| Objective:By exploring the changes of S1P,HGF and CDH5 related factors in the cell barrier function pathway in sepsis,the therapeutic effects of SIP and EPC on sepsis and ARDS were clarified,so as to provide a new idea for the treatment of sepsis and ARDS.Methods:1.Clinical partThe peripheral blood was collected from 10 patients(Sepsis group)and 10 healthy subjects(NC group)who were admitted to the respiratory department of our Hospital from April 2019 to December 2019 and met the diagnostic criteria for Sepsis and ARDS.The expressions of SIP,CDH5,HGF,IL-1β,IL-10,TNF-α and PCT in serum of peripheral blood of the two groups were detected within 24 hours after admission.2.The animal part(1)Subculture and in vivo imaging of endothelial progenitor cells:a large number of EPC adherent growth could be seen under microscope on the 7th day of culture,labeled EPC could be seen under fluorescent labeling,and fluorescence of labeled EPC could be seen in the chest of the LPS+EPC group.(2)Establishment of sepsis and EPCs transplantation models:First,the EPCs previously identified in our research group were resuscitated,sub cultured and fluoresced for later use.Rats were intravenously injected with LPS solution(1mg/ml,5mg/kg),and 200ul EPC was injected into the tail vein of rats in the LPS+EPC group 1 hour later.Rats in the LPS group and the LPS+EPC group were fluorescence tracers on in vivo imaging apparatus after 10%phenobarbital shallow anesthesia.(3)Retention of rats specimens:Rats were indwelling 5 days after the model was constructed,the rats were fixed on a clean surgical plate after abdominal anesthesia with chloral hydrate(0.05g/kg),anaesthetized and dissected,and the specimens were retained.He staining was performed on the lung tissues:according to Mikawa method,semi-quantitative pathological score was performed on the lung tissues:0 in the NC group,14 in the LPS group,and 6 in the LPS+EPC group.Rapid frozen sections were made to observe the pathological changes and fluorescence localization in each group under microscope.The expression levels of cytokines SIP,CDH5,HGF,IL-1β,IL-10,TNF-α and PCT were detected by ELISA in peripheral blood and lung tissue.The protein expressions of CDH5,Rac-1,GTP-Rac-1,c-Met,P-c-Met(Y1003),P-c-Met(Y1234+1235),SPHK1,SPNS2 and S1PR1 were detected by Western blot.The mRNA expression levels ofc-Met,Rac-1,SPHK1,SIPR1 and SPNS2 were detected by RTPCR.Results:1.Clinical partThe expressions of SIP,CDH5,HGF,IL-1β,IL-10,TNF-α and PCT in peripheral blood of NC group and Sepsis group were detected by ELISA.(1)The expressions of CDH5,HGF,IL-1β,IL-10,TNF-α and PCT in sepsis group were significantly higher than those in NC group(t=2.913,3.580,4.856,4.205,4.293,3.385,P<0.05,respectively).SIP significantly decreased(t=4.871,P<0.05).Blood HGF concentration was negatively correlated with S1p in the Sepsis group(r=0.7947,P=0.0060).There was a positive correlation between CDH5 and NC group and Sepsis group(r=0.7683,P=0.0094).Blood SIP concentration was negatively correlated with CDH5 in the sepsis group(r=-0.9349,P<0.05).(2)ROC curve:AUC of SIP,HGF,CDH5,PCT,IL-1β,IL-10 and TNF-α were 0.9500,0.8850,0.8100,0.8750,0.9200,0.9300,0.9000,respectively.All the above factors have predictive value for the diagnosis of sepsis,and SIP has the highest AUC,which indicates that SIP has better diagnostic effect than other factors.2.The animal part(1)Subculture and in vivo imaging of endothelial progenitor cells:a large number of EPC adherent growth could be seen under microscope on the 7th day of culture,labeled EPC could be seen with fluorescence labeling,and the fluorescence of labeled EPC could be seen with obvious expression in the chest of the LPS+EPC group.(2)HE staining of lung tissue was performed,and semi-quantitative score of lung tissue was performed according to Mikawa method.NC group score:(1.0±0.894);The score of the LPS group was(14 ± 1.26),and that of the LPS+EPC group was(6.0 ± 1.41).Rapid frozen sections of lung tissue:in the LPS group,no distribution of labeled EPC was found in the lung tissue.Rapid frozen sections of lung tissue in the LPS+EPC group showed the distribution of labeled EPC in lung tissue.