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Rye (Secale cereale), a valuable relative of wheat, contains abundant powdery mildew resistance (Pm) genes. Using physical mapping, transcriptome sequencing, barley stripe mosaic virus-induced gene silencing, ethyl methane sulfonate mutagenesis, and stable transformation, we isolated and validated two coiled-coil, nucleotide-binding site and leucine-rich repeat (CC-NBS-LRR) alleles, PmTR1 and PmTR3, located on rye chromosome 6RS from different triticale lines. PmTR1 confers age-related resistance starting from the three-leaf stage, whereas its allele, PmTR3, confers typical all-stage resistance, which may be associated with their differential gene expression patterns. Overexpression in Nicotiana benthamiana showed that the CC, CC-NBS, and CC-LRR fragments of PMTR1 induce cell death, whereas in PMTR3 the CC and full-length fragments perform this function. Luciferase complementation imaging and pull-down assays revealed distinct interaction activities between the CC and NBS fragments. Our study elucidates two novel rye-derived Pm genes and their derivative germplasm resources and provides novel insights into the mechanism of age-related resistance, which can aid the improvement of resistance against wheat powdery mildew.
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Ascomicetos , Secale , Secale/genética , Resistência à Doença/genética , Triticum/genética , Proteínas de Repetições Ricas em Leucina , Ascomicetos/fisiologia , Nucleotídeos , Cromossomos de Plantas/genética , Sítios de Ligação , Doenças das Plantas/genéticaRESUMO
A highly enantio- and diastereoselective approach has been developed for the synthesis of chiral phosphono dihydropyranones. This approach is enabled by Pd/chiral isothiourea relay catalysis under mild reaction conditions, starting from readily available benzyl bromides, CO, and α-ketophosphonates. The cascade reaction involves the generation of a ketene intermediate from Pd-catalyzed carbonylation of benzyl bromide and subsequent chiral Lewis base catalyzed formal [4 + 2] reaction. Phosphono lactone products can also be transformed to chiral 1,5-diester products in good yield and high stereoselectivity.
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Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease that seriously threatens wheat yield and quality. To control this disease, host resistance is the preferred measure. However, wheat breeding is a complex process with elusive exchange and recombination of the traits from their parents. Increased resistance often leads to a decline in other key traits, such as yield and quality. Developing breakthrough germplasms with harmonious powdery mildew resistance and other key breeding traits is attractive in wheat breeding. In this study, we developed an ideal wheat breeding line AL46 that pyramided its hexaploid triticale parent-derived desirable yield traits and its wheat parent-derived powdery mildew resistance gene Pm2. Sequential genomic in situ hybridization (GISH), multicolor GISH, multicolor fluorescence in situ hybridization, and molecular marker analyses revealed that AL46 was a wheat-rye T1RS·1BL translocation line. Genetic analysis combined with function marker detection and sequence alignment were used to confirm that AL46 carried the Pm2 gene. Then, we evaluated the powdery mildew resistance and comprehensive traits of AL46, and just as we designed, AL46 showed harmonious powdery mildew resistance with some key breeding traits. This study not only developed an ideal wheat germplasm resource but also provided a successful example for pyramiding breeding, which could be a promising direction for wheat improvement in the future.
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Secale , Triticum , Triticum/genética , Hibridização in Situ Fluorescente , Secale/genética , Resistência à Doença/genética , Melhoramento Vegetal , Erysiphe/genéticaRESUMO
Chronic obstructive pulmonary disease (COPD) is a serious threat to human health, but an effective targeted therapy for COPD is still lacking at present. During the progression of COPD, the epithelial mesenchymal transition (EMT) ensures the remodeling of pulmonary epithelial cells, and it could not be precisely targeted due to its complex and elusive mechanism. In this study, we determined that the TLR2/MMP9 axis is upregulated in the pulmonary monocytes in cigarette smoke (CS)-induced COPD mice. Using a co-culture system, we identified that the TLR2/MMP9 axis in pulmonary monocytes promotes the EMT of pulmonary epithelial cells. Further, our results confirmed that miR-144-3p inhibits TLR2 expression in monocytes by directly binding to the 3'UTR of TLR2. Finally, we proved that circRERE works as a sponge to antagonize miR-144-3p and promote TLR2 expression in monocytes. Thus, our results conclude that the circRERE/miR-144-3p/TLR2/MMP9 axis in COPD pulmonary monocytes is critical for CS-induced COPD and circRERE may serve as a potential target for COPD.
