| Research background:Anxiety disorder is one of the most widespread mental diseases,which seriously endangers people's life and health.One in nine people worldwide has had an anxiety disorder.At present,the anti-anxiety drugs mainly used in clinical practice have slow onset of efficacy and large side effects,and have no therapeutic effect on some patients with anxiety.There are more and more indications that the abnormal physiological function of the prefrontal cortex is associated with the occurrence of anxiety disorders.In addition,anxiety disorders are associated with abnormal changes in the immune system,which are mainly manifested as abnormal changes in cytokines and other immune-related proteins.Therefore,an anti-anxiety drug with quick effect,small side effects,regulatory effect on the nervous and immune system,and more effective is urgently needed to be developed to deal with this mental disease that seriously endangers people's physical and mental health.Objective:1.To test whether Cordycepin can quickly and stably relieve anxiety in animal behavior experiments.2.To detect the regulatory effect of cordycepin on proinflammatory state and anti-inflammatory state in the prefrontal cortex of the mice.3.To detect the effect of cordycepin on the expression of IL-4 in the prefrontal cortex.4.The correlation between the expression level of IL-4 and the anti-anxiety effect of cordycepin was analyzed.5.The specific regulation of Cordycepin on the expression level of IL-4 in neurons,microglia,and astrocytes in the prefrontal cortex was detected.6.To determine the importance of IL-4 signaling pathway in the anti-anxiety process of Cordycepin.7.To determine the importance of the IL-4 signaling pathway in the regulation of Cordycepin effects on the pro-inflammatory state and anti-inflammatory state of the prefrontal cortex and the regulation of the expression of anti-inflammatory proteins.Research methods:1.Mice(CD-1,7-8 weeks)are randomly assigned into 5 groups:normal saline group(negative control),the cordycepin(3 mg/kg)low dose group,the cordycepin(5 mg/kg)medium dose group,cordycepin(12.5 mg/kg)high-dose group,and imipramine(15 mg/kg)group(positive control))in a single drug injection after 45 minutes(fast)or after 5 days of continuous drug injection(long-term).Anxiety behavior was tested using elevated pluz maze.2.Western blot was used to detect the effect of cordycepin on the expression of iNOS,a proinflammatory state marker protein,and Arg-1,an anti-inflammatory state marker protein in the prefrontal lobe of mice,and immunofluorescence method was used to verify the effect of cordycepin on the expression of iNOS and Arg-1 in the prefrontal cortex of mice.3.Western blot and immunofluorescence were used to verify the effect of cordycepin on the expression level of IL-4 in the prefrontal cortex of mice.4.Linear regression analysis was performed to analyze the correlation between the IL-4 expression levels in the frontal lobe and the behavioral data,45 minutes after a single injection or continuous injection for five days.5.Immunofluorescence co-localization was used to detect the effect of cordycepin on co-localization of IL-4 and microglia marker protein Ibal,neuronal marker protein NeuN,and astrocyte marker protein GFAP in the prefrontal cortex of mice,and to analyze the specific regulatory effect of cordycepin on the expression level of IL-4 in these cells.6.The specific IL-4 antagonist RIL-4Ra was used to block the IL-4 signaling pathway in the brain of mice and simultaneously injected with cordycepin(12.5mg/kg),and then the effect of the drug on the anxiety-like behavior of mice in the elevated plus maze was detected.7.We used the RIL-4Ra to block IL-4 signaling pathways in the brain,followed by Western blot or immunofluorescence method to detect whether the RIL-4Ra would also block the cordycepin effects on proinflammatory iNOS,anti-inflammatory Arg-1,cytokines IL-10,and brain derived neurotrophic factor(BDNF).Imipramine was used as the positive control drug.Results:1.45 minutes after a single injection,the time spent in the open arms,the ratio of time in the open arms to time in the closed arms,the distance travelled in the open arms and the ratio of distance travelled in the open arms to distance travelled in the closed arms of the EPM revealed strong anxiolytic effects in the high-dose 3'-dA group,only the time spent in the open arms was increased in the imipramine group.After constitutive,daily injections for 5 days,all anxiolytic behavioural parameters revealed strong chronic anxiolytic effects in the 3'-d A groups,as well as in the imipramine group.2.After a single injection of the drug for 45 minutes,both Western blot and immunofluorescence detection results showed that the expression of Arg-1,an anti-inflammatory marker protein,in the cordycepin-treated group was increased in the prefrontal cortex of mice.The expression of iNOS,an proinflammatory marker protein,did not change significantly,and the expression of these two labeled proteins in the prefrontal cortex of mice in the imipramine group did not change.After 5 days of continuous injection,the expression of anti-inflammatory Arg-1 was increased in the prefrontal cortex,and the expression of proinflammatory iNOS,was decreased in both the cordycepin and imipramine groups.3.Both Western blot and immunofluorescence analysis showed that the expression of IL-4 in the prefrontal cortex of the mice in the cordycepin-treated group increased after the single injection of the drug for 45 minutes and/or after the continuous injection for 5 days,while the expression of IL-4 in the prefrontal cortex of the mice in the imipramine group increased only after the injection for 5 days.4.After 45 minutes of a single injection or 5 days of treatment,the up-regulation of IL-4 expression in the prefrontal cortex of mice induced by cordycepin was positively correlated with the anxiety improvement parameters of corresponding individual mice in the elevated plus maze or black and white box behavioral tests.5.After 5 days of continuous injection of cordycepin or imipramine,the expression of IL-4 in the neurons in the prefrontal cortex of mice was significantly increased,while the expression of IL-4 in microglias and astrocytes in the prefrontal cortex of mice was not significantly changed.6.The anti-anxiety effect of cordycepin or imipramine in mice with elevated plus maze was eliminated by blocking the IL-4 signaling pathway with RIL-4Ra.7.After blockage of IL-4 signaling pathway by RIL-4Ra,the increase of the anti-inflammatory Arg-1 by cordycepin or imipramine disappears,and the inhibition of pro-inflammatory factor iNOS also disappeared.RIL-4R? also blocked cordycepin induced increase of IL-10 BDNF expression.Conclusion:Cordycepin rapidly up-regulated the expression of IL-4 in the prefrontal cortex,affect the balance of pro-inflammation and anti-inflamation in the prefrontal cortex,and enhanced the expression of anti-inflammatory signaling pathways and molecules related to functional repair of nerve injury in this region,thus producing anti-anxiety effects.The anti-anxiety effect of cordycepin was faster than that of the clinical drug imipramine,and showed stable efficacy after long-term injection.This study indicated that cordycepin regulated neuroimmune-related functions and produce anti-anxiety effects,with fast onset and small side effects,and it has the potential to be developed into a new anti-anxiety drug.At the same time,it also provides a direction for the study of the drug theoretical mechanism for the treatment of anxiety disorders by improving neuroimmune-related dysfunction. |
PDF Full Text Request