Investigation On Protective Effect Of Artesunate On CLP Immunosuppressive Model Mice And Its Mechanisms

ObjectiveSepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection,involving in a series of clinical problems such as infection,inflammation,immune,coagulation and tissue damage,etc.Although medical technology is gradually improved nowadays,morality rate of sepsis remains high.According to incomplete statistics,sepsis affects more than 31.5 million patients and 19.4 million are severe sepsis cases,leading to about 5.3 million deaths annually worldwide each year and is ranked as the leading cause to death for clinical critical patients.The pathophysiology process of sepsis is tightly related to pathogenic bacteria invade,representing as infection and the body’s immune disorders.The initial symptom of body is excessive inflammatory response,representing as cytokine storm phase.Approximately 30% of patients die at this stage.Because of sustained immune disorders,body enters the phase of sepsis immunosuppression,about 70% of deaths in sepsis occurs at this stage because of secondary infection and multiple organ dysfunction syndrome(MODS).This is closely related to the immunosuppression and weakened resistance of the body to secondary hit of pathogens of the patients in later stages of sepsis.There are two death peaks of sepsis patients,systemic inflammatory response syndrome(SIRS)and immunosuppression period of sepsis immunomodulatory disorders.Sepsis immunotherapy drug research aimed at SIRS but did not achieve the goal to reduce the mortality rate.Currently,researchers begin to focus on immunomodulatory drugs in the purpose of improving immunosuppressive state.Ideal sepsis treatment drugs should have two-way adjusting effects of anti-cytokine storm and reverse immunosuppression.There are two different opinions about the change rules of pro-inflammatory and anti-inflammatory release during the immunosuppressant state of sepsis.One opinion is that the nature of sepsis immunosuppressive state is compensatory anti-Inflammatory response syndrome(CARS),which came along with the appearance of SIRS.The features of this stage is the overproduction of anti-inflammatory cytokines such as IL-10,TGF-β,IL-4 and etc.The other opinion thinks that immunosuppressive state appears when body are completely through cytokine storm.The feature of this is the overall reduction of pro inflammatory cytokines and anti-inflammatory cytokines caused by comprehensive inhibit of immune system.Now,research on sepsis immunosuppression pathophysiological mechanisms and therapeutic drugs is based on animal models which can reflect clinical sepsis immun osuppression pathological physiological characteristics.Currently,sepsis immunosuppre ssion models widely recognized by academic world include endotoxin tolerance and cecal ligation puncture(CLP)models.In the early stage of the study,a mouse endo toxin tolerance model was established in our lab.However,the model of endotoxin tolerance model cannot fully represent the pathophysiological characteristics of clinica l immunosuppressive state.Although CLP is regarded as the golden standard for experimental sepsis models,the turning point of over-inflammation to immunosuppression is differently reported in different labs,which confused us.Additionally,there is no systematical investigation about the turning point.Therefore,CLP model was firstly established herein,and then a two-hit model was established using Pseudomonas aeruginosa(PA)as secondary hit.Based on above two models,the effect of artesunate(AS)was investigated,AS is an artemisinin derivatives containing sesquiterpene structure.Long-term clinical application has shown its low toxicity and good safety feature.Previously our group has found that AS has significant therapeutic effect for sepsis mice models including sepsis induced by CLP and different bacteria(Escherichia coli,Pseudomonas aeruginosa,and methicillin-resistant Staphylococcus aureus,etc).In addition,AS also could improve the survival rate of LPS tolerance mice,increase serum,lung and spleen TNF-α,IL-6 and IL-1β released from the mice,and so on.Therefore,the subsequent aim of this investigation is to observe the therapeutic effect of AS on CLP sepsis-induced immunosuppressive model mice based on above animal models of CLP sepsis immunosuppression and the possible mechanism of AS.Methods1.Establishment and evaluation of sepsis-induced immunosuppressive mice model1.1 Establishment of CLP model with different mortalities in mice: to establish CLP mice model using different surgeries which can lead 40% mice to die by observing the moralities of mice for 7 days.1.2 Determination of the