| ObjectiveSepsis is a life-threatening organ dysfunction due to a dysregulated host response to infection.Sepsis-associated encephalopathy(SAE)is commonly seen in systemically ill patients.The syndrome is defined by diffuse cerebral dysfunction that accompanies sepsis in the absence of direct CNS infection,structural abnormality or other types of encephalopathy,as detected by clinical or standard laboratory tests.SAE patients always have high mortality,50% mortality of patients in ICU was related with SAE.SAE induced long-term cognitive function is one of the major nerve damage,resulted low quality of life of patients.Previous studies have found that extracellular nucleotides,proinflammatory cytokines,oxidative stress may be the main cause of pathological changes in sepsis encephalopathy.Resolvins are one of the endogenous lipid proinflammatory resolving mediators,of which Resolvin D1(Rv D1)can improve the survival rate of sepsis mice,reduce the inflammation of lung,kidney and other vital organs.Previous studies have found that extracellular nucleotides,proinflammatory cytokines,oxidative stress may be the main cause of pathologic changes in sepsis encephalopathy.Resolvins are a kind of endogenous pro-resolving lipid mediators,which is metabolized by ?-3 fatty acid dicyclohexaenoic acid(DHA).Resolvin D1(Rv D1)is one of them,studies have confirmed that Rv D1 can improve the survival rate of sepsis mice,have role of protection and treatment to the lungs,kidneys and other vital organs.It is speculated that Rv D1 can improve the prognosis of SAE mice,thus providing a new direction for the treatment of SAE patients.The aim of this study is to explore that Rv D1 can treat mice with SAE by reducing the activation of microglia,reduce the level of proinflammatory cytokines,and finally improve the behavior of mice.Methods Part one Establish the model of SAE1?A total of 20 male C57BL/6 mice were divided into 2 groups randomly by the table of randomization: Control group,CLP group,n=10 for each group.Each mouse was trained with Morris Water Mazing(MWM)for 5 consecutive days before operation.The sepsis model was established the second day after training in CLP group.The MWM wasperformed the second day after operation.2?A total of 12 male C57BL/6 mice were divided into 2 groups randomly by the table of randomization: Control group,CLP group,n=6 for each group.The IBA-1immunohistochemistry staining results of hippocampus were observed and compared in different groups.Part two The protection of Rv D1 on mice with SAE1?A total of 24 male C57BL/6 mice were divided into 4 groups randomly by the table of randomization: Control group,CLP group,low dose of Resolvin D1(CLP+Rv D1-L group,Rv D1 0.1?g/per mouse)and high dose of Resolvin D1(CLP+Rv D1-H group,Rv D1 1?g/per mouse),n=6 for each group.Different dose of Resolvin D1 was given through tail vein immediately post operation.The blood was harvested 24 h after operation.The levels of cytokines were assessed.2?A total of 30 male C57BL/6 mice were divided into 3 groups randomly by the table of randomization: Control group,CLP group,treatment of Resolvin D1 group(CLP+Rv D1 group),n=10 for each group.Resolvin D1 1?g per mouse was given in group CLP+Rv D1.Morris Water Mazing(MWM)was performed the second day after operation.3?A total of 18 male C57BL/6 mice were divided into 3 groups randomly by the table of randomization: Control group,CLP group,treatment of Resolvin D1 group(CLP+Rv D1 group),n=6 for each group.Rv D1 1?g per mouse was given in group CLP+Rv D1.The IBA-1 immunohistochemistry staining results of hippocampus were observed and compared in different groups.Part three The possible mechanism of Rv D1 on the treatment of SAE mice1?A total of 24 male C57BL/6 mice were divided into 3 groups randomly by the table of randomization: Control group,CLP group,treatment of Resolvin D1 group(CLP+Rv D1 group),n=8 for each group.Rv D1 1?g per mouse was given in group CLP+Rv