Value of neutrophil to lymphocytes and platelets ratio for predicting 28-day mortality in sepsis patients / 中华危重病急救医学

Objective:To evaluate the value of neutrophil to lymphocyte and platelet ratio (N/LPR) for predicting 28-day mortality in sepsis patients.Methods:A retrospective analysis was conducted. The clinical data of 154 sepsis patients admitted to intensive care unit (ICU) of the Affiliated Hospital of Jiangsu University from June 2017 to June 2020 were enrolled. The time of first diagnosis of sepsis in ICU was taken as the research starting point, and the death or 28 days as the end point. The 28-day outcomes of patients were recorded. The counts of peripheral blood neutrophil (NEU), lymphocyte (LYM) and platelet (PLT) were collected from all the enrolled patients within 3 days after diagnosis of sepsis. The ratios of N/LPR and NEU/LYM (NLR) were calculated respectively. The differences of N/LPR and NLR between survival group and death group were compared. Receiver operating characteristic (ROC) curve analysis was used to analyze the value of N/LPR and NLR on predicting the 28-day mortality of sepsis patients. According to the best cut-off value of ROC curve analysis, the 28-day mortality of patients with sepsis was analyzed by subgroup analysis, and the 28-day cumulative survival of patients with sepsis was analyzed by Kaplan-Meier survival curve.Results:Of the 154 sepsis patients, the patients with age < 18 years, pregnancy, blood disease, taking aspirin or other antiplatelet drugs within 1 week, taking leucocyte drugs within 1 week, length of ICU stay < 3 days and incomplete data were excluded. Finally, 50 patients were enrolled. Among them, 30 patients survived on the 28th day and 20 died. Compared with the survival group, the levels of N/LPR and NLR in the death group were significantly increased (N/LPR: 23.85±11.99 vs. 12.41±5.25, NLR: 17.83±8.69 vs. 10.75±3.63), with statistical differences (both P < 0.01). ROC curve analysis indicated that the area under ROC curve (AUC) of N/LPR for predicting 28-day death of sepsis patients was 0.827, it was higher than that of NLR (AUC = 0.762). Base on N/LPR≥15.48 as a predictor of cut-off value of death in 28 days of sepsis patients, the sensitivity was 75.0% and the specificity was 80.0%, respectively. Base on NLR≥10.65 as a predictor of cut-off value of death in 28 days of sepsis patients, the sensitivity was 75.0% and specificity was 56.7%, respectively. Subgroup analysis showed that the 28-day mortality in the patients with N/LPR≥15.48 ( n = 21) was significantly higher than those with N/LPR < 15.48 ( n = 29; 71.4% vs. 17.2%, χ 2 = 14.901, P < 0.01); and the 28-day mortality in the patients with NLR≥10.65 ( n = 28) was also significantly higher than those with NLR < 10.65 ( n = 22; 53.6% vs. 22.7%, χ 2 = 4.884, P < 0.05). The results were consistent with Kaplan-Meier survival curve analysis. Conclusion:Peripheral blood N/LPR has a good predictive value for 28-day mortality of sepsis patients, and which is better than NLR.

Peripheral 5-hydroxytryptophan aggravates lung injury in septic mice by inducing the formation of neutrophils extracellular trap / 中华危重病急救医学

