The gut microbiome is an ecosystem that involves complex interactions. Currently, our knowledge about the role of the gut microbiome in health and disease relies mainly on differential microbial abundance, and little is known about the role of microbial interactions in the context of human disease. Here, we construct and compare microbial co-abundance networks using 2,379 metagenomes from four human cohorts: an inflammatory bowel disease (IBD) cohort, an obese cohort and two population-based cohorts. We find that the strengths of 38.6% of species co-abundances and 64.3% of pathway co-abundances vary significantly between cohorts, with 113 species and 1,050 pathway co-abundances showing IBD-specific effects and 281 pathway co-abundances showing obesity-specific effects. We can also replicate these IBD microbial co-abundances in longitudinal data from the IBD cohort of the integrative human microbiome (iHMP-IBD) project. Our study identifies several key species and pathways in IBD and obesity and provides evidence that altered microbial abundances in disease can influence their co-abundance relationship, which expands our current knowledge regarding microbial dysbiosis in disease. Gut microbiome alterations have been linked to inflammatory bowel disease (IBD) and obesity. Here, the authors characterize the metagenomes of four large human cohorts and perform co-abundance network analysis showing that dysbiosis in disease is marked by the altered co-abundance relationships, suggesting that pathway coabundance networks are more heterogeneous than species network. In recent years, associations have been identified between gut microbiome composition and the development of certain human diseases, including diabetes7, cardiovascular disorders9, obesity11 and chronic gastrointestinal disorders like inflammatory bowel disease (IBD)14. Most associations to human diseases have been linked to lower microbial diversity, altered microbial composition and differing abundances of certain microorganisms and pathways19. However, the gut microbiome is an ecosystem in which microbes can exchange or compete for nutrients, signalling molecules or immune-evasion mechanisms through complicated ecological interactions that are far from fully understood22. Enthusiasm has thus been rising to decipher these microbial interactions in order to detect key microbes in health and disease24. One way of doing this is to create co-abundance networks based on correlations, a method that has the potential to study interactions between microbes and thereby generate hypotheses for experimental validation at a later stage24.The human gut harbours a diverse community of microorganisms that interact closely with both the host and each other. Gut microorganisms are involved in digestion and degradation of nutrients, maintenance of digestive tract integrity, stimulation of the host immune system and modulation of the host metabolism29 and applied to infer microbial taxonomic networks in healthy individuals and in individuals with extreme longevity, gestational diabetes, Crohn’s disease and colorectal cancer35. These studies have identified microbial genera that are potentially key in health and disease, e.g. Porphyromonas and Bacteroides in gestational diabetes33. However, these previous studies were either based on 16S rRNA sequencing data, which yields limited information on microbial species and pathways, or carried out in small cohorts34. A further limitation of 16S sequencing is that it can only identify microbial networks up to genus level. As different bacterial species can have very different functional properties, analysis at genus level cannot fully capture the biochemical interactions between microbes. In consequence, the importance of metabolic network construction from metagenomics data has recently been highlighted37.Various network inference tools have been developedn = 496), an obesity cohort , and 2 population-based cohorts  and 500 Functional Genomics ). We compare the microbial taxonomic and functional networks under different host health conditions and identify potential key species and pathways that shape host-associated microbial networks  < 0.05, Fig. Metagenomic data of the 2379 participants from the four cohorts was processed using the same pipeline. Principle coordinate analysis showed that microbial composition and functional profiles are largely overlapped, although we observed a significant shift in species composition in the IBD cohort , as compared to only 3 species and 117 pathway co-abundance relationships specific to the population-based cohorts LLD and 500FG  and 21,882 (59.0%) pathway co-abundance could be replicated at P < 0.05  species co-abundances and 24,165 (65.1%) pathway co-abundances showed no significant difference in their co-abundance strengths between our IBD cohort and the iHMP-IBD cohort  cohort: Of the 2554 species and 37,699 pathway co-abundances established in our IBD cohort, we were able to assess 2090 species co-abundances and 37,106 pathway co-abundances in 77 IBD individuals from the iHMP-IBD14. We therefore hypothesized that this could also be the case for co-abundance relationships. We assessed whether IBD co-abundances (including IBD co-abundances at FDR < 0.05 and IBD-specific co-abundances) could be related to the disease subtypes [ulcerative colitis  vs. Crohn’s disease ], disease location [ileum (n = 212) vs. colon (n = 286)] and disease activity [inflammation (n = 121) vs. no inflammation (n = 377)]  and proton pump inhibitors  was higher in patients with IBD than in the general population cohorts (1.1% and 8.4%) , which is also known as vitamin K2. The co-abundance relationship for this pathway in IBD (r = 0.1) was weaker than in other cohorts (r = 0.3)  cycle I), two vitamin biosynthesis pathways  and an amino acid biosynthesis pathway (PWY-5505: L-glutamate and L-glutamine biosynthesis) Fig. 47. For i.3) Fig. , despites42 Fig. . AccordiQ test P > 0.05, Supplementary Fig. Replication of 300OB network in LLD obese individuals: 1107 species and 37,886 pathway co-abundances were detected in the 300OB cohort Fig. . These eQ test FDR < 0.05, Supplementary Fig. Dorea longicatena and Dorea formicigenerans and negative co-abundances of Lachnospiraceae bacterium 9.1.43BFAA with Coprococcus comes and D. longicatena.Microbial networks in relation to obesity-related diseases: The 300OB cohort was set up to study cardiovascular disease in obese individuals, including 139 patients with atherosclerotic plaque and 159 obese controls  and the biosynthesis pathway of aspartate  and two disease cohorts (IBD and 300OB). We report 3454 species and 43,355 pathway co-abundance relationships that were significant in at least one cohort. Among them, the effect sizes of 38.6% of species co-abundances and 64.3% of pathway co-abundances were significantly different between cohorts. In particular, 113 species co-abundances and 1050 pathway co-abundances showed IBD cohort-specific effects and 281 pathway co-abundances had specific effects in the 300OB cohort. Our study provides evidence that microbial dysbiosis can be reflected in alterations in microbial co-abundance.E. coli and O. formigenes51. A higher abundance of the pathogenic species E. coli has previously been associated with IBD51, likely due to an increased release of oxidized haemoglobin into the intestinal lumen as a result of chronic inflammation of the gastrointestinal walls53. Consistent with this, we replicated high abundances of E. coli and low abundances of anaerobic metabolism pathways in IBD. E. coli also showed strong positive co-abundance with other inflammation-inducing species in IBD, including streptococcus species such as S. mutans49, S. vestibularis41 and S. infantis42. In contrast, these co-abundances were either weak or negative in our population-based and obesity cohorts. We further identified A. graevenitzii as a key species in IBD. Although no evidence supports a direct role for Actinomyces in the pathogenesis of IBD, A. graevenitzii has been associated with coeliac disease in children54 and can induce actinomycosis56, with both conditions sharing similar abdominal pathologies with IBD57. Two case reports have also suggested that Actinomyces may aggravate the intestinal injuries caused by inflammation59.Our study yielded several findings. We identified three species and four pathways in IBD and one pathway in 300OB that served as key players in disease-specific co-abundance networks. Key IBD-associated species included E. coli is known to be an important species for the biosynthesis of menaquinone, a growth-promoting factor for a variety of microorganisms in the gut microbiota48. In line with this, we found that 18.8% of the menaquinone biosynthesis pathway in IBD patients was attributed to E. coli, which is two times higher than in the two population-based cohorts (Wilcoxon test P < 3.0×10−11). Another notable IBD key pathway is the tetrahydrofolate pathway, which is responsible for folic acid derivative biosynthesis and supplementation of folic acid. This pathway has been shown to reduce the risk of colorectal cancer in IBD patients60. Interestingly, previous research has shown that oral intake of L-glutamine attenuates the colitis induced by dextran sulfate sodium in mice61. We identified a negative co-abundance with L-glutamine biosynthesis and biosynthesis of other amino acids like L-isoleucine and L-methionine. Previous research showed that both these amino acids play an important role in the immune system63. L-glutamine has been tested as supplement in patients with IBD but did not show improvements in clinical outcomes like disease activity scores64. Our results show large numbers of connections for L-glutamine with other pathways such as the biosynthesis of other amino acids. These pathways might also be of interest when exploring L-glutamine as an intervention for IBD.The top key functional pathway in IBD was the reductive TCA cycle pathway (P23-PWY), which had 76 IBD-specific co-abundances. Interestingly, the key IBD species D. longicatena, D. formicigenerans, L. bacterium 9.1.43BFAA and C. comes. Notably, D. Longticatena and Lachnospiraceae species have been linked to atherosclerotic cardiovascular disease9.In obesity, we identified the allantoin degradation pathway as a key pathway, showing obesity-specific co-abundance relationship to 85 pathways, mainly negative correlations with biosynthesis of oxaloacetate/aspartate amino acids. These pathways are related to insulin secretion and glucose metabolism. However, their co-abundance relationships did not show significant differences between patients and non-diabetic individuals, which is likely due to a power issue as there were only 35 diabetic patients in 300OB. Instead, we found three species co-abundances related to presence of atheriosclerotic plaque, involving 65, may yield different pictures of microbial community and functional profile, thereby identifying different co-abundance networks. Thus such in silico-based network inferences require further functional validation. Although bacterial genes are believed to be expressed uniformly66, previous studies have also shown that meta-transcription can exert dynamic changes in response to environmental perturbations that cannot be detected at the metagenome level68. Thus, in order