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Objective@#To investigate the association of SNPs in TET2 gene with the susceptibility and prognosis of sepsis.@*Methods@#Ninety-nine patients diagnosed with sepsis and 107 controls were enrolled in the study. The septic patients were further divided into survivors (56 cases) and non-survivors (43 cases) according to the outcome of 28-day hospitalization. Patients without sepsis after major surgery were enrolled as the controls. The genotypes of the five loci (rs6839705, rs7670522, rs7679673, rs7698522 and rs10010325) with high minor allele frequency in the TET2 were screened according to the existing research reports and the SNP database of the NCBI website. The five loci were detected by TaqMan probe based allelic discrimination assays using real-time polymerase chain reaction (PCR). The data were calculated for genetic association study through χ2 test and Fisher’s exact probability method.@*Results@#There was no significant difference in genotype frequencies of the five tested SNPs in TET2 gene between septic patients and controls or between survivors and non-survivors in septic patients (P > 0.05). Furthermore, the allelic frequencies of the five SNPs between septic patients and controls or between survivors and non-survivors in septic patients also had no significant difference (P > 0.05).@*Conclusions@#This study showed that the five SNPs in TET2 gene (rs6839705, rs7670522, rs7679673, rs7698522, and rs10010325) were not associated with the susceptibility and prognosis of sepsis, which needs to be further confirmed by large-sample studies.
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Objective To investigate the association of SNPs in TET2 gene with the susceptibility and prognosis of sepsis.Methods Ninety-nine patients diagnosed with sepsis and 107 controls were enrolled in the study.The septic patients were further divided into survivors (56 cases) and non-survivors (43 cases) according to the outcome of 28-day hospitalization.Patients without sepsis after major surgery were enrolled as the controls.The genotypes of the five loci (rs6839705,rs7670522,rs7679673,rs7698522 and rs10010325) with high minor allele frequency in the TET2 were screened according to the existing research reports and the SNP database of the NCBI website.The five loci were detected by TaqMan probe based allelic discrimination assays using real-time polymerase chain reaction (PCR).The data were calculated for genetic association study through x2 test and Fisher's exact probability method.Results There was no significant difference in genotype frequencies of the five tested SNPs in TET2 gene between septic patients and controls or between survivors and non-survivors in septic patients (P ＞ 0.05).Furthermore,the allelic frequencies of the five SNPs between septic patients and controls or between survivors and non-survivors in septic patients also had no significant difference (P ＞ 0.05).Conclusions This study showed that the five SNPs in TET2 gene (rs6839705,rs7670522,rs7679673,rs7698522,and rs10010325) were not associated with the susceptibility and prognosis of sepsis,which needs to be further confirmed by large-sample studies.
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Objective RY-15, a specific agonist of Sphingosine 1-phosphate Receptor 3, was synthesized for investigating the function and mechanism of S1PR3 in bacterial clearance. Methods Measure the ability of RY-15 with FITC to enter the THP-1 cell after coculture for 5 min, 10 min, 20 min, 30 min through confocal microscopy. The function of GY-5 and RY-15 in bacterial clearance was observed by gentamicin protection test. The phosphorylation level of ERK and p-ERK in THP-1 cell was detected by Western Blot after GY-5 and RY-15 stimulation for different times. Results According to confocal microscopy, RY-15 started to enter the THP-1 cell after stimulating for 10 min and the effect of entering cell was very obvious after stimulating for 30 min. Compared to GY-5 group, live bacteria in the macrophage were largely decreased in the RY-15 group( P<0.05). Conmpared to GY-5 group, the p-ERK level raised largely at different poins. Conclusions RY-15, a specific agonist of Sphingosine 1-phosphate Receptor 3, can promote bacterial clearance through entering cell and the phosphorylation level of ERK is a possible mechanism.
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Objective To study the effects of the myristoyl-glycine modified peptide which derived from the second intracellular loop of sphingosine 1-phosphate receptor 3 (S1PR3) on activation of mitogenactivated protein kinases (MAPKs) pathway.Methods The phosphorylation levels of JNK and ERK in THP-1 cells were detected by western blot after GPS-725.017 stimulation.Statistical data analysis was conducted by multivariate analysis of variance.Results Western blot showed that 10 min after 30 μmol/L or 50 μmol/L GPS-725.017 stimulated,phosphorylation of ERK significantly increased in comparison with the solvent-treated group [30 μmol/L group:(3.10 ± 0.27) vs.(7.98 ± 0.45),P ＜ 0.01;50 μmol/L group:(4.78 ±0.44) vs.(25.98 ±2.32),P ＜0.01];after 50 μmol/L GPS-725.017 stimulated THP-1 cells for 5 min,10 min,20 min or 30 min,p-ERK or p-JNK level raised at different time points (P ＜0.01vs.solvent group).Conclusions GPS-725.017,a kind of myristoyl-glycine modified peptide derived from S1 PR3,could traverse cytomembrane and activate MAPKs pathway.This study provides an implication of targeting S1PR3 for clinical therapy on inflammatory diseases or sepsis.
