Regulatory Effect Of Rapamycin On Neuronal Autophagy In Septic Mice

Objective(s):The incidence of sepsis and sepsis-associated encephalopathy(SAE)is increasing worldwide.For the study of its related mechanism,more and more evidence shows that the action of inflammatory mediators on neurons and nerve cells is a key factor in the development of long-term cognitive impairment in SAE.There is also evidence that up-regulation of autophagy can protect neurons from neuronal damage caused by inflammation associated with neurodegenerative diseases such as Alzheimer’s disease and Huntington’s chorea.Other studies have revealed the possible relationship between neuronal injury and mammalian rapamycin target(m TOR)-autophagy signaling pathway.In some studies,rapamycin(RAPA)can effectively reduce abnormal Aβ and tau protein deposition and improve learning and memory by promoting autophagy flux in neurodegenerative diseases.In many recent studies,rapamycin is also often used as an immunosuppressant in the treatment of monogenic diseases and tumors,including cerebral cavernous malformations,chronic lymphoblastic leukemia,colorectal cancer and so on.However,whether rapamycin can save acute inflammation caused by sepsis is still unknown,and the underlying mechanisms involved are not fully clear.The purpose of this study is to explore the relationship between acute inflammatory model of sepsis induced by LPS and autophagy,and whether rapamycin can protect neurons from acute inflammation caused by sepsis by regulating autophagy induced by LPS.Methods:First,24 mice were randomly divided into 4 groups(8mg/kg,10mg/kg,12mg/kg,16mg/kg)for LPS intraperitoneal injection concentration gradient pre-test,and the intraperitoneal injection concentration of LPS in septic mice was determined according to the survival rate of mice.In order to study the changes of autophagy in acute inflammation of septic mice,25 mice were randomly divided into 5 groups(Sham group,12 h group,24 h group,48 h group and 72 h group).The morbid score of septic model,the level of serum inflammatory factors and the expression of autophagy related protein were analyzed,and the most obvious time point of autophagy was inferred.In order to study the protective effect of Rapamycin on acute inflammation in septic mice and its related mechanism,32 mice were randomly divided into Sham group,LPS group,LPS+Nacl group and LPS+RAPA group.The latter two groups were intraperitoneally injected with Nacl and Rapamycin half an hour before LPS administration.After administration of LPS,according to the most obvious time point of autophagy selected in the previous part of the experimental results,the morbid score of sepsis model and behavioral open field test were performed,and the serum levels of inflammatory factors,HE and Nissl staining,immunofluorescence staining and autophagy-related protein expression were analyzed.Results:1.In the pre-experiment,according to the survival rate,the 10mg/kg dose of LPS was selected as the intraperitoneal dose of septic mouse acute inflammation model.2.In the first part of the experiment,the results showed that compared with Sham group,the morbid score and serum inflammatory factor levels of septic mice in 12 h,24 h,48 h and 72 h groups increased from 12 h(P <0.01),reached the highest level at 24 h and then decreased(P <0.01),and remained at a high level at 72 h(P <0.05).Compared with Sham group,the expression of autophagy-related proteins changed in12 h group,24 h group and 48 h group,but not in 72 h group.The expression of LC3II/I,ATG5 and ATG7 decreased at different time points(P <0.01),while the expression of p62 and p-m TOR/m TOR increased(P <0.01).3.In the second part of the experiment,the results showed that the morbid score and the level of serum inflammatory factors in LPS group and LPS+Nacl group were significantly higher than those in Sham group(P < 0.01),which was reversed by Rapamycin administration,and the action ability and exploration ability in LPS group and LPS+Nacl group were significantly inhibited compared with Sham group(P <0.01),which was alleviated by rapamycin.The results of HE staining and Nissl staining of brain tissue showed that rapamycin could also effectively alleviate the morphological changes of cortex and hippocampus induced by LPS and reduce the number of neuronal damage,and the results of immunofluorescence staining also showed the close relationship between neuronal Neu N and autophagy protein LC3,and the decrease of autophagy flux induced by LPS was closely related to neurons,which could be reversed by rapamycin and better protect neurons.The expression of autophagy-related proteins in the cortex and hippocampus of septic mice was analyzed.ATG5,ATG7 and LC3II/I decreased significantly after LPS stimulation(P< 0.01),while the levels of p62 and p-m TOR/m TOR increased(P < 0.01).Early administration of Nacl could not alleviate this phenomenon,but this phenomenon was reversed after administration of Rapamycin.Conclusion(s):Rapamycin can reduce the systemic inflammatory response caused by sepsis to some extent,restore the ability of action and exploration in mice,and alleviate the abnormal morphological changes and neuronal damage of neurons in the cerebral cortex and hippocampus of septic mice.Its regulatory effect on the brain protection of septic mice may be related to the restoration of the inhibited autophagy activity in the cortex and hippocampus of septic mice.