to understand the microbial ecosystem in terms of functional interaction in diseases, we need complementary approaches like meta-proteomics and meta-metabolomics that provide a more direct readout of the functional properties of the gut microbiome. Furthermore, the cross-sectional design of this study makes it hard to assess the stability of our findings over time. However, we did observe similar findings for the iHMP-IBD cohort for 98.2% of species co-abundances and 99.4% of pathway co-abundances between two time points spanning 1 year (Cochran-Q test P > 0.05). This implies that co-abundance relationships are largely consistent over time.Altogether, our analyses show that microbial dysbiosis in disease may not be driven solely by differences in abundance level, it may also reflect shifts in microbial interactions that are mirrored in co-abundance analyses. Particularly when applied to metagenomics sequence data, pathway-based co-abundance networks provide further insights into functional dysbiosis in IBD and obesity. However, we also acknowledge several limitations of our study. This is an in silico network analysis based on correlation in bacterial abundance levels. Even with the large sample size, our study is still undersized for making comparisons to the number of interactions assessed. In recent years, many different network tools have been developed to tackle the statistical challenges in inferring networks for compositional data. In this study, we applied two independent methods, SparCC and SpiecEasi, to establish microbial co-abundance networks based on MetaPhlan and HUMAnN2 annotation. Our analysis can thus be biased due to these annotation tools. Other annotation tools, e.g. mcSEEDIn addition, due to our study design, we cannot disentangle cause from consequence. Longitudinal studies are therefore warranted and should be combined with functional validation. Moreover, especially in the context of IBD, which is a heterogeneous disease, we had limited ability to pinpoint co-abundance networks to specific disease characteristics like the subtypes CD and UC. This is probably due to the lack of power to detect this by subgrouping our cohorts. Larger cohorts with well-documented disease characteristics are needed in the future.This study presents the microbial network analysis to examine both microbial species and functional pathways based on metagenomics sequencing. Our data show that dysbiosis of the gut microbial ecosystem in disease can not only be assessed by the altered abundance level but can also be seen at the level of microbial interaction, at least in terms of co-abundances. We have also identified IBD- and obesity-specific species and pathways that potentially play important roles in regulating the microbial ecosystem in disease, and these disease-specific microbial interactions extend our current knowledge about the role of the microbiome in disease.70. In short, the LLD cohort is a large prospective cohort study from the north of the Netherlands71. LLD contains 58.20% females and 41.80% males, the mean age (SD) of participants is 45.04 (13.60) years and their mean BMI is 25.26 (4.18)  years and their mean BMI is 22.70 (2.72)  years and their mean BMI is 30.73 (3.48)  years and their mean BMI is 25.55 (5.17)  cohort consists of patients with IBD recruited at the specialized IBD outpatient clinic of the University Medical Center Groningen in the NetherlandsAll participants signed an informed consent form prior to sample collection. Institutional ethics review board (IRB) approval was available for all four cohorts: the LLD (ref. M12.113965) and the IBD (IRB-number 2008.338) cohorts were approved by the UMCG IRB and the 500FG study  and 300OB (NL46846.091.13) cohorts were approved by the Ethical Committee of Radboud University Nijmegen.75.All participants from the four cohorts were asked to collect faecal samples at home and to place them in their home freezer (−20 °C) within 15 min after production. Subsequently, a nurse visited the participant to pick up the faecal samples on dry ice and transfer them to the laboratory. Aliquots were then made and stored at −80 °C until further processing. The same protocol for faecal DNA isolation and metagenomics sequencing was used for all four cohorts. Faecal DNA isolation was performed using the AllPrep DNA/RNA Mini Kit . After DNA extraction, faecal DNA was sent to the Broad Institute of Harvard and MIT in Cambridge, MA, USA, where library preparation and whole-genome shotgun sequencing were performed on the Illumina HiSeq platform. From the raw metagenomic sequencing data, low-quality reads were discarded by the sequencing facility and reads belonging to the human genome were removed by mapping the data to the human reference genome (version NCBI37) with Bowtie2 (v2.1.0)76, and the relative abundances of metabolic pathways were determined using the HUMAnN2 pipeline (v0.10.0)77, which maps DNA/RNA reads to a customized database of functionally annotated pan-genomes. HUMAnN2 reported the abundances of gene families from the UniProt Reference Clusters78 (UniRef90), which were further mapped to microbial pathways from the MetaCyc metabolic pathway database80. In total, we detected 698 species and 489 pathways present in at least 1 of the 4 cohorts. To deal with sparse microbial data in the network analysis, we focused on species/pathways present in at least 20% of samples in at least one cohort. This provided a confined list of 134 species and 343 pathways for use in the network analysis. Together these accounted for, on average, 86.9% and 99.9% of taxonomic and functional compositions, respectively.The relative abundance of gut microbial taxonomic units was determined using MetaPhlan (v2.7.2)84. In particular, the problem can be more serious in a microbial community with low compositionality85. We therefore first assessed the inverse Simpson index of microbial composition for the effective number of species (neff). Our analysis showed high compositionality in both functional pathway composition  and species composition . Following the suggestion of Weiss et al.85, based on their assessment of the performance of eight different methods , we decided to use the SparCC method because it has been proven to be able to infer linear relationships with high precision for high diversity compositions with neff < 13. Species composition data from MetaPhlan was converted to predicted read counts by multiplying relative abundances by the total sequence counts and then subjected to a Python-based SparCC tool29. For pathway analysis, the read counts from HUMAnN2 were directly used for SparCC. Significant co-abundance was controlled at FDR 0.05 level using 100× permutation. In each permutation, the abundance of each microbial factor was randomly shuffled across samples. To reduce indirect associations, we further applied SpiecEasi (v1.0.6), which infers the microbial network underlying graphical model using the concept of conditional independence38. In this way, we obtained 3454 species and 43,355 pathway co-abundances that were detectable by both methods Co-occurrence network inference: presence and absence of each bacterial species and metabolic pathway were treated as binary traits. The pair-wise co-occurrence relationship between two microbial factors (species or pathway) in each cohort was assessed using Pearson’s chi-squared test. If the number of co-occurrence pairs was greater than the number of co-exclusion pairs, the two microbial factors were considered to be a co-occurrence. If the number of co-occurrence pairs was less than the number co-exclusion pairs, the two factors were considered to be a co-exclusion. Permutation (100×) was conducted to determine significance at an FDR < 0.05. In each permutation, the presence and absence of each microbial factor was randomly shuffled across samples. At the species level, we detected 6015 co-occurrence relationships that were found in at least one cohort, with 3423 found in LLD, 1845 in 500FG, 1199 in 300OB and 4701 in IBD (Supplementary Data Q tests to assess the heterogeneity of effect sizes and directions across the four cohorts for each co-abundance (correlation coefficient generated by SparCC) and co-occurrence (odds ratio). Here we treated each cohort as one study and conducted the Cochran-Q test using the metagen function from the package meta (v4.9.5) in R, which calculates the squared difference between individual study effects and the pooled effect using inverse variance weighting86. For each co-abundance, the P values from the Cochran-Q test were recorded, and co-abundances with significant heterogeneity were controlled at the FDR 0.05 level determined by permutation (100×). In this case, all samples from the four cohorts were randomly shuffled across cohorts, i.e. shuffling the cohort labels but keeping the correlation structure of species and pathways intact. Co-abundances with a Cochran-Q test FDR < 0.05 were considered heterogeneous, while co-abundances with Cochran-Q test P > 0.05 were considered stable. We also summarized species co-abundance co-abundances based on microbial genus and pathway co-abundance co-abundances based on metabolic category.Network heterogeneity analysis: To assess the variability of networks among the four cohorts, we conducted Cochran-Q test FDR < 0.05), we further assessed whether these relationships showed cohort specificity, i.e. whether the effect size of co-abundance/co-occurrence in one cohort was very different from that in the other three. In this analysis, effect size for co-abundance was the SparCC correlation coefficient and the odds ratio for co-occurrence. We adopted interquartile ranges (IQRs) based the outlier detection method . IQR was then calculated, and we assessed whether the smallest or largest effect size fell outside of Q1 − 2 × IQR or Q3 + 2 × IQR. If only one met the condition, we called this co-abundance specific and assigned it to the corresponding cohort . This insured that the partial correlation determined between A and B was independent of the covariant C. To assess the impact, we compared the correlation coefficient between SparCC correlation and partial correlation for all co-abundances and found comparable effect size  and 99% of the pathways (340 out of 343) found in our IBD cohort were also detected in the first sample collection in iHMP-IBD. The differences in co-abundance strength between the IBD cohort and the iHMP-IBD cohort were assessed using Cochran-Q test. A significant P > 0.05 was applied to define replicable co-abundances. We also investigated the stability of microbial networks in iHMP-IBD by comparing the microbial co-abundances in the first and last sample collection from the same participants using the same approach.To replicate microbial networks in IBD, we used data from 77 IBD patients from the iHMP-IBD as a replication cohortQ test heterogeneity between the discovery and replication cohorts was not significant at P > 0.05.To replicate microbial networks in 300OB, we selected 134 obese individuals from the LLD cohort with matched age and BMI. Here we considered a co-abundance to be replicable if the Cochran-72 and 35 were diabetic. To assess the co-abundance related to disease sub-phenotypes, we split the cohorts based on disease subtypes or medication use and inferred microbial