The Protection Of Resolvin D1 And Its Mechanism On Mice With Sepsis-associated Encephalopathy

ObjectiveSepsis is defined as the uncontrolled systemic inflammatory response syndrome(SIRS)induced by infections,which can cause the damage of liver,kidney,brain,lung or other organs and even may lead to multiple organ dysfunction syndrome(MODS).The brain plays a pivotal role in sepsis,acting as both a mediator of the immune response and a target for the pathologic process.Sepsis-associated encephalopathy(SAE)refers to a clinical spectrum of diffused neurological dysfunction that arises in the context of sepsis.The most common clinical feature of SAE is the alteration of consciousness,including confusion,anxiety,delirium,coma and long-term cognitive impairment.SAE is one of the most important reasons that cause the mortality of patients in intensive care unit(ICU).Although the pathophysiology of SAE is incompletely understood,it is thought to involve endothelial activation,blood–brain barrier leakage,inflammatory cell migration,and neuronal loss with neurotransmitter imbalance.The cerebral inflammation and oxidative injury of brain mediated by microglia may be highly related to the cause of SAE.It has been reported that inhibiting the activation of microglia can reduce the inflammatory response and improve the long-term cognitive function of mice with SAE.Resolvins are endogenous pro-resolving lipid mediators.Resolvin D1(Rv D1),which is metabolized by w-3 fatty acid docosahexaenoic acid(DHA),has been reported as its strong effect of promoting the regression of inflammation in many diseases of mice,such as acute lung injury,pathological pain,acute kidney injury and acute pancreatitis.Previous studies have shown that Rv D1 can improve the survival of septic mice and reduce the pulmonary inflammatory reactions of mice with acute lung injuries.However,whether Rv D1 has protective effect on SAE has not been reported at present.Thus,the aim of this study is to explore whether Rv D1 can alleviate inflammation,improve the clinical behaviors of SAE mice and the underlying mechanism of its function.Methods1.Establish the model of SAEThe model of SAE was established by operating cecal ligation and puncture(CLP)on male C57B/6 mice,which were 6-8 weeks old.The changes of cognitive functions were confirmed by Morris Water Mazing(MWM).Hippocampal tissues of the mice were taken for IBA-1 immunohistochemical staining and then the results were analyzed and compared.2.Rv D1 can improve the cognitive functions of SAE miceThe mice were trained in water maze for 5 consecutive days before the operation of CLP.After training,the mice were randomly divided into three groups: the sham group,the model group(CLP group)and the Rv D1 treatment group(CLP+Rv D1 group).The CLP+Rv D1 group was given 100 ng Rv D1 by intravenous injection(caudle vein)immediately after CLP,while the Sham group and the CLP group were given 0.9% normal saline at the same dose.Morris water maze test was performed on the second day after operation and the data was recorded.3.Rv D1 can inhibit the inflamation of SAE mice(1)The effect of different doses of Rv D1 on inhibiting inflammationThe mice were randomly divided into five groups: the Sham group,the model group(CLP group)and different doses of Rv D1 treatment groups(CLP + Rv D1 1 ng;CLP + Rv D1 10 ng;CLP + Rv D1 100 ng).Rv D1 was given by intravenous injection(caudle vein)immediately after CLP,while the Sham group and CLP group were given 0.9% normal saline at the same dose.Peripheral blood of the mice was collected to detect the levels of inflammatory factors 24 hours after the operation.