This study conducted transcriptome sequencing of goat-mammary-gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages to reveal the expression characteristics and molecular functions of circRNAs during mammary involution. A total of 11,756 circRNAs were identified in this study, of which 2528 circRNAs were expressed in all three stages. The number of exonic circRNAs was the largest, and the least identified circRNAs were antisense circRNAs. circRNA source gene analysis found that 9282 circRNAs were derived from 3889 genes, and 127 circRNAs’ source genes were unknown. Gene Ontology (GO) terms, such as histone modification, regulation of GTPase activity, and establishment or maintenance of cell polarity, were significantly enriched (FDR < 0.05), which indicates the functional diversity of circRNAs’ source genes. A total of 218 differentially expressed circRNAs were identified during the non-lactation period. The number of specifically expressed circRNAs was the highest in the DP and the lowest in LL stages. These indicated temporal specificity of circRNA expression in mammary gland tissues at different developmental stages. In addition, this study also constructed circRNA–miRNA–mRNA competitive endogenous RNA (ceRNA) regulatory networks related to mammary development, immunity, substance metabolism, and apoptosis. These findings help understand the regulatory role of circRNAs in mammary cell involution and remodeling. The mammary gland is a complex exocrine gland composed of many different types of cells that distinguish mammals from other animals by its unique anatomy . It can Mammary gland involution is regulated by multiple factors, including some growth factors, proteases, hormones, coding genes, and non-coding RNAs (ncRNAs) ,5,13. ciHowever, the expression characteristics and molecular functions of circRNAs in goat-mammary-gland involution are still unclear, although current studies have characterized circRNAs in both lactating and non-lactating mammary gland tissues of sheep  and cattThe methods and experimental procedures used in this study were approved and directed by the animal protection and ethics committee of Shandong agricultural university (protocol number: SDAUA-2018-048) . All expn = 3, 240 days postpartum), dry period , and late gestation . The specific tissue collection method was conducted as follows: after intravenous injection of sodium pentobarbital (100 mg/kg), the muscles relaxed, and the heart and respiration stopped; the goats were then rapidly dissected; and mammary gland tissue was harvested. Immediately after washing the mammary tissue with normal saline (0.9%), the harvested tissue was placed into an RNase-free cryovial and stored in liquid nitrogen.Nine healthy Laoshan dairy goats raised at the Qingdao Aote goat farm  were used in this study. Similar standards of housing and management were provided for all goats. An open-air natural sports field was provided to keep the breeding environment clean, hygienic, and dry. The goats were fed according to the standard feeding process of the farm. The goats were all in the third parity, with an age range of 3 years and 8 months to 4 years and 1 month; the weight range of the goats was 50.16 to 56.41 kg; and the specific information of the goats can be obtained in https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 15 January 2023) and high-throughput sequencing reads were processed using Trimmomatic software (version 0.39) [Total RNA was extracted using TRIzol-based RNAiso Plus . The quality of RNA was analyzed using a NanoDrop 2000C . Only when the RNA integrity number (RIN) was > 8 were the RNA samples used for subsequent experimental analysis. Nine sequencing libraries  were constructed using the TruSeq RNA Library Prep Kit v2 , and they were sequenced on the HiSeq 2500 platform (Illumina). After generating raw data, the sequencing data quality was assessed using FastQC software   to removon 0.39) , CIRIquaon 0.39) , CIRCexpon 0.39) , and CIRon 0.39)  to identon 0.39)  or BWA [on 0.39)  softwareon 0.39) .Low-expression circRNAs were filtered and transcripts with expression levels ≥ 2 were retained. Principal component analysis (PCA) was performed on all circRNAs and the expressed circRNAs in each mammary-gland-developmental stage were counted. circRNAs were classified according to their position information in the genome, and circRNAs types are displayed in a Venn plot. In addition, the length distribution of circRNA is displayed by bar graph. Gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG)-pathway-enrichment analysis were performed on circRNA-derived genes using clusterProfiler software . Goat gep value (p.adj) < 0.05 and the absolute value of log2 (fold change) ≥ 1, circRNAs were considered differentially expressed. According to the ranking of p.adj, the 10 most significantly up-regulated and down-regulated circRNAs were screened and are displayed in a volcano map. The differentially expressed circRNAs were then sorted according to the expression level at each developmental stage, and the top 10 differentially expressed circRNAs at each developmental stage  were selected and are displayed using histograms. The R package pheatmap was used to draw a heatmap to display the expression characteristics of all differentially expressed circRNAs.Differential expression analysis  of mammary gland tissues at different stages was performed using edgeR software . When th® Fast qPCR Mix (Takara) was used for the real-time quantitative reverse-transcription PCR (RTqPCR) assay. Primers were designed using NCBI Primer Blast software, and the specific steps are as follows: after obtaining the circRNA sequences, the sequence (100 bp) at the 3′ end was placed in front of the 100 bp at the 5′ end of the sequence. The new sequence (200 bp) was uploaded to the NCBI Primer Blast website, the length of the PCR product was set to 90–120 bp, and the divergent primer of the circRNA was obtained. The geometric mean of 2 carefully selected reference genes (GAPDH and MRPL39) was used as an accurate normalization factor [To verify the accuracy and reliability of transcriptome data, we randomly selected 14 differentially expressed circRNAs for real-time quantitative reverse-transcription PCR (RTqPCR) assay and loop junction site verification. Total RNA was extracted from goat-mammary-gland tissue using TRIzol-based RNAiso Plus . Linear RNA was removed using Ribonuclease R . Reverse-transcription reactions were performed using the RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. PCR reactions were performed using the 2xTaq MasterMix kit . PCR products were confirmed by 2% agarose gel electrophoresis. Bands approximately 100 bp in length were isolated and analyzed by Sanger sequencing. In order to determine the expression level of circRNAs, TB Greenn factor . Relativn factor .miRanda software  was usedThe function corr.test in the R package psych was used to calculate the Pearson correlation between miRNA and circRNA, miRNA and mRNA, and circRNA and mRNA. The correction method used was FDR. Relationship pairs with a correlation value ≥ 0.8 and an FDR < 0.05 between circRNA and mRNA, a correlation ≤ −0.8 and an FDR < 0.05 between miRNA and circRNA, and a correlation value ≤ −0.8 and an FDR < 0.05 between miRNA and mRNA were screened. Considering the target genes results predicted by miRanda software and the expression correlation between miRNAs and circRNAs or mRNAs comprehensively, GO functional annotation and KEGG-pathway-enrichment analysis were performed on mRNAs that compete with differentially expressed circRNAs for miRNA binding. The signaling pathways and genes related to physiological processes, such as mammary gland development, cell apoptosis, immunity, and substance metabolism, were screened out. STRING software  was usedt-test was used to test the significance of the difference between the experimental group and the control group; p < 0.05 indicated a significant difference. Tukey’s honestly significant-difference test was used to analyze gene expression levels at different developmental stages. Figures presented in this work were generated using R software (version 4.2.0) unless otherwise stated.Student’s Statistical analysis of the sequencing data found that 75,670,537 ± 2,451,052, 80,547,045 ± 4,443,268, and 73,765,766 ± 1,723,917 clean reads (mean ± SE) were obtained from the sequencing library of goat mammary glands during late lactation (LL), dry period (DP), and late pregnancy (LG), respectively. The results of the reads’ alignment with the goat reference genome showed that the alignment rate of each sequencing library was ≥ 95% . Raw seqLALBA, CSN1S1, CSN1S2, CSN2, and CSN3) affecting protein content and composition in milk could produce circRNAs, of which 10 circRNAs were derived from CSN1S2 and 8 circRNAs were derived from CSN2. GO functional analysis of the 3889 cicrRNA-derived genes found  in the lactation term were down-regulated during the DP. This may be related to the lack of milk