Suppressive effect of CORM-2 on platelet α-granule exocytosis in sepsis via SNARE/Munc18b complex formation / 中华危重病急救医学

Objective To investigate the suppressive effect of carbon monoxide-releasing molecule Ⅱ (CORM-2) on LPS induced platelet α-granule exocytosis in sepsis via soluble N-ethylmaleimide-sensitive factor attached protein receptor/mammalian uncoordinated 18b (SNARE/Munc18b) complex formation.Methods Blood was collected from healthy volunteers' cubital vein, then platelets were isolated by differential centrifugation. Platelets were randomly divided into 5 groups. The control group did not undergo any treatment, the LPS group received 10 mg/L LPS simulation, the CORM-2 group and iCORM-2 group underwent LPS simulation and immediate administration of CORM-2 (10μmol/L and 50μmol/L) or iCORM-2 (50μmol/L), respectively. Samples were incubated in a CO2-incubator at 37 ℃, 95% humidity, and 5% CO2. Platelet α-granule contents were detected by using standard enzyme linked immunosorbent assay (ELISA), including platelet factor 4 (PF4), platelet derived growth factor-BB (PDGF-BB), and matrix metalloproteinase-2 (MMP-2). The expression of P-selectin was detected by flow cytometer. Transmission electron microscope and immunofluorescence microscope was used to assess platelet α-granules distribution. Expressions of Munc18b and SNARE proteins including vesicle-associated membrane protein-8 (VAMP-8), synaptosomal-associated protein-23 (SNAP-23) and syntaxin-11 (STX-11) were detected by Western Bolt. The SNARE/Munc18b complex formation was detected by immunoprecipitation.Results Compared with the control group, levels of PF4, PDGF-BB, MMP-2 and P-selectinin LPS-induced platelets were found to markedly elevated, while CORM-2 (10μmol/L and 50μmol/L) could decrease platelet α-granule contents exocytosis: [PF4 (μg/L): 7.69±0.58, 6.03±0.71 vs. 10.13±0.82; PDGF-BB (μg/L): 112.71±1.79, 102.91±5.86 vs. 128.78±1.39; MMP-2 (ng/L): 32.94±2.73, 27.58±3.36 vs. 53.26±1.21; P-selectin: (17.14±0.57)%, (15.35±0.68)% vs. (23.78±0.62)%; allP < 0.01]. Transmission electron microscope and immunofluorescence microscope showed that the extent of platelet α-granules assembled to platelet plasma membrane was significantly decreased following CORM-2 treatment. Compared with the control group, the expressions of Munc18b and SNARE proteins and SNARE/Munc18b complex formation in LPS-stimulated platelets were significantly increased, while CORM-2 (10μmol/L and 50μmol/L) inhibited these elevations (Munc18b/GAPDH: 0.80±0.08, 0.69±0.01 vs. 0.99±0.09; VAMP-8/GAPDH: 0.72±0.09, 0.50±0.12 vs. 1.18±0.14; SNAP-23/GAPDH: 1.18±0.22, 0.63±0.10 vs. 1.90±0.08; STX-11/GAPDH: 0.76±0.02, 0.57±0.08 vs. 1.16±0.23; VAMP-8/ Munc18b: 0.65±0.09, 0.53±0.07 vs. 1.21±0.20; SNAP-23/Munc18b: 0.85±0.07, 0.55±0.09 vs. 1.26±0.08; STX-11/ Munc18b: 0.78±0.05, 0.61±0.10 vs. 1.39±0.16; allP < 0.01). Above all, the data showed a dose dependent change.Conclusion We could suggest that CORM-2 suppressed α-granule exocytosis in LPS-stimulated platelets and the potential mechanisms might involve SNARE/Munc18b complex formation.

Suppressive effect of exogenous carbon monoxide on abnormal platelet exocytosis and its molecular mechanism in sepsis / 中华危重病急救医学

Objective To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism. Methods Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation. Results ① Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P < 0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P < 0.05]. ② Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P < 0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P < 0.05]. ③ Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P < 0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group. Conclusions LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.