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MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Animais , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Metaloproteinase 9 da Matriz , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
BACKGROUND: Plant height (PH), spike length (SL) and spike compactness (SCN) are important agronomic traits in wheat due to their strong correlations with lodging and yield. Thus, dissection of their genetic basis is essential for the improvement of plant architecture and yield potential in wheat breeding. The objective of this study was to map quantitative trait loci (QTL) for PH, SL and SCN in a recombinant inbred line (RIL) population derived from the cross 'PuBing3228 × Gao8901' (PG-RIL) and to evaluate the potential values of these QTL to improve yield. RESULTS: In the current study, Five, six and ten stable QTL for PH, SL, and SCN, respectively, were identified in at least two individual environments. Five major QTL QPh.cas-5A.3, QPh.cas-6A, QSl.cas-6B.2, QScn.cas-2B.2 and QScn.cas-6B explained 5.58-25.68% of the phenotypic variation. Notably, two, three and three novel stable QTL for PH, SL and SCN were identified in this study, which could provide further insights into the genetic factors that shape PH and spike morphology in wheat. Conditional QTL analysis revealed that QTL for SCN were mainly affected by SL. Moreover, a Kompetitive Allele Specific PCR (KASP) marker tightly linked to stable major QTL QPh.cas-5A.3 was developed and verified using the PG-RIL population and a natural population. CONCLUSIONS: Twenty-one stable QTL related to PH, SL, and SCN were identified. These stable QTL and the user-friendly marker KASP8750 will facilitate future studies involving positional cloning and marker-assisted selection in breeding.
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Melhoramento Vegetal , Triticum , Triticum/genética , Mapeamento Cromossômico , Locos de Características Quantitativas/genética , FenótipoRESUMO
Powdery mildew and leaf rust, caused by Blumeria graminis f. sp. tritici and Puccinia triticina, respectively, are widespread diseases of wheat worldwide. The use of resistant cultivars is considered the most economical, environment-friendly, and effective method to control these diseases. In the present study, a collection of 2,978 wheat accessions consisting of 1,394 advanced breeding lines, 1,078 Chinese cultivars, 291 introduced cultivars, 132 lines containing alien chromosomes, and 83 landraces was tested for reactions to powdery mildew and leaf rust. The results indicated that 659 wheat accessions (22.1%) were highly resistant to a widely prevalent B. graminis f. sp. tritici isolate, E09, at the seedling stage, and 390 were consistently resistant to the mixture of B. graminis f. sp. tritici isolates at the adult plant stage. Meanwhile, 63 accessions (2.1%) were highly resistant to leaf rust at the adult plant stage, of which 54 were resistant to a predominant and highly virulent P. triticina race, THTT, at the seedling stage. Notably, 17 accessions were resistant to both powdery mildew and leaf rust. To detect known genes for resistance to powdery mildew and leaf rust, these accessions were tested with gene-specific or tightly linked markers for seven powdery mildew genes (Pm genes; Pm2, Pm4, Pm5, Pm6, Pm8, Pm21, and Pm24) and 10 Lr genes (Lr1, Lr9, Lr10, Lr19, Lr20, Lr24, Lr26, Lr34, Lr37, and Lr46). Of the 659 powdery mildew-resistant accessions, 328 might carry single Pm genes and 191 carry combined Pm genes. Pm2 was detected at the highest frequency of 59.6%, followed by Pm8, Pm6, Pm21, Pm4, and Pm5, whereas Pm24 was not detected. In addition, 139 accessions might contain unknown Pm genes different from those tested in this study. In the 63 accessions resistant to leaf rust, four leaf rust genes (Lr genes; Lr1, Lr10, Lr26, and Lr34) were detected in 41 accessions singly or in combination, whereas six genes (Lr9, Lr19, Lr20, Lr24, Lr37, and Lr46) were not detected. Twenty-two accessions might contain unknown Lr genes different from those tested in this study. This study not only provided important information for rationally distributing resistance genes in wheat breeding programs, but also identified resistant germplasm that might have novel genes to enrich the diversity of resistance sources.
