| ObjectiveThrough a preliminary study to explore the effect of NF-κB signal pathway inhibitor PS1145 on inflammatory response in sepsis,to study the role of glycocalyx on vascular dysfunction of sepsis and its relationship with NF-κB signal of vascular endothelial cells,it could provide new ideas and methods for the treatment of sepsis.Methods1.The animals were randomly dividied into three groups according to the treatment:a blank control group(abbreviated as group C,receiving the solution by intraperitoneal injection of equal dose 0.9%saline,n=5);a experimental group by tail vein injection of IkB kinase specific blocker PS1145(50 mg/kg)which inhibites NF-κB signaling pathway to the LPS mouse(served as group P,n=5);and Sepsis group(definition as group Lipopolysaccharide by intraperitoneal injection of lipopolysaccharide(10 mg/kg),n=5).And the experiment was divided into three groups:group C,group L and group P.2.Hemodynamic indexes,including Systolic pressure were monitored among the three mouse groups after the tail vein injection of Norepinephrine(300 ng/kg,iv)and acetylcholine(600 ng/kg,iv)to mouse,to assess the reactivity to vasoconstrictor,Vasodilator of mice model.3.The plasma levels of syndecan 1 were monitored by the ELISA assay in mice model to reflect the glycocalyx shedding damage.4.The expression of eNOS and iNOS in the mesenteric artery were detected by Western-Blot assay;Plasma levels of NO in mice model were monitored by the ELISA assays.The relationship between vascular dysfunction was further studied in sepsis and NF-κB signaling pathway in vascular endothelial cells.5.The EBD values of myocardial tissue,lungs and mesenteric tissue of mice were measured to evaluate the changes of vascular permeability between three groups.6.The wet and dry weight ratios of myocardial tissue,lungs and mesenteric tissue from three mice groups were measured to assess the edema of these tissues.7.The plasma levels of TNF-ɑin three mice groups were detected to reflect their inflammatory response by the ELISA assay.Results1.General situation of mice after modeling:Compared with group C,mice in group L showed decreased activity,apathy,erect hair,and increased purulent discharge in eyes,which were worse than in group P(?~2=12.9,p=0.002).2.Hemodynamic monitoring results of three groups of mice model(1)Repeated variance analysis showed time effect on the systolic baseline blood pressure between the three groups(F=118.02,p=0.000).The difference was statistically significant in mean blood pressure values between the three groups over time(F=23.926,p=0.000).1 hour after model establishment,baseline systolic blood pressure in group L(102.4±1.0 mmHg)was lower than in group C(113.3±1.528mmHg,p=0.000)andgroup P(104.0±1.0 mmHg,p=0.004);2 hours after model establishment,baseline systolic blood pressure in group L(102.0±1.0 mmHg)was significantly lower than that in group C(114.3±0.928 mmHg,p=0.001)and slightly lower than that in group P(102.67±0.577 mmHg,p>0.05).6 hours after model establishment,baseline systolic blood pressure in group L(85.67±1.528 mmHg)was significantly lower than that in group C(112.33±2.082 mmHg,p=0.003)and group P(89.0±1.0 mm Hg,p=0.002).(2)The repeated measures analysis of variance indicated no time effect on the amplitude of norepinephrine systolic pressure between the three groups(F=9.489,p=0.095).The vasoconstrictor reactivity monitoring results between three mice groups:1 hour after the the establishment of model:after norepinephrine(300 ng/kg)injection,the increase in baseline systolic blood pressure was lower in the group L(8.88±0.177 mmHg)than in the group C(14.65±0.495 mmHg,p=0.006)and group P(10.93±0.099 mmHg,p=0.005),which showed no significant difference between the2nd and 6th hours.