| Background:Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis(Mtb)and is the second main cause of death caused by a single infectious source and the 13 th leading cause of death globally,and China is one of the countries with a high incidence of tuberculosis,which poses a serious threat to public health security,as well as its prevention,control and treatment is a huge challenge for us.During the treatment of tuberculosis,many patients develop drug resistance to Mtb due to mutations in genes encoding drug targets or drug-activating enzymes,making tuberculosis prevention and control more challenging.Patients with drug-resistant tuberculosis(DR-TB)often have long hospital stays,poor prognosis,high treatment costs,low cure rates,and high mortality rates,placing a heavy burden on their families and society.Recently,the effectiveness of global DR-TB control is not as expected and deviate from WHO’s goal of ending the global tuberculosis epidemic by 2035.Therefore,how to treat DR-TB is the key issues that need to be addressed.Exosomes are vesicles secreted by cells,20-200 nm in diameter,and involved in cellular communication and immune response to kill Mtb by macrophages.Proteins are one of the important components of exosomes,and can be used to unravel the mechanisms of exosome biogenesis,identify the origin cells of exosomes in body fluid samples,and develop biomarkers for disease diagnosis.Proteomics plays an important role in early diagnosis,prognosis determination and progression monitoring of disease.In recent years,with the rapid development of proteomics technologies,Label-free proteome quantification,has been playing an important role in disease diagnosis and therapeutic target identification.However,the role of exosome-mediated protein components in the formation of DR-TB remains unclear.In summary,this study initially elucidated the the drug resistance situation of tuberculosis patients in the region through analysis of the drug resistance status of tuberculosis in Shanxi Province in several aspects,such as comparing the trends in the rates of resistance to different anti-tuberculosis drugs,hospitalization days,and treatment costs among patients with DR-TB and non-drug-resistant tuberculosis(NDR-TB)in Shanxi Province from 2017 to 2022;meanwhile,this study selected exosome proteomics as the entry point,screened the differential proteins in plasma exosomes of DR-TB patients and NDR-TB patients and performed functional analysis to explore the possible pathogenesis of DR-TB and find the early diagnosis markers of DR-TB,which will help to solve the problem of difficult prevention and control and treatment of DR-TB,and provide a new idea and a certain theoretical basis for its prevention and treatment.Objective:(1)To provide a basis for more targeted measures to curb the development of DR-TB by analyzing the drug-resistant status of tuberculosis in Shanxi Province from 2017-2022.(2)To analyze the immunological pathogenesis of DR-TB from the perspective of exosome proteomics and explore potential biological markers for the diagnosis and treatment of DR-TB.Methods:(1)The information of 3091 inpatients with positive Mtb smears who underwent anti-tuberculosis drug susceptibility testing at the Fourth People’s Hospital of Taiyuan,Shanxi Province from 2017-2022 was collected,and information such as age and gender composition,resistance rates to different anti-tuberculosis drugs,hospitalization days,and treatment costs were statistically analyzed(2)Plasma samples were collected from DR-TB patients(n=17)and non-drug-resistant(NDR-TB)patients(n=33)using grouped case-control study design.Exosomes were extracted by differential ultra-high speed centrifugation.After the detection of morphological characteristics,particle size and immunological markers of exosomes,differentially expressed proteins in plasma exosome of DR-TB and NDR-TB patients were screened by Label-free quantitative proteomics.Functional analysis of differentially expressed proteins was performed by GO and KEGG pathway enrichment analysis,then the protein-protein interaction network was mapped.Results:(1)The total drug resistance rate among tuberculosis patients admitted to the Fourth People’s Hospital of Taiyuan,Shanxi Province from 2017 to 2022 showed an overall increasing trend,with only a slight decrease in 2020.(2)The age of DR-TB patients was slightly older compared with that of NDR-TB patients,and the proportion of patients older than 65 years old was the smallest in all age groups;among both DR-TB patients and NDR-TB patients,the proportion of male patients was higher than that of female patients,and the drug resistance rate was higher in male patients than in female patients.(3)The total drug resistance rate of Mtb in Shanxi Province from 2017-2022 was38.9%,and the total multi-drug resistance rate was 13.9%,with isoniazid resistance rate(26.6%)> streptomycin resistance rate(26.5%)> rifampicin resistance rate(14.7%)>ethambutol resistance rate(9.7%)> para-aminosalicylic acid resistance rate(4.8%)>levofloxacin resistance rate(4.7%)> moxifloxacin resistance rate(3.9%)> capreomycin resistance rate(2.7%)> amikacin resistance rate(1.9%)> protionamide resistance rate(1.8%).(4)The rates of streptomycin resistance and rifampicin resistance from 2017 to 2022 had no significant changes,and the resistance rates of other tested drugs significantly changed and basically increased,with a significantly higher resistance rate of second-line anti-tuberculosis drugs from 2021 to 2022.(5)The total treatment cost and average daily treatment cost of DR-TB patients were both higher than those of NDR-TB patients,and there was no significant difference in the number of hospitalization days.(6)Among patients for plasma exosome proteomic analysis,DR-TB patients were older and relatively less educated than NDR-TB patients(7)The exosomes were typical cup-like,20-200 nm,and CD9 and CD63 proteins positive.(8)Compared with the NDR-TB group,16 up-regulated proteins and 10down-regulated proteins were present in the DR-TB group.(9)The up-regulated proteins were mainly involved in the biological processes of fibroblast migration and morphogenesis of an epithelium.The down-regulated proteins were mainly active in phospholipid binding,phosphatidylcholine-sterol O-acyltransferase activator activity and lipase inhibitor activity,and participate in the biological processes of cholesterol efflux,cholesterol metabolic process and lipoprotein biosynthetic process.(10)The up-regulated proteins were mainly enriched in nitrogen metabolism related pathways and the down-regulated proteins were mainly enriched in cholesterol metabolism related pathways.(11)Apolipoproteins family(APOA1,APOB,APOC1)were key proteins in protein-protein interaction network.Conclusion:(1)In the first part of this study,through statistical analysis of the characteristics of age and gender,resistance rates to different anti-tuberculosis drugs,hospitalization days,and treatment costs of inpatients in the Fourth People’s Hospital of Taiyuan,Shanxi Province from 2017-2022,it was found that the drug resistance status of tuberculosis in Shanxi Province has been serious in recent years,with a higher drug resistance rate in male patients and a higher drug resistance rate to second-line anti-tuberculosis drugs.The drug resistance rate to second-line anti-tuberculosis drugs has increased significantly in the past two years,and patients with DR-TB have been faced with a heavier economic burden.(2)In the second part of this study,26 differential proteins in the plasma exosomes of DR-TB and NDR-TB patients were screened by Label-free quantitative proteomics technology,mainly related to cholesterol metabolism,and key proteins such as APOA1,APOB and APOC1 were screened by protein-protein interaction network.It is suggested that DR-TB with current high incidence,may be related to the involvement of exosomes in the pathogenesis of DR-TB by reducing cholesterol efflux,promoting macrophage foaming;altering lipid raft components in cell membrane and affecting drug uptake. |
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