Mechanism Study Of Inhibition By CBN, A Natural Product From Angelicae Pubescentis Radix, On Inflammation In AECOPD Mice Induced By NTHi

Chronic obstructive pulmonary disease(COPD)refers to an obstructive pulmonary disease characterized by persistent airway inflammation and respiratory airflow limitation,which is a progressive disease,morbidity rate is colse to 10% among people over 45 years old,the fourth fatality rate and the fifth disability rate in the world.Controlling airway inflammation during acute exacerbation of Chronic obstructive pulmonary disease(AECOPD)is the main method to treat COPD currently Airway inflammation is the result of body immune system response to pathogenic microorganism infection in the airway.Currently,it is generally believed that nontypeable haemophilus influenza(NTHi)is related to the occurrence of AECOPD closely.NOD1 receptor pathway is a pattern recognision receptors(PRRs),which significantly activated after bacterial infection.Mediates the release of large amounts of inflammatory cytokines and participate in tissue and organ damage by multiple pathways.Because of the entry of NTHi into respiratory epithelial cells,antibiotics show poor killing effect on it,so(Inhaled corticosteroid(ICS)is the main anti-inflammatory therapy in clinical,which still fails to control inflammation effectively.Chinese herbal medicine has a long history in the treatment of various inflammatory diseases.itsDeveloping safe and effective anti-inflammatory drugs from traditional Chinese herbal medicine and its natural products is a research focus in the development of new anti-inflammatory drugs all over the world.Angelicae Pubescentis Radix is the dried root of Angelica pubescens Maxim.f.biserrata Shan et Yuan,umbelliferae apiaceae.It is a traditional Chinese medicine for dispelling wind,removing dampness,relieving arthralgia and pain.Modernpharmacological studies about Angelicae Pubescentis Radixhas showed anti-inflammatory,anti-tumor,analgesic,platelet aggregation inhibition and antihypertensive effects.Columbianadin(CBN)is a natural coumarin isolated from report,also show inhibit effect in reducing mast cell-mediated allergic inflammatory reaction by supressing the production of proinflammatory cytokines in MH-S of human mast cell line stimulated by LPS,thus.Therefore,CBN has a certain inhibitory effect on inflammatory factors and has the potential to be a new anti-inflammatory candidate dru.,But whether CBN can control airway inflammation of AECOPD has not been reported.The research group has established a NTHi-induced mouse model of AECOPD previously.Based on this model,this paper will carry out research on the inhibitory effect and mechanism of natural product-CBN on airway inflammation in NTHi-induced AECOPD mice.Objective:In order to develop a safe,effective and well-defined new natural drug for anti-AECOPD airway inflammation,and provide theoretical basis and data support for pharmacology and toxicology of it.Our research studied the anti-inflammatory effects of CBN in vitro by LPS induced THP-1 cell model.Experiment on acute toxicity of oral administration in mice to observe the toxic effect of CBN.Finally,the mechanism of CBN against AECOPD will be discussed by PCR Array,gene silencing and overexpression,molecular docking technology and metabonomics.Method: 1.Effect of CBN on LPS induced THP-1 cell(1)Stimulating THP-1 cells with LPS to simulating bacterial infection in vitro,establishing LPS-induced THP-1 cells inflammation model,and using enzyme linked immunosorbent assay(ELISA)to measure the effects of low dose(30 μg/ml),medium dose(50 μg/ml)and high dose(100 μg/ml)CBN on the secretion of cytokines TNF-α,IL-1β and monocyte chemotactic protein-1(MCP-1)in inflammatory cell models.