(3)The expression of SIP,CDH5,HGF,IL-1β,IL-10,TNF-α and PCT in peripheral blood and lung tissue of rats were detected by ELISA.Serum concentrations of HGF and IL-10 in the LPS group and the LPS+EPC group were higher than those in the NC group,with significant differences among the three groups(F=35.1,202,P<0.0001,respectively),and significantly increased in the LPS+EPC group compared with the LPS group(t=4.00,2.37,P<0.05,respectively).The serum concentrations of IL-1β,PCT,TNF-α and CDH5 in LPS and LPS+EPC groups were higher than those in NC group,and there were significant differences among the three groups(F=30.6,156,156,95.7,P<0.05,respectively).The LPS group was significantly higher than the LPS+EPC group(t=4.43,14.4,12.4,5.41,P<0.05,respectively).Serum concentrations of SIP in NC and LPS+EPC groups were higher than those in LPS group,with differences among the three groups(F=17.5,P=0.0001),and the levels in LPS+EPC group were significantly higher than those in NC group(t=2.46,P=0.0335).The results of variance analysis were as follows:the concentrations of HGF and IL-10 in the lung tissues of rats in the LPS and LPS+EPC groups were higher than those in the NC group,with significant differences among the three groups(F=120,256,P<0.0001,respectively),and the expression of HGF and IL-10 in the LPS+EPC group was significantly higher than that in the LPS group(t=3.12,2.39,P<0.05,respectively).The concentrations of IL-1β,PCT and TNF-α in LPS and LPS+EPC groups were higher than those in NC group,with significant differences among the three groups(F=51,181,210,P<0.0001,respectively),and the expression of IL-1β,PCT and TNF-α in LPS group was significantly higher than that in LPS+EPC group(t=4.93,13.6,13.2,P<0.05,respectively).The concentrations of SIP and CDH5 in the lung tissues of NC and LPS+EPC groups were higher than those in the LPS group,with differences among the three groups(F=160,40.3,P<0.0001,respectively),and the expression of SIP and CDH5 in the LPS+EPC group was significantly higher than that in the NC group(t=9.96,2.44,P<0.05,respectively).(4)The protein expressions of CDH5,P-c-Met(Y1003),P-c-Met(Y1234+1235),GTP-Rac-1,SPHK1,SPNS2 and S1PR1 in rat lung tissues were detected by Western blot.The concentration of S1PR1 in lung tissue of LPS and LPS+EPC group was higher than that of NC group,and there was a significant difference among the three groups(F=179,P<0.0001),and the concentration of S1PR1 in LPS+EPC group was significantly higher than that in LPS group(t=5.97,P=0.0001).The concentrations ofSPHK1,SPNS2,CDH5,P-c-Met(Y1234+1235)and GTP-Rac-1 in the lung tissues of NC and LPS+EPC groups were higher than those of LPS group,and there were significant differences among the three groups(F=226,26.4,40.7,11.6,23.7,P<0.01,respectively),and LPS+EPC group was significantly higher than NC group(t=4.94,2.26,3.53,2.27,3.5,P<0.05,respectively).The concentrations of,respectively(Y1003)in the lung tissues of rats in the LPS and NC groups were both higher than those in the LPS+EPC group,with significant differences among the three groups(F=17.6,P=0.0002),and the concentrations in the LPS group were significantly higher than those in the NC group(t=2.87,P=0.0177).