turning point of CLP sepsis immunosuppression: based on the CLP model mice with morality of 40%,2.5 × 107 CFU/10 g body weight of PA was injected via caudal vein injection on D0.5,D1,D2,D3,D4,D5,D6 and D7 after CLP surgery,respectively.The moralities of mice within 7 days were observed.1.3 Determination of the sensitivity of CLP mice to different doses of PA challenge: CLP mice were intravenously injected with different dosage of PA(2.5 × 108,5.0 × 107,2.5 ×107,5.0 ×106,1.0 ×106,5 ×105,and 1.0 ×104 CFU/10 g,total injection volume was 0.1 mL/10 g)at 24 h(D1)after CLP surgery,and then the mortality rate in each group was observed for 7 days after injection of PA.1.4 Detection of the levels of inflammation-related cytokines in mice: serum,lung and spleen were collected to detect the levels of inflammation-related cytokines in each group(NS,Sham,CLP,sham+PA,CLP+PA)using ELISA method.1.5 Observation of the dynamic changes of inflammation-related cytokines: to detect the changes of inflammation-related cytokines in serum,lung and spleen at different time-points after CLP surgery: sham(0,2,8,12,and 24 h),CLP(0,2,4,24,and 48 h),sham+PA(4,24,and 48 h after PA injection),and CLP + PA(4 and 24 h after PA injection)using ELISA method.The pro-inflammatory cytokines were TNF-α,IL-6 and IL-1β,and anti-inflammatory cytokines were IL-10,IL-4 and TGF-β.1.6 Detection of the numbers of blood cells: the absolute value and percentage of five kinds of white blood cells(neutrophil,lymphocyte,monocyte,osinophile granulocyte and basophile granulocyte)of venous blood in each mice group were detected using small animal blood analyzer by retinal vein plexus draw blood and anticoagulant tubes collecttion 4 h later.1.7 Determination of CFU count: the number of bacteria in serum,spleen and lung in each mice group(NS,sham,CLP,sham+PA,and CLP+PA)was detected using plate count method.2.Therapeutic effect of AS on CLP sepsis-induced immunosuppressive mice model2.1.Observation of mortality: by observing the mice mortality on D7 to make sure the influence of AS on CLP sepsis-induced immunosuppressive mice injected with PA.2.2.Detection of cytokines levels: the influence of AS on pro-inflammatory cytokines such as TNF-α,IL-6 and IL-1β,and anti-inflammatory cytokines such as IL-10,IL-4 and TGF-β levels of serum,lung and spleen from sepsis-induced immunosuppression mice injected with PA were detected using ELISA assay.2.3 Detection of the amounts and proportions of blood cells: the influence of AS on the amounts and proportions of blood cells of CLP sepsis-induced immunosuppression mice were detected using small animal blood analyzer.2.4 Observation of bacteria counts: the influence of AS on the counts of bacteria in blood,lung and spleen of CLP sepsis-induced immunosuppression mice injected with PA was observed using plate count method.3.Preliminary study of protective mechanisms of AS3.1.In vitro studies(1)Establishment and evaluation of an immunosuppressive cell model: LPS tolerance cell model was established and used as an in vitro model of sepsis immunosuppression.To evaluate the cell model,the release of TNF-α was detected using ELISA assay.The expression of MyD88 and TLR4 in cells were detected using WB method.(2)Influence of AS on TNF-α and IL-6 release from LPS tolerance model cells: TNF-α and IL-6 released from LPS tolerance cells were detected using ELISA assay.(3)Effects of AS on the inflammation and autophagy-related key molecules: The expression of TLR4 and BECN1 in inflammatory cells and autophagy-related proteins were detected by WB method.3.2.In vivo studies(1)Effects of AS on the inflammation and autophagy-related key molecules in CLP sepsis-induced immunosuppressive mice: using immunohistochemistry to detect the influence of AS on autophagy and inflammatory pathway related molecules such as BECN1,TLR4,MyD88,TRAF6 expression and NF-κBp65 activation in lung and spleen of mice.(2)Effects of AS on the inflammation and autophagy-related key molecules in CLP sepsis-induced Immunosuppressive mice injected with PA: using WB method to detect the influence of AS on the inflammation and autophagy-related key molecules such as BECN1,TLR4,MyD88,and TRAF6 expression and NF-κBp65 activation in lung and spleen of mice.Results1.Establishment and evaluation of sepsis-induced immunosuppressive mice model1.1 Different needles and distances to the end of cecum produced different mortalities.The results showed there was no mice death in sham and CLP1 group,but the mortality was 10.0%,36.3% and 45.4% for CLP2,CLP3,and CLP4,respectively,the method(the distal 1.0 cm of the cecum was ligated and punctured once with a No.16 steel needle)used in CLP4 group was selected