(2)Analysis inflammation-related differential expressed genes about inflammation by PCR Array,Combining with KEGG database,the gene pathway with the highest enrichment degree of differentially expressed genes was screened.qPCR was used to detect the expression of key genes in the selected gene pathway and verify the regulatory effect of CBN2.CBN supressed the inflammation in LPS induced THP-1 cell by NOD1 pathwayTransfect NOD1 siR NA and pc DNA3.1 overexpression plasmid into THP-1 cells.Detect the NOD1 gene expression by qPCR to determine the transfection efficiency.Then,qPCR and Western blot were used to detect the expression levels of NOD1,RIP2 and NF-κB P65 genes and proteins after the effect of CBN in the cell inflammation model,ELISA was used to detect the expression levels of TNF-α,IL-1β and MCP-1 in the cell supernatant,and flow cytometry was used to detect the cell apoptosis.3.Effect of CBN on AECOPD mice induced by NTHiCBN Oral intragastric administration twice.The single dose is 2500 mg/kg and the total dose is 5000 mg/kg.Observations the mice within 1 hour and 4 hours after administration,and once a day for 14 days thereafter.Weighing the animals on days 0,3,7 and 14,and dissect all animals at the end of the observation period.(2)Study on lung function effect of CBN on AECOPD mice.Mice COPD model was established after 16 weeks of smoke stimulation,and NTHi(108 CFU/ml)was injected into trachea at the 15 th weekend of smoke stimulation to establish a mice AECOPD model.CBN(50,100 and 200 mg/kg)was given by gavage for 7 days,and the pulmonary function of mice was detected by the small animal pulmonary function instrument: forced expiration volume in 100ms(FEV0.1),functuional residual capacity(FRC)and airway resistance(RI).(3)Flow cytometry was used to detect the changes in the number of BALF neutrophils before and after CBN treatment.HE staining was used to detect the degree of inflammatory infiltration in lung tissue.ELISA was used to detect theexpression of inflammatory cytokines TNF-α,IL-1β and MCP-1 in BALF.qPCR,Western blot and immunohistochemistry were used to detect the changes in the expression levels of NOD1,RIP2 and NF-κB genes and proteins in lung tissue.Meanwhile,molecular docking was used to simulate the binding mode and affinity of CBN and receptor protein.4.Metabonomics research on the metabolites in serum of AECOPD mice affected by CBNNon-target metabonomics research on serum samples of mice with AECOPD intervened by CBN.Combine UPLC-QTOF-MS technology with multivariate statistical to analysi,small molecular endogenous metabolites and related metabolic pathways in serum of normal group,mice with AECOPD model group and mice with CBN high dose group were studied to further clarify the possible mechanism of CBN from the overall level.Result: 1.Effect of CBN on the inflammation of LPS induce THP-1 cell and screening the differentially expressed genes(1)After treated by three dose groups of CBN in LPS-induced THP-1 cells,the secretion of TNF-α,IL-1β,MCP-1 and other pro-inflammatory cytokines decreased significantly(p<0.01),suggesting that CBN has great anti-inflammatory effect in vitro.(2)PCR Array analysis showed that 46 differentially expressed genes were regulated in LPS-induced THP-1 cells after CBN action,and they were mainly concentrated on the NOD1/NF-κB pathway,suggesting that the anti-inflammatory effect by influencing the pathway and further inhibiting the production of inflammatory cytokines.2.CBN supressed the inflammation in LPS induced THP-1 cell by NOD1 pathwayTHP-1 cell model with NOD1 gene silencing and overexpression was established.When NOD1 gene was silenced,the regulatory effect of 50 μg/ml CBN on the expression level of RIP2 and NF-κB decreased or disappeared(p>0.1),the regulatory effect on the levels of inflammatory cytokines TNF-α,IL-1β and MCP-1 in cell supernatant also decreased or disappeared(p>0.1),and the inhibitory effect of CBN on LPS-induced apoptosis of THP-1 cells decreased(p < 0.05).After overexpression of NOD1 gene,CBN significantly reduce the expression of RIP2 and NF-κB in THP-1 cells induced by LPS(p<0.01),and also reduce the secretion level of inflammatory cytokines in cell supernatant(p<0.01).LPS-induced apoptosis of THP-1 cells was also significantly inhibited(p<0.01),which proved that NOD1 signaling pathway plays a role in CBN anti-inflammation.3.Effect of CBN on AECOPD mice induced by NTHi(1)Acute toxicity test of CBN mice by intragastric administration: Under test condition,CBN was administered to mice by intragastric administration twice in one day at a dose of 2500 mg/kg.After 14 days,there was no obvious toxic reaction to animals,and its LD50 was more than 5000 mg/kg.