| Objective: To clarify that Chaipu decoction may regulate the p38 mapk signaling pathway and inhibit the m RNA and protein expressions of eotaxin,TARC and IL-33 by down-regulating the expression of 14-3-3 protein.Methods: Twenty-four BALB /c mice(4-6 weeks)were first given adaptive feeding for one week,and then divided into normal group(A),liver depression and asthma group(B),and chaipu decoction group(C)by random number table,with 8 mice in each group.Stage 1: Mice in group A were fed normally.During the period from day 1 to 28,mice in group B and C were stimulated by chronic unpredictable stress,as follows:At the same time,the living conditions of mice were changed by means of wet padding and cage tilt,etc.,and the body weight increase of mice every weekend and the immobile time of forced swimming were recorded in detail to evaluate whether the model of liver depression was successful.Stage 2: Mice in group B and C were treated by intraperitoneal injection of a mixture of 10μg ovalbumin(OVA)and 400 ug aluminum hydroxide on the 1st and 7th days.On the 14 th day,mice in each group were placed independently in a semi-closed atomization chamber,and1%OVA was selected for atomization treatment,once a day,for half an hour each time,for 7 days.When the mice showed shortness of breath,cyanosis of the mouth,and wheezing,the asthma model was established successfully.Mice in group A were treated with the same amount of normal saline.It is particularly noted that each group was treated differently with gastric injection half an hour before atomization.Group A and B received 0.3ml normal saline,while group C received 0.3ml(1.5g/kg)Chaipui decoction.One hour after the last administration,10%chloral hydrate(400mg/kg)was injected into the abdominal cavity of mice in each group for the purpose of anesthesia.Blood samples were taken through the eye socket(to ensure the aseptic state of blood collection),placed at low temperature for one hour,and then centrifuged(4℃,2500r/min,25min).The serum was isolated and stored at-20℃ for later use.The sera were divided into groups and labeled as drug-containing sera in Chaipu decoction group,liver depression asthma group and normal group.The lung tissues of mice were taken for HE staining image analysis.Normal airway epithelial cells of mouse bronchus were cultured and subcultured until the cells grew to 70%～80% of the surface area of the growth matrix.The bronchial airway epithelial cells(NHBEC)with good growth were divided into three groups: normal control group(A),liver depression and asthma serum group(B),and Chaipu decoction group(C).Group A consisted of bronchial epithelial cells + 2m L normal serum.Group B consisted of bronchial epithelial cells + 2m L serum of liver depression and asthma group.Group C consisted of 2m L drug-containing serum of bronchial epithelial cells + Chaipu decoction group.Serums of each experimental group were prepared at a concentration of 20%,and the same amount of medium was added in each group.Culture for 24 hours,cell culture medium and cells were collected.The morphologic changes of cells were observed by living cell tracking analysis microscope.The expressions of 14-3-3 protein,p38 mapk,eotaxin,TARC and IL-33 m RNA in mouse airway epithelial cells were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The expressions of 14-3-3 protein,p38 mapk,eotaxin,TARC and IL-33 in mouse airway epithelial cells were detected by western blot(WB).The concentrations of eotaxin,TARC and IL-33 in the supernatant of mouse airway epithelial cell culture were determined by enzyme-linked immunosorbent assay(ELISA).Results: 1.Liver depression model: Before modeling and on the 7th day of modeling,there was no significant difference in body weight among the three groups,and after intervention,the body weight gain of mice in Ganyu asthma group and Chaipu decoction groupwas slower than that in normal group(P<0.05);However,there was no significant difference in body weight between Ganyu asthma group and Chaipu decoction group.Before modeling,there was no significant change in immobility time of normal group,liver depression and asthma group and chaipu decoction group,7 days before the end of modeling,the immobile time of Ganyu asthma group and Chaipu decoction groupincreased compared with the normal group(P<0.05),there was no significant difference in swimming immobility time between Ganyu asthma group and Chaipu decoction group.2.Changes of lung tissue and airway structure in mice: the lung tissue and airway structure of group A mice were normal,no special changes were observed,and no obvious infiltration of eosinophils or other inflammatory cells in the canal wall was observed.When observing group B,the bronchial lumen was obviously narrow and the edge was thickened,and even some parts were fractured.The infiltration of inflammatory cells and the proliferation of bronchial epithelial cells were observed around the bronchial lumen.No obvious cell proliferation or defect was observed in the lung tissue of mice in group C,and the canal wall was basically complete with only a few inflammatory cells around it.3.RT-qPCR detection: the relative expression levels of 14-3-3protein,p38 mapk,eotaxin and IL-33 in the liver depression and asthma group were significantly higher than those in the normal group(P<0.01);The relative expression levels of 14-3-3 protein,p38 mapk,eotaxin and IL-33 in Chaipu decoction group were significantly lower than those in Gan-yu asthma group(P<0.01).The relative expression level of TARC in liver depression asthma group was significantly higher than that in normal group(P<0.05);The relative expression level of TARC in the chaipu decoction group was significantly lower than that in the Ganyu asthma group(P<0.05).4.WB detection:The protein expression levels of 14-3-3 protein p38 mapk,eotaxin,TARC and IL-33 in liver depression and asthma group were significantly higher than those in normal group(P<0.01);The relative expression levels of p38 mapk,eotaxin,TARC and IL-33 in Chaipu decoction group were significantly lower than those in Gan-yu asthma group(P<0.01).The relative expression level of 14-3-3 protein in Chaipu decoction group was significantly lower than that in Ganyu asthma group(P<0.05).5.ELISA test: the concentrations of eotaxin and IL-33 in the liver depression asthma group were significantly higher than those in the normal group(P<0.01);The concentration of TARC in the liver depression asthma group was significantly higher than that in the normal group(P<0.05);The concentrations of eotaxin,TARC and IL-33 in the chaipu decoction group were significantly lower than those in the Ganyu asthma group(P<0.01).Conclusions: 1.The serum of liver depression and asthma induced the expression of 14-3-3 protein,p38 mapk,Eotaxin,TARC and IL-33 in airway epithelial cells of mice.2.Chaipu decoction containing serum can down-regulate the expressions of 14-3-3 protein,p38 mapk,eotaxin,TARC and IL-33 in airway epithelial cells of mice. |
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