(5)The mRNA expression ofc-Met,Rac-1,S1PR1,SPNS2 andSPHK1 in NC group,LPS+EPC group and LPS group were detected by RT-PCR.There was no difference in c-Met mRNA between the LPS group and the NC group(F=1.23,P=0.3204),and there was no significant difference between the LPS+EPC group and the LPS+EPC group(t<2,P>0.05).The mRNA expressions of Rac-1 and S1PR1 in LPS and LPS+EPC group were higher than those in NC group,with significant differences among the three groups(F=22.56,19.2,P<0.001,respectively),and the expression of LPS+EPC group was significantly higher than that in LPS group(t=2.73,P<0.05).The mRNA expressions ofSPHK1 and SPNS2 in the lung tissues of NC and LPS+EPC groups were higher than those of LPS group,with significant differences among the three groups(F=40.5,35.4,P<0.0001,respectively),and the expression of LPS+EPC group was significantly higher than that of NC group(t=4.52,3.74,P<0.05,respectively).The serum HGF concentration of rats was significantly higher than that of S1P in peripheral blood and the protein p-c-Met(Y1234+1235),SPNS2 and SPHK1 in lung tissue in LPS+EPC group(r=0.9188,0.9362,0.9428,0.8560,P<0.05,respectively).and negatively correlated with each other in LPS group(r=-0.9220,-0.8573,-0.8774,-0.9322,P<0.05,respectively).CdH5 and P-c-Met(Y1003)were negatively correlated in the LPS+EPC group(r=-0.9320,-0.8945,P<0.05,respectively),but positively correlated in the LPS group(r=0.8788,0.8227,P<0.05,respectively).The serum S1P concentration of rats was correlated with the protein P-c-Met(Y1234+1235),SPNS2,SPHK1 and GTP-Rac-1 in the lung tissues of the LPS group(r=0.8744,0.9120,0.8388,0.9606,P<0.05,respectively)and LPS+EPC groups(r=0.9407,0.8533,0.8405,0.9176,P<0.05,respectively)were positively correlated with blood CDH5(r=-0.8143,-0.8453,P<0.05,respectively)and the protein P-c-Met(Y1003)in lung tissues(r=-0.8354,0.9635,P<0.05,respectively)were negatively correlated;There was a positive correlation between S1PR1 and lung protein in the LPS+EPC group(r=0.8327,P<0.05),and a negative correlation between S1PR1 and lung protein in the LPS group(r=0.9594,P<0.05).There was a negative correlation between blood CDH5 concentration and lung protein GTP-Rac-1 in the LPS group and the LPS+EPC group(r=-0.8662,0.8771,P<0.05,respectively).There was a negative correlation between S1PR1 and lung protein in the LPS+EPC group(r=-0.8293,P<0.05),and a positive correlation between S1PR1 and lung protein in the LPS group(r=0.8829,P<0.05).The concentrations of protein p-c-Met(Y1234+1235)in lung tissue of rats were significantly higher than those of SPNS2 and SPHK1 in lung tissue of rats in LPS group(r=0.9750,0.8448,P<0.05,respectively),and in LPS+EPC group(r=0.9286,0.8499,P<0.05,respectively)were positively correlated.The concentration of protein p-c-Met(Y1003)in lung tissue of rats was significantly higher than that of protein SPNS2 and SPHK1 in lung tissue of rats in LPS group(r=-0.9188,-0.8757,P<0.05,respectively),and in LPS+EPC group(r=-0.9473,-0.9242,P<0.05,respectively)were negatively correlated.Conclusion:1.Clinical studies had shown that in sepsis,the expression level of SIP in peripheral blood was significantly decreased,while the expression of HGF and CDH5 was significantly increased.The HGF concentration in peripheral blood was positively correlated with CDH5 in the Sepsis group.The SIP concentration in peripheral blood was negatively correlated with CDH5 in the Sepsis group.SIP,HGF,CDH5,PCT,IL-1β,IL-10 and TNF-α in peripheral blood played a diagnostic role in the sepsis and ARDS,among which SIP was more obvious than others.2.After EPC transplantation,the expressions of CDH5,IL-1β,TNF-α and PCT in the peripheral blood of septic rats were significantly decreased,while the expressions of SIP,IL-10 and HGF were significantly increased.3.In rat lung tissue,c-Met participates in physiological activities through phosphorylation of different subtypes,which was mainly manifested in sepsis as P-c-Met(Y1003),while EPC transplantation can promote the expression of P-c-Met(Y1234+1235).4.The expression of S1PR1 increased in sepsis and further increased after EPC transplantation.5.sepsis in vascular endothelial barrier function was impaired,after transplantation of EPC can repair endothelial barrier function,its mechanics may be through the EPC paracrine HGF,again through the HGF/P-c-Met sthen through the S1P/S1PR1/GTP-Rac-1 promote restoration of the EC barrier function,which have the effect of treatment of sepsis and ARDS. |
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