co-abundance using SparCC. Cochran-Q test was applied to assess the differential microbial co-abundances at FDR < 0.05.Patients in the IBD and 300OB cohorts have different disease subtypes, and both cohorts had higher proportions of drug users than our population cohorts. In particular, the IBD cohort contained 276 patients with CD and 189 with UC. Within the IBD cohort, 126 patients took PPIs and 97 took antibiotics. In the 300OB cohort, 53.4% (159 out of 298) had an atherosclerotic plaque detected by ultrasound77, we further looked into the contribution of species to each pathway and reported the top contributor (species). To show the functional relationship between species and pathways , we also checked the correlation (Spearman) between microbial species and pathway abundance after adjusting for age, sex and read depth using a linear regression model89. FDR was further calculated based on 100× permutation.Since the pathway abundances reported by HUMAnN2 are computed at both community and individual species level90 in R. For species networks, species belonging to the same genus were clustered together. For pathway networks, pathways from the same metabolic category were presented in a sub-circle, and categories with a limited number of pathways (<4) were grouped into the other category. Classification of pathways was based on the MetaCyc metabolic pathway database80.Cohort-specific networks based on cohort-specific co-abundances were visualized using a circle plot or heatmap with hierarchical clustering analysis (ward.D clustering based on Minkowski distance). Both species and pathways networks were visualized using the package igraph (v1.2.4.1)Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Supplementary Data 15Supplementary Data 16Reporting Summary


To explore the oxidative stress mechanism of modified Buyang Huanwu decoction (MBHD) in intervention of vascular dementia (VD) based on systems biology strategy.  In this study, through the reverse virtual target prediction technology and transcriptomics integration strategy, the active ingredients and potential targets of MBHD treatment of VD were analyzed, and the drug-disease protein-protein interaction (PPI) network was constructed. Then, bioinformatics analysis methods are used for Gene Ontology (GO) enrichment analysis and pathway enrichment analysis, and finally find the core biological process. After that, in animal models, low-throughput technology is used to detect gene expression and protein expression of key molecular targets in oxidative stress-mediated inflammation and apoptosis signaling pathways to verify the mechanism of MBHD treatment of VD rats. Finally, the potential interaction relationship between MBHD and VD-related molecules is further explored through molecular docking technology. κB signaling pathway-mediated inflammation, oxidative stress, angiogenesis, etc. Among them, SIRT1, NF-κB, BAX, BCL-2, CASP3, and APP may be important targets for MBHD to treat VD. Low-throughput technology  detects oxidative stress-mediated inflammation and apoptosis-related signaling pathway molecules. The molecular docking results showed that 64474-51-7, cycloartenol, ferulic acid, formononetin, kaempferol, liquiritigenin, senkyunone, wallichilide, xanthinin, and other molecules can directly interact with NF-κB p65, BAX, BCL-2, and CASP3.  There are a total of 54 MBHD components, 252 potential targets, and 360 VD genes. The results of GO enrichment analysis and pathway enrichment analysis showed that MBHD may regulate neuronal apoptosis, nitric oxide synthesis and metabolism, platelet activation, NF- The active compounds of MBHD interact with multiple targets and multiple pathways in a synergistic manner, and have important therapeutic effects on VD mainly by balancing oxidative stress/anti-inflammatory and antiapoptotic, enhancing metabolism, and enhancing the immune system. Hedysarum multijugum Maxim. (Huang Qi), Angelicae sinensis Radix [Dang Gui or Angelica sinensis (Oliv.) Diels], Pheretima Aspergillum (E. Perrier) (Di Long or Pheretima), Chuanxiong Rhizoma (Chuan Xiong or Ligusticum striatum DC.), Acoritataninowii Rhizoma , Panax notoginseng (Burk.) F. H. Chen ex C. Chow (San Qi), and Polygalae Radix  with a ratio of 120 : 15 : 15 : 10 : 10 : 9 : 10 [Vascular dementia (VD) is a cognitive impairment syndrome caused by a series of cerebrovascular factors, which cause blood circulation disorders and impaired brain function . The res: 9 : 10 . Clinica: 9 : 10 , and its: 9 : 10 . The pro: 9 : 10 –14. HoweCurrently, network pharmacology is considered as a new model for drug development , 16. Thihttp://lsp.nwu.edu.cn/tcmsp.php) [http://tcm.cmu.edu.tw/zh-tw/) [http://www.megabionet. org/tcmid/) [The compound information of MBHD comes from TCMSP (msp.php) , TCM@Tai/zh-tw/) , and TCM/tcmid/) . The TCM/tcmid/) . The sta/tcmid/) –27.https://pubchem.ncbi.nlm.nih.gov/) to obtain their SMILES formula. Those SMILES formulas of the components were input into the SwissTargetPrediction Database (http://www.swisstargetprediction.ch/) to predict the potential targets of each component [http://omim.org/) [http://www.genecards.org) [The predicted compounds are standardized by PubChem (omponent . The OMIim.org/)  and Generds.org)  were utihttps://string-db.org/) searches and predicts gene/protein interactions based on biological databases and web resources [https://david-d.ncifcrf.gov) for Gene Ontology (GO) enrichment analysis and pathway enrichment analysis.In molecular biology, the interactive gene/protein search tool STRING (esources . Proteinesources . In thisesources . In thisOne hundred and twenty (120) male specific pathogen-free (SPF) grade SD rats were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.  2013-0004), weighing 250-300 g and raised in the Experimental Animal Center of Hunan University of Chinese Medicine. The breeding environment is at room temperature (24 ± 1) °C, relative humidity (50 ± 5)%, and 12 h/12 h alternate day and night. The rats started the experiment after 2 weeks of adaptive feeding. The entire experimental procedure was approved by the ethics committee of Hunan University of Chinese Medicine (HCM-00150023).Hedysarum multijugum Maxim. , Angelicae sinensis Radix (Dang Gui or Angelica sinensis (Oliv.) Diels; specimen number: 201504212), Pheretima Aspergillum (E.Perrier) , Chuanxiong Rhizoma , Acoritataninowii Rhizoma , Panax notoginseng (Burk.) F. H. Chen ex C. Chow , Polygalae Radix  with a ratio of 120 : 15 : 15 : 10 : 10 : 9 : 10. All herbs are certified by Associate Professor Liu Lin of Hunan University of Chinese Medicine to be authentic products specified in the 2015 edition of Pharmacopoeia of the People's Republic of China. The equivalent dose of MBHD rats is 17.01 g/kg through the conversion of the human/mouse coefficient. Oxiracetam capsules were purchased from CSPC. The clinical equivalent dose for rats is 0.216 g/kg.Modified Buyang Huanwu decoction (MBHD) consists of The herbs of MBHD were mixed, soaked in 5 times the volume of distilled water for 2 hours, and boiled for 0.5 hours on strong fire and 1 hour on slow fire. Then, the filtrate was collected, and the filtrate was extracted again with 3 times the volume of distilled water according to the above steps, and the filtrate was mixed twice and concentrated by a rotary evaporator. The final drug concentration was 5.1 g of crude drug/ml.κB which inhibits the protein subunit (IκBα), and NF-κB p65 were purchased from Proteintech . Ordinary agarose was purchased from Biomiga, Inc.; TRIzol reagent was purchased from Sigma, Inc.; mRNA reverse transcription kit and SYBR Premix Ex Taq were purchased from Takara, Inc.; BSA was purchased from Huami, Inc.; PVDF membrane was purchased from Amersham, Inc.; HRP-coupled goat anti-rabbit IgG was purchased from Sigma, Inc.; PV-9000 two-step detection kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; reverse transcription cDNA kit and SYBR dye were purchased from Takara, Inc. The primers were designed and synthesized by Shanghai Shenggong Biology Co., Ltd. according to the nucleic acid sequence searched by NCBI.Rabbit anti-rat polyclonal antibodies SIRT1, nuclear factor MT-200 Morris water maze was purchased from Chengdu Taimeng Technology Co., Ltd.; Motic B5 image analysis system was purchased from McAudi Industrial Group; PCR amplification machine (2400 PCR system) was purchased from PerkinElmer, Inc.; Eppendorf desktop low-temperature microcentrifuge was purchased from Eppendorf, Inc.; horizontal electrophoresis was purchased from Bio-Rad, Inc.; Gel Doc1000 gel imaging analysis system was purchased from Bio-Rad, Inc.; Mixer Gentus was purchased from MicroBio, Inc.; J2-21 high-speed centrifuge, J6-HC centrifuge GS-15R, and high-speed desktop centrifuge were purchased from Beckman, Inc.; clean bench was purchased from Suzhou Purification Equipment Co., Ltd.; automatic plate washer was purchased from Beijing Pulang New Technology Co., Ltd.; microscopic imaging system of Mike Audi was purchased from Motic, Inc.; CFX96 real-time PCR system was purchased from Bio-Rad, Inc.; RE-5002 rotary evaporator was purchased from Ride Instruments Co., Ltd.; SMART 3.0 Small Animal Behavior Video Collection and Analysis System was purchased from Panlab, Inc.Twenty rats were randomly selected as the sham operation group by only freeing bilateral common carotid arteries without ligation. The remaining 100 rats underwent bilateral common carotid artery ligation (2-VO) method to replicate the VD rat model according to Reference . The MorRats in MBHD high-, medium-, and low-dose groups were intragastrically administered with MBHD 17.0, 34.0, and 51.0 g/kg, respectively. Rats in the positive control group were intragastrically administered with oxiracetam 0.216 g/kg. The remaining 3 groups were given equal volume of distilled water. The drug intervention lasted 30 days.The learning and memory ability of rats was tested by Morris water maze. The rats were trained for 5 consecutive days. On the 6th day, the water platform was removed, the rats were put into the water from 4 quadrants, and the Motic B5 image analysis system was used to record the number of times the rats crossed the platform in 120 s.HE staining was used to detect histopathological changes in the hippocampus of rats. The hippocampal tissue samples of each group of rats were fixed with 4% paraformaldehyde and dehydrated by ethanol gradient, sliced, stained, and finally observed under a microscope.The freshly collected rat hippocampus tissue was placed in a 1.5 ml EP tube, wrapped with gauze, quickly frozen in liquid nitrogen at −70°C, placed in a sufficient amount of dry ice, and sent to Beijing Boao Jingdian Biotechnology Co., Ltd. for mRNA extraction, purification, and detection. The qualified RNA was detected by Agilent mRNA chip hybridization, and image scanning and data analysis were performed to obtain the differential expression information of mRNAs between the two groups.The total RNA of the rat hippocampus was extracted by the TRIzol method. Total RNA is reverse transcribed into cDNA according to the instructions of the reverse transcription cDNA kit. The qRT-PCR analysis was performed using the CFX96 real-time PCR system according to the method described in the kit instructions, and the specific primers are shown in After the rat hippocampus tissue was fixed with 4% paraformaldehyde, section deparaffinized, 3% hydrogen peroxide treatment, antigen restoration, etc., the primary antibody (1 : 200) was added and placed overnight at 4°C, and then, HRP-IgG secondary antibody (1 : 400) was added. Finally, after operations such as color development, dehydration, and mounting, 5 fields of view were randomly selected under a light microscope, and the average gray value of the Motic image analysis system was used to determine the average gray value. The smaller the gray value, the higher the positive protein expression.