| Purpose: Depression is a common chronic mental illness.With the acceleration of the pace of modern life and the attack of COVID-19 in recent years,people have increased pressure on their health,life and study,and the risk of depression is also increasing year by year.The pathogenesis of depression is complex and there are several hypotheses include classical monoamine hypothesis,receptor hypothesis,neuroendocrine hypothesis and immune-inflammatory hypothesis about the development of depression.In recent years,the activation of immune-inflammatory response and the abnormality of inflammatory cytokines in the pathological process of depression have attracted more and more attention.The immune inflammation hypothesis holds that neuroinflammation may be closely related to depression.Under the effect of chronic stress for a long time,the immune system function disorders,peripheral proinflammatory factors and inflammatory mediators abnormally invade the central nervous system,causing the activation of central glial cells and the release of inflammatory factors disorders,leading to neuroinflammation,and ultimately leading to depression.So far,no drugs based on the inflammatory activation hypothesis have entered the clinic.Euparin is a benzofuran compound extracted from Eupatorium chinense L.Previous studies have shown that Euparin has antidepressant effect and anti-inflammatory activity.This project aims to construct an inflammatory model of depression from the perspective of neuroinflammation.Further study on the intervention effect of Euparin on neuroinflammation and depression by inhibiting the activation of NLRP3 inflammasome,and explore its specific mechanism,so as to provide a new way for the research and development of antidepressant drugs.Methods:(1)The establishment of a neuroinflammatory model of BV2 cells: MTT method was used to determine the effects of Euparin and LPS on the proliferation of BV2 cells,and an in vitro neuroinflammation model was constructed by combined induction of BV2 cells with LPS and ATP.The expression levels of inflammation-related proteins in the cell supernatant were determined by Griess method and Western blot method to select the best modeling conditions.(2)The inhibitory effect and specific mechanism of Euparin on neuroinflammation of BV2 cells: After the inflammatory response of BV2 cells induced by LPS+ATP,the expression levels of inflammatory mediators NO,IL-18 and IL-1β in the cell supernatant were measured by Griess method and ELISA method after Euparin administration;the effect of Euparin on the expression of NLPR3/Caspase-1/GSDMD signaling pathway was analyzed by Western blot;the effects of Euparin on LPS-induced NLRP3 inflammatory in BV2 cells were detected by immunofluorescence co-localization.The expression levels of inflammation-related proteins in the cell supernatant were measured after blocking the NLRP3 site using MCC 950.NO,IL-18 and IL-1β levels as well as the expression levels of inflammation-related proteins.(3)The establishment and detection of mouse depression model: a mouse depression model was established using the chronic unpredictable mild stimulation method(CUMS)and LPS induction method,and the degree of depression in mice was measured by behavioural experiments.The serum levels of cytokines IL-18 and IL-1β were analyzed by ELISA.Western blot analysis of NLPR3/Caspase-1/GSDMD signaling pathway related protein expression levels in mouse hippocampal tissue,to explore the best method of depression model(4)The effect of Euparin on depression: the behavioral assaies was used to examine the effect of Euparin on depression-like behavior of mice,and the levels of IL-18 and IL-1β in serum of mice after Euparin administration were measured by ELISA and the expression levels of NLPR3/Caspase-1/GSDMD signaling pathway related proteins in hippocampal tissues of mice were analyzed by Western blot method.Results:(1)Neuroinflammatory modeling results of BV2 cells: MTT results showed that LPS had no significant toxic effect on the proliferation of BV2 cells in the concentration range of 0μg/m L ～ 5 μg/m L(P>0.05),so follow-up experiments were conducted in this concentration range.The detection of NO expression level by Griess method showed that NO secretion in cell supernatant was the highest at 0.5 and 1 μg/m L of LPS.On the basis of these concentrations,the effect of ATP on modeling effect was discussed.Western blot analysis showed that BV2 cells were stimulated by LPS at 1 μg/m L for 6 h.Then,when ATP was added to stimulate BV2 cells for 1 h,the expression of inflammation-related proteins was the highest(P<0.01).Therefore,LPS 1 μg/m L was selected to stimulate BV2 cells for 6 h,and then ATP was added to stimulate BV2 cells for 1 h.(2)The inhibitory effect of Euparin on neuroinflammation and its mechanism: After the combined induction of BV2 cells with LPS+ATP,the results of NO and IL-18 and IL-1β assay showed that Euparin could significantly reduce the secretion of NO and inflammatory factors IL-18 and IL-1β in the supernatant of BV2 cells(P<0.01),and the results of Western blot method showed that Euparin could significantly reduce the expression of NLRP3,ASC,Caspase-1,GSDMD,IL-18,IL-1β and Pro-IL-1β expression in BV2 cells(P<0.01).The results of immunofluorescence co-localization showed that Euparin could inhibit the expressions of NLRP3,ASC and Caspase-1 in LPS-induced BV2 cells which indicated that Euparin on neuroinflammation may be related to the inhibition of the assembly and activation of NLRP3 inflammasome.Euparin showed synergistic effects with NLRP3 site inhibitors in blocking NLRP3 activation after the use of NLRP3 site inhibitors.This suggests that Euparin has an inhibitory effect on neuroinflammation and that the specific mechanism may be through inhibition of the NLPR3/Caspase-1/GSDMD pathway,thereby inhibiting the immune response of microglia.(3)Establishment and detection results of mouse depression model: by observing the daily status of mice and behavioural experiments,it was found that the mice in CUMS+LPS combined group showed the most obvious depression state,and the detection of inflammatory factors IL-18,IL-1β and Western blot analysis showed that: The expression levels of inflammatory factors IL-18 and IL-1β in serum and NLPR3/Caspase-1/GSDMD signaling pathway in hippocampus of CUMS+LPS combined group were the highest(P<0.05).The results showed that CUMS+LPS was the most effective method to establish a depression model in mice.(4)The intervention effect of Euparin on depression: the results showed that compared with the depression model group,the depression behavior of mice treated with Euparin was significantly reduced(P<0.05),the levels of IL-18 and IL-1β in mice serum were decreased(P<0.05),and the levels of NLRP3/Caspase-1/GSDMD pathway-related protein expression was significantly reduced(P<0.05).Indicating that Euparin can improve the depressive-like behavior of LPS+CUMS mice,and its mechanism may be by inhibiting the activation of NLPR3/Caspase-1/GSDMD signaling pathway to inhibit the neuroinflammatory response in the brain of mice,and then play an anti-depression role in mice.The results of Nissler staining in the CA1 region of the hippocampus showed that Euparin improved the neuronal status in the brain of depressed mice and reduced the pathological damage to neurons in the hippocampus,suggesting that Euparinthin had a protective effect on hippocampal neurons of depressed mice,indicating that Euparin had a protective effect on hippocampal neurons in depressed mice.Conclusion:Euparin exhibited significant antidepressant activity,which confirmed that its mechanism of action is related to inhibiting the activation of NLPR3/Caspase-1/GSDMD signaling pathway,inhibiting the secretion of inflammatory mediators NO and inflammatory cytokines IL-18 and IL-1β.These results indicate that Euparin has a good application prospect in the prevention and treatment of depression. |
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