| Objective:1.Based on network pharmacology method,the main active components of Qushi tongluo Formula and its potential targets and mechanism of action against Mycoplasma pneumoniae(MP)were studied;2.To study the effects of Qushi tongluo Formula drug-containing serum on the expression of IL-6,TNF-?,miR-155 and miR-146 a in MP-infected RAW264.7 cells;3.Discuss the risk factors and diagnostic value of children's RMPP(damp-heat closed lung syndrome),and provide reference for the early diagnosis of the disease.Methods:1.Network pharmacology analysis of Qushi tongluo Formula: Based on TCMSP database,the active ingredients of 10 drugs in Qushi tongluo Formula were determined by Ooral Bioavailability(OB?30%)and Drug-Likeness(DL?0.18);the target prediction model in the database was used to further predict the potential target of each drug in Qushi tongluo Formula;OMIM database and Genecards database were searched to obtain MPP disease-related targets in children,the interaction network diagram of “drug component target disease” was constructed by using Cytoscape software,and the enrichment analysis of Go function and KEGG pathway was carried out by using R language.2.Experimental study:(1)Preparation of medicated serum of Qushi tongluo Formula : The experimental rats were random Ly divided into 8 groups,blank group,low,medium and high dose of Chinese medicine,azithromycin group,and low,medium and high dose of Chinese medicine plus azithromycin group.After one week of adaptive feeding,the 8 groups of rats were respectively intervened.The blank group was given sterile distilled water,the low,medium and high dose group of Chinese medicine was given low,medium and high dose Qushi tongluo Formula,the azithromycin group was given azithromycin granule solution,and the low,medium and high dose Chinese medicine plus azithromycin group was given low,medium and high dose Qushi tongluo Formula plus azithromycin granule solution.Each group was administered for 7 consecutive days,2 hours after the last administration,chloral hydrate was injected into the abdominal cavity for anesthesia,and blood was taken from the abdominal aorta under aseptic conditions.After standing at room temperature,the supernatant was centrifuged to take the supernatant,further sterilized,filtered and sterilized,and stored at-80°C for use after labeling.(2)Establish MP-induced RAW264.7 cell inflammation model: Through cell culture technology,set up a blank group,a model group,a serum group containing low,medium and high doses of Chinese medicine,a serum group containing azithromycin,and a serum group containing low,medium and high doses of Chinese medicine plus azithromycin.Observe the cell survival rate of Qushi tongluo Formula in vitro from the indirect inhibition of MP experiment,therapeutic inhibition of MP experiment,preventive inhibition of MP experiment,prevention + treatment of inhibition of MP experiment.(3)The ELISA method was used to detect the expression levels of IL-6and TNF-? in the cells after the intervention of the drug-containing serum in each group in the indirect MP experiment,and the q RT-PCR method was used to detect the expression changes of miR-155 and miR-146 a.3.Clinical research: Collect and screen the cases of hospitalized children with damp-heat closed lung syndrome diagnosed as MPP and RMPP in the Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2018 to December 2020,and develop a retrospective analysis table of clinical cases,and their clinical symptoms and experiments Perform statistical analysis on the characteristics of laboratory examinations,and explore the micro-diagnostic indicators of the onset of children's RMPP damp-heat closed lung syndrome.Results:1.(1)In the indirect MP inhibition experiment,the cell survival rate of each drug containing serum treated for 24 hours was higher than that of the model group,and the cell survival rate was higher than 93%.The cell survival rate of the low,medium and high dose group of traditional Chinese medicine and the low,medium and high dose group of traditional Chinese medicine plus azithromycin was concentration dependent.The cell survival rate of the azithromycin group was similar to that of the medium dose group of traditional Chinese medicine and the low,medium and high dose group of traditional Chinese medicine plus azithromycin.The results indicated that all the administration groups showed a better inhibition of MP and protection of cells.(2)In the treatment and anti MP experiment,the cell survival rate of each drug containing serum group was higher than that of the model group after 24 hours,and the cell survival rate was higher than85%.Among them,the cell survival rate of low,medium and high dose group and low,medium and high dose group with azithromycin was slightly concentration dependent.The cell survival rate of azithromycin group was higher than that of high dose group and low,medium and high dose group with azithromycin.The results indicated that all the drug administration groups showed good inhibitory effect on MP and cell protection.