production during the DP. The number of genes up-regulated during the DP in wound healing was more than the number of down-regulated genes, which may be related to tissue remodeling during the DP. In the LL vs. LG group  genes that produced 1 circRNA transcript, and only 45 (1.2%) genes could produce 10 or more circRNA transcripts . The genes found A, histones found B endocytes found  in the Les found , mammaryLG group , mammaryNC_030813.1:85981695|85987343 was the largest at 130.2-fold. In the DP vs. LG comparison   and RTqPCR (log2 fold change) of 14 circRNAs were significantly positively correlated  . Agarose < 0.01) . The res < 0.01)  found thA total of 23,574 target regulatory relationships were predicted between 330 miRNAs and 218 differentially expressed circRNAs by miRanda software. There were 441,782 targeted regulatory relationships between miRNAs and 12,056 protein-coding transcripts . ThroughINSR, KIT, GNB4, GNB5, CSF1, ITGA2, and ITGAV are all involved in the regulation of the PI3K-Akt signaling pathway. Combining the relationship between target regulation and expression level, a mammary-gland-development-related ceRNA regulatory network, including 11 circRNAs, 9 miRNAs, 37 genes, and 38 transcripts, was constructed , and their expression levels are significantly negatively correlated  . NC_030816.1:83819802|83825048 was significantly positively correlated with XM_018051136.1 , both of which were highly expressed during the DP  D,F. chi-g the DP A–C.FASN, ELOVL5, and ACACA are involved in the regulation of fatty acid metabolism  and FABP3 (NM_001285701.1 and XM_013971605.2) were highly expressed at LL. PEMT is highly expressed at LG. SLC7A5 and SLC1A1 are involved in the amino-acid-transmembrane-transport process, and they are highly expressed at LL. In addition, only 3 hub gene transcripts were highly expressed during the DP, and the expression levels of the other 12 hub gene transcripts were decreased during the DP. Similarly, only three circRNAs were highly expressed during the DP, while other circRNAs were down-regulated during the DP , and NC_030818.1:70611932|70614030 can compete for binding to chi-miR-148a-3p. chi-miR-30b-5p can target and regulate MAP3K5 (XM_005684842.3). NC_030816.1:19670043|19696979, NC_030820.1:37909806|37923941, and NC_030821.1:56966260|56986994 can compete with chi-miR-30b-5p for binding. Hub genes MAP3K5, IL7R, BTK, IKBKG, TNFRSF1A, and IL2RA were highly expressed during the DP. CSF2RB was low expressed during the DP. In addition, it was found through bioinformatics prediction that chi-miR-148a-3p can target and regulate TNFRSF1A (XM_018048606.1), and NC_030818.1:70611932|70614030 can compete for binding to chi-miR-148a-3p. chi-miR-30b-5p can target and regulate MAP3K5 (XM_005684842.3); NC_030816.1:19670043|19696979, NC_030820.1:37909806|37923941, and NC_030821.1:56966260|56986994 could compete with chi-miR-30b-5p for binding. In addition, a total of six genes, including IL2RA, IL7R, CSF2RB, JAK3, AKT3, and IL3RA, were enriched in the JAK-STAT signaling pathway. Among them, JAK3 (XM_018051403.1) was targeted and regulated by chi-miR-30b-5p. NC_030821.1:56966260|56986994 and NC_030831.1:54852737|54883065 can compete for binding to chi-miR-30b-5p.A total of 27 genes related to apoptosis were screened, and 7 genes were identified as the hub genes of the PPI network . These gg the DP . CSF2RB circRNAs are a new class of endogenous non-coding RNA molecules formed by covalent bonds, and they have been shown to play an important role in various physiological activities of the mammary gland . HoweverThis study found that circRNAs were expressed in mammary gland tissues at different developmental stages with time specificity A. This icircRNAs have similar structural features as regulators of gene expression. The number of circRNAs derived from exons was the largest  in this study D. This iCSN1S1, CSN1S2, CSN2, and CSN3) and alpha-lactalbumin-encoding gene (LALBA) produced circular RNAs in mammary gland tissues at LL and LG, but their expression levels were not expressed during the DP (CSN1S1 and CSN1S2, which may be related to the active expression of genes encoding milk protein and casein during lactation. Similarly, genes encoding casein and lactalbumin were also detected to produce circRNAs in the mammary gland tissues of sheep [The functions of circRNAs’ source genes are rich and involve various physiological processes and signaling pathways . It is kg the DP . In addiof sheep  and cattof sheep ,27 durinINSR was identified as a hub gene in the mammary-gland-development ceRNA network. The INSR-encoded protein could regulate the uptake and release of glucose, as well as the synthesis and storage of carbohydrates, lipids, and proteins [NC_030816.1:83819802|83825048 and NC_030829.1:30515090|30520814-chi-miR-148b-3p-INSR may regulate the growth state of goat-mammary-gland cells.Accumulating evidence suggests that circRNAs function as miRNA sponges, preventing miRNAs from binding to target genes, thereby regulating mammary gland development and lactation physiological processes ,22,53,54proteins . In addiproteins ,56,57. Fproteins . TherefoOne of the characteristics of mammary gland involution is extensive apoptosis of mammary cells . PreviouNC_030808.1:40225918|40234775, NC_030809.1:106427830|106442766, and NC_030812.1:113584493|113589629-chi-miR-130b-5p-FAPB3 may be related to lipid metabolism. On the one hand, FABP3 is considered to be involved in the uptake of long-chain fatty acids, intracellular metabolism, and material transportation; on the other hand, it can also inhibit the growth of mammary epithelial cells [FABP3 was highly expressed at LL (SLC7A5, SLC1A1, SLC38A1, SLC7A7, and SLC7A4 are involved in amino acid transmembrane transport. In this study, a circRNA–miRNA–mRNA ceRNA network related to the above amino acid synthesis and transport was constructed. B3GALT4 and B3GNT5 are involved in carbohydrate metabolism [B3GNT5, and chi-miR-423-3p can target and regulate B3GALT4. NC_030810.1:84946448|84965332 can compete with chi-miR-30b-5p, and NC_030815.1:76944795|76962878 can compete with chi-miR-423-3p. Research on circRNAs that can regulate the metabolism of substances in the mammary gland has been extensively conducted on dairy goats cows [Secretion composition of mammary gland changes during involution . The mamed at LL , which mats cows . In conc2-induced macrophage activation through the HECTD1/ZC3H12A-dependent ubiquitin-proteasome pathway [NC_030808.1:142388405|142433828, NC_030812.1:12090221|12092129, NC_030814.1:56550226|56581054, NC_030814.1:75848324|75868791, and other circRNAs can compete for binding to chi-miR-29c-3p. Meanwhile, a regulatory loop between miR-29c-3p and gene Myc exists in cancer cells to counteract the cytotoxicity of natural killer cells [CCNL2 axis as a novel prognostic biomarker associated with immune infiltration in prostate cancer [CLEC7A, CD36, and DOCK2, were also identified in the immune-related ceRNA network. This plays an important role in removing apoptotic cells and residual milk in involutional mammary gland tissue [The presence of inflammatory cells in the involutional mammary gland helps to remove apoptotic cells and residual milk, thereby promoting mammary gland remodeling . The imm pathway . Further pathway . In thiser cells . Other se cancer . In addid tissue , indicatThis study also has some limitations. First, as explained in our previous work ,30, the In this study, a total of 11,756 circRNAs were identified in the non-lactating goat-mammary-gland group by Illumina sequencing. Among them, 218 circRNAs were differentially expressed in non-lactating mammary glands, with the highest number of circRNAs specifically expressed in the DP and the least number of circRNAs specifically expressed in LL. On the one hand, this shows that the expression of circRNAs changes at the transcriptional level during mammary gland involution; on the other hand, it indicates the temporal specificity of circRNAs expressed in mammary gland tissues at different developmental stages. GO functional analysis of cicrRNA-derived genes found that GO terms, such as histone modification, regulation of GTPase activity, positive regulation of organelle organization, and establishment or maintenance of cell polarity, were significantly enriched, which reflected the diversity of circRNAs’ source gene functions. Both genes that encode lactalbumin and casein can produce circRNAs, indicating that circRNAs may regulate milk protein synthesis. In addition, this study also constructed circRNA–miRNA–mRNA ceRNA regulatory networks related to mammary gland development, immunity, substance metabolism, and cell apoptosis, indicating that circRNAs can affect physiological processes, such as cell apoptosis, growth, substance metabolism, tissue remodeling, and intercellular connection and communication. Taken together, these findings provide new insights into the role of circRNAs in regulating goat-mammary-gland involution and cellular remodeling.


