| Objective:The dual advantages of local and overall regulation of traditional Chinese medicine(TCM)nasal preparation are shown in treating allergic rhinitis(AR).Modified Yupingfeng naristillae(MYN),the external treatment method of invigorating qi and promoting blood circulation proposed by Professor Tian Li,is an innovation in the dosage form and usage of classical prescriptions.As a non-chemical drug therapy for AR,it can make up for the limitations of existing chemical drug therapy,which can not only alleviate nasal mucosa inflammation,but also carry out systematic regulation,with remarkable curative effect,but its mechanism is still unclear.As the dysfunction of epithelial barrier is the key pathological link of allergic diseases,local inflammation of nasal mucosa is an important factor leading to chronic injury of epithelial barrier of AR,and P38 mitogen-activated protein kinase(p38MAPK)/peroxisome proliferator-activated receptor?(PPAR?)pathway plays an important role in local inflammation,and the barrier function of epithelium can be weakened by TSLP.Therefore,the nasal mechanism of MYN from the perspective of repairing dermatitis injury is explored in this subject,as well as the mechanism of MYN regulating TSLP and its subtypes through p38 MAPK/PPAR?pathway,providing scientific basis for clinical promotion and application of MYN.Method:Experiment 1:21 SD rats were randomly divided into Normal group,AR+Normal saline(NS)group(hereinafter referred to as NS group)and AR+MYN group(hereinafter referred to as MYN group).Allergic rhinitis rat models were established except Normal group.Normal group and NS group were given 0.9%saline naristillae,and MYN group was given MYN naristillae.After 30 minutes of intervention,NS group and MYN group were stimulated by 5%ovalbumin solution.Observation after treatment:(1)Pathological changes and eosinophil count of rat nasal mucosa;(2)the gene transcription of claudin-1,ZO-1 and TSLP in nasal mucosa was detected by Real-time PCR(RT-PCR);(3)the expression of p38 MAPK and PPAR?were detected by Western blotting,and the mechanism of MYN improving dermatitis injury on nasal mucosa of AR rat model was explored from two aspects of epithelial barrier repair and local anti-inflammation.Experiment 2:It is completed in two stages.In the first stage,after subculturing Human Nasal Epithelial Cells(HNEp C),the sensitization model of HNEp C was established with dust mite antigen Derp1.Based on this model in vitro,the following three contents were explored:(1)determining the best intervention concentration and time of p38 MAPK pathway and PPAR?pathway blockers;(2)the safe concentration range of MYN intervention in HNEp C was determined by MTT assay;(3)the best therapeutic concentration and time of MYN intervention in Derp1-induced HNEp C model were determined by dose-effect experiment,which laid the foundation for the second stage experiment.In the second stage,HNEp C was divided into TCM group,TCM with p38MAPK blocking group,TCM with PPAR?blocking group,TCM with PPAR?blocking group+p38mapk blocking group,model group and protocell group.Except the protocell group,the other groups were sensitized by HNEp C in vitro with Derp1.TCM group was intervened by MYN;TCM with p38 MAPK blocking group,TCM with PPAR?blocking group,TCM with PPAR?blocking group+p38 MAPK blocking group were intervened by TCM after dropping corresponding blockers,while model group was intervened by culture medium,but protocell group was not intervened.After culture:(1)the expression of p-PPAR?,PPAR?,p-p38 MAPK and p38 MAPK were detected by WB;(2)RT-PCR was used to detect the expression of TSLP,lf TSLP and sf TSLP,to explore the mechanism of MYN interfering TSLP and its subtypes through p38 MAPK/PPAR?pathway,and to further clarify the therapeutic mechanism of MYN in improving nasal mucosa inflammation and alleviating inflammatory injury of epithelial barrier.Results:Experiment 1(1)Pathological observation of rat nasal mucosa:in Normal group,the tissue structure of nasal mucosa was clear,continuous and complete,and the ciliated columnar epithelium of respiratory part was normal and arranged neatly.In NS group,the tissue structure of nasal mucosa was damaged to varying degrees,the epithelial cilia almost completely fell off,and a small amount of residual lodging disorder;epithelial swelling,epithelial gap enlargement,partial epithelial necrosis and shedding,residual epithelial cell defect.In MYN group,the structure level of nasal mucosa was clear,and a small number of cilia were disordered;epithelial cell swelling is not obvious,intercellular space is normal,and goblet cells are occasionally slightly enlarged in some areas.