| Objective: To explore the molecular mechanism of Shu-feng Tong-luo Recipe on bronchial asthma cells by studying the effect of medicated serum on Eotaxin / CCR3 pathway of mouse eosinophils in vitro,so as to provide further theoretical basis for clinical application of Shu-feng Tong-luo recipe.Methods: The primary culture of bone marrow-derived eosinophils in mice was conducted,and the fluid was changed on the third day of culture.After twelve-fourteen days,we should collected the third generation cells of culture.Divided all the cells into blank group,as CCL11 group,CCL11+ comparison serum group,CCL11+ medicated sera of TCM low-dose group,CCL11+ medicated sera of TCM medium-dose group,CCL11+ medicated sera of TCM high-dose group,CCL11+SB328437 group,CCL11+PD98059 group,CCL11+SB203580group,and CCL11+ Wortmannin group.Each receptor antagonist group was pretreated with corresponding antagonist at 37℃ for 20 min,and then CCL11 was added for induction.The comparison serum group was added with no drug serum from mice,and the medicated sera of TCM on each dose groups were added with drug serum from mice with disparate concentration gradients,respectively.Each antagonist was not treated.After induction and incubation for 24 h,the survival rate of > was confirmed by trypan blue staining to be93%.The cells were separated from the medium,centrifuged(1000rpm)for 5min,and the supernatant was absorbed and discarded.After washing with PBS,the supernatant was centrifuged again(700rmp)for 5min,and then discarded.Detection :(1)General morphological observation and HE staining of EOS cultured in vitro.(2)Western Blotting was used to detect the protein content of CCR3 and ERK phosphorylation in mice EOS.(3)The expression levels of CCR3,p38 MAPK genes in EOS were detected by RT-PCR.Result:(1)General morphological observation and he staining of EOS cultured in vitro: EOS cultured from bone marrow was differentiated and matured.Microscopic observation showed that the cells were mainly round and grew in suspension in the medium.The overall distribution was more uniform,and some of them proliferated faster,showing "colony-like growth".Hematoxylin eosin(he)staining showed that there were many round cells in the 200× field of vision,arranged closely,and purple particles could be seen in the cells.Under the400 × field of vision,the dark purple nucleus in the cytoplasm could be seen clearly,and most of them were in the shape of two leaves and glasses.(2)Western Blot method was used to detect the protein content of CCR3 and ERK phosphorylation in EOS of mice:(1)Compared with blank group,the content of CCR3 was increased in CCL11 group and CCL11+ comparison serum group(P > 0.05).After intervention with medicated sera of TCM,the CCL11+ medicated sera of TCM low-dose group was lower than the CCL11+ comparison serum group(P > 0.05).Compared with CCL11 group and CCL11+ comparison serum group,CCL11+ medium dose serum group was significantly decreased(P < 0.05).CCL11+ medicated sera of TCM high-dose group was significantly lower than CCL11 group and CCL11+ comparison serum group(P < 0.05),and the comparison was statistically significant.CCL11+SB203580,SB328437 and PD98059 groups were significantly lower than those of CCL11 and CCL11+ comparison serum group(P < 0.05).(2)The ERK content level was make a comparison with blank group,CCL11 group and CCL11+ comparison serum group was increased significantly(P < 0.01),with statistical significance between groups.After intervention with Chinese medicine serum,the level of CCL11+ medicated sera of TCM in low-dose group was decreased compared with that in comparison serum group(P > 0.05).The level of CCL11+ medicated sera of TCM medium dose group was decreased compared with that of CCL11 group(P > 0.05),and significantly decreased compared with that of CCL11+ comparison serum group(P < 0.05);CCL11+ medicated sera of TCM high-dose group was significantly lower than CCL11 group and CCL11+ comparison serum group(P < 0.05),and the comparison values between the two groups has statistically significant;Make a comparison with CCL11 group and CCL11+comparison serum group,the level of ERK in CCL11+ antagonist groups was significantly decreased(P < 0.05),and the comparison values between the two groups has statistically significant.(3)Make a comparison with the blank group,the level of p-ERK in CCL11 group and CCL11+ comparison serum group was significantly increased(P < 0.05).After medicated sera of TCM intervention,CCL11+ medicated sera of TCM low-dose group was significantly lower than CCL11 group and CCL11+ comparison serum group(P < 0.05);CCL11+ medicated sera of TCM medium dose group was significantly lower than CCL11 group and CCL11+ comparison serum group(P < 0.05),and the comparison between groups was statistically significant.CCL11+SB203580 and SB328437 groups were higher than those in CCL11 group and CCL11+ comparison serum group(P < 0.05).CCL11+PD98059 group was lower than CCL11 group and CCL11+ comparison serum group(P < 0.05),and the comparison was statistically significant.(3)The expression levels of CCR3,p38 MAPK genes in mice EOS were detected by RT-PCR:(1)Compared with blank group and CCL11+ comparison serum group,CCL11 group gene levels of CCR3 were increased obviously(P < 0.05).After the intervention of TCM,the CCL11+ medicated sera of TCM in each groups were obviously lower than those in CCL11group(P < 0.05);CCL11+ medicated sera of TCM medium-dose group was evidently lower than those of CCL11+ comparison serum group(P < 0.05);The comparison was evidently significant.Make a comparison with CCL11 group,CCL11+SB203580,PD98059,Wortmannin group were evidently decreased(P < 0.05).(2)CCL11 group the gene level of P38 MAPK was higher than that in CCL11+ comparison serum group and blank group(P < 0.05).After intervention with Chinese medicine serum,the CCL11+ medicated sera of TCM high-dose group was evidently lower than that of CCL11 group(P < 0.01);make a comparison with CCL11+ comparison serum group,it was evidently decreased(P <0.05);Comparison values between the two groups has statistical criteria.CCL11+SB328437,SB203580 and PD98059 groups were lower than those of CCL11 and CCL11+ comparison serum group(P < 0.05).There was no statistical difference in the comparison of antagonists among the three groups.Conclusions:1.CCL11 can induce the increase of CCR3 protein content and up-regulate the level of ERK phosphorylation in eosinophils in vitro;The expression levels of CCR3,P38 MAPK genes were up-regulated.2.Shu-feng Tong-luo Decoction can effectively reduce the expression of CCR3 protein and ERK phosphorylation level of eosinophils induced by CCL11 in vitro.3.Shu-feng Tong-luo Decoction can effectively reduce CCR3,P38 MAPK gene expression levels of eosinophils induced by CCL11 in vitro.4.The mechanism of shu-feng tong-luo prescription on bronchial asthma may be that it can inhibit Eotaxin/CCR3 by inhibiting the levels of CCR3,P38 MAPK,ERK and p-ERK,so as to achieve the purpose of improving bronchial asthma inflammation. |
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