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Basidiomycota , Triticum , Genes de Plantas , Melhoramento Vegetal , Doenças das Plantas/genética , Triticum/genéticaRESUMO
OBJECTIVE: To investigate the relationship between hepatitis B virus (HBV) infection and risk of pancreatic cancer. METHODS: Various English and Chinese language literature databases, including PubMed, Web of Knowledge, Embase, Cochrane Library and the Chinese National Knowledge Infrastructure, were searched for case-control studies comparing rates of HBV infection and pancreatic cancer. The RevMan meta analysis software, version 5.0, was used to perform the meta-analysis of the 6 included studies. RESULTS: Compared with the control group, the pancreatic cancer group had a significantly higher rate of positivity for hepatitis B surface antigen (HBsAg) (8.87% vs.5.86%, odds ratio (OR) =1.24, 95% confidence interval (CI):1.06 to 1.47, P =0.009) and a lower rate of patients never exposed to HBV (defined as HBsAg(-)/hepatitis B core antibody (anti-HBc)(-) (69.4% vs.77.1%, OR =0.68, 95% CI:0.51 to 0.92, P =0.01). There was no significant difference between the two groups in the rate of hepatitis B e antigen positivity (P =0.55). CONCLUSION: HBV-infected patients with HBsAg(+) status are at greater risk of developing pancreatic cancer; however, since most of the research studies evaluated were conducted in Asians, the generalizability of this conclusion is unknown.
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Hepatite B/epidemiologia , Neoplasias Pancreáticas/epidemiologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B , Humanos , Neoplasias Pancreáticas/virologia , Fatores de RiscoRESUMO
A dual rhodium/isothiourea catalytic system was developed for the enantiodivergent transformation of terminal alkynes. Under synergistic rhodium/isothiourea dual catalysis, terminal alkynes can be creatively utilized as precursors for C1-ammonium enolate species, which subsequently participate in [4 + 2] and [2 + 2] annulation reactions with α,ß-unsaturated ketimines or ketones, respectively. A wide range of chiral lactams and lactones were obtained in excellent yields and stereoselectivities (up to >20:1 dr, 98% ee).
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Due to the differences in land cover and natural surroundings within cities, residents in various regions face different thermal risks. Therefore, this study combined multi-source data to analyze the relationship between urban heat risk and local climate zones (LCZ). We found that in downtown Shenyang, the building-type LCZ was mainly found in urban centers, while the natural- type LCZ was mainly found in suburbs. Heat risk was highest in urban centers, gradually decreasing along the suburban direction. The thermal risk indices of the building-type LCZs were significantly higher than those of the natural types. Among the building types of LCZs, LCZ 8 (open middle high-rise) had the highest average thermal risk index (0.48), followed by LCZ 3 (0.46). Among the natural types of LCZs, LCZ E (bare rock and paved) and LCZ F (bare soil and sand) had the highest thermal risk indices, reaching 0.31 and 0.29, respectively. This study evaluated the thermal risk of the Shenyang central urban area from the perspective of LCZs and combined it with high-resolution remote sensing data to provide a reference for thermal risk mitigation in future urban planning.
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Clima , Temperatura Alta , Cidades , Planejamento de Cidades , Monitoramento Ambiental , TemperaturaRESUMO
Urban ventilation corridors introduce fresh air into urban interiors and improve urban livability, while mitigating the urban heat island (UHI) effect. However, few studies have assessed the impact of urban ventilation corridors on UHI intensity (UHII) from the perspective of the local climates of different cities. Therefore, this study integrated multisource data to construct ventilation corridors from the perspective of local climate zone (LCZ) and analyzed its impact on UHII. The results showed the following: (1) the average UHII of constructed LCZs was higher than that of natural LCZs, among which the building type LCZ10 (heavy industry) had the highest intensity (5.77 °C); (2) in extracted ventilation corridors, the pixel number of natural LCZs was substantially larger than that of constructed LCZs, among which LCZE (bare soil/paved) was the largest; and (3) for natural LCZs, the average UHII of each LCZ was lower within the ventilated corridors than within the non-ventilated corridors (except for LCZG [water]), with the UHII of LCZB (scattered trees) exhibiting the greatest mitigation effect. Quantitative research on the composition and function of ventilation corridors can not only assess the ability of ventilation corridors to mitigate UHIs, but also provide a reference for urban ventilation corridor planning.
