| Background:The competition in modern society is fierce and the life pace is accelerating.Continuing state of stress will have a serious impact on the normal physiological functions of the human body and results in a series of physical and mental health problems.Prolonged fatigue can cause decreased learning and memory capability,decreased concentration and work efficiency which would seriously influence people's normal life and may even trigger safety problems.Moreover,military personnel are constantly under long-time and high-intensity work in no matter warfare or non-war military operations.During such operations,the injuries result from fatigue and stress are quite obvious.Therefore,it is of important social and practical significance to in-depth study the mechanism in fatigue development and to develop anti-fatigue drugs and therapies.It is a complicated process in fatigue development and so far there is no definite conclusion about its mechanism.Current point of view is that multiple systems such as nervous system,immune system and endocrine system and many factors all take part in fatigue development.Many clinical studies and animal experiments have found that the immune system was excessively activated in fatigue patients.The aberrantly activated immune system causes increased levels of inflammatory cytokines,such as IL-1?,these inflammatory cytokines could induce fatigue-like symptoms after acting on central nervous system.Inflammasome is the hot spot of the inflammation research in recent years.The NLRP3 inflammasome is an important part of the innate immunity.It could recognize pathogen-associated molecular patterns and damage-related molecular patterns through its own NOD-like receptors,which in turn would activate the downstream signal pathway and induce adaptive immune response.When activated,NOD-like receptors could recruit the apoptosis-associated speck-like protein containing a CARD and the caspase precursor pro-Caspase-1 to form a multiprotein complex,named NLRP3 inflammasome.The activated NLRP3 inflammasome can cleave pro-Caspase-1 to produce activated Caspase-1,which further cleaves IL-1? and IL-18 precursors and produces active IL-1? and IL-18,which would be extracellular secreted and participate inflammatory reaction.The normal function of inflammasome is to protect the body from pathogen infection and stress damage,but exaggerated activation can also cause amplification of inflammation and organ damageIt has been found that serum IL-1? levels are abnormally elevated in chronic fatigue syndrome patients,and IL-1? itself is also the main product of NLRP3 inflammasome.This association strongly suggests that the NLRP3 inflammasome may be involved in fatigue development.However,whether NLRP3 inflammasome is involved in the occurrence and development of fatigue and the specific mechanisms involved in the occurrence of fatigue haven't been reported in the literature.So,to explore the role of NLRP3 inflammasome in the pathophysiology of fatigue and its possible molecular mechanisms is of great significance for the comprehensive illustration of the mechanism of fatigue and the development of new prevention and treatment strategies.Objective:In this project,we established acute and chronic mice fatigue model to study NLRP3 inflammasome activation in the central nervous system.With the help of Nlrp3 knockout mice,we further explored the exact role of NLRP3 inflammasome in the two kinds of fatigue model.Reactive oxygen species(ROS)is also going to be measured to demonstrate the mechanism of NLRP3 inflammasome activation.Finally,we used the omega-3 unsaturated fatty acids in the above fatigue models in order to study its intervention effect and to verify the potential therapeutic targets of NLRP3 inflammasome and ROS in the process of fatigue.Methods:First of all,the study explored the establishment of acute fatigue mice model: a lipopolysaccharide(LPS)-induced mouse fatigue model and chronic fatigue mice model: repeated forced swimming-induced mouse fatigue model.The fatigue model was tested by using locomotor activity assessment and rota-rod test as behavioral indicators to measure the fatigue state.Then we chose the mouse central nervous system as a target organ to study whether the NLRP3 inflammasome was activated.And then,Nlrp3 knockout mice were subjected to the same fatigue model modeling process and behavioral indicators including locomotor activity assessment and rota-rod test were measured.Meanwhile,the generation of ROS in the brains of wild-type and knockout mice was also examined to explore the specific mechanism of NLRP3 inflammasome activation.Finally,we used the omega-3 unsaturated fatty acid-DHA with definite anti-oxidation and anti-inflammatory effects to intervene in mice forced by swimming with forced forced swimming to further verify the value of NLRP3 inflammasome as a target for fatigue therapy.Results:1?NLRP3 inflammasome is involved in LPS and additional forced swimming induced mouse fatigue model1.1 After the intraperitoneal injection of LPS and additional forced swimming,mice showed significant fatigue-like behaviors,as shown by decresed locomotor activity and shorted on bar time in rota-rod test,which represent the successful establishment of the fatigue model.1.2 In the fatigue mice,their serum IL-1? and IL-6 levels were significantly increased.The NLRP3 and IL-1? mRNA and protein expression levels were significantly increased in mice brain and immunofluorescence results showed NLRP3 inflammasome was assembled and activated.1.3 Under the same LPS injection and additional forced swimming,the fatigue-like behavior exhibited by Nlrp3 knockout mice was significantly reduced compared to wild-type mice.NLRP3 knockout mice also showed significantly reduced Caspase-1 activation in the brain tissue,accompanied by a significant decrease of IL-1?.2?NLRP3 inflammasomes is involved in forced swimming-induced mouse fatigue model2.1 Repeated forced swimming was conducted by forced swim for 10 mins every 12 hours in 14 consecutive days.Repeated forced swimming could induce significant fatigue-like behavior in wild-type mice.As compared,the Nlrp3 knockout mice fatigue-like behavior was significantly reduced.2.2 In this model,NLRP3 inflammasome was significantly activated in wild-type mouse prefrontal cortex brain region and IL-1? levels were significantly increased.The fatigue-like behavior of Nlrp3 knockout mice was also significantly reduced with the reduction of IL-1? levels in the prefrontal cortex brain regions and serum were also significant.2.3 The levels of reactive oxygen species(ROS)in brain of wild-type and NLRP3-knockout mice and serum malondialdehyde(MDA)were significantly increased under repeated forced swim-induced fatigue and there was no significant difference between the two genotypes.3?Effects of omega-3 unsaturated fatty acids on mice fatigue induced by repeated forced swimming3.1 Omega-3 unsaturated fatty acids could significantly improve the fatigue-like behavior of mice induced by repeated forced swimming challenge.3.2 Omega-3 unsaturated fatty acids could significantly inhibit the activation of NLRP3 inflammasome,the IL-1? increase and reactive oxygen species(ROS)production in the prefrontal cortex brain regions of the mice induced by repeated forced swimming,thus to improve tht fatigue-like behaviors.Conclusions:The NLRP3 inflammasome was involved in the fatigue-like behavior of mice in both of the animal models;NLRP3 inflammasome played an important role in fatigue pathogenesis;Omega-3 unsaturated fatty acids could effectively inhibit the generation of reactive oxygen species and NLRP3 inflammasome activation in the brain,thereby improving the fatigue-like behavior of the mice. |
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