Effects Of The NLRP3 Inflammasome On Depressive-like Behavior And Its Molecular Mechanism

BackgroundAs increasingly fierce competition in our society, accelerating the pace of work and life, people are exposed to more and more pressures and challenges(stress). Continuous and excessive stress seriously affects our body, triggering a series of diseases that have serious harm to human health and well-being. Stress has become an important cause of many human causes and major diseases, and it has particularly close relationship with the incidence of depression. Significant and long-lasting low mood is the main feature of depression. Because of its high incidence, high disabling rate as well as heavy burden on families and society, depression has caused widespread attention. There are more than 350 million people worldwide suffering from depression. The incidence of depression in China is about 3%-5%. Depression is a major factor leading to the global burden of disease. Therefore, the in-depth research of pathogenesis of stress-induced depression for the development and clinical application of new antidepressants and exploring effective prevention strategy has very important social and economic significance.The pathogenesis of depression is very complex. To date, the specific pathogenesis of depression remains unclear. There are neural circuit theory and neurotransmitter theory which hypothesize that the incidence of depression is mainly due to quenched imbalances of brain neurotransmitters, like norepinephrine, choline, serotonin and dopamine or dysfunction of the hypothalamus-pituitary-adrenal axis. In recent years, the cytokine theory is getting more and more attention for its role in the development of depression. The cytokine theory, which is also known as the inflammatory response hypothesis, is based on the existence of immunological changes in patients with depression. It proposes that the activation of inflammatory cytokines produced by the immune system plays an important role in the pathogenesis of depression. This view has been proved by the results of a series of clinical studies and animal experiments. Moreover, anti-inflammatory strategies are gradually becoming a new direction for the prevention and treatment of depression.A variety of cytokines involved in the occurrence of depression, in which IL-1β plays an important role in the pathogenesis of depression. IL-1β is also closely related with stress induced emotional and behavioral abnormalities. Maturation of IL-1β needs the IL-1β converting enzyme, which is an important component of the inflammatory multi- protein complex. Because of its pivotal role in the innate immune system, inflammasome has been an extremely widespread concern. Inflammasome is constituted of NOD-like receptor, adapter protein ASC and effector protein Caspase-1, which can promote maturation of IL-1β, IL-18 and IL-33. Inflammasome can be activated by a variety of factors, such as infection, ROS, injury and metabolites, ATP and so on. However, there is no direct evidence reported in the literature regarding if stress can activate the inflammasome.Although foreign researchers speculate in reviews of the inflammatory mechanisms of depression that inflammasome may play a key role in the pathogenesis of depression, the molecular mechanisms of how inflammasome involved in the development of depression is not clear. These molecular mechansisms include the downstream signaling pathways of inflammasome, activation of inflammasome in target tissue and target cells. In-depth study of mechanisms of stress-induced activation of inflammasome and the underlying molecular mechanisms of stress-induced depression will not only help explain the pathogenesis of depression, but also help to provide new ideas and experimental evidence for the prevention and treatment of stress-induced diseases. ObjectiveThis study aims to confirm that inflammasome is one of the key targets of LPS and stress-induced depression-like behavior in mice by establishing acute and chronic mouse models of depression. Our study plans to clarify the signal transduction pathways and molecular mechanisms of how stress lead to activation of inflammasome, explore new intervention targets of depression, and provide new strategies for the prevention and treatment of depression as well as other stress related disorders.To investigate if inflammasome is activated in acute and chronic mouse model of depression. First, to detect the expression of inflammasome protein by western blot and detect m RNA expression of inflammasome by real-time quantitative PCR. Second, to observe if there is significant improvement of LPS and stress-induced depression-like behavior after suppression of inflammasome.To investigate the signal transduction mechanisms of how LPS and stress lead to activatiton