| Research purposesAnxiety is a common emotional response of the body to external stimuli.Appropriate anxiety can increase people’s sense of vigilance and crisis,and is beneficial to the survival of the body;but excessive anxiety can affect people’s quality of life,and can lead to anxiety,or even appear complications such as sleep disorders,depression and obesity.However,the mechanism of anxiety disorder is still unclear.Therefore,studying the regulation mechanism of anxiety is of great value to the prevention and treatment of anxiety.Recent studies have shown that cytokines not only regulate immune function,but also participate in the regulation of anxiety.IL-3 3 is a newly discovered stress alert cytokine,which belongs to the IL-1 family and plays an important role in innate immunity,inflammation and allergy.Studies have shown that IL-33 is highly expressed in the brain and is involved in the development and differentiation of the nervous system.But its exact effect on anxiety is unclear.This project established IL-33 gene knockout mice,analyzed the influence of IL-33 gene on anxiety,and confirmed the effect of IL-33 in the amygdala on anxiety by overexpression IL-33 in the amygdala.Through a variety of cell and molecular biological experimental methods,the specific molecular mechanism is explored.Research methodⅠ.The effect of IL-33 on the anxiety of mice1.In vivo experiments have shown that IL-33 is specifically involved in anxiety in the amygdala:In the LPS-stressed mice model,analyze the anxiety behavior of LPS-stressed mice through anxiety-related experiments-open field test(OFT)and elevate plus maze(EPM),and WB was used to detect the expression of IL-33 in four brain regions in the hippocampus,hypothalamus,prefrontal lobe and amygdala of mice.2.Preparation of IL-33 systemic knockout mice:The knockout effect of Il-33 KO mice was tested by first-generation sequencing,RT-PCR and WB3.Detect the anxiety behavior of WT and Il-33 KO mice under normal state and LPS induction state:OFT and EPMⅡ.The behavioral mechanism of Il-33 KO mice affecting anxiety1.Detection of the relationship between anxiolytic behaviors of Il-33 KO mice and brain neurotransmitters:RT-PCR was used to detect the expression of glutamate receptors and y-aminobutyric acid receptors in the hippocampus,prefrontal lobe and hypothalamus;WB detection the expression of y-aminobutyric acid in hippocampus,prefrontal lobe,hypothalamus and amygdala;injection of FG7142(inverse agonist ofγ-aminobutyric acid receptor)by intraperitoneal to Il-33 KO mice to detect mice anxiety behavior.2.Exploring the relationship between GABA and BDNF in 11-33 KO mice brain:Using RT-PCR and WB to detect the level of BDNF in hippocampus,hypothalamus,prefrontal lobe and amygdala of 6-8W of WT and Il-33 KO mice mRNA and protein levels.3.Further explore the expression of inflammatory factors in the brains of Il-33 KO mice:RT-PCR was used to detect the inflammatory factors levels in the hippocampus,hypothalamus,prefrontal lobe and amygdala of 6-8W of WT and Il-33 KO mice.Ⅲ.In vitro experiments to explore whether IL-33 overexpression affects BDNF through the activation of NF-κB1.Explore the relationship between IL-33 and BDNF and inflammatory factors:use RT-PCR and WB methods to detect the expression of neurotrophic factors after mouse IL-33 recombinant protein stimulates BV2;RT-PCR and ELISA methods were used to detect the expression of inflammatory factors in cells and cell supernatants.2.Explore the relationship between IL-33,NF-κB and BDNF and inflammatory factors:After IL-33 recombinant protein and NF-κB inhibitor(PDTC)co-stimulate BV2,WB detects the expression of P-P65 and BDNF,RT-PCR to detect the expression of inflammatory factors and BDNF.IV.In vivo experiments to explore whether IL-33 depends on the activation of NF-κB,which affects the BDNF-GABA axis and thus affects anxiety1.Overexpression of adeno-associated virus-IL-33 in the amygdala:Firstly,the adeno-associated virus GFP-IL-33 of