Few studies evaluated possible treatments to anti-senescence effects. The included studies used in vitro models of senescence in musculoskeletal tissues, providing powerful tools to evaluate age-related changes and pathologies, also contributing to the development of new therapeutic approaches.Ageing is an irreversible and inevitable biological process and a significant risk factor for the development of various diseases, also affecting the musculoskeletal system, resulting from the accumulation of cell senescence. The aim of this systematic review was to collect the in vitro studies conducted over the past decade in which cell senescence was induced through various methods, with the purpose of evaluating the molecular and cellular mechanisms underlying senescence and to identify treatments capable of delaying senescence. Through three electronic databases, 22 in vitro studies were identified and included in this systematic review. Disc, cartilage, or muscle cells or tissues and mesenchymal stem cells were employed to set-up in vitro models of senescence. The most common technique used to induce cell senescence was the addition to the culture medium of tumor necrosis factor (TNF)α and/or interleukin (IL)1β, followed by irradiation, compression, hydrogen peroxide (H Human ageing is an irreversible and inevitable biological process characterized by the physiological deterioration of physical functions and an increasing of pathological conditions starting from the third decade of age ,2. In thAgeing is a significant risk factor for the development of various diseases, also affecting the musculoskeletal system, with factors such as intervertebral disc degeneration (IDD), osteoarthritis (OA), osteoporosis (OP), and sarcopenia. These pathologies result from the accumulation of cell senescence and degenerative stimuli, both from inside the cell and from the microenvironment, contributing to progressive tissue degeneration . Cell seDuring ageing, an imbalance arises between the production of reactive oxygen species (ROS) and the ability to repair tissue damage through endogenous antioxidant defenses , causingTwo modalities of induction of cellular senescence are known: the replicative senescence and the stress-induced premature senescence (SIPS). Cell senescence is caused, in the first case, by telomere attrition after repeated proliferation while, in the second case, by subtoxic doses of extrinsic or intrinsic stimuli such as irradiation, oxidative stress, drugs, oncogenic stress, and metabolic or epigenetic changes .When a cell becomes senescent, its proliferation and replication cease , leadingRecent hypotheses suggest that eliminating senescent cells, possibly using chemical agents like senolytic compounds, can improve tissue homeostasis and delay age-associated pathologies .Over the past decade, in vitro models that simulate specific pathologies or scenarios have gained prominence. These models align with the 3R  principles for animal use for scientific purposes because they are performed using advanced in vitro systems rather than in vivo ones . ActuallIn the literature, some in vitro models are usually employed to induce cell or tissue senescence: (1) primary cells cultured in monolayers under senescence-inducing conditions; (2) three-dimensional (3D) tissue models , which better replicate the native tissue microenvironment compared to cell cultures ; (3) culThe aim of this systematic review was to collect the in vitro studies conducted over the past decade in which cell senescence was induced through various methods. The objective was to evaluate the molecular and cellular mechanisms underlying senescence and to identify treatments capable of delaying senescence.This review investigates different techniques to induce senescence in musculoskeletal cells and tissue cultures, as well as the in vitro methodologies used to assess cell senescence. The perspective of our review is to encourage further research exploring the characteristics of senescent cells and potential therapeutic treatments in this context.For the selection and analysis of the relevant papers, a PICO question (Population of interest (P), Intervention (I), Comparators, and Outcomes (CO)) was formulated.The Population considered was in vitro studies in which musculoskeletal system cells or tissues were employed. The Intervention was all in vitro cultures in which cell or tissue senescence was induced. The Comparator was cells cultured in normal conditions, without the induction of senescence. The considered primary Outcome was cell senescence evaluation through cellular, molecular, biochemical, and histological techniques. In addition, a secondary outcome was the effect of some treatments used to reduce cell senescence when present.The search was performed on 15 July 2023 and included research published from 15 July 2013 to 15 July 2023 according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.Three electronic databases  were used to identify relevant papers using the following MESH with Boolean operators: ((“In Vitro Models”[Mesh]) AND (“Aging”[Mesh] OR “Cellular Senescence”[Mesh])) AND . The limit was the language (English) for all the databases.www.mendeley.com, accessed on 17 July 2023), relevant articles were screened using the title and abstract by two reviewers (FV and DC), and articles that did not meet the inclusion criteria, previously reported, were excluded. The included full-text articles were retrieved and reviewed by the two authors, and any disagreement was resolved through discussion until consensus was reached or with the involvement of a third author (VB). The researchers involved in the process of reviewing the papers used an Excel spreadsheet to independently perform the screening and data extraction. The following information was extracted from each paper to summarize the evidence reported in each study: (a) cells/tissues, (b) in vitro model of senescence, (c) evaluations, (d) main results, and (e) references (Ref.)  .The initial literature search retrieved 151 studies from PubMed, 38 from Scopus, and 23 from Web of Science, for a total of 212 articles. There were 186 identified papers after the removal of duplicates (26 records). Reading the titles and abstracts, 18 papers were excluded, and 168 full texts were evaluated for inclusion. Reviews or book chapters (n = 10), in vivo studies (n = 10), and non-inherent studies because they employed cells from other tissues different from musculoskeletal ones, or because the authors did not induce cell senescence (n = 132), were excluded, for a total of 152 studies. The remaining 16 articles were considered eligible. A further search was performed by reading the reference lists of these eligible articles, and a further six papers were added. Therefore, a total of 22 articles were included in the present systematic review .In the studies included in this review, nucleus pulposus (NP), annulus fibrosus (AF) cells or discs ,28,29,30In vitro cell senescence was evaluated through the assessment of cell proliferation, apoptosis, and cell cycle phases ,36,37,38Most studies 12/22 studies) employed NP or AF cells or discs harvested from rats, pigs, humans, bovines, and rabbits  studies ,28,29,30In four studies, cells or discs were treated with pro-inflammatory cytokines, such as TNFα and/or interleukin1β (IL1β) ,20,21,22More precisely, the addition of both TNFα (20 ng/mL) and IL1β (20 ng/mL) to the culture medium increased the ROS, SA-β-Gal, p16, p53, and degrading enzymes’ gene expressions and reduced cell proliferation, TE activity, and matrix proteins. In addition, this combination also altered the distribution of cells across cell cycle phases; the percentage of cells in G0/G1 phase was higher than that in S phase .Another study focused solely on TNFα, using concentrations of 10 ng/mL for cells and 200 ng/mL for disc cultures. The authors observed reduced cell viability, TE activity, and matrix gene expressions and increased SA-β-Gal activities, ROS levels, and p53 and p16 gene expressions [A study tested different TNFα concentrations  in cell culture, observing dose-dependent decreases in cell proliferation, TE activities, and matrix proteins, as well as dose-dependent increases in p53, p16, and AKT gene expressions. Similar results were observed in groups treated with TNFα for 3 days with 3 days of recovery or with six consecutive days of treatment .Finally, a 3D co-culture model was set-up with NP cells, cultured in presence of TNFα (20 ng/mL), and calcium alginate gel balls, empty or containing normal BMSCs or NP cells.The addition of TNFα increased SA-β-Gal activities in NP cells, whereas the addition of balls with BMSCs reversed the senescence phenotype. This intervention reduced MMP9 protein expression and p16, p21, and p53 gene expressions and increased zinc metallopeptidase STE24 (ZMPSTE24) protein levels .Four studies induced senescence in