acutelung injury in rats. Conclusion: This effect might be related to anisodamine’s blockage of α1--AR andsuppression of PLA2, prevention of membranous phospholipids from degradation. and the reduction ofarachidonic acid release.
文摘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.
文摘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.
文摘A fraction of cobra （Naja naja atra） venom has been discovered to have protective effect on rat cultural cerebellar granule neurons （CGNs） from apoptosis induced by removing serum and reducing the extracellular potassium concentration from 25 to 5 mM. This component has been purified and identified as secreted phospholipase A2 （cobra sPLA2）. In order to study the relationship between the protection on CGNs and enzymatic activity of phospholipase A2, CGNs stained by Hoechst 33258 were quantified to determine survival rate under the fluorescence microscopy; the protective potencies on apoptosis of cultural CGNs were compared among cobra sPLA2, the cobra sPLA2 modified in carboxylate groups with water soluble carbodiimide and semicarbazide, and the heated cobra sPLA2 at 80℃ for 30 rain. The results showed that the CGN survival rate was unaffected significantly both in modified cobra sPLAz whose enzymatic activity of PLA2 had decreased by 80%, and in cobra sPLA2 adding 7, 7-Dimethyleicosadienoic acid, an inhibitor of sPLA2 at concentration of 10-fold IC50; contrary, the neuronal survival rate fell about 60% in heated cobra sPLA2, although its PLA2 activity only decreased by 10%. The protection on CGNs were also found in some of sPLA2s derived from venoms of bee, Naja naja mossambica, Crotalus atroxalso and Vipera Ammodytes Ammodytes but could not be found in other sPLA2s from bovine pancreas and Streptomyces violaceoruber. Above results suggest that the protection on CGNs of cobra sPLA2 is independent of its enzymatic activity.
基金financially supported by National Natural Science Foundations of China(No.31471615)Scientific Research Start-up Fund for Introduced Talents of Anhui Polytechnic University(2022YOO068)Natural Science Research Project of Colleges and Universities in Anhui Province(Grant 2022AH050971).
文摘Phospholipase A1(PLA1)is a kind of specific phospholipid hydrolase widely used in food,medical,textile.However,limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS.In this study,we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation.Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells,resulting in improved cell growth and increased protein solubility of the protein.The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS(ΔN27 PlaS),highlighting their binding capabilities.Furthermore,in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 andΔN27 PlaS,exhibiting high affinity.Molecular docking analysis provides insights into the hydrogen bond interactions betweenΔN27 PlaS and PlaA1,identifying key amino acid residues crucial for their binding.Finally,the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test.Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1,offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.