μl RIPA lysis buffer and 4 μl PMSF. The tissue was ground and centrifuged under low temperature conditions, and the supernatant was collected. After collecting the protein, the protein concentration was measured with a microplate reader, and the protein concentration was calculated according to the standard curve. Then, SDS-PAGE electrophoresis, film transfer, and other operations were carried out. After ECL color development, observation and photographing are carried out in the gel imaging system. The image analysis software IPP6.0 is used to analyze the integral optical density value of the protein.100 mg of tissue was added to 200 MBHD sample: MBHD 6.94 ml was adjusted to 10.0 ml with water. Standard sample: ferulic acid standard 9.4 mg was placed in a 100 ml volumetric flask and dissolved with methanol-1% glacial acetic acid (1 : 1) and diluted to the mark. Negative control solution: after Chuanxiong Rhizoma in MBHD was removed, other herbs were prepared as a negative control solution according to the MBHD preparation method.μm). Mobile phase is methanol-1% glacial acetic acid (36 : 64). Column temperature is 25°C. Flow rate is 1 ml/min. Detection wavelength is 322 nm.Chromatography column is Hypersi1 C18 : y = 3.782x − 4.4 and the linear range: 18.8 ng-188 ng. Ferulic acid: tR = 9.40 ± 0.12 min, RSD = 1.4%(n = 8), peak area precision RSD = 2.0%(n = 4)  .x ± s). The sample is tested for the homogeneity of variance first. When the variance is uniform, the one-way ANOVA test is used for analysis, and the LSD method is used for multiple comparisons between groups. When the variance is not uniform, the nonparametric rank sum test is used for analysis. The Kruskal-Wallis H test is used to compare the total difference, and then, the Mann–Whitney U test is used to compare the two groups.Statistical analysis was performed using SPSS 17.0. Measurement data are expressed as the mean ± standard deviation (http://scifinder.cas.org), and the structure of the compound was reconstructed in ChemBioDraw strictly according to the structure on PubChem. These compounds are then saved in the “mol2” file format and their energy is minimized. The PDB database (https://www.rcsb.org/) is used to retrieve the 3D structure of SIRT1, NF-κB p65 (RELA), BAX, BCL-2, and CASP3, and download the file in the “pdb” format [The molecular structure of each compound was searched in PubChem  of 5 termined .μl each, and the content of ferulic acid was determined according to the above chromatographic conditions  was determined according to the external standard method  and 808 edges. In this network, nodes closing to the center show more interactions with compounds than peripheral nodes. This indicates that many targets are regulated by multiple compounds, but some can be modulated by only one compound  .The MBHD potential targets, VD genes, and their PPI data were input into Cytoscape to construct MBHD-VD PPI network. This network is composed of 512 nodes  and 8468 edges . The tarThe MBHD-VD PPI network was analyzed by MCODE, and fourteen clusters were obtained . The tarκB signaling pathway-mediated inflammation, oxidative stress, and angiogenesis. Cluster 2 is related to vasoconstriction, cell proliferation, and synaptic transmission. Cluster 3 is related to the differentiation of foam cells and the anabolism of cholesterol. Cluster 4 is related to cell proliferation, platelet activation, and vasodilation. Cluster 5 is related to synaptic transmission, triglyceride, and other lipid metabolisms. Clusters 6, 8, 9, and 14 are related to redox. Cluster 10 is related to apoptosis, platelet degranulation, neuronal apoptosis, synaptic transmission, and coagulation. Cluster 11 is related to apoptosis and nerve cell migration. Clusters 7, 12, and 13 failed to return any VD-related biological processes. The biological processes of cluster 1 are shown in Cluster 1 is related to neuronal apoptosis, nitric oxide synthesis and metabolism, platelet activation, NF-The targets and genes in MBHD-VD PPI network were input into DAVID to perform pathway enrichment analysis and return fourteen core VP-related pathways . The topHedysarum multijugum Maxim. totally regulate 29 targets (which is the most), while Chuanxiong Rhizoma regulate 28 targets. Earthworm Lumbricus regulate 16 targets; Panax notoginseng (Burk.) F. H. Chen ex C regulate 10 targets; Polygala tenuifolia control 18 targets; Acoritataninowii Rhizoma control 10 targets; Angelicae sinensis Radix regulate 12 targets. This once again suggests that Hedysarum multijugum Maxim. plays a major role in BHD intervening VP.In this network, κB pathways [κB signaling and enhance anti-inflammatory Nrf-2/HO-1 signaling, thereby improving cognitive function [κB and MAPK pathways [α pathway, thereby reducing OGD/R-induced damage [Hedysarum multijugum Maxim., astragaloside IV has a variety of pharmacological effects, including anti-inflammatory, antifibrosis, antioxidative stress, antiasthma, antidiabetic, and immune regulation [κB signaling pathway, signaling pathway Nrf2 antioxidant signaling pathway, PI3K/Akt/mTOR signaling pathway, and many other signaling pathways [TCM network pharmacology is based on computer network modeling analysis and target prediction. It analyzes the mechanism of TCM at the system level and reflects its overall thinking . In recepathways . Formonopathways . It may function . Calycospathways . In an id damage . As the gulation . The sigpathways .κB signaling pathway-mediated inflammation, oxidative stress, angiogenesis, etc. Current studies have shown that the pathogenesis of VD mainly includes (1) hypoperfusion and hypoxia caused by microvascular circulatory disorders, (2) increased blood-brain barrier permeability caused by endothelial dysfunction, (3) inflammation and oxidative stress damage the nutritional interaction between neurovascular unit cells, (4) ROS and inflammation inhibit the survival of neurons (neuron apoptosis), and (5) the destruction and demyelination of myelin tablets caused by the oxidative and proinflammatory environment caused by cerebral ischemia and hypoperfusion and the breakdown of the blood-brain barrier [Meanwhile, the further research  showed that the mechanism of MBHD to treat VD is mainly related to neuronal apoptosis, nitric oxide synthesis and metabolism, platelet activation, NF-includes  hypoperf barrier , 50.The normal group and sham operation group: Before the operation, the rats had bright hair, red lips, bright red eyes, vigorous spirit, frequent fighting, and strong appetite. Within 3 days after the operation, the spirits were sluggish, less active, and appetite decreased, but gradually recovered, the mental state became better, the movement was agile, the voluntary activities increased, and the hair, lips, nails, eyeballs, appetite, etc. recovered to a good state.The model group: Before modeling, the experimental rats' hair, lips, nails, eyeballs, spirit, and appetite were generally in good condition. After the model was created, various degrees of lethargy, slow response, slow movement, dull fur, dark purple lip nails, increased sleep, and poor appetite appeared. Some rats have different degrees of drooping eyelids, diminished eye fissures, and slight sunken eyeballs; a small number of rats are irritable, more active, irritable, have unstable walking, and turning in circles. Moreover, the self-cleaning ability of rats is reduced, and the hair becomes sparse, dry, and erected or lacks gloss, and occasionally, there are transient paroxysmal convulsions in rats. In some cases, the abdomen gradually swelled into a spherical shape, accompanied by obvious reduction in eating and defecation, rough and dull coat color, gradual weight loss of limbs, and death.The treatment groups: In the early stage after the successful modeling of the rat, the general condition and behavior of the rat are similar to the VD model group. After a period of drug administration, compared with the model group, the diet, spirit, and activity gradually recovered, and the general situation was better than that of the model group.P > 0.05). There was a significant difference in the escape latency between the model group and the sham operation group at the same time point (P < 0.05), indicating that the model was successful. Compared with the model group, the escape latency in the positive group and the MBHD high- and medium-dose groups at the same time point has significant differences (P < 0.05), suggesting that MBHD and oxiracetam can improve learning and memory in VD ratsPositioning navigation experiment: the escape latency of rats in each group was shortened with the extension of training time. There was no significant difference between the blank group and the sham operation group in the escape latency at the same time point (P < 0.05). Compared with the model group, the number of times the rats crossed the platform was significantly increased (P < 0.05) in the oxiracetam group and the MBHD high- and medium-dose groups, suggesting that the memory ability of the rats was improved after drug intervention , and inflammatory cell infiltration increased. Compared with the model group, the hippocampal neuron pyramidal cells of the oxiracetam group and the MBHD high-, medium-, and low-dose groups were arranged more neatly, the outline was clear, and the inflammatory cell infiltration was significantly reduced .Agilent mRNA was used to detect the mRNA expression profile of rats in the MBHD group and model group. The sample similarity is reflected by sample principal component analysis (PCA). The PCA results show that the overall variance contribution rate is 51.17%.The data of the drug intervention group is relatively clustered (except GZ18), and the data of the model group is relatively divergent. Through the analysis of the two sets of gene chip data, a total of 469 differentially expressed mRNAs were screened, of which 180 were upregulated and 289 were downregulated. Among them, the most upregulated was Slc39a12 (FC = 3.874), and the most downregulated was Liph (FC = 5.986). The differentially expressed mRNA volcano map is shown in The top 20 upregulated and downregulated mRNAs were input into STRING to obtain their PPI data and construct a PPI network , and theκB p65, and IκBα protein was brown or tan. The results showed that the positive expression of SIRT1 and IκBα in the hippocampus of the model group was decreased, and the positive expression of NF-κB p56 was significantly increased (P < 0.05). After drug intervention, the positive expression of SIRT1 and IκBα increased, and the positive expression of NF-κB p56 decreased significantly (P < 0.05) . The Bcl-2-positive cells expressed in the hippocampus of VD rats were significantly decreased (P < 0.05), while the Bax- and caspase-3-positive cells were significantly increased (P < 0.05). After drug intervention, Bcl-2-positive cells increased significantly (P < 0.05); Bax- and caspase-3-positive cells decreased significantly (P < 0.05) , while the level of IκBα mRNA was significantly increased (P < 0.05). After drug intervention, the levels of SIRT1 and IκBα mRNA in the hippocampus increased significantly (P < 0.05), and the level of NF-κB mRNA decreased significantly (P < 0.05)  .P < 0.05). After drug intervention, the levels of SIRT1 and Bcl-2 mRNA expressed in the hippocampus were significantly increased, and the levels of Bax and caspase-3 mRNA were downregulated (P < 0.05)  .