the astrocyte-specific expression vector(GFAP)was overexpressed in the WT by stereotactic brain positioning.In the mice amygdala,14 days later,immunohistochemistry was used to detect the expression of GFP in the amygdala of mice,and the expression of IL-33 in the amygdala of mice in different groups was detected by WB.2.The effect of IL-33 overexpression and the addition of NF-κB inhibitor on the anxiety behavior of mice:OFT and EPM.3.The effect of IL-33 overexpression and the addition of NF-κB inhibitor on mice neurotrophic factor BDNF:WB was used to detect the expression of BDNF in the mice amygdala.4.The effect of IL-33 overexpression and the addition of NF-κB inhibitor on neurotransmitters in mice brain:RT-PCR detects the expression of GABA receptors in the hippocampus,hypothalamus and prefrontal lobe of mice;WB detects the expression of GAD67 in hippocampus,hypothalamus,prefrontal lobe and amygdala.ResultⅠ.The effect of IL-33 knockout on anxiety in mice and its mechanism1.IL-33 expression is significantly increased in the amygdala of LPS-induced anxiety model miceTo study the relationship between IL-33 and anxiety,we established an anxiety model with LPS chronically stimulated mice.WB was used to detect the expression of IL-33 in the hippocampus,hypothalamus,prefrontal lobe and amygdala in different brain regions of mice.It was found that after LPS stress,the expression of IL-33 in the amygdala of mice was significantly increased,but it had no significant effect on the expression of IL-33 in the hippocampus,hypothalamus and prefrontal lobe.It is suggested that the expression of IL-33 in the amygdala is related to anxiety behavior.2.Preparation and identification of IL-33 knockout(Il-33 KO)miceIn order to explore the effect of IL-33 on mice anxiety,IL-33 system gene knockout mice were prepared using CRISPR/Cas9 technology.First,through first-generation sequencing,the deletion at the gene level of Il-33 KO mice was detected;then RT-PCR and WB methods were used to detect IL-33 at the mRNA and protein levels in the mice brain,lung,liver,spleen and intestines.The results showed that IL-33 was expressed in all tissues of wild-type(WT)mice,but the mRNA and protein expression of IL-33 were absent in all tissues of Il-33 KO mice.It shows that IL-33 knockout(Il-33 KO)mice were successfully prepared.3.The effect of IL-33 knockout on anxiety in mice under physiological conditionIn order to study the effect of IL-33 on the anxiety of mice,OFT and EPM were used to detect the effect of IL-33 knockout on the anxiety behavior of mice under physiological conditions.OFT found that 11-33 KO mice took longer to enter the central area than the WT mice;EPM found that Il-33 KO mice stayed in the open arms longer than the WT mice.It shows that the knockout of IL-33 significantly reduces the anxiety of mice under physiological conditions.4.The effect of IL-33 knockout on the anxiety behavior of mice induced by LPSIn order to further determine the effect of IL-33 on anxiety behavior,the effect of IL-33 knockout on anxiety was analyzed in the LPS-induced mice anxiety model.Dates show that Il-33 KO mice can reduce LPS induced anxiety compared to WT mice.It shows that IL-33 knockout can alleviate the anxiety induced by LPS.Ⅱ.The mechanism of IL-33 affecting anxiety behavior1.The effect of IL-33 knockout on the expression of neurotransmitters and receptorsIt is known that the inhibitory neurotransmitter y-aminobutyric acid(GABA)and its receptors in the brain play an important role in regulating anxiety.In order to explore the mechanism that affects anxiety after IL-33 knockout,we used RT-PCR to detect the expression of glutamate receptors and y-aminobutyric acid receptors(GABAARal-3)in the prefrontal lobe,hippocampus and hypothalamus.WB was used to detect the expression of GAD67 in the prefrontal lobe,hippocampus,hypothalamus and amygdala.The results are as follows:(1)The effect of IL-33 knockout on the expression of excitatory neurotransmitter glutamate receptors:RT-PCR was used to detect the expression of glutamate receptors in the prefrontal lobe,hippocampus and hypothalamus of WT and Il-33 KO mice.Results showed that the expression of glutamate receptors in the brain area of mice after IL-33 was knocked out had no obvious effect.