D1.The levels of proinflammatory cytokines in the peripheral blood were detected in the mice.The hippocampus was used to detect the m RNA level and analyze the results.2?BV2 cells(microglia cells)were divided into 3 groups: Control group,LPS group,Treatment of Resolvin D1 group(LPS+ Rv D1 group).Cells were harvested 24 h afterLPS stimulation.The levels of cytokines were assessed by ELISA and PCR.Results Part one Establish the model of SAE1?The latency of CLP group was shortened from the second day after operation,and there was no significant difference.The latency CLP group(47.42 ± 9.23)was significantly longer than that of the control group(30.11 ± 4.12)on the fifth day after operation(P <0.05).2?In the number of crossing the platform,the CLP group(0.65 ± 0.40)was significantly lower than that of the control group(1.54 ± 0.34)(P <0.05).The target quadrant time in CLP group(28.01 ± 6.70)was extended.Part two The protection of Rv D1 on mice with SAE1?The levels of IL-6,IL-1? and TNF? in CLP + Rv D1-L group and CLP + Rv D1-H group were lower than those in CLP group(P <0.05).The levels of proinflammatory cytokines in CLP + Rv D1-H were lower than those of CLP + Rv D1-L group(P <0.05).Subsequent experiments may use higher doses as intervention doses.2?The latency of CLP group(51.36±9.02)was longer than that of CLP+Rv D1 group(51.78±9.17)and control group(45.63±7.53)on the second day after operation.On the third day,the latency of CLP+Rv D1 group(40.83±6.37)was shorter than that of CLP group(43.25±5.37)(P<0.05),and the latency was longer than that of Control group(39.83±7.80),On the fourth day after operation,the latency of CLP+Rv D1 group(32.57±19.86)was significantly shorter than that of CLP group(45.75±5.48)(P<0.05),but there was no significant difference compared with control group(33.80±21.18).3?In the space exploration experiment,when counting the number of crossing the platform,CLP group(0.88±0.14)and CLP+Rv D1 group(1.42±0.14)were lower than that of control group(1.52±0.24)(P<0.05).And noting to the target quadrant time,CLP group(16.5±1.85)vs Control group(33.45±5.19)and CLP+Rv D1(26.85±4.06)were visibly shorter(P<0.05),and the CLP+Rv D1 group was significantly higher than that in the CLP group(P<0.05),CLP+Rv D1 group and Control group compared to no significant difference.4 ? As is showed in IBA-1 immunohistochemistry staining,compared to CLP group(39.50±4.95),the number of positive cells were remarkably reduced in CLP+Rv D1group(16.5±3.42)(P<0.05).Meanwhile,the degree of activation of microglia is alsoreduced.At the same time,Mean option density(MOD)of each group at CA1 and CA3 area of hippocampus.Compared to CLP group(0.020±0.0048),the MOD of Control group and CLP+Rv D1 group(0.0076±0.0025)are lower(P<0.05).Part three The possible mechanism of Rv D1 on the treatment of SAE mice1?The levels of IL-6,IL-1? and TNF? in peripheral blood of CLP + Rv D1 group were significantly lower than those in CLP group(P <0.05).The expression of IL-6,IL-1? and TNF? m RNA in hippocampus of CLP + Rv D1 group was also significantly lower than that in CLP group(P <0.05),suggesting that the protective effect of Rv D1 on SAE was not only related to the reduction of systemic inflammatory response,but also may be dependent on the brain to reduce the inflammatory response.2?The secretion of IL-6,IL-1? and TNF? in LPS + Rv D1 cells was significantly lower than that in LPS group(P <0.05).The expression of IL-6,IL-1? and TNF? m RNA in BV2 cells was significantly lower than that in LPS + Rv D1 group(P <0.05).ConclusionCLP can be a stable operationof the sepsis-associated encephalopathy mouse model.Rv D1 can improve the cognitive function of SAE mice from both spatial learning and spatial memory.The protection of SAE may be associated with the elimination of systemic inflammatory response,reduce intracranial microglia inflammatory response level at the same time. |
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