| Objective:The imbalance of inflammatory reaction and ant-inflammatory caused by immune dysfunction is one of the important reasons which lead to the progress of sepsis.At the same time,the immunotherapy of sepsis has become the research hot spot.However,the immune state of sepsis is different in the early,middle and late period of disease progression.The response of body is also various in the different pathogen infected sepsis.Up to now,the new guidelines of immunotherapy in sepsis is still no clear direction.At present,the research of immunotherapy for thymosin-?1 is plentiful.Nevertheless,there are some issues is uncertan such as opportunity,dose,schedule and so on.Review the past,the application of thymosin-?1 research in sepsis are mainly concentrated in severe sepsis of endotoxin mediated model.However,the gram-positive bacteria compound with gram-negative bacteria infected sepsis is found abundant.There are complex coordination and interaction between endotoxin and exotoxin.Out research is little about the application of thymosin-?1 in endotoxin compound with exotoxin induced sepsis model.In addition,any Immunomodulation should take the potential harmful effects to dody into consideration and try to avoid it.Due to the lack of timely and reliable monitoring means for the immune status in clinical,which lead to the boundary between mild and seriousness of sepsis is not clear.Immunosuppression may not have happened in mild patients.How about the impact on the prognosis of sepsis in the application of thymosin-?1 in this immune state?There is no reliable basis of evidence-based medicine.This experiment established enterotoxin B combined with endotoxin attack sepsis in mice model to simulate the gram-positive bacteria compound with gram-negative bacteria infected sepsis.The modol belongs to the mild sepsis,the objective is to study the immune effects of the different dose of thymosin-?1 in the mice model of enterotoxin B combined with endotoxin induced sepsis.Method:Firstly,we established SEB and LPS composite infection sepsis in mice model.After giving the male BALb/c mice abdominal cavity injection of SEB(500ug/kg,400ug/kg,300ug/kg)2hours,then injected LPS(1500ug/kg,1000ug/kg,500 ug/kg),at the same time setting the PBS blank group.We had obsered the survival and symptoms of all mice until 72 hours,and record survival every 8 hours at the same time.Finally,we used the 300ug/kg SEB+1000ug/kg LPS to establish the model.Nest,we randomly divided 90 mice into three groups,which is control group,low-dose group and high-dose group.Then,each group of mice were randomly divided into three group of 2h,4h,6h,thus each group has 10 mice.After establishing models,low-dose group high-dose group mice were respectively gived abdominal subcutaneous injection of thymosin-?1 100ug/kg and 2000ug/kg,while the control group mice were given isometric PBS.We had observed and recorded the symptoms until to the corresponding end time of 2h,4h,6h.Then leaved the serum stored after centrifugal blood,and dissected to store the lungs and other tissues for HE.We had mesured serum concentration of IL-6,IL-10,TNF-? in ELISA method.Results were expressed as the mean ± standard error of the mean(SEM).Statistical calculations were performed with the one way analysis of variance(ANOVA)using SPSS19.0 software.Differences in measured variables between experimental and control groups were assessed by Kruskal-Wallis H Test.P<0.05 was considered statistically significant.Results:1 Effect of different dose thymosin-?1 on the serum concentration of IL-6In 2 hour after established the models,the concentration of IL-6 in low-dose and high-dose group is significantly higher than the control group,with statistical significance(P<0.05),while the difference between low-dose and high-dose group is no statistical significance(P>0.05).In 4 hour and 6 hour after established the models,the difference in concentration of IL-6 among control group,low-dose and high-dose group is no statistical significance(P>0.05).2 Effect of different dose thymosin-?1 on the serum concentration of IL-10In 2 hour after established the models,the concentration of IL-10 in low-dose and high-dose group is significantly lower than the control group,with statistical significance(P<0.05),while the difference between low-dose and high-dose group is no statistical significance(P>0.05).In 4 hour and 6 hour after established the models,the difference in concentration of IL-10 among control group,low-dose and high-dose group is no statistical significance(P>0.05).3 Effect of different dose thymosin-?1 on the ratio of IL-10/IL-6In 2 hour after established the models,the ratio of IL-10/IL-6 in low-dose and high-dose group is significantly lower than the control group,with statistical significance(P<0.05),while the difference between low-dose and high-dose group is no statistical significance(P>0.05).In 4 hour and 6 hour after established the models,the difference on the ratio of IL-10/IL-6 among control group,low-dose and high-dose group is no statistical significance(P> 0.05).4 Effect of different dose thymosin-?1 on the serum concentration of TNF-?In 2 hour after established the models,the concentration of TNF-?in low-dose and high-dose group is significantly lower than the control group,while the concentration of TNF-? in high-dose group is significantly higher than low-dose and group,both with statistical significance(P<0.05).In 4 hour after established the models,the concentration of TNF-?in low-dose and high-dose group is significantly lower than the control group,with statistical significance(P<0.05).while the difference between low-dose and high-dose group is no statistical significance(P>0.05).In 6 hour after established the models,the concentration of TNF-?in low-dose and high-dose group is significantly lower than the control group,with statistical significance(P< 0.05),while the difference between low-dose and high-dose group is no statistical significance(P>0.05).5 Effect of different dose thymosin-?1 on clinical manifestations in miceWithin 4 h after building the control group,all mice show piloerection,shortness of breath,not a diet,diarrhea,depression,lose interest in the environment,since the ability of self cleaning disappear,eyes a scab phenomenon such as seepage,and so on.Observed until the end of 6 h,there is no relief of symptoms.Compared to control symptoms,symptoms are relatively mild symptoms relief appear early in low-dose group and high-dose group: within 4 h after drug use,the extent of the shortness of breath,diarrhea in low-dose group and high-dose group mice is relatively light.Within 5 ~ 6 h h after drug use,in small dose group and high dose group,most of the mice have better mental state,increased activities,relief of breath shortness,the recovery of the ability of self cleaning,even for individual diet phenomenon occurring in mice,while the control group did not have obvious relief.There is similar symptoms between low-dose group and high-dose group.6 Effect of different dose thymosin-?1 on histopathological manifestation of viscera damage in miceLung tissue pathology: the control group appears part of the destruction of alveolar walls,with pulmonary interstitial hemorrhage phenomenon.Small dose group and high dose group appear pulmonary interstitial vascular congestion expansion,withnot pulmonary interstitial hemorrhage;Liver tissue pathology: liver cell in control group become edema and color dodge,while the lesion in small dose group and high dose group is lighter;Renal pathology: control group in renal interstitial vascular congestion,while the lesion in small dose group and high dose group is lighter;Heart tissue pathology: control group in occasional angiectasis hyperemia,morphologic features in small dose group and high dose group are basic normal.Conclusions:1 Thymosin-?1 could improve the composite SEB and LPS mediated the prognosis of sepsis in mice.2 Thymosin-?1 could enhance the immune response of mice in the early inflammatory response,which play a role in the immunotherapy.3 Different doses of thymosin-?1 effect had no signifficant difference,and increase the dose not get better returns. |
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