| ObjectiveSepsis is systemic Inflammatory response syndrome(SIRS) caused by infection factors. Septic shock, multiple organ dysfunction syndrome(MODS) caused by further development of sepsis is the main cause of death in critically ill patients in clinical. Its mortality is as high as 30% to 70%.Pathophysiology of sepsis includes cytokine storm stage and immunosuppression stage. For a long time, cytokine storm has been considered the main feature of sepsis. Therefore cytokine storm and MODS caused by it became the research focus of sepsis and its prevention and treatment measures. But many anti-Inflammatory treatment strategies failed. In recent years, reports from clinical showed that although sepsis patients can survival the early inflammatory storm stage, but many will be die of late secondary infections and MODS. It’s closely related to patients who under the immunosuppression stage with reduction resistance to second hit of pathogenic microorganisms. Monocytes / macrophages are the first defense line of body against pathogenic microorganisms. They play an important role in the identification, removal of pathogenic microorganisms as well as antigen processing and presentation. Their suppression is closely related to the reduction of body’s resistance to second hit.Bacterial endotoxin(lipopolysaccharide) is a major pathogen molecules cause sepsis. LPS tolerance would lead over immunosuppression, making the body more vulnerable to pathogens second hit. Through the establishment of LPS tolerance model can simulate the clinical immune suppression state in patients with sepsis. It also plays an important role to find an effective sepsis treatment drugs.Antimalarial artesunate(AS) is an artemisinin derivatives containing sesquiterpene structure, has good water solubility, low toxicity. Our group was found in the previo us study AS has significant anti-Inflammatory effect.Based on its anti-Inflammatory effects, we found AS had significant protective effect on LPS, heat-killed Escherichia coli(EC) and Staphylococcus aureus(SA), live EC and methicillin-resistant SA(MRSA) challenged mice as well as cecal ligation and puncture(CLP) sepsis model mice. This effect may be related to inhibition of TLR4, TLR9 expression, inhibition of LPS level and reduction of NF-κB activity.In addition to its anti-Inflammatory effect, it has been reported that AS has immunomodulatory effects. To investigate whether AS have septic immunosuppression treatment effect by its immunomodulatory effect, This study intended to observe the protective effect of AS on LPS tolerance second hit model mice and its possible mechanism on the basis of establishing LPS tolerance and second hit mice, cells model. Providing experimental evidence for looking for drugs effective in the treatment of sepsis and broaden clinical indications of AS.Methods1. Establish and evaluate of LPS tolerance mice model1.1. Initial establishment and evaluation of LPS tolerance mice model(1) Initial establishment of the model: Mice were injected intraperitoneally(i.p.) with LPS(1 mg/kg/day for 5 days). LPS(50 mg/kg) was administered intravenously(i.v.) in the 6th day.(2) Eevaluation of the model: By observing the mice 7 d mortality; serum TNF-α and IL-6 release level; sensitivity to EC; change of the number of bacteria in the blood.1.2. Optimize and evaluate of LPS tolerance mice model(1) Optimization of the model: Mice were i.p. injected with different times and doses of LPS, and then i.v. injected with LPS(50 mg/kg). By observing the changes in mouse body weight and state to determine the best establish programs of tolerance model.(2) Eevaluation of the model: By observing the mice 7 d mortality; serum, lung and spleen TNF-α, IL-6 and IL-1β release level; Sensitivity to Pseudomonas aeruginosa(PA).2. Protective effect of AS to LPS tolerance and second hit model mice(1) Cytokines detection: Detect the influence of AS on serum, lung and spleen TNF-α, IL-6 and IL-1β released by LPS tolerance model mice using ELISA assay.