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Clima , Temperatura Alta , Cidades , Solo , ÁguaRESUMO
Tourism development has influenced industrial structure changes and has become a major driving force for China's new urbanization. However, the development will negatively impact natural resources and the ecological environment and will become an essential driving factor for land use change. Therefore, understanding the impact of tourism urbanization is crucial for sustainable local development. This study selected the Dachangshan Island in the Changhai County, Dalian, China, as the study area, because it is the only coastal island-type border county in China. During the study period, changes in local environmental factors were analyzed based on land use data, Landsat 5 and Landsat 8 data of 2009, 2014, and 2019. The results showed that: (1) the overall land surface temperature (LST) in the research region shows an increasing trend; the LST in 2014 and 2019 increased by 6.10 and 5.94 °C, respectively, compared with 2009. With respect to specific land types, impervious surfaces maintained a high land surface temperature (25.44, 32.38, and 31.86); however, surface temperatures for cropland, forest, grassland, and water bodies remained stable. (2) The land use land cover (LULC) change analysis from 2009-2019 indicates that impervious surfaces and cropland increased by 0.5653 km2 and 0.9941 km2, while the areas of forest, grassland, and water bodies decreased. The results also showed that forests (-1.3703 km2) are most affected by urbanization. (3) The results of the landscape index calculation showed that the variation at the patch scale is different for different LULC types. The patch density of impervious surfaces decreased, but the aggregation index increased over time, while the patch density of the forest increased continuously. At the landscape scale, overall patch type and distribution remained stable. The purpose of this study is to explore the environmental changes of islands and provide a reference for the sustainable development of islands.
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Monitoramento Ambiental , Turismo , Monitoramento Ambiental/métodos , Temperatura , Urbanização , ÁguaRESUMO
In the present study, a new series of different heterocycles was synthesized through base-free Knoevenagel condensation of various aldehydes and active methylene-containing compounds using the hydrothermal developed Ag@TiO2 as a heterogeneous catalyst. The catalyst was synthesized by mixing TiO2 (P25) with AgNO3 and hydrothermally treated in ethanol at 180 °C for 12 h. The developed Ag@TiO2 catalyst was directly applied for Knoevenagel condensation, and the optimized procedure involved stirring the aldehydes and active methylene-containing compounds with Ag@TiO2 in ethanol at 65 °C. The reaction scope was investigated for various aromatic and heterocyclic aldehydes with active methylene-containing compounds, and the isolated yields were significantly high. The reusability of the catalyst was investigated for up to five cycles, where an insignificant decrease in the catalyst's reactivity was observed. Also, the reaction could proceed in water as a solvent, and the isolated yield was 40%. Hence, this protocol features mild reaction conditions, a facile procedure, and clean reaction profiles.
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High urban temperatures affect city livability and may be harmful for inhabitants. Analyzing spatial and temporal differences in surface temperature and the thermal impact of urban morphological heterogeneity can promote strategies to improve the insulation of the urban thermal environment. Therefore, we analyzed the diurnal variation of land surface temperature (LST) and seasonal differences in the Fifth Ring Road area of Beijing from the perspective of the Local Climate Zone (LCZ) using latest ECOSTRESS data. We used ECOSTRESS LST data with a resolution of 70 m to accurately interpret the effects of urban morphology on the local climate. The study area was dominated by the LCZ9 type (sparse low-rise buildings) and natural LCZ types, such as LCZA/B (woodland), LCZD (grassland), and LCZG (water body), mainly including park landscapes. There were significant differences in LST observed in different seasons as well as day and night. During daytime, LST was ranked as follows: summer > spring > autumn > winter. During night-time, it was ranked as follows: summer > autumn > spring > winter. All data indicated that the highest and lowest LST was observed in summer and winter, respectively. LST was consistent with LCZ in terms of spatial distribution. Overall, the LST of each LCZ during daytime was higher than that of night-time during different seasons (except winter), and the average LST of each LCZ during the diurnal period in summer was higher than that of other seasons. The LST of each LCZ during daytime in winter was lower than that of the corresponding night-time, which indicates that it is colder in the daytime during winter. The results presented herein can facilitate improved analysis of spatial and temporal differences in surface temperature in urban areas, leading to the development of strategies aimed at improving livability and public health in cities.