(2)The effect of IL-33 knockout on the expression of inhibitory neurotransmitterγ-aminobutyric acid receptor:RT-PCR was used to detect the expression of GABA receptors in different brain regions of WT and Il-33 KO mice.The results showed that IL-33 knockout significantly up-regulates the expression of GABAARal in the hypothalamus,but has no significant effect on the expression of GABAARal in the prefrontal lobe and hippocampus;and IL-33 knockout has no significant effect on the expression of GABAARa2,3 in various brain regions.It shows that IL-33 affects the expression of GABAARal in the hypothalamus.(3)The effect of IL-33 knockout on the expression of y-aminobutyric acid:As GABA protein is unstable and difficult to detect,we used WB to detect the key enzyme GAD67 that synthesizes GABA in different brain regions of WT and Il-33 KO mice.GAD67 can express directly reflects the level of GABA.WB results showed that IL-33 knockout significantly increased the expression of GAD67 in the hippocampus,hypothalamus and prefrontal lobe,but had no significant effect on the expression of GAD67 in the amygdala.It suggests that the anti-anxiety of Il-33 KO mice is related to the increase of GABA synthesis in hippocampus,hypothalamus and prefrontal lobe.(4)GABA receptor antagonist blocking the interaction of GABA and its receptor can reverse the anti-anxiety effect of IL-33 knockout:FG7142 is an anxiety-causing drug that weakens the function of GABA channels and reduces the interaction of GABA with its receptors,.After intraperitoneal injection of FG7142 into Il-33 KO mice,we tested the anxiety behavior and the results showed that the anxiolytic behavior of 11-33 KO mice disappeared.It shows that Il-33 KO mice exerts anti-anxiety effects by up-regulating the GABA and its receptors.2.The effect of IL-33 knockout on the expression of BDNF in the brain areaStudies have shown that the BDNF projection in the amygdala up-regulates the synthesis of GABA in anxiety-related brain areas and exerts an anti-anxiety effect.In order to explore the anti-anxiety mechanism of IL-33 knockout,we used RT-PCR and WB to detect the expression of neurotrophic factors in the hippocampus,hypothalamus,prefrontal lobe and amygdala of WT and Il-33 KO mice.The results showed that at the mRNA level,there was no change in BDNF in the four brain regions;at the protein level,the expression of BDNF in the amygdala of Il-33 KO mice was specifically up-regulated,but there was no significant change in BDNF expression in other brain regions.This indicates that IL-33 may selectively negatively regulate the expression of BDNF in the amygdala.3.The effect of IL-33 knockout on the expression of cytokines in the brainIt has been reported that inflammatory factors(IL-1 β,IL-6 and TNF-α)can promote anxiety by inhibiting BDNF and other pathways.We further used RT-PCR to detect the mRNA levels of inflammatory factors in four brain regions of mice.The results showed that the expression of inflammatory factors in the hippocampus,hypothalamus and prefrontal lobe of WT and Il-33 KO mice did not change significantly,but in the amygdala,The expression of the inflammatory factors(IL-1βand TNF-α)of Il-33 KO mice has dropped significantly.It shows that IL-33 positively regulates the expression of inflammatory factors in the mice amygdala.