κB, and IκBα in the model group (P < 0.05), further suggesting the success of the model. After drug intervention, SIRT1 and IκBα protein levels increased significantly (P < 0.05), and NF-κB protein levels decreased significantly (P < 0.05) . After drug intervention, the level of Bcl-2 protein expressed in the hippocampus increased significantly, and the protein levels of Bax and caspase-3 decreased significantly (P < 0.05) , BAX, BCL-2, and CASP3 molecules. It is generally believed that when the conformation of ligand and receptor is stable, the lower the energy, the greater the possibility of interaction between ligand and receptor. The compounds in the compound-compound target network of MBHD are arranged in descending order of degree, and the top compound of each herb is randomly selected. Finally, 15 compounds  were randomly selected for molecular docking with SIRT1, RELA, BAX, BCL2, and CASP3 , the inflammatory factors bind to cytokine receptors, and IκBα is phosphorylated and then degraded by the ubiquitin-dependent proteasome to promote the transfer of NF-κB into the nucleus. This activates the transcription of a series of cytokines and chemokines, which leads to inflammation [κB requires posttranscriptional modifications such as methylation, acetylation, and phosphorylation. In this process, RelA/p65 is a subunit of NF-κB, and its deacetylation effect needs special attention. Studies have reported that SIRT1 has a deacetylation effect on p65. SIRT1 inhibits the transcriptional activity of NF-κB and the expression of proinflammatory cytokines, which can be achieved by deacetylation of p65 by overexpression or the activator resveratrol [κB in the hippocampus of VD mice [κB subunit RelA/p65, thereby inhibiting its transcriptional activity, reducing the production of inflammatory factors, and preventing damage caused by inflammation [κBα protein in the hippocampus of rats were significantly reduced, while the level of NF-κB protein was significantly increased. After drug treatment, the level of SIRT1 and IκBα protein in the hippocampus increased significantly, and the level of NF-κB protein decreased significantly. This suggests that MBHD may have a certain ameliorating effect on neuronal inflammation in VD rats through the SIRT1/NF-κB p65 inflammatory pathway.Recent studies have found that the SIRT1/NF-diseases . Patholodiseases . NF-κB mytokines . IκBα isveratrol . SIRT1 cveratrol . Wei et  VD mice . Studiesammation , 69. TheIn addition, the apoptosis signal pathway mediated by SIRT1 participates in the programmed cell death of neurons in VD rats and damages the function of neural units. Current research shows that cell necrosis and apoptosis coexist in cerebral infarction lesions after hypoxia-ischemia. The central area of ischemia mainly exists in the form of cell necrosis, and apoptosis is a major way of cell death in the ischemic penumbra around the infarct . The funκB p65 signaling pathway and then improve the learning and memory abilities of VD rats.In summary, MBHD may protect hippocampal neurons by regulating the SIRT1/NF-The active compounds of MBHD interact with multiple targets and multiple pathways in a synergistic manner, and have important therapeutic effects on VD mainly by balancing oxidative stress/anti-inflammatory and antiapoptotic, enhancing metabolism, and enhancing the immune system.


Yuzhi Zhixue Granule (YZG) is a traditional Chinese patent medicine for treating excessive menstrual flow caused by ovulatory dysfunctional uterine bleeding (ODUB) accompanied by heat syndrome. However, the underlying molecular mechanisms, potential targets, and active ingredients of this prescription are still unknown. Therefore, it is imperative to explore the molecular mechanism of YZG.The active compounds in YZG were screened by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The putative targets of YZG were collected via TCMSP and Search Tool for Interacting Chemicals (STITCH) databases. The Therapeutic Target Database (TTD) and Pharmacogenomics Knowledgebase (PharmGKB) databases were used to identify the therapeutic targets of ODUB. A protein–protein interaction (PPI) network containing both the putative targets of YZG and known therapeutic targets of ODUB was built. Furthermore, bioinformatics resources from the database for annotation, visualization and integrated discovery (DAVID) were utilized for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, molecular docking was performed to verify the binding effect between the YZG screened compounds and potential therapeutic target molecules.The study employed a network pharmacology method, mainly containing target prediction, network construction, functional enrichment analysis, and molecular docking to systematically research the mechanisms of YZG in treating ODUB. The putative targets of YZG that treat ODUB mainly involved PTGS1, PTGS2, ALOX5, CASP3, LTA4H, F7 and F10. The functional enrichment analysis suggested that the produced therapeutic effect of YZG against ODUB is mediated by synergistical regulation of several biological pathways, including apoptosis arachidonic acid (AA) metabolism, serotonergic synapse, complement and coagulation cascades and C-type lectin receptor signaling pathways. Molecular docking simulation revealed good binding affinity of the seven putative targets with the corresponding compounds.This novel and scientific network pharmacology-based study holistically elucidated the basic pharmacological effects and the underlying mechanisms of YZG in the treatment of ODUB. Ovulatory dysfunctional uterine bleeding (ODUB), mainly occurring in women of childbearing age, is a common gynecological disease worldwide. Clinically, patients often have symptoms that include abnormal increases in menstrual flow, prolonged menstruation, dull pain in the lower abdomen, and a dark brown menstrual blood color. Clinically, the main causes of ODUB are luteal dysplasia and luteal atrophy, which lead to irregular shedding of the endometrium and prolonged menstruation. Sometimes, patients may have continuous bleeding before or after menstruation, and large menstrual volume –4. ODUB Traditional Chinese medicine (TCM) has been widely used for the treatment and prevention of various diseases, especially in Asian countries, including China, Japan, and South Korea . In TCM,Sanguisorba officinalis L.(Diyu), Gardenia jasminoides Ellis (Zhizi), Cirsium japonicum Fisch.ex DC. (Daji), Sophora japonica L.(Huaihua), Rehmannia glutinosa Libosch.(Dihuang), Paeonia suffruticosa Andr. (Mudanpi), Rubia cordifolia L.(Qiancao), Polygonum bistorta L.(Quanshen), Scutellaria baicalensis Georgi (Huangqin), Paeonia lactiflora Pall. (Baishao), and Angelicasinensis(Oliv.) Diels (Danggui), which have the functions of clearing heat, cooling blood, and stopping bleeding. Its main traditional function is to treat ODUB caused by heavy menstruation and syndromes belonging to blood fever in TCM. Patients can be accompanied by dry mouth and upset, red tongue and yellow skin. In the YZG formula, Diyu is used for cooling blood and hemostasis, and Zhizi can cool blood, clear away the heat syndrome and agitation. Diyu and Zhizi are concerned as the “Monarch” medicine in the prescription. Daji, Platycladus orientalis(L.)Franco (Cebaiye), Huaihua, Quanshen, Huangqin and Eclipta prostrate L. (Mohanlian) are mainly used to cool blood and stop bleeding, which can enhance the ability of “Monarch” medicine on cooling blood and stopping bleeding, alleviating bleeding with heat syndrome. They are regarded as the “Minister” medicine in the prescription. Qiancao, Agrimonia pilosa Ledeb.(Xianhecao) can treat blood leakage; Typha angustifolia L.(Puhuang) is good for removing blood stasis and stopping bleeding; Dihuang and Mudanpi are good at clearing heat, cooling blood. These Traditional Chinese medicine can cooperate with “Monarch” medicine and “Minister” medicine to treat ODUB with fever syndrome. They can be identified as “Assistant” medicine. The Baishao and Danggui that can nourish the blood and kidney play a role in“Guide”medicine. It can reconcile the properties of various drugs in the prescription and enhance the nourishing effect. The combination of all medicines can not only strengthen the effect of hemostasis, regulate menstruation and clearing away heat, but also nourish yin and blood, preventing physical weakness. ODUB belongs to the premenstrual and prolonged periods in TCM. TCM theory indicates that the deficiency of Yin fluid and endogenous heat will show menstrual blood without containment, further inducing hypermenorrhea and menostaxis. YZG has the function of nourishing Yin and clearing heat, cooling blood and hemostasis. Hence, it can be used to treat ODUB, which will achieve an excellent effect [YZG is composed of 17 kinds of TCM, including t effect , 12. HowNetwork pharmacology is a novel discipline based on network theory and systems biology principles, which is deemed as an effective tool for systematically revealing complex network relationships , 14. TCMhttps://tcmspw.com/tcmsp.php) [In this research, all the chemical components of YZG were identified from the traditional Chinese medicine system pharmacology database and analysis platform  . TCMSP, msp.php) . The actmsp.php) , 20.https://stitch.embl.de/) is a database of known and predicted interactions between compounds and protein, and is based on text mining and molecular docking techniques [https://www.UniProt.org/), which contains a large number of protein sequences and detailed annotated information, was used to find the gene name corresponding to the proteins; only human targets were reserved [To identify the compound targets of YZG, we chose two widely used databases: STITCH and TCMSP. STITCH (chniques . STITCH reserved .https://www.cytoscape.org/) to construct a compound-predicted target network map. Cytoscape is a bioinformatics analysis software that visualizes biological pathways and intermolecular interaction networks. It provides a basic set of data integration, analysis and visualization capabilities for complex network analyses [All the compounds that possess targets are were numbered according to their molecular ID number, and a series of numbers with their relevant targets are were imported into the Cytoscape 3.6.1 software , is a database that provides information about nucleic acid targets and the therapeutic effects of proteins [https://www.pharmgkb.org/) is a database that collects complete genotypic and phenotypic information related to the pharmacogenomics and systematically categorizes this information [The therapeutic target database  database, with the species limited to “Homo sapiens” and confidence scores higher than 0.7 [All the targets associated with excessive menstruation were input into the STRING . DAVID, a high-throughput and integrated data-mining environment, analyses gene lists derived from high-throughput genomic experiments. P-value < 0.01 and FDR < 0.05 were considered as the criteria for difference screening [To shed light on the potential mechanism of YZG and its effects on ODUB, the functional pathways of YZG related to excessive menstruation diseases were analyzed using the KEGG pathway and GO enrichment analyses based upon the database for annotation, visualization and integrated discovery (DAVID) version 6.8 catechins, sitosterol, kaempferol and quercetin exist in a variety of TCM in YZG, suggesting that these compounds may be necessary for therapeutic effects.We used Cytoscape to construct a compound–compound target network that comprised 285 nodes (72 compound nodes and 213 target nodes) and 654 edges. The blue nodes represent the compound molecules, and the orange nodes represent the compound targets. Each edge represents the interaction between the compound and its target  were chosen according to the P-value, as shown in Fig. Through molecular docking experiments, the numerical results of the binding energies from Vina docking were collected. The docking details are shown in Table Platycladus orientalis (L.), Franco (Cebaiye), Cirsium japonicum Fisch.ex DC. (Daji), Cirsium setosum (Willd.) MB. (Xiaoji) in cooling and hemostatic effects. The effective active ingredients of TCM for clearing heat and stopping bleeding also contain organic acids, phenols, and tannins. TCMs, including such as Cebaiye, Daji, Xiaoji, Rubia cordifolia L. (Qiancao), and Sanguisorba officinalis L. (Diyu) can achieve hemostasis by shortening bleeding and clotting times. In contrast, Qiancao are mainly used to increase the number of platelets and enhance their aggregation to ensure hemostasis [To holistically explain the interaction mechanisms among the different drugs, compounds and main targets in YZG, a drug-compound-target-pathway network was constructed established based on Cytoscape software. Red indicates the drugs; yellow indicates the compounds; pink indicates the potential targets; and green indicates the pathways. The red lines are connected to the potential therapeutic pathway, which has the highest degree of value, its key targets and the components with the highest docking scores Fig. . In YZG,mostasis –38. HoweUnlike Western medicines, TCM treats diseases by regulating the overall bias of the body; hence, TCM often appears in the form of prescriptions containing many herbs and ingredients . YZG hasProstaglandin-endoperoxide synthase (PTGS), also known as cyclooxygenase, is the essential enzyme in prostaglandin biosynthesis. There are two isozymes of PTGS: constitutive PTGS1 and inducible PTGS2, which can catalyze AA to generate unstable prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2), respectively; the latter is quickly converted to various prostaglandins (PGs) by different enzymes. Under the action of isomerase and synthetase, PGH2 produces the relatively stable prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), while under the action of thromboxane synthase or prostaglandin synthetase, PGH2 produces thromboxane A2 (TXA2) and prostacyclin (PGI2). PTGS1 regulates angiogenesis in endothelial cells and is thought to be involved in cell–cell signaling and maintaining tissue homeostasis. PTGS2, a rate-limiting enzyme for AA to synthesize PGs, plays an important role in a series of reproductive processes involving ovulation, fertilization, implantation, and childbirth –42. AA, Another AA lipoxygenase family member is 5-lipoxygenase (5-LO), which is encoded by the ALOX5 gene and is a starting catalytic enzyme that bypasses the inflammatory mediators leukotriene (LTs) and lipoxins (LXs). It is widely present in various tissues and blood cells of mammals and has the effect of promoting angiogenesis. Moreover, 5-LO can catalyze the AA biosynthesis of LTs and LXs, whose products have a variety of biological functions and play an important role in the occurrence and development of diseases. AA is converted into 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5-LO and 5-lipoxygenase activated protein (5-FLAP). 5-HPETE generates leukotriene A4 (LTA4), leukotriene B4 (LTB4), leukotriene (LTC4), and leukotriene D4 (LTD4) under the catalysis of the enzyme. Among them, LTC4, LTD4 and leukotriene E4 (LTE4) can increase the permeability of capillary venules and promote the contraction of vascular smooth muscles .The caspase-3 protease encoded by the CASP3 gene is the main terminal cleaving enzyme that plays an irreplaceable role in apoptosis. The endometrial functional layer proliferates and exfoliates from the basal layer periodically to form a menstrual cycle, and its mechanism is closely related to apoptosis. Therefore, it is speculated that AA and other compounds in YZG act on the CASP3 gene to treat dysfunctional uterine bleeding through the apoptosis pathway. CASP3 gene balances apoptosis and proliferation, improves the cell cycle, and regulates the normal and cyclic growth, proliferation, and apoptosis of endometrial tissues, which impels the endometrium to shed completely and stops bleeding in time .Beta-sitosterol, a sterol, is a key compound with the highest degree value in YZG. It is found in a wide range of TCMs, such as Cebaiye, Puhuang, Baiji, Danggui, Huaihua. β-Sitosterol inhibits the abnormal proliferation of cells, changes the cell cycle, and plays a role in estrogen regulation, and acts on the hypothalamus-pituitary-ovarian axis and regulates the menstrual cycle. Under normal circumstances, the neuroendocrine mechanism mainly regulates normal menstrual formation through estrogen and progesterone. Studies have shown that the levels of estrogen and progesterone in patients with menstrual dysfunctional uterine bleeding decrease, leading to higher levels of estrogen receptor (ER) and progesterone receptor (PR) expression on the endometrium. PR acts on VEGF, resulting in reduced VEGF secretion and microvascular formation disorders, and leads to abnormal uterine bleeding. β-Sitosterol can play a role in the estrogen effect, bind to the receptor, and regulate the secretion of VEGF. Estrogen can also increase the expression of the cyclic PTGS2 protein in the vaginal and endometrial cells of mice, and PTGS2 is an important rate-limiting enzyme for PGI2. Elevated PTGS2 can inhibit the expression of PGI2. The reduction in PGI2 can reduce the inhibitory effect of platelets and enhance the coagulation effect, thereby achieving hemostasis –53.Leukotriene A4 hydrolase (LTA4H) is a protein that belongs to the peptidase M1 family. LTA4H participates in AA metabolism and is a key enzyme in the AA-mediated inflammatory metabolic network. LTB4 is one of the strongest leucocyte chemokines in the body. It can mediate vascular injury by inducing the release of lysosomal enzymes and reactive oxygen species and participating in systemic lupus erythematosus, skin and kidney tissue inflammation, and tissue damage. Therefore, it is speculated that AA can regulate tissue repair and the inflammatory response by acting on the LTA4H gene, thereby creating a good internal environment in the uterus that is conducive to the shedding and reconstruction of the endometrium .Flavonoids, including skullcapflavone II, salvigenin, and rivularin in YZG can all act on factor VII (FVII) and factor X (FX) receptors. The FVII receptor is considered to be a classic coagulation factor that plays a key role in the coagulation mechanism. When tissues and blood vessels are damaged, tissue factor (TF) is released. TF and FVII or activated FVII (FVIIa) form a complex (TF-FVIIa). This complex can activate FX and factor XI (FXI), and TF released into the blood can significantly promote the coagulation reaction process. Consequently, we can speculate that flavonoids may upregulate F7 and other related genes through the complement and coagulation cascade pathways to increase the release of coagulation factors, enhance coagulation, and accelerate platelet aggregation to achieve hemostasis .There are many flavonoids in YZG that can act on PTGS1 and PTGS2 receptors. It is well known that the PTGS enzymes are related to the production of TXA2. TXA2 is one of the strongest vasoconstrictive and platelet aggregation agents. Platelets can generate TXA2 to make platelets aggregate. PGI2 is an important vasodilator that has a strong antiplatelet aggregation effect. Normally, TXA2 and PGI2 are in a state of dynamic equilibrium in the body. If stimulated by changes in hormone levels, the synthesis of PGI2 increases, and the synthesis of TXA2 decreases, resulting in the inhibition of platelet aggregation and vasodilation, leading to increased bleeding and prolonged menstrual cycles , 57. AniAlthough our study basically discussed the molecular mechanisms of YZG, there still exist some limitations. First of all, the data of the study came from existing databases so that the authenticity and integrity of the result rely on the data. Next, the result may not reflect all the real cell network characteristics in the organism. What’s more, further experiments confirming the results of this prediction in YZG are required because our study was performed based on data analysis.In the present study, we applied a network pharmacology approach to predict, elucidate, and confirm the potential mechanisms of YZG on ODUB by network construction, integrating target prediction, module analysis, molecular docking and enrichment analysis. The therapeutic efficacy of YZG against ODUB is likely mediated via the regulation of seven targets, namely, PTGS1, PTGS2, ALOX5, CASP3, F7, F10, and LTA4H. In addition, the molecular docking simulation demonstrated a good affinity of PTGS1, PTGS2, ALOX5, CASP3, F7, F10, and LTA4H with their corresponding compounds. GO enrichment analysis illustrated that the targets of YZG against ODUB might be closely associated with the regulation of blood coagulation, regulation of blood coagulation regulation of hemostasis, platelet activation, prostaglandin biosynthetic process, estrogen receptor binding, regulation of heat generation. Additionally, the KEGG pathway enrichment analysis suggested that YZG may simultaneously act on a variety of pathways, including the apoptosis, AA metabolism, serotonergic synapse, complement and coagulation cascade, and C-type lectin receptor signaling pathways. In summary, it can be speculated that the mechanism of YZG in treating ODUB may involve three aspects. First, because the active ingredients regulate endometrial blood vessel contraction, promote platelet aggregation, and induce angiogenesis, YZG is able to promote hemostasis. Second, the active ingredients in YZG exert estrogen and progesterone-like effects that can regulate the menstrual cycle and menstrual flow by acting on the hypothalamus-pituitary-ovary axis. Third, normal menstrual bleeding may be achieved by promoting the apoptosis of the endometrium and causing it to regularly shed. Our results provide a preliminary prediction of the mechanisms related to the therapeutic effect of YZG against ODUB and provide a significant basis and reference for further exploration.


