| ObjectiveAirway mucus hypersecretion is an important pathological feature of various chronic respiratory diseases.Airway mucus is difficult to remove mainly due to the increase of mucin secretion and the change of physical and chemical properties of the main components.Around the mucin and hydration,this study based on interleukin-1β(IL-1β)induced human airway epithelial cells H292 secreted mucin5AC(MUC5AC),screening mucus hypersecretion model in vitro,and investigated the effect of clearing heat and resolving phlegm method and the representative fomula Qingqi Huatan Pill in the regulation of NF-κB/miRNA-494 signaling pathways of H292 cell mucus hypersecretion model,and exploring the molecular mechanism of clearing heat and eliminating phlegm.Method1 Establish mucus hypersecretion model of H292 cellMTT was used to detect the effects of different concentration of IL-1β(1.25ng/mL,2.5ng/mL,5ng/mL,10ng/mL)on the cell growth of H292 cells were detected at different time(6h,12 h,24h,48h).ELISA detection content of mucin MUC5 AC and inflammatory cytokine interleukin-8(IL-8)when H292 cells cultured at different time(6h,12 h,24h,48h)with different concentrations of IL-1β(1.25ng/mL,2.5ng/mL,5ng/mL,10ng/mL).The expression of MUC5 AC mRNA in H292 cells cultured at 24 h with different concentrations of IL-1β(2.5ng/mL,5ng/mL,10ng/mL)was detected by PCR.2 The effect of Qingqi Huatan Pill on NF-κB/miRNA-494 Signaling PathwayAccording to the model screening results,the experiment was set up six groups:normal group,model group,inhibitor group,Qing Qi Huatan Wan high dose group,Qing Qi Huatan Wan low dose group,and baicalin group.ELISA was used to detect the MUC5 AC and IL-8 and TNF-α levels.The expression of MUC5 AC mRNA,CFTR mRNA and miRNA-494 in each group was detected by PCR.WB detected the expression of p65 and IκB proteins in each group.Result1 Establish mucus hypersecretion model of H292 cells1.1 Effect of IL-1β on the growth of H292 cellsCompared with the normal group,there were no statistically significance in the OD values of the cells in different groups when the different concentrations of IL-1βwere incubated in H292 cells for 6 h,12 h,24 h,and 48 h,respectively(P>0.05).1.2 Effect of IL-1β on mucin and inflammatory factor secreted in H292 cellsCompared with the normal group,the MUC5 AC levels in IL-1β 1.25ng/mL,5ng/mL,10ng/mL group were increased when IL-1β effected 6h(P<0.05);the MUC5 AC levels in IL-1β 1.25ng/mL,2.5ng,5ng/mL,10 ng/mL group were increased at 12h(P<0.05);the mucin MUC5 AC levels in IL-1β 1.25ng/mL,2.5ng/mL,5ng/mL,and 10ng/mL group were significantly increased at 24 h,and the IL-1β 5ng/mL group was the most significant(P<0.05);the MUC5 AC levels in IL-1β 1.25ng/mL,2.5ng/mL groups were significantly increased,and 5ng/mL,10ng/mL groups were significantly increased at 48h(P<0.01),the remaining groups were not statistically significant.Compared with normal group,the IL-8 levels in IL-1β 1.25ng/mL,2.5ng/mL,5ng/mL,10ng/mL group were increased when treated with IL-1β for 6h(P<0.01);the IL-8 levels in IL-1β 2.5ng/mL,5ng/mL,10ng/mL group were increased at 12h(P<0.05);the IL-8 levels in IL-1β 1.25ng/mL,2.5ng/mL,5ng/mL,10ng/mL group were increased at 24 h,and the IL-1β 5 ng/mL group was the most significant(P<0.05);the IL-8 levels in IL-1β 2.5 ng/mL,5 ng/mL,10 ng/mL group were increased at 48h(P<0.05),the other groups were not statistically significant.1.3 Effect of IL-1β on MUC5 AC mRNA expression in H292 cellsCompared with the normal group,the expression of MUC5 AC mRNA was significantly up-regulated in IL-1β 2.5ng/mL,5ng/mL,and 10ng/mL groups after interfering with H292 cells for 24 h with different concentration of IL-1β,and the most significant was in the IL-1β 10 ng/mL group(P<0.05).2 Effect of Qingqi Huatan Pills on NF-κB/miRNA-494 signaling pathway in high secretion model of H292 cells2.1 Effect of Qingqi Huatan Pill and baicalin on the growth of H292 cellsCompared with the model group,the OD values of Qingqi Huatan Pills in the gradient concentrations of 250μg/mL,500μg/mL,and 1000μg/mL groups were significantly increased,and the Qingqi Huatan Pills 1000μg/mL group was the most significant(P<0.05);There was a significant difference in the OD value of the Qingqi Huatan Pill 2000 μg/mL group(P<0.05);there was significant difference in the decrease of OD value between the baicalin 200μg/mL group and the 400μg/mL group(P<0.05),the change of the OD value in the other groups was not statistically significant.2.2 Effect of Qingqi Huatan Pill,baicalin each group on mucin and inflammatory factors level in H292 cellsCompared with the model group,MUC5 AC levels in the culture supernatant of the inhibitor group decreased,and MUC5 AC levels in the cell cytoplasm increased significantly(P<0.05);Qingqi Huatan Pills culture supernatant and MUC5 AC levels in cell cytoplasm significantly decreased(P<0.05);MUC5AC levels in the culture supernatant of Qingqi Huayu Pill low-dose group and baicalin group decreased(P<0.05),and there was no significant difference in the other groups.Compared with the model group,the IL-8 secretion level increased and the TNF-α secretion level decreased in the inhibitor group(P<0.01);The secretion levels of IL-8 and TNF-α in the baicalin group were decreased(P<0.05),and the other groups had no statistical significance.2.3 Effect of Qingqi Huatan Pill,baicalin each group on MUC5 AC mRNA,CFTR mRNA and miRNA-494 expression in H292 cellsCompared with the model group,MUC5 AC mRNA expression was decreased,and CFTR mRNA expression was increased in the inhibitor group(P<0.05);The expression of CFTR mRNA and miRNA-494 in high dose group of Qingqi huatan Pills was significantly increased(P<0.05);The expression of CFTR mRNA was significantly increased in the baicalin group(P<0.05),but there was no significant difference in the other groups.2.4 Effect of Qingqi Huatan Pills on the expression of p65 and IκB proteins in H292cellsCompared with the model group,in the inhibitor group,Qingqi Huatan Pill high-dose group,Qingqi Huatan Pill low-dose group,and the baicalin group both decreased,the expression of P65 protein and IκB protein was increased(P<0.05).Conclusion1 IL-1β has no inhibitory effect on the growth of H292 cells and can induce H292 cells secrete inflammatory cytokines IL-8,and up-regulate MUC5 AC gene expression and protein secretion.IL-1β 5ng/mL can be secreted to induce H292 cells for 24 h to establish a mucus hypersecretion model.2 Qingqi Huatan Pill,a representive of clearing heat and resolving phlegm method can not directly inhibit the gene expression of mucin MUC5 AC,but can up-regulate the gene expression level of CFTR,miRNA-494,and reduce the expression of MUC5 AC protein.3 The mechanism of up-regulating the expression of CFTR gene and decreasing the expression of MUC5 AC protein in Qingqi Huatan Pills may not be closely related to the NF-κB/miRNA-494 pathway. |
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