Objective:To observe the effect of peripheral 5-hydroxytryptophan (5-HT)-induced neutrophil extracellular trap (NET) on lung injury in septic mice.Methods:Wild-type (WT type) and Tph1 knockout (KO) C57 mice (6-8 weeks) were selected and divided into WT mice sham group, WT mice sepsis group, Tph1 KO mice sham group and Tph1 KO mice sepsis group according to the random number table method. Mice in the sham group received sham surgery (only open the abdominal cavity to flip the cecum without ligation and puncture, and then close the abdominal cavity); the mice in the sepsis group received cecal ligation and puncture (CLP) to establish sepsis model. The mice were sacrificed 12 hours after the operation, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in bronchialalveolar lavage fluid (BALF) were detected by enzyme linked immunoadsordent assay (ELISA); at the same time, the lung tissues were collected, and the pathological changes of lung tissues were observed under light microscope, and the production of NET in lung tissues was observed by immunofluorescence microscope. Results:The pathological results suggested that the lung tissue structure in sham groups was intact without exudation, while the alveolar structures of mice in the sepsis groups were damaged, with obvious exudation in the alveolar cavity and thickened alveolar walls accompanied by a large number of inflammatory cell infiltration, and the degree of lung injury in the sepsis group of WT mice was more severe than that of the sepsis group of Tph1 KO mice. ELISA results showed that there was no statistically significant difference in the contents of TNF-α and IL-6 in mice BALF from different strains of the sham group; while the contents of TNF-α and IL-6 in BALF of septic mice group were significantly higher than those in sham group [WT mice: TNF-α (μg/L) was 158.20±28.46 vs. 14.00±3.28, IL-6 (μg/L) was 304.98±21.78 vs. 57.70±12.30; Tph1 KO mice: TNF-α (μg/L) was 85.88±20.13 vs. 14.95±1.53, IL-6 (μg/L) was 169.50±45.61 vs. 55.05±12.68, all P < 0.01], and the above index levels in the sepsis group of WT mice were significantly higher than the sepsis group of Tph1 KO mice [TNF-α (μg/L): 158.20±28.46 vs. 85.88±20.13, IL-6 (μg/L): 304.98±21.78 vs. 169.50±45.61, both P < 0.01]. Immunofluorescence staining showed that a very small amount of NET formation was detected in the mice lungs from the sham group; a large amount of NET formation was detected in the lung tissues in the sepsis group, which were significantly higher than those in sham group [WT mice: (34.75±7.27)% vs. (1.75±0.96)%, Tph1 KO mice: (14.25±5.74)% vs. (2.50±1.29)%, both P < 0.01], and the amount of NET produced in the lung tissues of the WT mice sepsis group was significantly higher than that of the Tph1 KO mice sepsis group [(34.75±7.27)% vs. (14.25±5.74)%, P < 0.01]. Conclusions:In sepsis, the increased production of inflammatory factors in the mice lung tissues induces to lung injury. The mechanism may relate to the increased production of NET in the lung tissues mediated by peripheral 5-HT synthesized by enterochromaffin cells and released into the blood; inhibiting the production of 5-HT in the peripheral blood can effectively reduce the production of NET in the lung tissues, thereby reducing lung injury.

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.

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>

Leflunomide in treating rheumatoid arthritis: a double-blind study / 中国新药与临床杂志

treating rheumatoid arthritis.　METHODS: Eighty patients with rheumatoid arthritis were randomly divided into two groups with each group having 40 patients. Group one (M 8, F 32; age 46 a± s 11 a; disease history 63 mo±48 mo) was treated with anti-inflammation sub-group No.1 and No.3. Group two (M 6, F 34; 44 a±9 a; disease history 45 mo±45 mo) was treated with sub-group No.2 and No.4. One week before the initiate of the study, the originally used non-steroid anti-inflammation drugs were stopped for all two-groups patients and each patient took 2 tablets of oxaprozin po qn. At the beginning of the study the patients received 2 tablets of anti-inflammation drugs No.1 daily and 6 tables of No.3 weekly in 1st group, and 2 tablets of anti-inflammation drugs No.2 daily and 6 tablets of No.4 weekly in 2nd group respectively.　RESULTS: In the leflunomide group, the total effect rate was 93 % and the remarkable improvement rate was 85 %. In the methotrexate group, the total effect rate was also 93 % and the remarkable effect rate was 83 %, P>0.05. Nine patients (23 %) in leflunomide group had adverse reaction as mainly skin itch, nettle-like rash, decrease of leukocytes, liver malfunction and others. Seventeen parients (43 %) in methotrexate group had adverse reaction as mainly responses of digestive tract, liver enzyme elevation, decrease of leukocytes, trichomadesis, manoxenia, and others.　CONCLUSION: Leflunomide has similar therapeutic efficacy to methotrexate. However, it has relatively less toxicity.