The Study On The Intervention Effect And Mechanism Of TAF On Chronic Obstructive Pulmonary Disease

Objective:To explore the intervention effect and mechanism of TAF in chronic obstructive pulmonary disease（COPD）by combining network pharmacology with in vivo and in vitro experiments.To clarify the anti-inflammatory mechanism of TAF on COPD,and to further explore the medicinal value of Trollius Altaicus on respiratory system diseases,and provide more new experimental basis datas and research ideas.Methods:（1）To use the network pharmacology technology,the Trollius chinensis Bunge-active ingredient-target gene network was drawn to search for COPD-related genes.And draw the wayne map to get the common genes of Trollius chinensis Bunge-COPD that could be studied,and locked the destination of anti-inflammatory targets that which need to be studied in basic experiments.（2）The rat COPD models were established by CS combined with LPS.The control group,COPD model group,model+low TAF Group,model+middle TAF group,model+high TAF group and model+positive control group were established.The low,middle and high TAF dose were given 125,250,500 mg/kg TAF intervention at the 45 day-model stage respectively,while the model+positive control group were given 1 mg/kg·bw dexamethasone for 5 days at the end of the model stage according to the dosage used like the actual COPD patients.To observe the general signs,lung X-ray results,lung function indexes,inflammatory cell level and pathological changes of lung of each rat group.To measure and analyse the changes of inflammatory factors just as IL-1β,IL-6,IL-8,IL-10,TNF-α,TGF-β1 in cell supernatant and BALF by ELISA.Western Blot and RT-PCR were used to detect the changes of TLR4,IKKα,p65,IL-1βprotein contents and IRAK-1,IKKɑ,p65,IL-1βm RNA expressions in rats of each experimental group,and to evaluate the anti-inflammatory effect of TAF on COPD rats based on TLR4/NF-κB.（3）CSE combined with LPS-induced inflammatory injury models of BEAS-2B cells were used as the study object,low,medium and high concentration of drug intervention group was determined by IC₅₀ of TAF,and dexamethasone was used as model+positive control group.The cell survival rates,cell apoptosis rates and the contents of inflammatory cytokines in cell culture supernatant were compared between groups,and the expression of IRAK-1,IKK,P65,IL-1βrelated protein and m RNA based on TLR4/NF-κB signaling pathway.To evaluate the intervention of TAF on CSE combined with LPS-induced inflammation of BEAS-2B cells.Results:（1）85 common genes with strong interaction between Trollius chinensis Bunge and COPD were screened out by network pharmacology.By the PPI map and KEGG enrichment results of drug-COPD,the 20 core targets identified as having anti-inflammatory associations between Trollius Chinensis and COPD diseases include TNF,Toll-like receptor,cancer-associated signaling pathways.And be sure to use TLR4/NF-κB pathway as the target in the follow-up study.（2）The results showed that TAF could maintain the weight gain,food utilization rate and defecation volume of rats exposed to CS/LPS,and reduce the cough,sputum and poor mental state,maintain the respiratory function of rats exposed to CS/LPS.Comparing the X-ray results,the wet-dry weight ratio of lung tissue and the pathological changes of lung,the results showed that TAF could significantly alleviate the pathological changes of lung parenchyma.By comparing the levels of inflammatory cells of rats in each group,the results showed that TAF could significantly decrease the counts of WBC,LYM and EOS.By comparing the contents of IL-1β,IL-6,IL-8,IL-10,TNF-αand TGF-β1 in the BALF and serum of rats in each group,and showed that TAF could effectively reduce the inflammation progression of body and lung in COPD rats,especially for lung.The expression of TLR4、IKKα、P65、IL-1βprotein and IRAK-1、IKKα、p65、IL-1βm RNA in lung tissues of rats were compared based on TLR4/NF-κB signal pathway,the results showed that TAF down-regulate inflammatory mediators related to TLR4/NF-κB pathway and played an inflammatory intervention role.（3）Successfully established the BEAS-2B cell inflammation model of CSE/LPS and the results showed that TAF could effectively maintain the stability of infected cells,prevent cell atrophy and cell disunion,increase the survival rate of cells significantly,reduce the cell apoptosis rate effectively,significantly decreased the levels of IL-1β（P<0.05）,IL-6（P<0.01）,IL-8（P<0.01）,TNF-α（P<0.01）and TGF-β1（P<0.05）,and up-regulated the levels of IL-10（P<0.01）respectively.The protein expression and m RNA expression of IRAK-1,IKK,p65 and IL-1βwere decreased significantly by the intervention of TAF（P<0.01）.Conclusion:There is a relationship between Trollius Chinensis and COPD in which active ingredients act on multiple inflammatory pathways.The prevention mechanism of Trollius Altaicus for COPD can be studied based on TLR and cancer-related pathways.Based on TLR4-NF-κB pathway,TAF could significantly inhibit the inflammation of rat COPD model induced by CS and LPS,and significantly interfere with the inflammatory injury of BEAS-2B cells induced by CSE and LPS for the characteristics of COPD,and prove the intervention effect of TAF.It provides a strong baseline evidence for the Trollius altaicus research on the prevention and treatment of COPD and the drug development and utilization.