Traditional Chinese medicine (TCM) was widely used in the treatment of recurrent respiratory tract infections (RRTIs) in East Asia, but its mechanism was not clear because of its complex prescription rules. This research prospectively collected 100 prescriptions of RRTI children treated with TCM. The characteristics of TCM in prescriptions were described and analyzed, and the rules of prescriptions were analyzed by hierarchical clustering and association rules. The results showed that the principle of RRTI was to pay equal attention to cold and mild, and six new meaningful prescriptions were obtained. Among them, the new prescription composed of Astragali Radix (Huangqi), Atractylodis Macrocephalae Rhizoma (Baizhu), Saposhnikoviae Radix (Fangfeng), Angelicae Sinensis Radix (Danggui), and Paeoniae Radix Rubra (Chishao) was an important method to treat RRTI. In order to explore the mechanism of the new prescription, the research obtained the action target of each herb of the core prescription on Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine, TCMIP v2.0. The target genes were enriched by Metascape, and 93 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were obtained. According to the classification and statistics of KEGG type, it was found that the new prescription mainly intervened in the metabolic pathway dominated by amino acid metabolism. In addition, there were also many interventions in the nervous system-, endocrine system-, and digestive system-related pathways. This study summarized the prescription rule of TCM in the treatment of RRTI, analyzed the mechanism of supplementing deficiency, and provided a new idea for the treatment of RRTI. Recurrent respiratory tract infections (RRTIs) are one of the most common diseases in children and adolescents . Data frAfter more than 2000 years of development, traditional Chinese medicine (TCM) has formed a complete health care system from diagnosis to prognosis, which has accumulated valuable experience for clinical application and medical research. The efficacy of TCM has been recognized by more and more people and countries. Researchers from many countries around the world have studied the efficacy of TCM in relieving symptoms , 10. AccData mining has been used to analyze the prescription patterns of TCM from clinical data and find the potential relationship between herbs and diseases. New prescription and core prescription were discovered based on the retrospective analysis of data of chronic kidney patients as well as frequency analysis and correlation evaluation of Chinese medicine prescriptions of chronic kidney diseases . The resThis study proposed a comprehensive method based on data mining and systematic pharmacology to analyze the rule of TCM prescription and reveal its mechanism. Clinical cases of children with RRTI were prospectively collected. These cases were prescribed by the chief physician of pediatrics from Dongzhimen Hospital, Beijing University of TCM. Then, the prescriptions of RRTI were analyzed by descriptive statistics, hierarchical clustering, and association rules to explore the medication rule of Chinese medicine prescriptions, and explore the core prescription, then retrieve the important targets from the database, and use Metascape to analyze Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Workflow is shown in Chinese Journal of Pediatrics. Diagnostic criteria : (1) the interval between the two infections should be more than 7 days; (2) if the number of infections of the upper respiratory tract was insufficient, the number of lower respiratory tract infections could be added; otherwise, it could not be added; (3) the frequency should be determined by continuous observation for more than one year; and (4) repeated pneumonia should be in accordance with the signs of pneumonia and imaging examination, and between the two diagnoses, the signs and imaging changes should completely disappear.  Inclusion criteria: (1) they should meet the diagnostic criteria of children's RRTI; (2) the age ranged from 1 to 17 years old; (3) it should be at least one week after the recovery of the acute infection; and (4) sign informed consent with the legal guardian or the tested child.  Exclusion criteria: (1) children with severe primary respiratory tract infections such as primary immunodeficiency and acquired immunodeficiency syndrome (AIDS). (2) There were serious primary diseases such as heart, liver, kidney, digestive system, and hematopoietic system. (3) Participants in other clinical trials.From July 2019 to January 2020, clinical information and Chinese medicine prescriptions of children suffering from RRTI were collected prospectively from Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine. RRTI diagnostic criteria referred to the Clinical Concept and Principles of Management of RRTI , which wThis study was reviewed by the Ethics Committee of Beijing University of TCM, with ethical batch number 2019BZHYLL0204, which was strictly implemented in accordance with the ethical system.The information of patients' age, gender, diagnosis, and Chinese medicine prescriptions was collected.People's Republic of China Pharmacopoeia (2015 Edition) and Chinese Medicine Dictionary in the process of herbs' entry [All data were entered into Microsoft Excel 2016, and the RRTI database of TCM was established. It included the patient's initials, hospital registration number, gender, age, diagnosis, and prescriptions, standardizing herbs as the official name according to the Chinese medicine classification of s' entry . The terThrough the descriptive analysis of the dosage, herbal properties, and taste of all prescriptions, the overall effect of prescriptions could be summarized and analyzed. Hierarchical clustering was applied to discover new prescriptions. Finally, this study used association rules to screen core prescriptions with strong relevance. GraphPad Prism 7 software was used to calculate the relationship between dosage, properties, and taste. RStudio version 3.5.1 was used for description analysis, hierarchical clustering, association rule analysis, and visual display.The characteristics of herbs were analyzed, including properties and taste. There are five items of herbal properties: cold, hot, warm, cool, and mild, and there are seven items of herbal taste: sour, bitter, sweet, pungent, salty, astringent, and light.A and B in the clustering algorithm formula represent different herbs. Euclidean method was used to calculate the similarity between herbs, and cluster analysis was carried out for the top 50 herbs.Compatibility of TCM referred to the combination of two or more kinds of herbs according to clinical needs and the nature and function of medicine. The hierarchical clustering algorithm was used to cluster each herb. According to the similarity measurement among the objects, stable and regular new categories could be obtained in many individualized prescriptions. A and B in the association rule formula are frequent itemset herbs of one or several herbs. The correlation coefficient between herbs can be obtained by calculation. The support degree is set to 0.25, and the confidence degree is set to 0.8:Apriori algorithm was a frequent itemset algorithm to form association rules. It was used to analyze the clear rules of TCM in RRTI treatment and obtain the core herbs. In the data, each herb was treated as an itemset, and each prescription was treated as a transaction. Find out the frequent itemset in prescriptions, mining association rules between herbs, and filter the top 15 rules based on “support” to get the core prescription.. http://www.tcmip.cn/TCMIP/index.php/Home/Login/login.html), was developed by the Chinese Academy of Chinese Medical Science. It mainly included five database resources from the Encyclopedia of Traditional Chinese Medicine (ETCM), “database of TCM prescriptions,” “database of TCM ingredients,” “database of TCM targets,” and “database of disease-related molecules.” [Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine, TCMIP v2.0 (ecules.”  In this http://metascape.org/gp/index.html#/main/step1) for the KEGG pathway. Upload the lists of genes and select H. sapiens species for analysis.. KEGG pathway analysis was carried out with the analysis mode of custom analysis [P value) > 5 and more than 5% targets falling into the category were retained. Using ggplot2 of RStudio, the remaining terms were drawn into bubble charts. Finally, the bubble charts of each herb were combined.The obtained targets were enriched and analyzed with Metascape , 292 for Atractylodis Macrocephalae Rhizoma (Baizhu), 196 for Saposhnikoviae Radix (Fangfeng), 232 for Angelicae Sinensis Radix (Danggui), and 106 for Paeoniae Radix Rubra (Chishao).A total of 93 KEGG pathways were obtained. Analysis of KEGG type showed that 26 signaling pathways were metabolic-related pathways. It was proved that the combined effect of core prescription herbs played a large part in intervening the metabolism process of the body. Astragali Radix (Huangqi), Saposhnikoviae Radix (Fangfeng), and Angelicae Sinensis Radix (Danggui) were mainly responsible for the fatty acid metabolism pathway, while, Atractylodis Macrocephalae Rhizoma (Baizhu) was mainly responsible for the amino acid metabolism pathway . In addiThere were seven pathways belonging to the nervous system, which were, namely, long-term potentiation, retrograde endocannabinoid signaling, glutamatergic synapse, serotonergic synapse, GABAergic synapse, dopaminergic synapse, and long-term depression. Astragali Radix (Huangqi) has a wide range of interventions in the middle and lower reaches of the nervous system pathway, such as Raf, PP1, AKT, PKA, Casp3, Cam, Can, Cox, and Cytp450. Atractylodis Macrocephalae Rhizoma (Baizhu) has a unique intervening effect on the protrusion anterior membrane and protrusion posterior membrane. GABAA, NMDAR, PKA, Cox, and CASP3 were the targets of most herbs. Through the intervention of multiple pathways, it could regulate the functions of neuroprotection, hyperpolarization decreased efficiency, arachidonic acid metabolism, and synaptic plasticity . See TabThe nature and function of TCM formed the fundamental basis in analyzing Chinese medicine and its clinical applications. In this study, a comprehensive descriptive analysis was used to analyze the herbs, prescriptions, dosage, herbal properties, and taste. Overall, the total frequency of cold herbs and warm herbs was similar. Cold herbs have the effect of clearing away heat, and warm herbs have the effect of supplementing benefits. This discovery reflected the concept of TCM to supplement missing substances and remove excess substances in the body to regulate homeostasis –28. FurtThe pathogenesis of RRTI is complex. In the noninfectious stage, it is usually associated with pharyngitis, allergic rhinitis, hyperhidrosis, dyspepsia, and other symptoms. There are also different emphases when prescribing TCM treatments. In this study, we found the relationship between herbs by cluster analysis and