(3)In the preventive MP inhibition experiment,the survival rate of cells treated with drug containing serum for 24 hours was higher than that in the model group,but the overall survival rate was lower,suggesting that the protective effect of drug containing serum on cells was not good.(4)In the experiment of preventive and therapeutic MP inhibition,the cell survival rate of each drug containing serum treated for 24 hours was higher than that of the model group,showing a good protective effect on the cells.Among them,the cell survival rate of high dose group,azithromycin group and low,medium and high dose group was more than 90%.2.(1)Detection of IL-6 in cell supernatant of each group: compared with the blank group,the results were statistically significant(P<0.05).Compared with the model group,except for the low-dose group of traditional Chinese medicine(P=0.373),the differences between the other groups were statistically significant(P<0.05).(2)TNF-? in cell supernatant of each group: compared with the blank group,the results were statistically significant(P<0.05).Compared with the model group,the results were statistically significant(P<0.05).3.Detection of miR-155 and miR-146 a in cells of each group: after drug intervention,the expression level of miR-155 in each treatment group decreased,while the expression level of miR-146 a increased.There were significant differences between the blank group and the model group(P<0.01),and there were significant differences between the administration groups and the model group,and between the administration groups(P<0.05).4.The incidence of MP infection is mostly male,and it is more common in preschool and school-age children,and it is easy to be combined with other pathogens in clinical practice.MPP damp-heat closed lung syndrome and RMPP damp-heat closed lung syndrome showed no significant difference in children's gender,age of onset,and combined pathogen infection(P>0.05);there were significant differences in children's hospitalization days and combined complications(P<0.05).Among them,the average hospital stay of children with RMPP damp-heat closed lung syndrome is relatively long,and they are prone to myocardial damage.Clinical test indicators: Two groups of children in WBC,N,L,M,EO,BA,RBC,HGB,AST,ALT,TP,ALB,PAD,ALP,UREA,CREA,?2-MG,CK,TNR,PT There was no statistically significant difference in PLT,APTT,and TT(P>0.05);the differences in PLT,hs-CRP,GLB,RBP,CKMB,LDH,FIB,DD were statistically significant(P<0.05),suggesting RMPP damp-heat closure Children with lung syndrome have higher inflammatory reactions and blood hypercoagulability,which are more likely to cause damage to myocardium and liver function.Conclusion:1.Qushi tongluo Formula has a good effect of inhibiting MP and protecting RAW264.7 cells.2.The occurrence and development of MP infection are closely related to the expression levels of inflammatory factors IL-6 and TNF-?.Qushi tongluo Formulacan inhibit MP and inhibit the inflammatory response by reducing the expression levels of IL-6 and TNF-?.The effect of azithromycin group was slightly better than that of Chinese medicine high dose group.The inhibition of IL-6 and TNF-? in low,medium and high dose Chinese medicine groups and low,medium and high dose Chinese medicine plus azithromycin group showed concentration dependence.3.Qushi tongluo Formula can reduce the expression of miR-155 and increase the expression of miR-146 a.It can inhibit MP,reduce inflammatory injury and regulate immune response by indirect regulating the expression levels of miR-155 and miR-146 a.And the low,middle and high dose group and the low,middle and high dose group with azithromycin showed a dosedependent effect.The effect of combined application of traditional Chinese medicine and azithromycin and azithromycin alone was better than that of traditional Chinese medicine group.4.The risk factors associated with children's RMPP(damp-heat closed lung syndrome)include PLT,hs-CRP,GLB,RBP,CKMB,LDH,FIB,D-D.Among them,LDH,FIB,D-D have higher specificity for predicting and diagnosing RMPP(damp-heat closed lung syndrome),which can be used for the predictive diagnosis of RMPP damp-heat closed lung syndrome.5.The mechanism of Qushi tongluo Formula in treating children with Mycoplasma pneumoniae pneumonia is mainly through regulating the cell cycle,inhibiting inflammation,regulating immune function,restoring immune homeostasis,and improving microcirculation to play an anti-pathogenic microorganism. |
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