Molecular Mechanism Of Shufeng Tongluo Prescription Drug-containing Serum's Effect On Mouse Eosinophil Degranulation Process In Vitro

[Objective]:To study the molecular mechanism of the effect of shufengtongluo formula containing serum on the degranulation of eosinophils in vitro.[Methods]: 1.The eosinophils of high purity mouse peripheral blood were extracted by magnetic bead sorting(MACS).2.Establish the experimental model in vitro by primary culture of eosinophils in peripheral blood of mice.3.The morphological characteristics of eosinophils were observed by diff-quick staining.4.Experimental groups: blank group,negative control group,LY294002(PI3K inhibitor)group,SB328437(CCR3 receptor antagonist),SB203580(p38 protein kinase antagonist),PD98059(MEK1 / MEK2 kinase antagonist),U0126(MEK1 / MEK2 kinase antagonist),PD184352(MEK1 / MEK2 kinase antagonist)The low dose group(10.2g /(kg · d)),the middle dose group(20.4g /(kg · d))and the high dose group(40.8g /(kg · d)).5.The effects of different concentrations of eotaxin / CCR3 signal pathway inhibitors on the proliferation of eosinophils in mice were detected by CCK-8 kit,and the granuloproteins released by the degranulation of eosinophils in mice were detected by ELISA,including MBP,ECP,EPO and eosinophils Expression of neurotoxin(EDN)derived from sex granulocyte.[Results]:(1)Compared with the control group,U0126 of 10 um,20um and 50 um had a certain inhibitory effect on EOS in peripheral blood of mice,and when the concentration was 20 um,the inhibitory effect was better(P < 0.01).After treated with SB203580,SB203580 of 20 um and 50 um had a certain inhibitory effect on eosinophils of mice,and when the concentration was 20 um,the inhibitory effect of SB203580 was better(P < 0.01),but the inhibitory effect of SB203580 of 10 um was too obvious(P > 0.05).When PD184352 and SB328437 were used as inhibitors of 10 nm,20nm and 40 nm,the inhibition effect of the three concentrations was better than that of 40nm(P < 0.01).LY294002 of 5um,10 um and 20 um can inhibit the proliferation of EOS in the peripheral blood of mice,and when the concentration is 20 um,the inhibition of LY294002 is more obvious(P < 0.01).When PD98059 of 50 um,80um and 100 um were given,80 um and 100 um had a certain inhibition effect,and when the concentration was 100 um,the inhibition effect was better(P < 0.01),but the inhibition effect of PD98059 of 50 um was not obvious(P > 0.05).The low,middle and high doses of shufengtongluo containing serum showed that the three concentrations of drugs had a certain inhibition,but the inhibition was more significant in the high dose group(P < 0.01).Therefore,it is concluded that 20umu0126,20umsb203580,40nmpd184352,20umly294002,100umpd98059,40nmsb328437 and the high dose of shufengtongluo containing serum are the best concentration to inhibit the value-added activity of eosinophils in mice,which will be used in the follow-up study;(2)Compared with the control group,the traditional Chinese medicine shufengtongluo prescription contained serum group and CCL11 + shufengtongluo serum group,CCL24 + shufengtongluo serum group can also inhibit the expression of eosinophil granule protein(EPO,MBP,EDN,ECP)in peripheral blood of mice in varying degrees(P < 0.01);and shufengtongluo serum group is better than CCL11 + shufengtongluo serum group and CCL24 + shufengtongluo serum group(P < 0.05),while CCL11 + shufengtongluo serum group Compared with CCL24 + shufengtongluo serum,there was no significant difference(P > 0.05).[Conclusion]: 1.Eotaxin / CCR3 signaling pathway plays an important role in the activation and degranulation of eosinophils in the peripheral blood of mice.The combination of eotaxin and its receptor CCR3 can activate eotaxin / CCR3 signaling pathway,produce downstream amplification effect,further promote the activation and degranulation of eosinophils in the peripheral blood of mice,release EPO,MBP,EDN,ECP and other cellular active substances,and induce asthma Asthma.2.Inhibition of eotaxin / CCR3 signaling pathway can significantly inhibit the activation and degranulation of eosinophils in peripheral blood of mice,and further inhibit the attack or aggravation of asthma.3.The serum containing shufengtongluo recipe can significantly reduce the expression of EPO,EDN,ECP and MBP in peripheral blood of mice,and then inhibit the attack or aggravation of asthma.Therefore,Shufeng Tongfang,a traditional Chinese medicine,may inhibit the activation of eosinophils and their degranulation function in mice by inhibiting eotaxin / CCR3 signal pathway,improve airway inflammation,and then inhibit the attack or aggravation of asthma.The regulation of eotaxin / CCR3 signaling pathway by Shufeng Tongluo method may be an important molecular mechanism for improving chronic airway inflammation in asthma.