Ⅲ.In vitro experiments to explore the direct effects of IL-33 on pro-inflammatory cytokines and BDNF1.IL-33 inhibits the expression of BDNFIn order to explore the effect of IL-33 on BDNF,the cultured microglia(BV2)were directly stimulated with IL-33 recombinant protein,and the direct effect of IL-33 on the expression of BDNF mRNA was detected by RT-PCR.The results showed that IL-33 protein can inhibit the expression of BDNF in BV2.We further used WB to prove the effect of IL-33 on the level of BDNF protein in BV2.The experimental results found that IL-33 protein can significantly inhibit the expression of BDNF after stimulating BV2.It shows that IL-33 can directly inhibit the expression of BDNF in microglia.2.IL-33 promotes the expression of pro-inflammatory cytokinesIn vitro experiments proved that IL-33 recombinant protein can promote the expression of inflammatory factors in BV2.First,RT-PCR was used to detect the influence of IL-33 at different time points and different concentrations in BV2 on the expression of inflammatory factor mRNA levels.The results showed that different concentrations of IL-33 recombinant protein can promote IL-1β,IL-6 and TNF-αmRNA expression in BV2;In addition,the ELISA method was used to detect the expression of inflammatory factors in the culture supernatant after IL-33 stimulated BV2,and it was found that IL-33 can promote the secretion of inflammatory factors-IL-6 and TNF-α in microglia.The above results indicate that IL-33 promotes the expression of inflammatory factors in microglia.3.IL-33 may through the NF-κB pathway to inhibit the expression of BDNFThe above results indicate that IL-33 promotes the release of inflammatory factors In order to further clarify whether IL-33 affects the NF-κB pathway in microglia,we used WB to detect the activation of P-P65 after IL-33 protein stimulates BV2.It was found that the expression of P-P65 increased with the stimulation time of IL-33.It shows that IL-33 can promote the activation of P-P65.In addition,we added IL-33 and PDTC,an inhibitor of NF-κB,to stimulate BV2 cells.WB results showed that PDTC can inhibit the increase of P-P65 protein caused by IL-33.It shows that the activation of P-P65 can be inhibited by PDTC.Furthermore,RT-PCR and WB results proved that NF-κB inhibitor can partially restore the reduction of BDNF caused by IL-33.It shows that IL-33 can affect the expression of BDNF through NF-κB pathway.Ⅳ.IL-33 in the amygdala can affect the BDNF-GABA axis and promote anxiety by activating NF-κB1.Overexpression of AAV-IL-33 in the amygdalaIn order to fully prove the unique anxiety-inducing effect of IL-33 in amygdala astrocytes.Firstly,WT mice were injected with a GFP-AAV-IL-33 and astrocyte-specific promoter into amygdala via brain stereotaxic.14 days later,immune-fluorescence staining was performed on frozen sections of mice brain tissue,and the results showed that the fluorescence of GFP was expressed in the amygdala of the mice The mice amygdala tissue were taken and the WB experiment were performed.The expression of IL-33 in the amygdala of mice with AAV-IL-33 increased.It shows that AAV-IL-33 was successfully overexpressed in the amygdala.2.The effect of IL-33 overexpression in promoting anxious behavior in mice depends on the NF-κB pathwayIn order to explore the effect of IL-33 overexpression on the anxiety behavior of mice,and the anti-anxiety effect of adding NF-κB inhibitor,we conducted an anxiety behavior experiment.The results showed that in the open field test,mice injected with AAV-IL-33 exhibiting anxious behavior,and at the same time injected NF-κB inhibitor-PDTC into the brain of anxious mice,the anxiety behavior of the mice was reduced.It shows that overexpression of IL-33 in amygdala astrocytes can cause anxiety in mice,and NF-κB inhibitor can alleviate anxiety in mice.3.IL-33 overexpression promotes BDNF expression depending on the NF-κB pathwayIn order to prove the effect of IL-33 and NF-κB inhibitor on BDNF,WB was used to detect the expression of BDNF in the amygdala of mice.The results showed that the protein expression of BDNF in the amygdala of mice overexpressing AAV-IL-33 decreased;and mice that overexpressed with IL-33 and then injected NF-κB inhibitor,the protein expression of BDNF in the amygdala increased,corresponding to the results of cell experiments.It shows that IL-33 overexpression promotes