(2)Epithelial lesion score:NS group was significantly higher than Normal group(P<0.05);MYN group was significantly lower than NS group(P<0.05),and there was no difference between NS group and Normal group(P>0.05).(3)RT-PCR results:compared with Normal group,the expression of claudin-1m RNA and ZO-1 m RNA in NS group decreased,which had significant statistical significance(p<0.05).Compared with NS group,claudin-1 m RNA expression in MYN group was higher,which had significant statistical significance(P<0.05),but there was no significant statistical significance in the increase of ZO-1 m RNA expression(P>0.05).Compared with Normal group,the expression of TSLP m RNA in NS group was higher,which had highly significant statistical significance(p<0.01).Compared with NS group,TSLP m RNA expression in MYN group was lower,which had significant statistical significance(P<0.05).(4)It was shown in Western blotting that the expression of p38 MAPK in NS group was higher than that in Normal group,which had highly significant statistical significance(P<0.01).The expression of PPAR?decreased,which had highly significant statistical significance(P<0.01).Compared with NS group,the expression of p38 MAPK in MYN group was lower,which had highly significant statistical significance(p<0.01);the expression of PPAR?is higher,which had highly significant statistical significance(P<0.01).(5)It could be seen in regression analysis that the regression equation between PPAR?and p38 MAPK was y=2.67-1.88x,R~2=0.40,P<0.01,and p38MAPK expression could be negatively affected by nasal mucosa PPAR?.The regression equation of TSLP m RNA and p38 MAPK could be expressed as:y=0.45+0.51x,R~2=0.40,P<0.01.TSLP m RNA expression could be positively affected by Nasal mucosa p38 MAPK.The regression equation of TSLP m RNA and PPAR?can be expressed as:y=2.02-1.29x,R~2=0.42,P<0.01.TSLP m RNA expression could be negatively affected by nasal mucosa PPAR?.Experiment 2Stage 1:(1)The best intervention time and concentration of Derp1 sensitization:at24h,the expression TSLP m RNA induced by derp1 came to the maximum,when the concentration was 500ng/ml.Compared with 0ng/ml and 100ng/ml groups,the difference was extremely significant(P<0.01).Compared with 0h and 6h groups,the difference was extremely significant(P<0.01),and compared with 12h group,the difference was significant(P<0.05).There is no significant difference between the other groups,and there is no statistical significance.(2)The best intervention time and concentration of blocking agent:at 12h,the transcription level of PPAR?gene was the lowest under the intervention of GW9662at 5?M.Compared with 0?M group,the difference was extremely significant(P<0.01);compared with 0h group,the difference was extremely significant(P<0.01),and compared with 24h group,the difference was significant(P<0.05).At 12 hours,the transcription level of p38 MAPK gene was the lowest under the intervention of5?M SB239063.Compared with 0?M group,the difference was extremely significant(P<0.01);compared with 0.1?M group,the difference was significant(P<0.05);compared with 0h group,the difference was significant(P<0.05);compared with 12h group,the difference was extremely significant(P<0.01).There is no significant difference between the other groups,and there is no statistical significance.(3)Inhibition rate of TCM on HNEp C:with the increase of concentration and time,the inhibition rate of MYN on HNEp C increased,and when the concentration was 0.131g/m L,the inhibition rate was less than 50%at 6h,12h and 24h.0.131g/m L was set as the upper line of safe concentration,and the IC30 of MYN at 24h was0.04g/m L by SPASS 25.0.Two times(0.08g/m L)and 0.5 times(0.02g/m L)of this concentration were taken to form safe concentration ranges of 0.131g/m L,0.08g/m L,0.04g/m L and 0.02g/m L.