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Objective To study the role of KRX-725,a specific agonist of Sphingosin-1 phosphate receptor 3,S1PR3),in the function and mechanism of S1PR3 in respect of bacterial clearance.Methods Twenty healthy male C57BL/6 mice were randomly (random number) divided into two groups:KRX-725 group and control group.Septic mice model were established by intraperitoneal injection of E.coli (3 × 106),then KRX-725 (10 mg/kg) or the vehicle was administered intratracheally.Forty-eight-hour survival rate (n =12),bacterial colony numbers in peritoneal cavity and blood (n =5),and lung injury (n =3)were compared between two groups.In vitro,the peritoneal macrophages were stimulated by E.coli (cell∶E.coli=1∶10) with KRX-725 or the vehicle treatment.The reactive oxygen species (ROS) levels were detected by CM-H2DCFDA in macrophages.The bacteria clearance function of KRX-725 was observed by gentamicin protection test.Survival rates were analyzed with the Log-rank test.A 2-tailed student's t test was used to compare difference between two independent groups.Results Compared with the control group,the 48-hour survival rate of KRX-725 group was significantly higher (P ＜ 0.05).Bacterialload in the blood and the peritoneal lavage fluid (PLF) was greatly decreased in the KRX-725 group (blood:t =3.17,P ＜0.05;PLF:t =4.07,P ＜0.01).The lung tissues injury was also obviously reduced in the KRX-725 group of 24 hours after the injection of E.coli (lung injury score:KRX-725 group 1.4 ± 0.25;control group 2.4 ± 0.25) (t =2.89,P ＜ 0.05).In vitro,KRX-725 could up-regulate the ROS levels in macrophage at 20 min and 30 min after E.coli injected intra-peritoneally (20 min fluorescent intensity:KRX-725 group 522.9 ± 38.76,control group 385.9 ± 15.90,P ＜ 0.05;30 min fluorescent intensity:KRX-725 group 519.7 ±25.02,control group 384.5 ± 15.28,P ＜0.01).The bacterial load in the KRX-725 treated macrophage were significantly decreased at 3 h and 6 h after E.coli injected intra-peritoneally (3 h:KRX-725 group 286.5 ±98.35,control group 710.8 ± 107.8,P ＜0.05;6 h:KRX-725 group 72.5 ±6.45,control group 205.8 ±66.76,P ＜0.01).Conclusion In vivo,KRX-725 could improve the survival rate of septic mice,decrease the bacterial lioad in the blood and PLF,and reduce the lung injury.In vitro,KRX-725 could up-regulate the ROS level in macrophages and accelerate the bacterial clearance.
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Objective The purpose of this research is to study the preventive and therapeutic effects of suramin on lipopolysaccharide (LPS)-induced mouse model of acute lung injury and its molecular mechanism.Methods A total of 24 healthy male C57BL/6 mice were randomly divided into two groups: Control group and suramin group.LPS (5 mg/kg, iv) induced acute lung injury model was used in this study.The severity of lung injury was evaluated using haematoxylin-eosin (HE) staining after the injection of LPS for 0, 24 and 72 hours.The expression of TNF-α and IL-6 mRNA levels were also detected by RT-PCR.In vitro, THP-1 cells were stimulated by LPS (100 ng/mL) with saline or suramin pre-treatment.The expressions of p-ERK1/2, p-JNK and p-P38 were analyzed by Western blot at 10 min, 20 min and 30 min after LPS insult.A 2-tailed Student's t test was used to compare difference between two independent groups.Results Compared with the saline group, the lung tissues injury were significantly decreased in the suramin group of 72 hours after the injection of LPS (saline 3.90 ±0.35;suramin 2.50 ±0.12) (t =7.668, P ＜ 0.01).The expressions of TNF-α (saline 8.35 ± 1.63;suramin 4.62 ± 0.70) (t =4.187, P＜0.01) andIL-6 (saline10.53 ± 2.10;suramin5.53±1.10) (t=4.224, P＜0.01) mRNA were also obviously reduced in suramin group after the injection of LPS for 24 hours.The expression levels of pERK1/2, p-JNK and p-P38 were obviously down-regulated by suramin at 10 min, 20 min and 30 min after LPS stimulation.Conclusion Suramin protected LPS-induced acute lung injury through down-regulating the expression of pro-inflammatory factors, which was closely relative to the inhibition of the MAPK pathway.