discs, NP, and AF cells through intermittent, dynamic, or static compression and elongation ,24,25,26Discs were loaded in bioreactors with intermittent compression at two different intensities (0.1 and 1.3 MP), showing higher SA-β-gal activities, elevated expressions of p16, p21, p53, catabolic enzymes, and p38 proteins, and reduced matrix proteins and tissue inhibitors of metalloproteinase TIMP3 gene expression at 1.3 MP than at 0.1 MP .In addition, cells that underwent 20% dynamic compression (1.0 Hz for 6 h/day) had reduced proliferation, increased SA-β-Gal activity, and expressions of p53, p16, nuclear factor kappa-light-chain-enhancers of activated B cells (NF-κB), and ROS proteins . One study compared static and dynamic compression  in disc culture, observing that static compression led to greater SA-β-gal, p16, and p53 protein expressions and reduced TE activity and matrix proteins compared to dynamic compression. Notably, dynamic compression seemed to revert the senescent phenotype .Moreover, two different magnitudes (5% and 20% elongation) of mechanical tension (1 Hz for 8 h/day for 12 days) were applied to AF cells. Cell proliferation, TE activity, S phase, matrix, and BCL1 and LC3 proteins were significantly reduced, whereas G0/G1 phase and p16 and p53 proteins were significantly increased, with 20% more elongation than with the 5% one . 2O2) added to NP cells at different concentrations (from 0.05 to 2 mM) increased ROS levels, SA-β-Gal activities, p53, p21, and p16 protein expressions, and pro-inflammatory cytokines’ gene expressions, accompanied by reduced cell viability and proliferation in a dose-dependent manner [In two studies, hydrogen peroxide  [Finally, two studies irradiated human and rat NP or AF cells with γ-rays ( Gy/min) ,30. In t Gy/min) .2O, and 20% O2, whereas the second consisted of serum-free 0.9 mg/mL glucose, 400 mOsm/Kg H2O, and 2% O2. Both media increased p16, p21, and MMP gene expressions and reduced collagen II (COLL II) gene expression. Additionally, the first medium also increased SA-β-Gal activity and reduced cell proliferation [In the second study, irradiations were given to cells cultured in two different media: the first composed by 10% fetal bovine serum (FBS), 4.5 mg/mL glucose, 300 mOsm/Kg HThe second larger group of studies (6/22 studies) employed chondrocytes obtained from humans ,32,34,36Three of these studies evaluated the effects of irradiations on chondrocyte senescence ,32,33.The results revealed that γ-rays (at 10 Gy) induced SA-β-gal, p16, and yH2AX proteins and that the addition of transforming growth factor beta 1 (TGFβ1) and basic fibroblastic growth factor (bFGF) further increased SA-β-gal, p16, yH2AX proteins, insulin-like growth factor binding protein 3 (IGFBP3), and C-C motif chemokine ligand 2/monocyte chemoattractant protein 1 (CCL2) gene expressions . SimilarFurthermore, a study compared the effects of monoenergetic C-ions (75–95 MeV/mm) with X-rays (225 kV) on chondrocytes cultured in a monolayer or in a collagen scaffold . The autTwo studies treated chondrocytes with IL1β (10 ng/mL) ,35. The Finally, micromasses of chondrocytes harvested from severely damaged cartilage areas displayed lower cartilage matrix depositions, telomere lengths, matrix gene expressions, and higher MMP13, p16, p21, and p53 protein levels than chondrocytes isolated from preserved cartilage areas .Other studies 3/22 studies) used C2C12 cell lines 2 studies or purchMyotubes derived from C2C12 cells were treated with 10% serum from older donors, showing lower myotube diameters and decreased nuclear factor I (NFI), AKT, p70S6K1 and eukaryotic translation elongation factor 2 (eEF2) proteins than myotubes cultured in sera from younger subjects . Similar−3 G; µG), or normal gravity at the fifth passage. Microgravity decreased myosin heavy chain (MHC) and desmin proteins, cell proliferations, and myotubes’ diameters, whereas it increased the myogenic factor 5 (Myf5) protein. Both microgravity exposure and the fifth in vitro passage led to increased SA-β-Gal activity [Finally, human myoblasts at the first passage were cultured under normal gravity (1 G), microgravity  added to NPMSCs cultures increased senescent cell morphologies, SA-β-Gal activities, and p16, p21, p53, IL6, MMP13, and ADAMTS5 gene expressions .As shown in In the context of senescent NP cells, treatment with resveratrol  was administered. This intervention resulted in reductions in ROS, SA-β-gal activities, expressions of p16, p53, catabolic enzymes, and NF-κB proteins, as well as a decrease in cells present in the G0/G1 phase. Additionally, resveratrol increased cell proliferation, TE activity, matrix proteins, and cells in the S phase ,24.−7 M) enhanced cell proliferation, TE activity, matrix protein levels, and reduced SA-β-Gal activity, ROS levels, as well as p53 and p16 protein expressions [In another study, treating senescent NP cells with 17-beta-estradiol (E2) -rich NP explants increased proliferations, expressions of carbonic anhydrase 12 (CA12), FOXF1, Pax-1, and matrix proteins, and decreased COLL I protein levels of senescent NPMSCs .Finally, senescent chondrocytes were cultured in the presence of 100 mM 4-phenylbutyric acid (4-PBA). The authors observed a decrease in GRP78 protein levels, SA-β-gal activity, cell apoptosis, and an increase in cell viability after treatment .This systematic review collected and analyzed in vitro studies from the literature that evaluated the existing in vitro models used to induce senescence in musculoskeletal cells and tissues over the last 10 years.Generally, an in vitro environment can provide information about cell–cell and cell–extracellular matrix interactions, expressions of surface receptors, and syntheses of proteins and growth factors. By increasing the in vitro model’s complexity, we are able to represent and recapitulate the characteristics of many pathologies through the use of cells or tissues from affected subjects. Moreover, complex in vitro models are functional for the gathering of preliminary data on the safety and cytocompatibility but also on the bioactivity and therapeutic efficacy of newly developed materials, medical devices, or therapies .In the present systematic review, 22 studies were found, and most of them regarded cells or tissues obtained from intervertebral disc (IVD) ,28,29,30Almost all the cells and tissues utilized in these studies were harvested from healthy animals 15/22) or human subjects (6/22). Rats were the most employed donors of cells  or human,29,33,35Primary human cells were collected from IVD or cartilage of healthy subjects ,29,34, fAt last, purchased cell lines of humans and mice were also employed, and they were chondrocytes , C2C12 cIn the studies of the present review, the above-mentioned cells and tissues were cultured prevalently in two-dimensional 2D) monolayers D monolay,37,38,39These cells and tissues were induced to senescence by various methods , each ofTNFα and IL1β are the two inflammatory cytokines often used to recreate an inflammatory microenvironment in some in vitro models of tissue degeneration, including OA and disc degeneration ,42,43. ISecondly, NP or AF cells ,30 and cTheir use to induce senescence in cells is based on ionizing radiation, such as γ-irradiation, which is a principal DNA-damaging agent , and C-iThen, intermittent, dynamic, or static compressions of discs and NP cells ,24,25 an2O2 at different concentrations [Other authors cultured NP cells with Htrations ,28 and mtrations .2O2 is a commonly used inducer of stress-induced premature senescence and DNA damage, given its role as a natural inducer of oxidative stress [2O2 treatment creates a short-period senescent phenotype, which leads to rapid apoptosis and cell death in days and easily triggers cell apoptosis rather than cell senescence. Indeed, He stress . HoweverThe ageing process is associated with changes in circulating endocrine factors, such as growth factors hormones and pro-inflammatory cytokines, which are known to moderate muscle mass or the Klotho factor . UnfortuOne study employed microgravity on myoblasts  because A study proposed to create a senescence phenotype in BMSCs by an extensive in vitro expansion up to passage 10 , the so 2O2 or sera, and microgravity are part of the first group, whereas the addition of inflammatory cytokines and several in vitro passages expansion belong to the second group. To summarize, the above-mentioned in vitro models of senescence can be essentially divided into two groups: stress-induced premature senescence and replicative senescence. Irradiation, mechanical stimuli, the addition of HSeveral different techniques, both cellular and molecular ones, were employed to evaluate cell senescence. The technique of choice for the evaluation of senescence in vitro remains the assessment of cell proliferation, viability, and apoptosis ,36,38,39Then, the production  ,37,38,39Last but not least, some studies also evaluated TE activity ,24,25,26TE is a protein that adds a telomere repeat sequence to the 3′ end of telomeres, regions of repetitive sequences at each end of the chromosomes that protect the end of the chromosome from DNA damage. During aging, TE activity is reduced, causing a shortening of telomere . Its actAs the phenotype of senescent cells displays variability, a single unambiguous biomarker for the identification of senescent cells has not been recognized yet. A combination of multiple biomarkers is usually accepted to distinguish between senescent and quiescent or terminally differentiated cells, reflecting the dynamicity of the senescence process. In addition, some biomarkers are not specific to senescent cells and can be present also in apoptotic cells or quiescent cells, for example. This represents a limitation in the isolation and characterization of senescent cells and a challenge for future research.The purpose of performing in vitro studies with senescence-induced cells is to find and test agents with the ability to counteract senescence.In the present review, just a few studies (5/22 studies) evaluated possible treatments to that effect: resveratrol ,24 and EResveratrol is a natural phenol produced by several plants in response to injury, such as attack of bacteria or fungi, and shows protective effects, including anti-inflammatory and anti-aging properties . In the As NP cells contain estradiol receptor (ER) , it was Finally, 4-PBA, which alleviates ER stress in both cell lines and animal models , reducedThese in vitro models provide a controlled and reproducible environment to investigate the cellular and molecular changes associated with senescence, ultimately contributing to our understanding of age-related musculoskeletal diseases and potential therapeutic interventions. However, it is important to note that in vitro models have their limitations, and findings from these models must be further validated using in vivo studies for translations to human health.Indeed, studying senescence in musculoskeletal tissues and cells using in vitro models has still some limitations, which can impact the translatability and relevance of the findings to the complex in vivo environment. Some of these limitations include the lack of: (1) tissue complexity, which can lead to oversimplification of the biological processes involved in senescence; (2) cellular heterogeneity; (3) mechanical loading; (4) immune system interactions; and (5) standardized protocols.In addition, a short-term culture may not capture the long-term effects and progression of senescence observed in aging musculoskeletal tissues and may not fully capture the genetic and epigenetic changes that occur during aging and senescence in vivo.Many senescence studies use immortalized cell lines, which may not fully represent the senescence process in primary cells. Using primary cells more often can provide more physiologically relevant insights into senescence mechanisms.Developing 3D models for senescence studies can provide more physiologically relevant data, and the combination of genomics, transcriptomics, proteomics, and metabolomics can provide a comprehensive view of senescence.While in vitro models are valuable, they should be validated in animal models to confirm the relevance of findings in vivo. This step is critical for translating laboratory discoveries into potential clinical applications.However, in vivo and clinical studies have their advantages and limitations, and they complement each other in aging research. The advantages in the in vivo models concern complexity, the possibility to perform longitudinal studies, and genetics, whereas the limitations regard species differences, ethical concerns, and high costs and time consumption. The advantages of human aging studies are their direct relevance, ethical considerations, and the possibility to perform clinical observations, but the main limitations are the ethical and practical constraints; the fact that long-term human aging studies can be time consuming, expensive, and subject to dropouts; and genetic diversity.In summary, in vitro models are useful for controlled experiments and initial insights, animal models allow for more complex biological contexts and genetic manipulation, and human studies are essential for understanding human-specific aging processes and interventions. A combination of these approaches, along with advanced computational modeling, can provide a more comprehensive understanding of the aging process and improve the development of strategies to promote healthy aging.Studies included in the present systematic review used in vitro models of senescence in musculoskeletal tissues, providing powerful tools to analyze the complex processes of aging in musculoskeletal tissues and to provide valuable insights into age-related changes and pathologies, ultimately contributing to the development of new therapeutic approaches to mitigate the effects of aging on musculoskeletal health.The cells and tissues that were mostly investigated were those obtained from IVD, followed by cartilage and muscle tissues.2O2 was relatively limited.The most employed method to induce cell senescence was the addition of inflammatory cytokines in the culture medium, followed by irradiation and compression. The addition of HUnfortunately, there are still few studies focused on therapeutic treatments including resveratrol, 4-PBA, and E2; therefore, it would be desirable that there will be more in vitro studies investigating therapeutic treatments in future research.


Streptomyces strains (378 and 549) that produce compounds triggering DNA damage were prioritized for subsequent chemical investigations. Metabolomics profiling involving HPLC-UV/vis, UPLC-QTOF-MS/MS, and molecular networking identified three types of bioactive metabolites belonging to the aromatic polyketide family, i.e., cosmomycin, kidamycin, and hedamycin. In-depth analysis of the metabolomic data unveiled some potentially novel anthracycline compounds. A genome mining study based on the whole-genome sequences of strains 378 and 549 identified gene clusters potentially responsible for cosmomycin and kidamycin biosynthesis. This work highlights the effectiveness of combining metabolomic and genomic approaches to rapidly identify bioactive chemicals within microbial extracts. The saline lakes on the Northern Tibetan Plateau present prospective sources for discovering novel actinobacteria and biologically active compounds.The Tibetan Plateau, known as the “Roof of the World” and “The Third Pole”, harbors numerous saline lakes primarily distributed in the Northern Tibetan Plateau. However, the challenging conditions of high altitude, low oxygen level, and harsh climate have limited investigations into the actinobacteria from these saline lakes. This study focuses on investigating the biodiversity and bioactive secondary metabolites of cultivable actinobacteria isolated from the sediments of four saline lakes on the Northern Tibetan Plateau. A total of 255 actinobacterial strains affiliated with 21 genera in 12 families of 7 orders were recovered by using the pure culture technique and 16S rRNA gene phylogenetic analysis. To facilitate a high-throughput bioactivity evaluation, 192 isolates underwent OSMAC cultivation in a miniaturized 24-well microbioreactor system (MATRIX cultivation). The antibacterial activity of crude extracts was then evaluated in a 96-well plate antibacterial assay. Forty-six strains demonstrated antagonistic effects against at least one tested pathogen, and their underlying antibacterial mechanisms were further investigated through a dual-fluorescent reporter assay . Two  Actinobacteria produce the majority of naturally occurring antibiotics, and approximately two-thirds of naturally derived antibiotics in current clinical use were produced by actinobacteria ,2. As maNitriliruptorales + ions at mass-to-charge ratios (m/z) of 1189.5878, 1173.5925, 772.3516, and 756.3598, respectively. Based on their spectral properties and comparing them to literature data, they were putatively identified as cosmomycin D (1), cosmomycin C (2), cosmomycin B (3), and cosmomycin A (4), respectively. Moreover, the characteristic UV/Vis absorption bands at about 233, 246, 294, 497, and 530 nm for compounds 1–4 were consistent with those of previously reported cosmomycins [m/z value and fragmentation pattern with reference data [m/z 595.3 [M + 2H]2+ and another in lower abundance at m/z 1189.6, corresponding to the molecular ion [M + H]+. The dissociation products from cosmomycin D were: 1075.5 ; 945.3 ; 831.3 ; 701.3 ; and 544.3 . The dissociation products from 1173.3 for cosmomycin C were 1059.1 (–114) → 945.1 (–114) → 831.2 (–114) → 701.2 (–130) → 544.3 (–157). Cosmomycin C revealed a similar fragmentation pathway with cosmomycin D, except for the