(2)The effect of Rv D1 on the cytokines of SAE miceThe mice were randomly divided into three groups: the sham group,the model group(CLP group)and the Rv D1 treatment group(CLP + Rv D1 group).The CLP + Rv D1 group was given 100 ng Rv D1 by intravenous injection(caudle vein)immediately after CLP,while the Sham group and CLP group were given 0.9% normal saline at the same dose.The peripheral blood and the hippocampal tissues of mice was collected to detect the levels of inflammatory factors 24 hours after the operation.4.The role of microglia in SAEThe mice were randomly divided into three groups: the sham group,the model group(CLP group)and the Rv D1 treatment group(CLP + Rv D1 group).The CLP + Rv D1 group was given 100 ng Rv D1 by intravenous injection(caudle vein)immediately after CLP,while the Sham group and CLP group were given 0.9% normal saline at the same dose.24 hours after the operation,the hippocampal tissues of mice were collected for IBA-1 immunohistochemical staining.BV2 cells were divided into three groups: the control group,the LPS group and the Rv D1 treatment group(LPS+Rv D1,Rv D1 100 n M).Rv D1 was added immediately after LPS stimulation.After 24 hours,the levels of inflammatory factors in the supernatant of the cells were detected,meanwhile,the the expression of relative proteins was also detected.Results1.Rv D1 could improve the cognitive function of SAE miceThe result of Morris water maze experiment showed that,comparing with the Sham group(46.4±2.2s),the CLP group(57.4±1.5s)had longer latency in concealed platform experiment(P<0.01)at the second day after operation.From the third day after operation,the CLP+Rv D1 group(49.8±0.8s)had shorter latency in the concealed platform experiment,comparing with the CLP group(54.4±1.3s)(P<0.05).In the space exploration experiment,comparing with the Sham group,the CLP group had fewer times of platform penetration(CLP vs Sham,0.8±0.4 vs 2.6±0.2,P<0.01)and shorter target quadrant time(CLP vs Sham,35.4±1.6s vs 44.2±3.2s,P<0.05).While comparing with the CLP group,the CLP+Rv D1 group had more times of platform penetration(CLP vs CLP+Rv D1,0.8±0.4vs 1.8±0.2,P < 0.05)and longer target quadrant time(CLP vs CLP+Rv D1,35.4±1.6s vs 43±1.4s,P<0.01).2.Rv D1 could reduce inflammation responses of SAE miceThe result of ELISA showed that,comparing with the Sham group,the expression of TNF-?,IL-1? and IL-6 in peripheral blood and the hippocampal tissues of the CLP group were significantly increased(P<0.05),while Rv D1 could decrease the levels of these cytokines.In the experiment of giving different doses of Rv D1 to SAE mice,we found that the levels of inflammatory factors in peripheral blood of mice decreased.With the increase of Rv D1 dose,the levels of TNF-?,IL-1? and IL-6 decreased dependently.The concentration of these cytokines decreased significantly when the dose was 100 ng(P < 0.05).3.Rv D1 could inhibit the activation of microglia in the hippocampus of SAE miceThe activation of microglia in the CLP group increased and was obvious in the results of IBA-1 immunohistochemical staining of hippocampus tissue(CLP vs Sham,37.6±4.3 vs 18.2±3.4,P<0.05),while the activation of microglia in CLP+Rv D1 group decreased and was unobvious in the results of IBA-1 immunohistochemical staining of hippocampus tissue(CLP vs CLP+Rv D1,37.6±4.3 vs 22.7±4.1,P<0.05).4.Rv D1 could reduce the inflammation of microgliaLPS could increase the expression of TNF-??IL-1? and IL-6 in BV2 cells(P<0.05),while after treated with Rv D1,the levels of these cytokines were decreased significantly(P<0.05).After stimulated by LPS,the phosphorylation levels of ERK,JNK,p38 and p65 in BV2 cells were significantly increased(P<0.05),and the phosphorylation levels of these proteins decreased after Rv D1 was added(P<0.05).ConclusionResolvin D1 has protective effect on mice with sepsis-associated encephalopathy,which can alleviate the inflammation of peripheral blood and hippocampus,and it can improve the cognitive function of the mice.Resolvin D1 can inhibit the activation of microglia and reduce the inflammatory factors.Its mechanism may be associated with the inhibition of ERK,JNK,p38 and NF-?B p65 phosphorylation,which may further regulate the release of cytokines such as TNF-??IL-1? and IL-6.