Effects of exogenous carbon monoxide-releasing molecule 2 intervention in vitro on formation of human neutrophil extracellular traps stimulated by endotoxin/lipopolysaccharide and its mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of exogenous carbon monoxide-releasing molecule 2 (CORM-2) on formation of human neutrophil extracellular traps (NETs) stimulated by endotoxin/lipopolysaccharide (LPS) and its relevant mechanism.</p><p><b>METHODS</b>Venous blood samples were collected from a healthy adult volunteer to isolate neutrophils. The neutrophils were divided into normal control (NC) group, LPS group, LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ inactive CORM-2 (iCORM-2) group according to the random number table. No treatment was given to the neutrophils in NC group. The neutrophils in LPS group underwent LPS stimulation (1 μL, 1 μg/mL). The neutrophils in LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ iCORM-2 group underwent the same LPS stimulation as that in LPS group and treatment of 10 μmol/L CORM-2, 50 μmol/L CORM-2, and 50 μmol/L iCORM-2, respectively, with the volune of 1 μL. After conventional culture for 1 h, the number of NETs was determined with propidium iodide staining method; the early cell apoptosis rate was determined with flow cytometer; the generation level of reactive oxygen species (ROS) was assessed with dihydrogenrhodamine 123 fluorescent probe staining method (denoted as mean fluorescence intensity); the expression level of phosphorylated extracellular regulated kinase 1/2 (p-ERK1/2) was determined by Western blotting. The sample numbers of each group in the 4 experiments were all 5. Data were processed with one-way analysis of variance and SNK test.</p><p><b>RESULTS</b>(1) The numbers of NETs per 400-time visual field in cells of LPS and LPS+ iCORM-2 groups were close to the number in NC group (with P values above 0.05). The number of NETs per 400-time visual field was significantly larger in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in NC and LPS groups (with P values below 0.05). The number of NETs per 400-time visual field in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05). (2) The early cell apoptosis rate was significantly increased in LPS, LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The early cell apoptosis rates in LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups were close to the rate in LPS group (with P values above 0.05). (3) The generation level of ROS was significantly higher in cells of LPS, LPS+ 10 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The generation level of ROS in cells of LPS+ 50 μmol/L CORM-2 group was close to that of NC group (P>0.05). The generation level of ROS was lower in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05), while the generation level of ROS in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05). (4) The expression levels of p-ERK1/2 in cells of LPS and LPS+ iCORM-2 groups (respectively 0.0311±0.001 and 0.0309±0.0018) were close to the level in NC group (0.0304±0.0046, with P values above 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups (respectively 0.7891±0.0201 and 1.2970±0.0056) than in NC group (with P values below 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05). The expression level of p-ERK1/2 in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05).</p><p><b>CONCLUSIONS</b>CORM-2 can obviously increase the production of NETs in LPS-induced neutrophils, and it might be attributable to the promotion of inhibition of ROS generation and phosphorylation of ERK1/2.</p>

Study of exogenous carbon monoxide-releasing molecules 2 on endotoxin/lipopolysaccharide-induced abnormal activation of platelets of healthy human donors / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism.</p><p><b>METHODS</b>Venous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 µg/mL) stimulation, and the PRP in iCORM-2 group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 µmol/L iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein I bα (GPIbα) and GPVI were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test.</p><p><b>RESULTS</b>Compared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPIbα and GPVI in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with P values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with P values above 0.05), while the levels of ATP in platelets in the 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased (with P values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2 groups were 0.550 ± 0.060, 1.437 ± 0.214, 1.210 ± 0.108, 0.720 ± 0.010, and 0.670 ± 0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950 ± 0.070, 1.607 ± 0.121, 1.420 ± 0.040, 1.167 ± 0.015, and 0.513 ± 0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with P values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with P values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased compared with those in LPS group (with P values below 0.05).</p><p><b>CONCLUSIONS</b>LPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.</p>

Perinatal management and outcomes of triplet and multiple pregnancy / 中华全科医师杂志

Objective To analyze clinical management and outcomes of perinatal fetuses born in triplet and multiple pregnancy.Methods Data of clinical management and outcomes of 55 perinatal fetuses born during 1996 to 2005 by women with triplet and multiple pregnancy were analyzed retrospectively.Results Antenatal check-up was performed regularly for 1/6 of those pregnant women during 1996 to 2000,as compared to that of 8/11 during 2001 to 2005(P<0.05).No significant difference in incidence of varied obstetric complications was found between those during 1996 to 2000 and during 2001 to 2005.Average gestational week at birth was(32.7±2.8)and(35.1±1.9)weeks(P<0.05),and averagebirth weight was(1561±471)grams and(1987±453)grams(P<0.01),respectively for them during 1996 to 2000 and during 2001 to 2005.Caesarean section was performed more for the pregnant women in the earlier first five years than in the later five years,but not reaching statistical significance.Incidence of neonatal complications significantly decreased in the later five years than that in the earlier five yeats,including hyaline membrane disease,neonatal asphyxia,infectious diseases,intracranial hemorrhage and pulmonary hemorrhage(P<0.05),but difference in incidence of apnea and low body temperature between the earlier and later five years did not reach statistical significance.Perinantal mortality was 8 in 35 births in the later five years,as compared to that of 12 in 20 births in earlier five years(P<0.01).Conclusions Outcomes of perinatal fetuses in triplet and multiple pregnancy can be improved by combined action of obstetricians and pediatricians,including regular antenatal examination,active prevention and treatment for pregnant complications and preterm infant diseases,earlier admission waiting for delivery to prolong gestational weeks and increase birth weight,and applying antenatal dexamethasone therapy.