(4)The best time and concentration of TCM intervention on sensitization model in vitro:At 24h,0.08g/ml MYN inhibited the transcription of TSLP gene from sensitized HNEp C most.Compared with Derp1 group,the difference was extremely significant(P<0.01);compared with Derp1+0.02g/ml TCM group,the difference was significant(P<0.05);compared with 0h and 6h groups,the difference was extremely significant(P<0.01).There is no significant difference between the other groups,and there is no statistical significance.Stage 2:(1)It is shown in Western blotting that the expression of PPAR?protein in the model group was significantly lower than that in the protocell group,with extremely significant statistical significance(P<0.01);compared with the model group,the expression of TCM group was significantly higher,with significant statistical significance(P<0.05).The expression of TCM+PPAR?inhibitor and TCM+double inhibitor group were significantly higher with extremely significant statistical significance(P<0.01).There is no significant difference between the other groups,and there is no statistical significance.Compared with the protocell group,the expression of p-PPAR?protein in the model group is significantly lower,with extremely significant statistical significance(P<0.01);compared with the model group,the expression of TCM group was higher,with significant statistical significance(P<0.05).The expression of TCM+P38inhibitor in TCM group was lower with significant statistical significance(P<0.05).Compared with the TCM group,the expression in the TCM+PPAR?inhibitor group was lower with significant statistical significance(P<0.05).The expression in TCM+P38 inhibitor group was higher with extremely significant statistical significance(P<0.01).There is no significant difference between the other groups,and there is no statistical significance.Compared with the protocell group,the expression of P38 protein in the model group was significantly higher,with extremely significant statistical significance(P<0.01).Compared with the model group,the expression in the TCM+PPAR?inhibitor group was significantly lower,with significant statistical significance(P<0.05).There is no significant difference between the other groups,and there is no statistical significance.Compared with the protocell group,the expression of P38 protein in the model group was significantly higher,with extremely significant statistical significance(P<0.01).Compared with the model group,the expression in the TCM group was significantly lower,with significant statistical significance(P<0.05)and TCM+P38inhibitor group was significantly lower,with significant statistical significance(P<0.01).Compared with the TCM group,the expression in the TCM+PPAR?inhibitor group was significantly higher,with significant statistical significance(P<0.05).There is no significant difference between the other groups,and there is no statistical significance.(2)RT-PCR detection:Compared with the protocell group,the expression of TSLP gene in the model group was higher,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of TCM group and TCM+P38 inhibitor group were lower with significant statistical significance(P<0.05).Compared with the protocell group,the gene expression of lf TSLP in the model group was higher,with significant statistical significance(P<0.01);compared with the model group,the expression in TCM group and TCM+P38 inhibitor group were lower(P<0.05);compared with the protocell group,the gene expression of sf TSLP in the model group was lower with highly significant statistical significance(P<0.01);compared with the model group,the expression in TCM+P38 inhibitor group was higher(P<0.05).There is no significant difference between the other groups,and there is no statistical significance.Conclusion:Experiment 1:(1)In the nasal mucosa of AR model rats,the expression of claudin-1 m RNA was down-regulated,and the epithelium was damaged.However,the expression of p38MAPK and TSLP m RNA increased,while the expression of PPAR?decreased,resulting in local inflammatory infiltration.(2)In nasal mucosa of AR model rats,MYN can inhibit the increase of TSLP-mediated eosinophils,control local inflammatory infiltration,up-regulate claudin-1 m RNA expression to repair dermatitis-induced injury by promoting PPAR?expression,down-regulating p38 MAPK expression.Experiment 2:Stage 1:(1)In vitro experiments,Derp1(500ng/ml)showed the best sensitization effect on HNEp C after 24h intervention.(2)The optimal inhibitory dose and duration of action of SB239063 and GW9662on p38 MAPK pathway and PPAR?pathway in HNEp C were 5?M and 12h,respectively.(3)The optimal effective concentration of MYN on Derp1-sensitized HNEp C was0.08g/ml,and the intervention time was 24 hours.Stage 2:(1)In vitro cell model of HNEp C sensitized by Derp1,p38 MAPK pathway was activated,PPAR?pathway was inhibited,TSLP m RNA and lf TSLP m RNA were up-regulated,while sf TSLP m RNA was down-regulated.(2)In Derp1-sensitized HNEp C,MYN can inhibit p38 MAPK pathway,and inhibit TSLP and lf TSLP gene expression,but has no obvious regulatory effect on sf TSLP,which depends on its activation of PPAR?pathway. |
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