(2) Mortality observation: By counting the mice 7 d mortality, observe the mortality rate influence of AS to LPS tolerance second hit model mice.(3) Bacterial clearance capability: observe the influence of AS on the number of bacteria in blood, lung and spleen of LPS tolerance second hit model mice by plate count method.3. Preliminary study of protective mechanisms of AS to LPS tolerance model mice3.1. In vitro studies(1) Establishment and evaluation of LPS tolerance cell model: RAW 264.7 cells were cultured with LPS(5 ng/mL) for 4 h and then treated with LPS(100 ng/mL). Detect the supernatant TNF-α, IL-6 release level by ELISA assay.(2) Cytokines and signal transduction molecule detection: By using ELISA assay detect the influence of AS on TNF-α, IL-6 released by LPS tolerance model cells and NF-κB p65, p50 activation; By using immunofluorescence detect the influence of AS on TLR4, TRAF6 expressed by LPS tolerance model cells.(3) Ability of bacterial clearance: Tolerance cells were re-cultured with PA(5.0×106 CFU/mL) to establish LPS tolerance second hit cells model. Observe the influence of AS on the number of bacteria in LPS tolerance second hit model mice by plate count method.3.2. In vivo studiesBy using Immunohistochemistry detect the influence of AS on TLR4, TRAF6 and NF-κB p65 expressed in lung and spleen of LPS tolerance model cells.Results1. Establish and evaluate of LPS tolerance mice model1.1. Initial establishment and evaluation of LPS tolerance mice modelModel mice all survived within 7 d, indicating generally successful model.Model mice serum TNF-α, IL-6 release level decreased compared to attack mice(50 mg/kg LPS); EC number caused tolerance mice compared to normal mice all died decreased; The number of bacteria in blood of EC attacked model mice increased compared to EC attacked nomal mice.1.2. Optimize and evaluate of LPS tolerance mice model(1) Best modeling program: mice were injected i.p. with LPS(0.3 mg/kg/day for 3 days). LPS(50 mg/kg) was then i.v. administered.(2) Model mice all survived within 7 d; Model mice serum, lung, spleen TNF-α, IL-6, IL-1β release level decreased compared to attack mice(50 mg/kg LPS); EC number caused tolerance mice compared to normal mice all died decreased.2. Protective effect of AS to LPS tolerance and second hit model mice2.1. AS could dose-dependent increase serum TNF-α, lung and spleen TNF-α, IL-6 and IL-1β released by LPS tolerance mice.2.2. AS could decrease the 7d mortality of LPS tolerance second hit model mice with a dose-dependent manner.2.3. AS obviously decreased LPS tolerance second hit model mice PA loads in the blood, lung and spleen.3. Preliminary study of protective mechanisms of AS to LPS tolerance model mice3.1. In vitro studies(1) LPS tolerance cell model successfully established. Model cells TNF-α, IL-6 release level decreased compared to attack cells(100 ng/mL LPS).(2) AS could concentration-dependent increase TNF-α, IL-6 released by LPS tolerance cells. AS could increase TLR4, TRAF6 expressed and NF-κB p65 activated in LPS tolerance cells.(3) AS obviously decreased PA loads in LPS tolerance second hit model cells.3.2. AS could increase lung and spleen TLR4, TRAF6 expressed and NF-κB p65 activated in LPS tolerance mice.Conclusions1. LPS tolerance mice model was established successfully by injected i.p. with LPS(0.3 mg/kg/day) for 3 days followed by i.v. injection with LPS(50 mg/kg). This model had good stability, high repeatability and shortest modeling period, minimum reagents consumption.2. AS could remarkable increase TNF-α, IL-6 and IL-1β level released by LPS tolerance mice. AS had remarkable protective effect to LPS tolerance second hit model mice, AS could increase scavenging capacity of LPS tolerance second hit model mice to PA.3. AS could remarkable increase TNF-α, IL-6 level released by LPS tolerance cells. AS could increase scavenging capacity of LPS tolerance second hit model cells to PA.4. AS protective effect to LPS tolerance second hit model mice may be related to the promotion of TLR4, TRAF6 expression and NF-кB p65 activation. |
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