(2)Study on lung function effect of CBN on AECOPD mice.FEV0.1 of AECOPD mice was significantly lower than that of the normal group,FRC and RI were significantly higher than that of the normal group,and were corrected by CBN.(3)The therapeutic mechanism of CBN to AECOPD.HE staining of lung tissue showed that CBN could significantly inhibit lung tissue inflammation caused by NTHi infection.The number of neutrophils in BALF of mice decreased significantly(p<0.01),and the secretion levels of TNF-α,IL-1β and MCP-1 in BALF also decreased(p < 0.01).The expression levels of NOD1,RIP2 and NF-κB genes and proteins in lung tissue were significantly decreased after CBN treatment(p<0.01),suggesting that CBN can inhibit NTHi-induced AECOPD in mice,which may be mediated by inhibiting the activation of NOD1-RIP2-NF-κB pathway,further inhibiting the production of inflammatory cytokines and neutrophils infiltration.In addition,molecular docking showed that CBN can be well embedded into the active pocket of RIP2 receptor,with better spatial and electrical complementation,forming a hydrogen bond with ASP-164 amino acid residue.4.Metabonomics research on the metabolites in serum of AECOPD mice affected by CBNNon-target metabonomics research on serum samples of mice with AECOPD intervened by CBN: Compared with normal control group mice,the content of many endogenous metabolites in serum of AECOPD model mice has changed significantly.CBN intervention can significantlycallback α-ketoglutaric acid,citric acid,alanine,5-HETE,glutamic acid,glutamine,leukotriene A4,12.The levels of 24 endogenous metabolites such as 13-EpOME,palmitic acid,16(R)-HETE,retinyl ester,19(S)-HETE,oleic acid,docosahexaenoic acid,lecithin,arachidonic acid,linoleic acid,20-hydroxy-leukotriene B4,vitamin A,etc.suggest that CBN may play an anti-mouse AECOPD role by regulating metabolic pathways such as arachidonic acid metabolism,glutamine metabolism,D-glutamic acid metabolism,linoleic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,retinol metabolism,triphosphate circulation,etc.Conclusion: 1、 CBN can significantly alleviate airway inflammation and lung function of AECOPD mice,showed therapeutic effects.2、 CBN therapy AECOPD mice by inhibiting the activation of NOD1-RIP2-NF-κB pathway and inflammatory cytokines.3、 Effect of CBN on AECOPD is related to the cytokines reduced by and regulate the levels of 24 endogenous metabolites such as arachidonic acid,vitamin A,etc.Arachis arachidis tetradilute acid metabolism,glutamine metabolism,D-glutamic acid metabolism,linoleic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,retinol metabolism and triphosphate circulation may be metabolic pathways that exert anti-AECOPD effects.Innovation:1.Our study observed the acute toxic reaction of coumarin natural product CBN in Angelicae Pubescentis Radix,providing theoretical data for evaluating the toxic and side effects of the natural active ingredient.2.Our study screened the in vivo and in vitro anti-AECOPD effects of CBN.It is clear that CBN can inhibit airway inflammation in NTHi-induced AECOPD mice.3.Discussed the mechanism of CBN’s anti-AECOPD airway inflammation.From gene,protein expression level to overall metabolic pathway,it is revealed that the NOD1/NF-κB pathway,arachidonic acid metabolism,glutamine and D-glutamic acid metabolism,linoleic acid metabolism,alanine,aspartic acid and glutamic acidmetabolism,retinol metabolism and triphosphate circulation may be the key signal pathway and metabolic pathway for CBN to exert anti-inflammatory effect.To sum up,the research on the inhibitory effect and mechanism of natural component CBN on airway inflammation induced by NTHi in AECOPD mice in this paper enriches the pharmacological and toxicological research of CBN,which can provide important theoretical and experimental basis for finding and developing new safe,effective and well-defined anti-AECOPD inflammatory drugs from traditional Chinese herbal medicines and their natural products,and also provide ideas and enlightenment for the research on the treatment and mechanism of other inflammatory diseases.