first product, 1059.1 (–114), which was in correspondence with the middle sugar in one trisaccharide chain lacking a hydroxyl group. It is worth noting that cosmomycins have been previously reported to possess antibacterial activities against gram-positive bacteria, show significant antitumor activity against human tumors, and can induce the differentiation of Friend leukemia cells + 1171.5754, C60H87N2O21, 8) [m/z [M + H]+ 1171.5754, C60H87N2O21, 9) [m/z [M + H]+ 1157.5942, C60H89N2O20, 10) + ion at m/z 740.3627 that displayed a neutral loss of oxygen (16 Da). Despite a thorough search within the database, no match was found for compound 15, indicating it is possibly a novel compound. However, these potential novel compounds of the cosmomycin family were only detected in trace amounts during the examination of the original chromatogram data of strain 378.Cluster B, consisting of 6 nodes with precursor masses ranging from 6) was found to be clustered with 25 nodes with precursor masses ranging from m/z 642 to 733 Da. Starting with the kidamycin, six additional derivatives of kidamycin were identified, including the 14,16-epoxy analogue of kidamycin  [m/z [M + H]+ 687.3245, C39H47N2O9, 17), saptomycin C1  [m/z [M + H]+ 719.3541, C41H55N2O9, 19) [m/z [M + H]+ 731.3520, C41H51N2O10, 20) [m/z [M + H]+ 733.3668, C41H53N2O10, 21) [16–21) are illustrated in In the molecular network of the kidamycin family  , dehydro2O9, 18) , dihydro2O9, 19) , 3”-O-acO10, 20)  and rubiO10, 21) . UPLC-MSThe draft genome sequences of strains 378 and 549 were analyzed via the bioinformatic tool antiSMASH7.0 to determine their putative biosynthetic capabilities. For stain 378, a total of 30 putative BGCs were detected, including three types of polyketide synthases , non-ribosomal peptide synthase (NRPS), class i-lanthipeptides, butyrolactone, etc. . Among tStreptomyces sp. SCSIO 1666, 97% with cosmomycin D BGC and 92% with cosmomycin B BGC from Streptomyces olindensis, 76% with cosmomycin C BGC from Streptomyces sp. CNT302, 85% with cinerubin B BGC from Streptomyces sp. SPB074, and 91% with rhodomycin BGC from Streptomyces purpurascens , a chain length factor (CLF), and an acyl carrier protein (ACP), which starts with a propionyl-CoA, as well as a gene at ctg1_79 encodes the putative propionyl-CoA: ACP acyltransferase, all of which is responsible for the C-21 backbone formation. In addition, there are three genes at ctg1_92, ctg1_95, and ctg1_105 encoding cyclases, three genes at ctg1_94, ctg1_96, and ctg1_110 encoding ketoreductase, three genes at ctg1_102, ctg1_104, and ctg1_106 encoding methyltransferse, one gene at ctg1_101 encoding monooxygenase, three genes at ctg1_77, ctg1_86, and ctg1_98 encoding glycosyltransferase genes, and a series of genes responsible for the generation of sugar units, all of which are involved in the tailoring process of bioactive metabolites from strain 378. As shown in As outlined above, strain 378 was found to produce cosmomycins that belong to the anthracycline group. AntiSMASH analysis revealed that strain 378 harbors a predicted T2PKS BGC, which exhibits 84% similarity with the cytorhodin BGC from urascens . All theStreptomyces griseoruber  were added to the isolation media. Interestingly, a large number of actinobacteria were isolated using media without NaCl. Possible reasons for this inconsistency could be due to the fact that many halotolerant actinobacteria can tolerate a wide range of salinities and grow well in the absence of NaCl, or that the surface layer of sediment samples has a higher salinity than other parts of the sample. In the MATRIX cultivation step, inhibition activities were observed more frequently in the medium FM4. Medium FM4 contained versatile nitrogen sources such as peptone, yeast extract, and soybean meal, with soybean meal as a slowly-utilizing nitrogen source contributing to prolonged secretion periods and increased yield of antibiotic products [w/v) to the FM4 medium may stimulate the growth and metabolism of actinobacteria in the saline environment.It is quite essential to utilize various media for bioprospecting the untapped actinobacterial resources. In the isolation process, eight different culture media were applied, and the modified PYG agar medium (M1) showed the highest recoverability and genera diversity. This finding suggests that M1 is suitable for isolating actinobacteria from saline lake sediments. Supplementing betaine and glycerol in M1 may enhance the microbial antioxidative defense system against salinity stress and contribute to better isolation of rare actinomycetes, as demonstrated in other studies ,103. To products . AdditioStreptomyces strains were comprehensively analyzed with multi-omics strategies. Two types of metabolites belonging to the aromatic polyketide family were unambiguously identified, including anthracyclines (cosmomycin) and angucyclines (kidamycin and hedamycin). The crude fractions extracted from both strains 378 and 549 exhibited an orange-red/red-purple color, displaying absorption within the visible light range of 400–500 nm. The chromophore responsible for this distinctive coloration originates from the tetracyclic anthracycline aglycone structure. This unique visible absorption spectrum can serve as a distinguishing marker or probe for the screening of anthracycline and angucycline compounds. Notably, the bacteriostatic effects of cosmomycin, kidamycin, and hedamycin have been reported to arise from their ability to intercalate with DNA in major and minor grooves, inducing DNA breaks via topoisomerase II poisoning [Multi-omics approaches have recently made big strides toward the effective exploration of microorganisms, accelerating the discovery of new bioactive compounds. In this research, bioactive components from two oisoning ,107,108.Streptomyces are inclined to be dismantled by programmed cell death (PCD)-like mechanisms, thereby releasing nutrients to accelerate the buildup of the aerial hyphae [Interestingly, these aromatic polyketide antibiotics may have a potential biological function in the life cycle of their producing strains inhabiting adverse environments, such as hypersaline environments. Under poor nutritional conditions, the vegetative or substrate mycelia of l hyphae . This phl hyphae ,111. On l hyphae ,113. TheStreptomyces strains. Furthermore, an in-depth analysis of the metabolomic data leads to the detection of some potentially novel anthracycline compounds, which highlights the effectiveness of metabolomics profiling and molecular networking in guiding the discovery of novel bioactive candidates. This study not only provides pioneering insights into the pharmaceutical potential of actinobacteria associated with saline lakes on the Northern Tibetan Plateau but also presents a practical approach for the efficient chemical dereplication of the complicated secondary metabolites produced by microorganisms.The current investigation has demonstrated that the saline lakes located on the Northern Tibetan Plateau represent a promising source of diverse actinobacterial strains for the production of bioactive metabolites. By implementing combinational strategies such as MATRIX cultivation, dual-fluorescent reporter assays, UPLC-MS/MS-based metabolomic analysis, molecular networking, and genome mining, we have successfully identified the aromatic polyketide antibiotics accountable for the antibacterial activities exhibited by two It is also important to acknowledge that this study has several limitations that warrant consideration. Firstly, the findings of this study are based on a limited number of sediment samples collected from four specific saline lakes located on the Northern Tibetan Plateau. Fluctuations in bacterial communities across different sampling locations and seasons are not investigated in this study. The generalizability of these findings to other geographical regions or different types of saline lakes on the Northern Tibetan Plateau remains uncertain. Secondly, although the OSMAC strategy is employed in this study, the selected culture conditions are far from exhaustive. To comprehensively explore the synthetic metabolic potential of the isolated strains, alternative strategies can be pursued. These may include the adjustment of salt ion composition and concentration in the culture medium, as well as the incorporation of various small-molecule compatible solutes, enzyme inhibitors, or growth factors. Thirdly, while this study detects potentially novel anthracycline compounds, further endeavors are needed to achieve precise chemical characterization and structural elucidation of these compounds, including NMR spectroscopy and other sophisticated analytical methods. Through this study, we hope to encourage more concerted efforts toward exploring this pristine and unexploited environment and discovering new actinobacterial species and prominent antibiotic candidates.


Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease are among the most common liver diseases worldwide, and there are currently no Food and Drug Administration (FDA)-approved treatments. Recent studies have focused on lifestyle changes to prevent and treat NAFLD. Omega-3 supplementation is associated with improved outcomes in patients with chronic liver disease. However, it is unclear whether Omega-3 supplementation can prevent the development of liver disease, particularly in individuals at an increased (genetic) risk.In this UK Biobank cohort study, we established a multivariate cox proportional hazards model for the risk of incident liver disease during an 11 year follow up time. We adjusted the model for diabetes, prevalent cardiovascular disorders, socioeconomic status, diet, alcohol consumption, physical activity, medication intake , and baseline characteristics.p = 7.6 × 10−9). This protective association was particularly evident for alcoholic liver disease , liver failure , and non-alcoholic liver disease . Interestingly, we were able to replicate the association with reduced risk of NAFLD in a subset with liver MRIs . In particular, women benefited from Omega-3 supplementation as well as heterozygous allele carriers of the liver-damaging variant PNPLA3 rs738409.Omega-3 supplementation reduced the risk of incident liver disease (HR = 0.716; 95% CI: 0.639, 0.802; Omega-3 supplementation may reduce the incidence of liver disease. Our study highlights the potential of personalized treatment strategies for individuals at risk of metabolic liver disease. Further evaluation in clinical trials is warranted before Omega-3 can be recommended for the prevention of liver disease. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease are two of the most prevalent liver diseases worldwide, affecting millions of people across the globe –3. DespiFor our study, we used the UK Biobank Resource with application number 71300. The study was approved by the Northwest Multicenter Research Ethics Committee and conducted in accordance with the Declaration of Helsinki and Istanbul after written informed consent was obtained. The UK Biobank (UKB) is a multicenter research study with 22 participating centers. Enrolment studies were conducted between 2006 and 2010. The respective date was set as baseline. Death or end of data collection in May 2021 was defined as the end of follow-up. After informed consent was obtained for clinical data collection and genotyping, a total of 502,511 individuals aged 37 to 73 years were enrolled in the study. The diagnoses included in our study were coded using the ‘International Classification of Diseases and Related Health Problem' (ICD-10). The latter were assigned to the patients and continuously updated via the hospital admission code. Death data were collected in the UKB via the National Death Register, including the leading diagnosis of death and the date of death.Briefly, Omega-3 supplementation was tracked in a questionnaire and validated by lipidomic data, which were measured in 96,701 individuals by nuclear magnetic resonance. We included participants without any baseline diagnosis of liver disease  and performed an analysis of reported Omega-3 intake with new diagnosis of liver disease over the 11-year study period. Detailed information can be found in Supplementary material .We excluded individuals with any diagnosis of pre-existing liver diseases , HIV infection (B20-B24), chronic hepatitis (B18) or missing body mass index (BMI) data . We alsoFurthermore, patients were excluded from the multivariate cox proportional hazards model. Patients who reported being unable to walk  were considered as “0 days/week walked”. Missing data for vegetable, fruit, fish and meat intakes were excluded. Likewise, missing data for alcohol consumption (in g/d) and socioeconomic status (Townsend Index) were excluded .Omega-3 intake was recorded using a numerical code . We used10(p/59) >3.1 were considered significant  rs738409, transmembrane 6 superfamily member 2 (TM6SF2) rs58542926, hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) rs72613567, and mitochondrial amidoxime reducing component 1 (MTARC1) rs2642438. We examined wildtype, heterozygous, and homozygous minor allele carriers.The UKB provided genetic analyses of 488,377 individuals. We investigated carriers of known liver-associated variants. Variants examined were patatin-like phospholipase domain-containing protein 3 (The primary outcome was the development of incident liver disease (K70-K77) or a new diagnosis of hepatocellular carcinoma (C22.0) after baseline. As a secondary outcome, we replicated the results using multivitamin supplements, vitamin C, or vitamin B12.Covariates included in the multivariate cox proportional hazards model were age, sex, BMI, ethnicity, diabetes mellitus, hypertension, ischaemic heart disease, dyslipidemia, number of medications taken, as well as intake of insulin, biguanides, statins and aspirin. In addition, we integrated physical activity with “Number of days/week walked”, “Number of days/week of moderate physical activity”, and “Number of days of vigorous physical actitivity”. We further included nutritional factors with the help of daily vegetable and fruit consumption, and weekly consumption of fish and meat. In addition, alcohol consumption (g/d) was included as a covariate to account for alcohol consumption below the pathological cut-off. We determined socioeconomic status using the Townsend Index as a covariate .p-value < 0.05. Statistical analyses and graphical visualization were performed using R version 4.1.2 , SPSS Statistics version 27 , Prism version 8.0.1 , and yEd Graph Editor version 3.21.1.In the multivariate cox proportional hazards model, we considered influencing confounders as covariates. We included the covariates: Age, sex, BMI, ethnicity, diabetes mellitus, hypertension, ischemic heart disease, dyslipidemia, number of medications taken, insulin, biguanides, statins, aspirin, “number of days/week walked”, “number of days/week of moderate activity”, “number of days/week of vigorous activity”, daily fruit and vegetable consumption, weekly fish and meat consumption, daily alcohol consumption in g/d, and the Townsend index as a socioeconomic factor. Results are expressed as mean ± standard deviation (±SD). The 95% confidence intervals (CI) are given in parentheses for the hazard ratios (HR). Statistical significance was set at A total of 411,156 individuals were included, 373,035 participants without intake of Omega-3 (non-users) and 38,121 with regular  intake of Omega-3 (Omega-3 users) . First, p = 7.6 × 10−9) (p = 4.3 × 10−3) as well as hepatic failure  were significantly reduced in Omega-3 users. In addition, we found a risk reduction for “other diseases of liver” (K76), including NAFLD . We replicated this association in a subgroup of UKB patients who underwent MRI. Here, Omega-3 intake was associated with less MRI-confirmed steatosis   . In part × 10−4) .p = 1.4 × 10−4), whereas women showed a 28.5% reduction   .It is important to consider potential confounding factors, such as other supplements or medications, that may have an impact on the outcome being studied, to improve the accuracy of the results and draw more valid causal inferences. Therefore, we studied the intake of multivitamins, vitamin C, or vitamin B12 nutritional supplements in the UKB. Vitamin C and vitamin B12 have shown associations with liver health in previous studies –15. NevePNPLA3 and TM6SF2 have been shown to increase the risk and severity of NAFLD, whereas single nucleotide polymorphisms (SNPs) in MTARC1 and HSD17B13 have protective effects  . Similar results were obtained for MTARC1 rs2642438, suggesting additive hepatoprotective effects or potential synergistic effects (Common genetic variants in  effects –19. Simi × 10−3) . Importa effects . Additio effects .p = 8.0 × 10−3). Stratified analysis by gender revealed a significant association in women , but not in men.Finally, we examined the individuals who received an analysis of the lipidomic data and evaluated the differences between Omega-3 users and non-users in more detail . The incDietary modification is a cornerstone in the treatment of NAFLD. However, research on how targeted dietary interventions can be used for primary prevention is limited. This study aimed to investigate the relationship between Omega-3 fatty acid consumption and the development of liver disease in a large and diverse population-based cohort, including both non-alcoholic and alcoholic liver disease. We found that regular Omega-3 fatty acid consumption was associated with a significant risk reduction in liver disease development, particularly for (non-)alcoholic liver disease.Notably, our investigation included a comprehensive analysis of both general and genetic risk factors for liver disease, and we verified