anxiety in mice by down-regulating the expression of BDNF in the amygdala,and NF-κB inhibitors can up-regulate the expression of BDNF in the amygdala to relieve anxiety in mice.4.IL-33 overexpression inhibits the expression of the inhibitory transmitter y-aminobutyric acid(GABA)and its receptor in the projection brain area,and inhibits NF-κB to reverse its effectIn order to prove that IL-33 knockout can promote the expression of BDNF in the amygdala and project other brain regions to promote GABA expression,we used RT-PCR and WB to detect IL-33 overexpression and mice after adding NF-κB inhibitor The expression of y-aminobutyric acid receptor(GABAARal-3)and GABA in the hippocampus,hypothalamus and prefrontal lobe,the results are as follows:(1)Overexpression of IL-33 and the effect of adding NF-κB inhibitor on the expression of y-aminobutyric acid receptorIn Il-33 KO mice,GABAARal in the hypothalamus will change,but the receptors in the hippocampus and hypothalamus do not change much.We further detected the expression of GABAARal-3 in the hypothalamus of mice overexpressing IL-33 by RT-PCR,and the results showed that GABAARal decreased in the hypothalamus of mice overexpressing IL-33,while mice overexpressing IL-33 added NF-κB inhibitor,the receptor GABAARal has a rising trend,and the hypothalamus GABAARa2 also changes to varying degrees.After overexpression of IL-33,GABAARal in the hippocampus and prefrontal lobe showed an upward trend.After adding NF-κB inhibitor,the expression of GABAARal decreased.This shows that the overexpression of IL-33 in the amygdala changes the expression of GABA receptors in the brain,and NF-κB inhibitors can restore the normal expression of GABA receptors.(2)Overexpression of IL-33 and the effect of adding NF-κB inhibitor on theexpression of y-aminobutyric acid synthase In Il-33 KO mice,WB results have shown that the GAB A content of hippocampus,hypothalamus and prefrontal lobe increases in mice.We further used WB to detect the content of GAD67 in different brain regions in mice overexpressing IL-33,and the results showed that GAD67 in the hippocampus,hypothalamus and prefrontal lobe of mice overexpressing IL-33 showed a downward trend,while overexpressing IL-33 and adding NF-κB inhibitor to mice,GAD67 in the hippocampus,hypothalamus and prefrontal lobe of the mice showed an upward trend.It shows that the overexpression of IL-33 in the amygdala in astrocytes reduces the expression of GABA in the hippocampus,hypothalamus and prefrontal lobe,while NF-κB inhibitor can increase the expression of GABA in the hypothalamus and prefrontal lobe.Conclusion1.IL-33 in the amygdala can regulate anxiety-like behavior in mice:Knockout ofIL-33 can reduce anxious behavior in mice,and overexpression of IL-33 in the amygdala promotes anxious behavior in mice.2.IL-33 in the amygdala can inhibit the expression of BDNF,thereby inhibitingthe synthesis of y-aminobutyric acid in the projection brain area and promoting anxiety.3.The negative regulatory effect of IL-33 on BDNF depends on the activation of the NF-κB pathway.Innovation and significance1.In the mechanism of IL-33,we found that IL-33 can inhibit the expression of BDNF by activating the NF-κB pathway2.Regarding IL-33’s regulation of anxiety behavior,we found that IL-33’s regulation of anxiety behavior is brain-specific:the high expression of IL-33 in the amygdala can promote anxiety by inhibiting the BDNF-y-aminobutyric acid axis3.The research results reveal the role of IL-33 in the pathogenesis of central nervous system and anxiety disorders,and provide new ideas for the development of anti-anxiety drugsLimitation1.Due to the limitation of conditions,no astrocyte-conditional knockout IL-33 mice have been obtained,and it is impossible to directly prove that IL-33 knockout of astrocytes can affect the anxiety behavior of mice2.Due to the limitation of specimen sources,this study did not involve clinical trials. |
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