and used in subsequent experiments.1.2 The morality on D7 of mice injected with PA on D1 after CLP surgery is significantly higher than that of CLP group(p<0.05),while the moralities of mice injected with PA on other days did not show such significant difference.Therefore,above results demonstrated D1 after the CLP surgery might be the turning point of over-inflammation to immunosuppression.1.3 Different dosages of PA given on D1 after the CLP surgery induced different mortalities of mice.Mice in CLP group challenged with PA on D1 had higher sensitivity than mice in sham group,demonstrating again D1 after the CLP surgery was the turning point of over-inflammation to immunosuppression.1.4 The levels of pro-inflammatory cytokines(TNF-α,IL-6 and IL-1β in serum,lungs and spleen)and anti-inflammatory cytokines(IL-10,IL-4 and TGF-β in lungs)didn’t obviously increase at the turning point in CLP mice challenged with PA.1.5 The time-curve of cytokines of mice with or without CLP surgery was different.Both kinds of cytokines presented a trend from rise to decline.Inflammatory cytokine and anti-inflammatory cytokine at 4 h after receiving bacteria showed again that the level of CLP+PA was obviously lower than that of sham+PA.1.6 The number of white blood cell in the vein blood cells of immunosuppression mice was significantly reduced and the percentage of five types of white blood cell was also obviously changed.1.7 The plate count result showed that there was no detection of bacterial colony in blood,spleen,lung of NS,sham,CLP.And PA bacterial colony in blood,spleen,lung of sham+PA was obviously less than that in CLP+PA.The results showed the scavenging ability of sham was much stronger than that of mice on D1 after CLP surgery,demonstrating again that mice on D1 after CLP surgery was under immunosuppressive state.2.The therapeutic effect of AS to CLP sepsis-induced immunosuppressive mice model.2.1 AS could decrease the 7 d mortality of CLP sepsis-induced immunosuppressive mice with secondary hit.2.2 AS could increase TNF-α,IL-6 and IL-1β in serum,lung and spleen,decrease IL-4,IL-10,TGF-β levels in Lung of CLP sepsis-induced immunosuppressive mice.2.3 AS almost had little effect on the total number of white blood cells in venous blood and the number and percentage of five types after the Sepsis immunosuppression model mice and Sepsis immunosuppression state mice with secondary hit.2.4 AS obviously decreased PA loads in the blood,lung and spleen of CLP sepsis-induced immunosuppressive mice with secondary hit.3.Preliminary study of mechanisms of AS3.1 In vitro studies(1)Establishment and evaluation of LPS tolerance cell model: RAW264.7 cells were cultured with LPS(0.01 ng/mL)for 4 h and then treated with LPS(10 ng/mL).The results showed LPS tolerance cells released low level of TNF-α using ELISA assay,and the results from WB experiments showed there were low expressions of TLR4 and MyD88 in LPS tolerance cell model.(2)AS(20 μg/mL)could increase the ability of immunosuppressive cells to release TNF-α.(3)AS(20 μg/mL)could increase autophagy and inflammatory pathway related proteins such as BECN1 and TLR4 expression in LPS tolerance cell.3.2.In vitro studies(1)The results of Immunohistochemistry detect showed that AS(10 mg/kg)could increase lung and spleen BECN1,TLR4,TRAF6 expressions and NF-κBp65 activation in CLP sepsis-induced immunosuppressive mice.(2)The results of WB detect showed that AS(10 mg/kg)could increase lung and spleen LC3,TLR4,TRAF6,and MyD88 expressions and NF-κBp65 activation in CLP sepsis-induced immunosuppressive mice;AS could increase lung and spleen BECN1,TLR4,and MyD88 expressions,and NF-κBp65 activation in CLP sepsis-induced immunosuppressive mice injected with PA.Conclusions1.the CLP sepsis model with 40% mortality was established,the turning point of over-inflammation to immunosuppression appeared at 24 h after CLP surgery.This model appeared better stability,higher repeatability,and short establishment cycle.2.AS could significantly increase proinflammatory cytokines such as TNF-α,IL-6 and IL-1β and anti-inflammatory cytokines such as IL-10,IL-4 and TGF-β levels in CLP induced sepsis immunosuppressed mice;3.AS could significantly improve the survival rate of CLP sepsis-induced immunosuppression mice with secondary hit model,and could improve the survival rate of CLP sepsis mice;AS had a significant therapeutic effect on sepsis-induced immunosuppression mice with secondary hit model.4.AS protective effect on CLP sepsis-induced immunosuppression mice might be related to the promotion of autophagy and inflammatory pathway related proteins LC3,BECN1,TLR4,MyD88,and TRAF6 expressions,and NF-κBp65 activation.