of inflammasome. First is to study whether and how they affect the inflammasome activation pathway(ROS, P2X7/pannexin-1, Cathepsin-B) by detecting ROS, P2X7 R, NF-κB expression changes in brain tissue of LPS and stress-induced depression animal models. Second is to use the screened target cells of inflammasome activation, to further observe possible pathways of inflammasome activation. MethodsAnimal models: Lipopolysaccharide and chronic unpredictable mild stress are used to establish acute and chronic mouse model of depression, respectively. Depression-like behaviors are detected by sucrose preference test, forced swimming test and tail suspension test, etc.Antagonists: YVAD(ip, 8 mg/kg) and VX-765(ip, 50 mg/kg) targeting the downstream signaling pathways protein Caspase-1 of the inflammasome complex. Inhibitors targeting the upstream signaling pathway of inflammasome such as ROS scavengers antioxidant hydrogen-rich water.Expression of inflammasome: Conventional methods like Western blot, and real-time quantitative PCR.Inflammatory cytokines: High-throughput multiprotein detection method as well as ELISA and WB are used to detect expression levels of depression related cytokines such as IL-1β, TNFα and IL-6.Signaling molecules: High throughput kits and Western blot are used to detect ROS, P2X7 R as well as key proteins of NF-kB pathway. Results Experiment 1 NLRP3 inflammasome is involved in acute depression 1. We used forced swimming test and sucrose preference test to observe LPS-induced behavior changes. After giving LPS 24 hours later, mice exhibited depression-like behavior. 2. The multi-plex protein chip and real-time quantitative PCR were used to detect IL-1β protein and m RNA expression levels in mice brain, both of which were significantly increased in LPS-treated mice. 3. The m RNA expression levels of the NLRP3 inflammasome, NLRP3, ASC and Caspase-1 were also significantly increased in the brain of LPS-treated mice. 4. Half an hour before the intraperitoneal injection of LPS, we injected a specific inflammasome blocker YVAD. The forced swimming test and sucrose preference test showed that the depression-like behavior induced by LPS was attenuated by the specific inflammasome inhibitor YVAD. Experiment 2 NLRP3 inflammasome is involved in chronic depression 1. After detecting sucrose preference and tail suspension test we found that mice exposed to CUMS displayed depressive-like symptoms, including decreased sucrose preference and increased immobility time. 2. Mice subjected to CUMS for 4 weeks also had significantly higher serum corticosterone levels, serum IL-1β levels and hippocampal active IL-1β protein levels. 3. Moreover, the hippocampi of CUMS exposed mice had significantly higher activity of caspase-1 detected by caspase-1 activity assay kit, which accompanied by higher protein levels of NLRP3 and ASC. 4. After daily intraperitoneal injection of inflammasome antagonist VX-765 before chronic stress for four weeks, we found that stress-induced increase of caspase-1 activity was significantly inhibited, while NLRP3 and the ASC protein levels also decreased significantly. 5. Pretreatment with the NLRP3 inflammasome inhibitor VX-765 decreased serum and hippocampal levels of IL-1β protein detected by the multi-plex protein chip, and significantly moderated the stress-induced depressive-like behaviors measured by sucrose preference test and tail suspension test. Experiment 3 Hydrogen-rich water can prevent chronic depression 1. Our study found that daily drinking of hydrogen-rich water significantly prevented depressive-like behavior induced by CUMS. And hydrogen-rich water significantly inhibited stress-induced weight loss. 2. CUMS induced elevation in IL-1β protein levels in the hippocampus and cortex was significantly attenuated after 4 weeks drinking of hydrogen-rich water. 3. Over-expression of caspase-1(IL-1β converting enzyme) production induced by CUMS in hippocampus and prefrontal cortex(PFC) was successfully suppressed by hydrogen-rich water detected by caspase-1 activation fluorescence detection kit. 4. Excessive reactive oxygen species(ROS) production in hippocampus and prefrontal cortex(PFC) after 4 weeks stress exposure was successfully suppressed by hydrogen-rich water detected by ROS reactive oxygen species detection kit. ConclusionsThe data of our first experiment suggests for the first time that the NLRP3 inflammasome is involved in LPS-induced mice depressive-like behaviors.The data of our second experiment suggests the NLRP3 inflammasome mediates stress-induced depression via immune activation.The data of our third experiment demonstrates that hydrogen-rich water exert its beneficial effects on depressive-like behavior by effectively inhibiting inflammasome activation, attenuating IL-1β levels and suppressing ROS production.