| Objective:Around the changes of CCL24/CCR3 eosinophil activation pathway,taking mouse eosinophils as the research object,the effects of Shufeng Tongluo prescription containing serum on CCL24/CCR3 pathway activated by eosinophils,CCR3 receptor protein content,total ERK phosphorylation level,CCR3 gene expression level and ERK gene expression level of eosinophils were observed from the cellular molecular level.To provide a certain basis for the clinical treatment of Shufeng Tongluo prescription in the future.Methods:1.In this experiment,64 SPF male wild-type C57BL/6 mice average weight 18-22g(8-12weeks)were selected and randomly divided into blank control group,low-dose group,medium dose group and high-dose group.After 2 weeks of adaptive feeding,except the blank control group,the other groups were given Shufeng Tongluo formula by gavage once a day for7 days to prepare the corresponding serum containing the drug.The blank group was given normal saline by gavage,and the mice in the traditional Chinese medicine group were given different concentrations of Shufeng Tongluo formula by gavage.2.Primary culture of eosinophils and establishment of experimental model in vitro.3.The morphological characteristics of eosinophils were observed by Wright staining.4.Each group was intervened with corresponding antagonists,and then induced by chemokine CCL24 for 1 hour.Finally,the eosinophils were divided into blank control group,CCL24+blank serum group,CCL24 group,CCL24+Chinese medicine low dose serum group,CCL24+Chinese medicine medium dose serum group,CCL24+Chinese medicine high dose serum group and CCL24+SB328437(CCR3 receptor antagonist),CCL24+SB203580(p38protein kinase antagonist),CCL24+PD98059(MEK1 / MEK2 kinase antagonist),CCL24+Wortmannin(pl3k protein antagonist).5.Detection:Western blot was used to determine the protein content of CCR3 receptor and the phosphorylation level of total ERK in eosinophils;RT-PCR was used to determine the gene expression levels of CCR3,ERK in eosinophils.Results:1)Western blot results showed as follows:(1)Comparison of CCR3 protein receptor content in eosinophils:Compared with the blank control group,the expression of CCR3 protein receptor in CCL24+ blank serum group was increased(P<0.05).Compared with CCL24+blank serum group,the expression of CCR3 protein receptor content in low-dose serum group was increased,and the protein expression in medium-dose and high-dose serum groups was decreased,and the comparison between low-dose and high-dose serum group and CCL24+blank serum group was significant(P<0.05).The expression of CCR3 protein receptor content in the antagonist group was decreased,and the difference was statistically significant compared with that in the antagonist group(P<0.05).Compared with CCL24 group,the protein expression of CCR3 in low-dose serum group was increased,while that in high-dose serum group was decreased(P<0.05).The protein expression of CCR3 in antagonist group SB203580,SB328437 and Wortmannin group was decreased with statistical difference(P<0.05),while the protein content of PD98059 group had no significant difference with comparison(P>0.05).(2)Comparison of ERK phosphorylation levels:1)ERK level: Compared with blank control group,ERK level in CCL24+blank serum group and CCL24 group was rised(P<0.05).Compared with CCL24+blank serum group,the expression of ERK level in the low,medium and high drug-containing serum groups of Chinese medicine was decreased,with statistical difference(P<0.05),and the expression of ERK level in the antagonist group was decreased(P<0.05).Compared with the CCL24 group,the expression of ERK levels in the serum group containing traditional Chinese medicines were all decreased,which was statistically different(P<0.05),and the level of ERK in each antagonist group was decreased(P<0.05).2)Comparison of p-ERK levels:Compared with blank control group,the levels in CCL24+blank serum group and CCL24 group were increased(P<0.05).Compared with CCL24+ blank serum group and CCL24 group,the p-ERK level increased in the low,medium,and high-dose serum groups of traditional Chinese medicine,and there was no significant difference in the comparison between the groups(P>0.05),while there was no significant difference in p-ERK levels in SB203580 group,SB328437 group and Wortmannin group(P>0.05).2)Comparison of RT-PCR results:(1)Comparison of CCR3 m RNA expression level: Compared with the blank control group,the expression level of CCR3 m RNA in the CCL24+blank serum group and CCL24 group increased(P<0.05).Compared with the CCL24+ blank serum group,CCR3 gene expression level was decreased in serum groups with low,medium and high doses of TCM,and there was significant difference between serum group with low and high dose of TCM(P<0.05),which was statistically significant;the expression level of CCR3 m RNA in the antagonist group was reduced,and there was a significant difference between the SB203580 group and the PD98059 group(P<0.05);Compared with CCL24 group,CCR3 gene expression level was decreased in serum groups with low,medium and high doses of TCM,and there was significant difference between serum group with low and high dose of TCM(P<0.05),which was statistically significant;the expression level of CCR3 m RNA in the antagonist group was reduced,and there was a significant difference between the SB203580 group and the PD98059 group(P<0.05).(2)Comparison of ERKm RNA levels:the ERKm RNA levels of CCL24+blank serum group and CCL24 group were up-regulated compared with blank control group(P>0.05).Compared with CCL24 + blank serum group,the expression of ERKm RNA level in medium dose serum group was decreased,and there was no significant difference(P>0.05);the gene expression level in the remaining groups increased,and there was no statistically significant difference compared with it(P>0.05).Compared with the CCL24 group,the expression of ERKm RNA in the low-dose,medium-dose group,and PD98059 group of the drug-containing serum group decreased,while the gene expression of other groups increased,and there was no statistical difference between the groups(P>0.05).Conclusions:1.Shufeng Tongluo Recipe can reduce the expression of CCR3 protein and the level of ERK in eosinophils.2.Shufeng Tongluo Decoction can reduce the expression level of CCR3 gene in eosinophils of mice.3.Shufeng Tongluo recipe had no effect on p-ERK level and ERK gene level in eosinophils of mice.4.Shufeng Tongluo Recipe can regulate CCL24 / CCR3 pathway by reducing the expression of CCR3 and ERK in eosinophils of mice,reduce or inhibit the attack of asthma,and improve the airway inflammation of Asthma. |
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