obtained 6 effective new prescriptions based on the theory of TCM. Prescription 1 mainly supplements benefits; prescription 2 has the functions of clearing stomach heat and reducing hyperhidrosis; prescription 3 could relieve fidgety and reduce thick turbinate; prescription 4 has the functions of clearing the lung and the pharynx; prescription 5 could unblock the nose; and prescription 6 could relieve coughs. Many of these symptoms match those that are displayed by children suffering from RRTI in clinical data. We speculated that RRTI has a certain pathophysiological relationship with the frequently occurring combined diseases or symptoms. In the process of treating RRTI, comprehensive treatment of TCM may produce better clinical effect. Association rule analysis revealed that Astragali Radix (Huangqi), Atractylodis Macrocephalae Rhizoma (Baizhu), Saposhnikoviae Radix (Fangfeng), Angelicae Sinensis Radix (Danggui), and Paeoniae Radix Rubra (Chishao) were closely related, and they were the core herbs in the treatment of RRTI. These herbs belonged to new prescription 1 in the cluster analysis, which was an important treatment module for RRTI, and were considered to be the main herbs to play the role of tonic. The results of meta-analysis show that Astragali Radix (Huangqi), Atractylodis Macrocephalae Rhizoma (Baizhu), and Saposhnikoviae Radix (Fangfeng) could improve the total clinical effective rate of RRTIs in children and reduce the incidence of respiratory infection .The mechanism of core prescription in the treatment of RRTI was studied by systematic pharmacology. Five kinds of herbs in the core prescription played a synergistic role and intervened RRTI through various ways. The whole prescription was mainly aimed at metabolism-related pathways, especially fatty acid metabolism- and amino acid metabolism-related pathways. In addition, it showed that the core prescription with warming and tonifying effects could intervene in the enrichment of related pathways such as the nervous system, endocrine system, and digestive system, which has been proved by previous research results –31. The Combined with data mining and systematic pharmacology, this research comprehensively analyzed the prescription patterns of traditional prescriptions to explain the mechanism of traditional prescriptions and provide useful new enlightenment for the treatment of RRTI. This study prospectively collected clinical data and prescriptions of patients to ensure the quality of research data, but to some extent, there were still limitations due to insufficient samples. Big data analysis can summarize the group symptoms and prescriptions of RRTI patients from a macroperspective, and the new prescriptions produced are meaningful for understanding the pathological mechanism of RRTI, but the new prescription is not suitable for everyone. The clinical prescription should be individualized on the basis of syndrome differentiation and treatment. The new prescription can provide effective treatment guidance for the most common clinical syndromes. For example, the patients with “deficiency of lung Qi and spleen Qi” and “gastrointestinal heat accumulation” can choose the combination of new prescriptions 1 and 2. It is beneficial for doctors to improve the clinical efficacy by adding or subtracting herbs in clinical manifestations. Through the analysis of the mechanism of the prescription of TCM in the treatment of RRTI, more information was also revealed about the pathogenesis of RRTI. It is generally believed that RRTI is closely related to the immune and inflammation system , 34, sucA new prescription of RRTI composed of Astragali Radix (Huangqi), Atractylodis Macrocephalae Rhizoma (Baizhu), Saposhnikoviae Radix (Fangfeng), Angelicae Sinensis Radix (Danggui), and Paeoniae Radix Rubra (Chishao) has been extracted. It mainly intervenes in metabolism-, endocrine system-, and nervous system-related pathways. Research analysis and results showed that the method and software used in this study could effectively analyze the mechanism and law of traditional Chinese medicine prescription.


Achromobacter xylosoxidans and pulmonary MALT lymphoma. We aimed to investigate the presence of virus or any infectious agents in pulmonary MALT lymphoma by using metagenomic next-generation sequencing (mNGS).Mucosa-associated lymphoid tissue (MALT) lymphoma is generally associated with chronic antigen stimulation: auto-antigens or of microbial origin. Only one study suggested association between All lung samples were centrally reviewed. The t  was evaluated by FISH analysis. The snap frozen large lung biopsies were analyzed by mNGS. After lung biopsies homogenization total nucleic acids (RNA and DNA) were extracted, amplified and classified according to their taxonomic assignment, after exclusion of host DNA.A. xylosoxidans sequence in 4/13 patients and in 4/10 controls.We included 13 samples from pulmonary MALT lymphoma  and 10 controls. The diagnosis of MALT lymphoma was confirmed for the 13 samples, 3 showed API2-MALT1 translocation (23%). No evidence of the presence of a specific pathogen was clearly identified in the group of patients with pulmonary MALT lymphoma. We identifiedThis study did not find evidence for a DNA or RNA virus, a fungi, a parasite or a bacteria associated with pulmonary MALT lymphoma either in the stroma or in tumor cells. Helicobacter pylori was initially detected in almost 90% but only 65-70% in recent series of gastric biopsies from patients with gastric MALT lymphoma, and the lymphoma is usually cured by antibiotics [Campylobacter jejuniinfection and small intestine MALT lymphoma as well as between hepatitis C virus infection and some cases of splenic MZ lymphoma [Borrelia burgdorferi infection and skin MALT lymphoma, and between Chlamydophila psittaci and ocular adnexal MALT lymphoma [Though rare, mucosa-associated lymphoid tissue (MALT) lymphoma is the most common pulmonary B-cell lymphoma. The disease is slow-growing with an asymptomatic chronic alveolar opacity visible on radiography . Interesibiotics . Other ilymphoma . Severallymphoma .Achromobacter xylosoxidans in 57/124 pulmonary MALT lymphomas vs15/82 controls (p = 0.004) [H. pylori-targeting antibiotics [Only one study suggested an infectious cause of pulmonary MALT lymphoma. Using a 16S RNA-based approach, the authors found DNA from = 0.004) . Chronic= 0.004) . Finally= 0.004) , and theibiotics .By using an agnostic metagenomic next-generation sequencing (mNGS) approach, we aimed to investigate without prior hypothesis the presence of infectious agents sequences associated with pulmonary MALT lymphoma, in conjunction with the presence of oncogenic alteration and auto-immune disease .et al.[From a retrospective cohort of 63 patients with a histological diagnosis of pulmonary MALT lymphoma from 1993 to 2008, monitored in three French hospitals, we enrolled 13 patients  with surgical biopsy to insure sufficient available amount of snap frozen lung tissue specimens including tumor cells and stroma for mNGS exploration [et al.. Using tde novo assembled with CLC (v. 9.5.3). Generated contigs and unassembled reads were then aligned with BLAST against a reference database  to refine taxonomic classification results. Raw data were deposited in the NCBI Sequence Read Archive under bioproject ID PRJNA664596.The snap frozen lung biopsies provided from pulmonary MALT lymphoma patients and control patients were subjected to mNGS. After lung biopsies homogenization and depletion of host DNA, total nucleic acids were extracted using TRIzol Reagent (Invitrogen) according to manufacturer´s recommendations except for DNA purification step, which was performed with QiaAmp DNA mini kit (Qiagen). RNA and DNA amplification were performed using QuantiTect Whole Transcriptome Kit and REPLI-g kit (Qiagen) respectively. All amplified nucleic acids were pooled in equal concentrations to prepare NGS libraries with TruSeq DNA PCR-free Library preparation kit. The run was performed on HiSeq 2500 sequencer (Illumina) resulting in 2 x 150 nucleotides paired-end reads. All raw data were first cleaned by trimming adapters, by removing low quality sequence reads, duplicates and host contaminant residues. The filtered reads were classified according to their taxonomic assignment with Kaiju (v. 1.4.1) and then All 13 MALT-lymphoma samples showed dense proliferation of small CD20+ lymphocytes infiltrating the lung. Characteristic lymphoepithelial lesions were observed in 9/13 cases (69%). Large cells were absent in all samples. Plasma cells were observed in 3/13 cases (23%). Staining for CD5, CD10, CD23, IgD, cyclin D1 and CD30 were always negative in tumoral cells. Expression of BCL2 was positive in the nine cases for which it was performed. The clonality of B-cells was available for five patients and always positive. All samples were tested for the presence of API2-MALT1 translocation by FISH, and 3 were positive (23%). None of the patients with auto-immune background presented an API2-MALT1 translocation.A. xylosoxidans sequence. These results did not support a significant involvement of A. xylosoxidans in the pathogenesis of this MALT lymphoma in this series [Chlamydia psittaci, although previous results reported its possible involvement in non-gastrointestinal MALT lymphomas [Pneumocystis jirovecii. Although a huge number of specific reads and contigs were retrieved, we could not confirm the result by specific PCR.The microbial diversity in the patients and controls lungs revealed by mNGS analysis was depicted by the global taxonomic classification of sequenced reads Fig. . No evids series . In addiymphomas , 8. RemaA. xylosoxidans or Chlamydiae [A. xylosoxidans could eventually participate in the occurrence of MALT lymphoma in an unidentified subgroup of patients. While previous studies used marker gene sequencing, such as 16S rRNA, a targeted sequencing assay limited by the breadth of detection, our study is the first looking for an agnostic metagenomic NGS approach to explore a potential link between pulmonary MALT lymphoma and all microbial agents i.e. bacteria, viruses, fungi and parasites. Actually, our study did not detect any evidence for a specific pathogen associated with pulmonary MALT lymphoma. The small number of specimens tested and the auto-immune background profile of some patients are a limitation of our study. The detection of Pneumocystis DNA without the demonstration of living stages of pathogen in the tissue was observed in only one patient but not in all other tissue specimens tested from MALT-lymphoma patients and controls. Considering the high prevalence of Pneumocystis colonization, detected in almost 25% of patient with chronic lung disease by PCR [Pneumocystis in pulmonary MALT lymphoma.This study did not find evidence for a pathogen associated with pulmonary MALT lymphoma. Previous study evidenced an increased prevalence of lamydiae , 9. We ce by PCR , we did In conclusion, we were not able to identify a pathogen as a causative factor of pulmonary MALT lymphoma.