(3)The repeated measures analysis of variance indicated no time effect on the amplitude of acetylcholine systolic pressure between the three groups(F=0.055,p=0.948).The vasodilators reactivity monitoring results between three mice groups:1hour after the the establishment of model,after acetylcholine(600 ng/kg)injection,the decrease of baseline systolic blood pressure was lower in the group L(4.72±0.395mmHg)than in group C(7.115±0.219 mmHg,p=0.001)and group P(7.94±0.077mmHg,p=0.003),and remained the similar trend in the 2nd and 6th hours.3.Detection of plasma SDC-1 by ELISA AssayRepeated variance analysis showed time effect on the concentration of SDC-1 in the three mice groups(F=865.445,p=0.001).1 hour post sepsis establishment,the plasma concentration of SDC-1 was significantly higher in the group L than in the group C(121.12±3.76 ng/ml vs 93.64±3.64 ng/ml,p=0.003),and lower than in the group P(144.30±6.3 ng/ml,p=0.108);2 hours post sepsis establishment,the concentration of SDC-1 in group L(224.76±6.83 ng/ml)was higher than both in the group C(98.80±0.35 ng/ml,p=0.002)and group P(197.54±3.91 ng/ml,p=0.001);6hours post sepsis establishment,the concentration of SDC-1 were higher in the group L(324.06±1.93 ng/ml)than in the group C(85.28±2.50 ng/ml,p=0.012)and in the group P(233.42.±1.64 ng/ml,p=0.001).4.Evaluation of vascular dysfunction related moleculars4.1 6 hours post sepsis establishment,western blotting showed that the expression of eNOS in mesenteric blood vessels was lower in the group L(3.46±0.07)than in the group C(5.124±0.03,p=0.013)and in the group P(4.80±0.09,p=0.042);the expression of iNOS was higher in the group L(0.572±0.003)than in the group C(0.331±0.001,p=0.002)and group P(0.538±0.005,p=0.003).4.2 ELISA assay of the plasma NORepeated variance analysis showed time effect on the NO concentration in the three mice groups(F=7.529,p=0.017).1 hour staining results the plasma concentration of NO was higher in the group L(0.21±0.04 mg/dl)than in the group C(0.18±0.08 mg/dl,p=0.044),but lower in the group P(0.33±0.01 mg/dl,p=0.047);2 hours post sepsis establishment,the concentration of NO was higher in the group L(1.23±0.03 mg/dl)than in the group C(0.34±0.04 mg/dl,p=0.021),and in the group P(0.58±0.07 mg/dl,p=0.019);6 h post sepsis establishment,the concentration of NO was higher in group L(0.37±0.07 mg/dl)than in the group C(0.15±0.03mg/dl,p=0.025)and in the group P(0.22±0.03 mg/dl,p=0.039).5.Evans blue dye staining of EBD6 h post sepsis establishment,Evans blue dye staining showed that the EBD concentration in myocardial tissue、lung tissue and mesenteric tissue was higher in the group L than in the group C(p>0.05)and in the group P(p>0.05).6.Analysis of wet-dry weight ratio(W/D)of myocardial,lung and mesenteric tissue6 h post sepsis establishment,the W/D ratio of the myocardial,lung and mesenteric tissue was greater in the group L than that in the group C(p<0.05)and in the group P(p<0.05).7.ELISA assay of the plasma TNF-ɑlevelsRepeated variance analysis showed no time effect on the TNF-ɑconcentration in the three mice groups(F=8.134,p=0.109).1 hour post sepsis establishment,the concentration of plasma TNF-ɑwas higher in group L(3695±29 pg/ml)than in the group C(2115±27 pg/ml,p=0.001),but lower in the group P(4435±31 pg/ml,p=0.081);2 hours post sepsis establishment,the concentration of plasma TNF-ɑwas higher in the group L(4099.99±22.56 pg/ml)than in the group C(3691±26 pg/ml,p=0.003)and in the group P(3983.33±27.8 pg/ml,p=0.017);6 hours post sepsis establishment,the concentration of TNF-ɑwas higher in the group L(3699.66±21.73pg/ml)than in the group C(3008.21±50.08 pg/ml,p=0.001)and in the group P(3499.9±16.02 pg/ml,p=0.011).Conclusions1.LPS results in serious vascular inflammation,decreased blood pressure,reduced reactivity to vasoconstrictor and vasodilator,increased endothelial glycocalyx damage shedding,increased vascular permeability,severe tissue edema in the mice sepsis model.2.Specific Ik B kinase blocker PS1145 inhibites NF-κB signaling pathway,which caused reduced vascular inflammation,improved response to vasoconstrictors and vasodilator,reduced endothelial glycocalyx damage shedding,improved vascular permeability,and reduced tissue edema in the LPS mice. |
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