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Monitoramento Ambiental , Água , Cidades , Monitoramento Ambiental/métodos , Estações do Ano , TemperaturaRESUMO
Developing effective and durable host plant resistance is crucial for controlling powdery mildew, a devastating disease caused by Blumeria graminis f. sp. tritici (Bgt). In the present study, we dissected the genetic basis of the adult plant resistance to powdery mildew using a recombinant inbred line (RIL) composed of 176 F9 RILs population derived from a cross between PuBing 3228 (P3228) and susceptible cultivar Gao 8901. P3228 exhibits stable adult-plant resistance to powdery mildew in the field over consecutive years. We identified two QTLs on chromosomes 7DS (QPm.cas-7D) and 1AL (QPm.cas-1A) contributed by P3228, and one QTL on 3DS (QPm.cas-3D) contributed by Gao 8901, which could explain 65.44%, 3.45%, and 2.18% of the phenotypic variances, respectively. By analyzing the annotated genes in the 1.168 Mb physical interval of the major QTL QPm.cas-7D, we locked a previously cloned adult-plant resistance gene Pm38 that was most probably the candidate gene of QPm.cas-7D. Sequence alignment analysis revealed that the candidate gene of QPm.cas-7D in P3228 was identical to the reported Pm38 sequence. Two haplotypes QPm-7D-R and QPm-7D-S were identified in the whole Pm38 genomic regions between P3228 and Gao 8901. To apply QPm.cas-7D in wheat breeding, we developed a kompetitive allele-specific PCR (KASP) marker Kasp5249 that is closely linked with these haplotypes. It is worth mentioning that the QPm-7D-R haplotype significantly decreased TKW and underwent negative selection for higher yields in China wheat breeding. In this study, we identified a major QTL QPm.cas-7D and revealed the relationship between its resistance and yield, which could be beneficial for further applications in wheat disease resistance and high-yield breeding.
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Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive fungal disease of wheat throughout the world. Utilization of effective powdery mildew resistance genes and cultivars is considered as the most economic, efficient, and environmental-friendly method to control this disease. Synthetic hexaploid wheat (SHW), which was developed through hybridization of diploid Aegilops and tetraploid wheat, is a valuable genetic resource for resistance to powdery mildew. SHW line YAV249 showed high levels of resistance to powdery mildew at both the seedling and adult stages. Genetic analysis indicated that the resistance was controlled by a single dominant gene, temporarily designated PmYAV. Bulked segregant analysis with wheat 660K single nucleotide polymorphism (SNP) array scanning and marker analysis showed that PmYAV was located on chromosome 2AL and flanked by markers Xgdm93 and Xwgrc763, respectively, with genetic distances of 0.8 cM and 1.2 cM corresponding to a physic interval of 1.89 Mb on the Chinese Spring reference genome sequence v1.0. Sequence alignment analysis demonstrated that the sequence of PmYAV was consistent with that of Pm4a but generated an extra splicing event. When inoculated with different Bgt isolates, PmYAV showed a significantly different spectrum from Pm4a, hence it might be a new resistant resource for improvement of powdery mildew resistance. The flanked markers GDM93 and WGRC763, and the co-segregated markers BCD1231 and JS717/JS718 were confirmed to be easily performed in marker-assisted selection (MAS) of PmYAV. Using MAS strategy, PmYAV was transferred into the commercial cultivar Kenong 199 (KN199) and a wheat line YK13 was derived at generation BC3F3 from the population of YAV249/4*KN199 due to its excellent agronomic traits and resistance to powdery mildew. In conclusion, an alternative splicing variant of Pm4 was identified in this study, which informed the regulation of Pm4 gene function.
