Mechanism Of Action Of Anti-Mycoplasma Pneumoniae By Fagopyrum Dibotrys(D.Don) Hara

Mycoplasma pneumoniae(MP)is one of the main pathogens of community acquired pneumonia(CAP)in children and adolescents,which can cause acute upper and lower respiratory tract inflammation and extrapulmonary complications.Macrolide antibiotics are the drugs of choice for the treatment of mycoplasma infections in children.However,with the abuse of macrolide antibiotics,the resistance rate of MP to these drugs is as high as 90% in Asia;the emergence of a large number of resistant strains of MP has brought challenges to clinical treatment,but there is still no Drugs for the treatment of resistant MP.Traditional Chinese medicine has unique advantages in the prevention and treatment of MP infection.In addition to directly inhibiting mycoplasma,it can also adjust the body’s state as a whole to achieve the purpose of treating both symptoms and root causes.Therefore,screening traditional Chinese medicines with anti-drug-resistant MP and exploring their potential anti-MP mechanisms can provide a theoretical basis for clinical use of traditional Chinese medicine treatment.Fagopyrum dibotrys(D.Don)Hara(F.dibotrys)is a kind of treatment for lung abscess.F.dibotrys is a traditional Chinese medicine for the treatment of lung abscess.It has significant antioxidant,anti-tumor and antibacterial effects.It also has certain curative effects on diseases such as bacillary dysentery,leprosy pneumonia,chronic bronchitis,and pelvic inflammatory disease.However,its anti-MP effect has not yet been reported.In the early stage,we established a MP isolation and culture system and established a strain line.We screened anti-MP drugs and found that the ethanol-extracted part of F.dibotrys(high polyphenol content)has a significant anti-MP effect,especially more sensitive to drug-resistant MP.Based on this,we further explored the anti-MP mechanism of F.dibotrys.This topic mainly explores the anti-MP effect of F.dibotrys polyphenol extract and its monomer from three levels of Mycoplasma pneumoniae,cell experiment and animal experiment:(1)The study of Mycoplasma pneumoniae,through growth and metabolism inhibition experiments,found that F.dibotrys is more The higher the content of polyphenols in the phenol extract,the better the anti-MP effect.Through CFU counting,it was found that 75% of F.dibotrys polyphenols have concentration and time-dependent inhibition of drug-resistant strains M19 and standard strains 15531 and 29342,and their MIC50 and MIC90 values are 0.625mg/m L and 1.25mg/m L,respectively,but they can kill M19 The effect is more obvious.Since the adhesion of MP to host cells is the key to its pathogenicity,MP adhesion proteins P1 and P30,and adhesion accessory proteins P65 and HMW3 play a vital role in this process.Through the q RT-PCR experiment,it was found that treatment of MP with 75% F.dibotrys polyphenols alone could significantly reduce the adhesion protein P1 and P30 of type I strains M19 and 29342,but the expression of adhesion accessory protein P65 and HMW3 m RNA increased slightly.It is known that macrolide antibiotics such as erythromycin can stop protein synthesis by binding to the 50 s subunit of bacterial nucleoprotein,inhibiting m RNA translocation and preventing peptide chain elongation.Trm B t RNA(guanosine(46)-N7)-methyltransfera(Trm B)is a t RNA methyltransferase,which participates in the biosynthesis pathway of N(7)-methylguanine-t RNA and is essential for t RNA transfer function.Impaired function will affect the protein translation process.After treatment of MP with 75% F.dibotrys polyphenols,it was found that the expression of Trm B was significantly reduced,and the function of t RNA was destroyed and the translation process was inhibited.After network pharmacology screening and biological experiment verification,the monomer luteolin has the best inhibitory effect on MP.After luteolin treatment of MP,it was found that it can also significantly reduce the expression of P1,P30 and Trm B m RNA,but the expression of P65 and HMW3 m RNA increased slightly.Further explore its anti-MP mechanism through proteomics sequencing.It was found that MP treated with luteolin had a total of 32 differentially expressed proteins,among which the results of Trm B were consistent with the experimental results,but further differential protein expression analysis and biological verification were needed.(2)Cell experiment study,MP infected cells with 75% F.dibotrys polyphenols and luteolin.Through q RT-PCR test,it was found that after treatment of MP with drugs,it can significantly reduce MP adhesion proteins P1,P30 and The expression of adhesion accessory proteins P65 and HMW3,thereby reducing the adhesion ability of MP.CARDS TX(Community Acquired Respiratory Distress Syndrome Toxin,CARDS TX)is a unique cytotoxin in MP.Its expression is up-regulated during MP infection,causing cell vacuolation and eventually cell death.In view of this,after treatment of MP with75% F.dibotrys polyphenols,it was found that it can significantly reduce the expression of M19 CARDS TX,thereby reducing the toxic effect on cells.At the same time,MTT testing found that treatment of infected A549 cells with drugs can significantly restore their proliferation activity.(3)Animal experiment research.After 5 days of infection of mice with 109CCU/ML MP,mice were treated with 169mg/kg 75% F.dibotrys polyphenol,50mg/kg roxithromycin,and 50mg/kg luteolin.Give intragastrically every day.It was found that the body weight of the mice had been decreasing after the infection,and the body weight of the mice gradually returned to normal mouse body weight after the administration.And there was no significant difference in the wet lung weight of mice in each group.In the lung tissues of each group,the MP coating was counted,and it was found that 75% F.dibotrys polyphenols had a clearance rate of 50.7% for M19,a clearance rate of 44.9% for luteolin,and a clearance rate of 25.2% for roxithromycin.Then the lung tissues of each group of mice were tested for cellular immune factors,and it was found that 75% of polyphenols and luteolin in the M19 model group could significantly reduce the levels of IL-1β、IL-6、TNF-α、HMGB1、and IL-17 A.The expression indicates that F.dibotrys and luteolin can improve inflammation in MP-infected mice.In summary,our research has found that F.dibotrys and its monomeric luteolin may reduce MP’s adhesion ability and virulence ability,thereby reducing the damage to cells;and may play an anti-MP effect by inhibiting protein translation process;in addition,The extract of 75% F.dibotrys polyphenols and luteolin can improve the inflammation of resistant and standard MP-infected mice.This topic provides a certain theoretical basis for the treatment of mycoplasma pneumonia and its drug-resistant MP.