Knockout Of CCR3 Gene Inhibits Proliferation,Migration And Degranulation Of Eosinophils In Mice Through PI3K/Akt Signaling Pathway

Section1 PI3K / Akt signaling pathway involved in eosinophil proliferation,migration and degranulation in miceObjective: To investigate the effect of PI3 K / Akt signaling pathway on the degranulation,proliferation and migration of EOSMethods:1.By culturing wild-type C57 BL / 6 mouse bone marrow cells for primary culture,the cells were induced to differentiate into mature eosinophils under the induction of SCF,rm-FLt3 and IL-5 cytokines2.Eosinophil morphological characteristics were observed by Wright’s stain and the eosinophil purity was examined by flow cytometry for the expression of eosinophil surface Siglec-F3.The optimal concentration of PI3 K inhibitor Ly294002(1uM,10 uM,20uM)and PI3 K agonist(740Y-P)of 1ug/ml,10ug/ml,20ug/ml and 50ug/ml were detected by CCK8.The experimental groups were as follows: the control group、the PI3 K agonist group(740Y-P)and the PI3 K inhibitor group(Ly294002).The CCK8 groups were used to observe the changes of eosinophil activity at 6h,24 h and 48 h.Western Blot detection of three groups of P-AKT,AKT protein expression;ELISA eosinophil degranulation EPO expression;Transwell detection of eosinophil migration changesResults: 1.At the same time point,compared with the control group,Ly294002 at 10 uM and 20 uM inhibited the proliferation activity of EOS.At 6 h,10 uM Ly294002 showed inhibition of EOS proliferation activity(p<0.05),and 20 uM Ly294002 inhibition.It is more obvious(p<0.01),while the concentration of 1uM has no significant inhibitory effect(p>0.05),and the inhibitory effect of each concentration tends to be stable(p>0.05)when the culture is continued for 24 h and 48h;however,the PI3 K agonist 740Y-P After treatment,at the same time point,compared with the control group,1ug/ml 740Y-P had no significant effect on the proliferation activity of EOS.When the concentration was 10 ug/ml,the culture time was 6 hours,which promoted the proliferation of EOS.<0.05),but when the concentration increased to 20ug/ml,50ug/ml,the proliferation activity of EOS could be significantly inhibited(p<0.05),and the inhibitory effect of each concentration at 24 h,48h stabilized(p>0.05).Through the above experiments,we finally screened for eosinophils for 6 hours with 10 uM PI3K inhibitor Ly294002 and 10ug/ml PI3 K agonist 740Y-P as the optimal concentration and time for subsequent experimental studies.2.The number of migrated cells in PI3 K agonist group was significantly higher than that in control group(p <0.01),while the number of migrated cells after adding PI3 K inhibitor was significantly lower than that in control group(p <0.01),the difference was statistically significant3.Eosinophil granule protein EPO expression,PI3 K agonist group than the control group(p <0.05),on the contrary,PI3 K inhibitor group EPO expression was significantly lower than the control group(p <0.01),the difference was statistically significant significance4.The expression of P-AKT and AKT in PI3 K inhibitor Ly294002 was significantly lower than that in control group(p <0.01).The PI3 K agonist 740Y-P expression was significantly higher than that in control group(p <0.05)The AKT expression did not changeConclusion: 1.Activation of PI3 K can promote eosinophil proliferation,migration and degranulation,blocking PI3 K can inhibit eosinophil proliferation,migration and degranulation2.PI3 K regulates the proliferation,migration and degranulation of eosinophils,mainly by regulating the expression of downstream phosphorylated AKT.Section2 Knockout of CCR3 inhibits mouse eosinophil proliferation,migration and degranulation via the PI3 K / Akt signaling pathwayObjective:To explore Knockout of CCR3 inhibits mouse eosinophil proliferation,migration and degranulation via the PI3 K / Akt signaling pathwayMethods: 1.Construction and identification of conditional knockout CCR3 gene mouse2.The wild-type mice(wt)and the CCR3 knockout(CCR3-/-)mouse bone marrow cells were extracted and induced to differentiate into mature eosinophils under the induction of SCF,rm-FLt3 and IL-5 cytokines3.Morphological features of both eosinophils were observed by Wright’s stain and the eosinophil purity was examined by flow cytometry for the expression of eosinophil surface Siglec-F4.The experimental groups were divided into three groups: wt EOS group、wt EOS + Eotaxin group(100ng / ml)、CCR3-/-Eos group and CCR3-/-Eos + Eotaxin group;The changes of eosinophil activity at 6h,24 h and 48 h were observed by CCK8.The expression of eosinophil granulocyte EPO was detected by ELISA.The migration of eosinophils was detected by Transwell assayResults:1.The expression of eosinophils,phosphorylated P-AKT and AKT in CCR3 knockout mice was lower than that in wild type(p <0.05).After addition of eotaxin,the wild-type eosinophils phosphorylated(P <0.05),while the AKT expression did not increase,while there was no significant change in Eotaxin,P-AKT and AKT expression in knockout CCR3 mice eosinophils(p> 0.05)2.Eosinophils in knockout CCR3 mice were significantly lower than eosinophils in wild type mice and significantly lower than wild type mice eosinophils at 48h(p <0.001).After addition of Eotaxin,wild type mice Eosinophil activity was higher than the other three groups and was significantly higher at 6h than wild-type eosinophils(p <0.001)3.The expression of eosinophil EPO in knockout CCR3 mice was significantly lower than that in wild type mice(p <0.01).The expression of EPO did not change significantly when Eotaxin was added,while the wild type eosinophils After adding Eotaxin to granulocytes,the expression of EPO increased,higher than the other three groups(p <0.05)4.The number of eosinophil migrating cells in CCR3 knockout mice was lower than that in wild type mice(p <0.05),while the number of migrating eosinophils in wild type mice was significantly increased after adding Eotaxin In the other three groups(p <0.01),knockdown of eotaxin elicited by eosinophils in CCR3 mice showed no significant changes in the number of migrated cells.Conclusion: 1.Eotaxin promotes proliferation,migration and degranulation of eosinophils via CCR3-activated AKT pathway2.Knockdown of CCR3 gene blocks AKT pathway and inhibits eosinophil proliferation,migration and degranulation.