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BACKGROUND: Transarterial chemoembolization (TACE) has the characteristics of minimally invasive, strong repeatability, and good curative effect, so it is commonly used in the nonoperative treatment of hepatocellular carcinoma (HCC). However, infection will occur after TACE, which not only increases the hospitalization time and medical expenses, but also affects the efficacy of TACE treatment. At present, there is a lack of analysis of the risk factors of infection after TACE of patients with HCC. In this study, meta-analysis was used to further explore the risk factors of postoperative infection in patients with HCC after TACE, and to provide strategies for infection prevention and intervention. METHODS: To search the literatures about the influencing factors of post-TACE infection in patients with HCC published from the establishment of PubMed, Embase, The Cochrane Library, Web of Science, China Biology Medicine Database, China National Knowledge Infrastructure, China Science and Technology Journal Database, and WANFANG to April 2021. Screening was carried out according to inclusion criteria and exclusion criteria. A meta-analysis was performed using RevMan 5.3 software. RESULTS: We disseminated the findings of this systematic review and meta-analysis via publications in peer-reviewed journals. CONCLUSION: This study systematically reviewed the existing evidence and determined the incidence and predictors of infection after TACE of patients with HCC. ETHICS AND DISSEMINATION: The private information from individuals will not be published. This systematic review also should not damage participants' rights. Approval from an ethics committee is not required for this study. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/26P5X.
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Carcinoma Hepatocelular/cirurgia , Quimioembolização Terapêutica/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Neoplasias Hepáticas/cirurgia , Infecção da Ferida Cirúrgica/epidemiologia , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Quimioembolização Terapêutica/métodos , Procedimentos Endovasculares/métodos , Humanos , Incidência , Fígado/irrigação sanguínea , Fígado/patologia , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/patologia , Metanálise como Assunto , Medição de Risco/estatística & dados numéricos , Fatores de Risco , Infecção da Ferida Cirúrgica/etiologia , Revisões Sistemáticas como AssuntoRESUMO
BACKGROUND: Tuberculosis is an infectious disease caused by mycobacterium tuberculosis. It may occur in multiple parts and organs of the patients body, and the lung is the most common. It is a major health threat worldwide. Hepatotoxicity is a common adverse reaction of commonly used clinical anti-tuberculosis drugs, as well as one of the important factors leading to poor prognosis of tuberculosis. Milk thistle is a traditional Chinese medicine extract derived from the mature fruit of Silybum marianum. Clinical practice shows that milk thistle has a good anti-liver injury effect and can be used to prevent anti-tuberculosis drug-induced liver injury. However, there is a lack of evidence-based medicine. The research carried out in this protocol is to systematically evaluate the efficacy and safety of milk thistle preventive treatment of anti-tuberculosis drug-induced liver injury, and to improve the evidence-based basis for clinical application of milk thistle in the preventive treatment of anti-tuberculosis drug-induced liver injury. METHOD: Computer search of English databases (PubMed, the Cochrane Library, Embase, Web of Science) and Chinese databases (CNKI, VIP, Wanfang, China Biology Medicine disc (CBMdisc)) was performed. From the establishment of database to October 2020, 2 researchers independently extracted and evaluated the data included in the randomized controlled clinical research of milk thistle preventive treatment of anti-tuberculosis drug-induced liver injury, and used RevMan5.3 software to conduct a meta-analysis of the included literature. RESULT: In this research, the efficacy and safety of milk thistle preventive treatment of anti-tuberculosis drug-induced liver injury were evaluated by indicators such as the incidence of liver injury, bilirubin levels, and liver enzyme levels. CONCLUSION: In this research, reliable evidence-based evidence for the clinical application of milk thistle in the preventive treatment of anti-tuberculosis drug-induced liver injury was provided. OSF REGISTRATION NUMBER: DOI: 10.17605/OSF.IO/VC3RM.