the participants' self-reported Omega-3 intake using lipidomic data.Our findings indicate that regular Omega-3 fatty acid consumption is associated with a significant reduction in the risk of liver disease, particularly NAFLD. This is consistent with previous studies demonstrating the potential benefits of Omega-3 supplementation in treating NAFLD , 8. We fPNPLA3 rs738409 minor allele carriers, regular Omega-3 intake showed a benefit. Minor allele carriers of the rare variant TM6SF2 rs58542926, which is harmful to the liver (HSD17B13 and MTARC1 variants, which have been linked to lower rates of NAFLD (MTARC1 rs2642438 and HSD17B13 rs72613567. Interestingly, our data indicate an additive synergistic effect of Omega-3 supplementation and protective variants of HSD17B13 and MTARC1. Replication of the results in the subgroup of individuals with lipidomic data confirmed this to a large extent, however, for some genetic variants the number of cases was too small to draw a conclusion. To prevent the lack of significance due to a small sample size, analyzing larger cohorts is recommended, particularly for homozygous minor allele carriers.In the UKB, we developed a unique approach to study the interaction between genetics and Omega-3 intake. Especially for heterozygous he liver , did notof NAFLD , 21, shoOne explanation for the hepatoprotective association between Omega-3 intake and liver disease may be that Omega-3 fatty acids improve insulin sensitivity, which is strongly associated with protection against NAFLD . In addiThis is the largest study to date to demonstrate a primary preventive effect in a prospective and well-characterized cohort. Moreover, gene-dietary interactions have not been studied at a population-based level before and this research may uncover personalized treatment strategies for individuals at risk for metabolic liver diseases. Deciphering gene-environment interactions in metabolic liver disease holds promise for the development of patient-tailored dietary strategies. Nevertheless, our study has limitations. First, we used ICD-10 diagnoses as outcomes in our study, which were continuously updated. In spite of this, we confirmed the negative association between Omega-3 supplementation and hepatic steatosis using MRI data. However, we recommend the replication of genetic findings in a larger cohort. Second, the frequency of Omega-3 intake was not available for analysis except for the statement that it was taken regularly. We attempted to mitigate these limitations by assessing the blood metabolites indicative of Omega-3 use which strongly supports the reported intake . FurtherOur study raises the question of whether Omega-3 supplementation should be recommended to people at a high risk of liver disease. Supplementation with Omega-3 fatty acids was particularly beneficial for women. However, these associations need to be confirmed in randomized trials before recommending Omega-3 for protection against liver disease.In conclusion, this study demonstrated the primary preventive associations of Omega-3 supplementation with the development of incident liver disease, which warrants further evaluation in clinical trials.https://www.ukbiobank.ac.uk.The data analyzed in this study is subject to the following licenses/restrictions: approved registration at UK Biobank is required. Requests to access these datasets should be directed to The studies involving human participants were reviewed and approved by the Northwest Multicenter Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.MV: conceptualization, methodology, software, formal analysis, writing—original draft, and validation. KC and ES: resources, writing—review and editing, and validation. KSS, LH, and MR: writing—review and editing. KMS: conceptualization, writing—review and editing, and funding acquisition. CS: conceptualization, software, writing—review and editing, supervision, funding acquisition, and validation. All authors contributed to the article and approved the submitted version.


ARF genes have not been investigated in flax (Linum usitatissimum L.), an important oilseed and fiber crop. In this study, we comprehensively analyzed the ARF gene family and identified 33 LuARF genes unevenly distributed on the 13 chromosomes of Longya-10, an oil-use flax variety. Detailed analysis revealed wide variation among the ARF family members and predicted nuclear localization for all proteins. Nineteen LuARFs contained a complete ARF structure, including DBD, MR, and CTD, whereas the other fourteen lacked the CTD. Phylogenetic analysis grouped the LuARFs into four (I–V) clades. Combined with sequence analysis, the LuARFs from the same clade showed structural conservation, implying functional redundancy. Duplication analysis identified twenty-seven whole-genome-duplicated LuARF genes and four tandem-duplicated LuARF genes. These duplicated gene pairs’ Ka/Ks ratios suggested a strong purifying selection pressure on the LuARF genes. Collinearity analysis revealed that about half of the LuARF genes had homologs in other species, indicating a relatively conserved nature of the ARFs. The promoter analysis identified numerous hormone- and stress-related elements, and the qRT-PCR experiment revealed that all LuARF genes were responsive to phytohormone  and stress  treatments. Finally, expression profiling of LuARF genes in different tissues by qRT-PCR indicated their specific functions in stem or capsule growth. Thus, our findings suggest the potential functions of LuARFs in flax growth and response to an exogenous stimulus, providing a basis for further functional studies on these genes.Auxin response factors (ARFs) are critical components of the auxin signaling pathway, and are involved in diverse plant biological processes. However,  AUX/IAA) family, auxin response factor (ARF) family, gretchenhagen 3 (GH3) family, and small auxin upregulated RNAs (SAURs) . Th. ThLuARFectively . These oFurther analysis based on Pfam and CDD databases indicated that 19 out of 33 LuARF proteins had the typical ARF structure composed of a conserved DBD domain (B3 and AUX_RESP), a variable MR domain, and a CTD (Aux/IAA), while the remaining 14 lacked the CTD . AnalysiL. usitatissimum, G. hirsutum, A. thaliana, G. max, Vitis vinifera, Eucalyptus grandis, and Medicago truncatula) and 3 from monocotyledons  to assess the evolution among the ARFs of diverse species  underwent tandem duplication (TD) and another eighteen gene pairs  underwent segmental/whole-genome duplication (SD/WGD) (a) and synonymous (Ks) values and used the Ka/Ks ratios to reveal the selective forces on the duplicated LuARF gene pairs. In general, Ka/Ks > 1 indicates a positive selection, Ka/Ks = 1 indicates a neutral evolution, and Ka/Ks < 1 shows a strong purifying selection [s values of these collinear genes varied from 0.05 to 0.23, implying that the duplication events happened close to the recent WGD of flax (Ks = 0.13) [s values of LuARF15/32 and LuARF20/32 gene pairs were about 0.7, indicating that the duplication happened close to the early WGD event (Ks = 0.77), and the divergence time was estimated as 57–60 Mya. In addition, all duplicated gene pairs’ Ka/Ks ratios were less than 0.4, which suggested a strong purifying selection pressure on the LuARF genes.We then analyzed the duplication events to elucidate the mechanism underlying (SD/WGD) . The gen(SD/WGD) . Furtherelection . We foun = 0.13) . The divARF genes, the collinearity relationships between flax and nine species (six dicots and three monocots) described above were analyzed . For example, LuARF13 and 24 had up to five syntenic gene pairs in cotton, and LuARF4, 13, and 25 had five pairs in barrel medic, indicating that these genes remained highly conserved during evolution. Also, LuARF4 and 13 had homologous genes in all the selected species, suggesting that these gene pairs might share a common ancestor and existed before the monocotyledon-dicotyledon split. LuARF25 had orthologs in all species except for Arabidopsis, while LuARF24 had orthologs in seven plants. Except for LuARF4, 13, 24, and 25, all other genes lacked orthologs in monocots but had homologs in dicots, implying that most genes might have appeared after the divergence of dicotyledons and monocotyledons.Further, to explore the evolution of analyzed . The anacis-acting elements in the promoter regions are essential in regulating the transcription of genes. To determine the potential function of LuARF genes, the promoter sequences of all LuARFs were analyzed using the PlantCARE database [LuARF promoters and a few basic elements (TATA-box and CAAT-box) . The number of hormone response elements in each LuARF ranged from 4 to 12, implying an uneven distribution pattern. LuARF1, 2, 6, 8, 10, 13, and 32 had all five phytohormone-related elements, while LuARF20 and 27 contained only two types. LuARF13 and 22, LuARF1, and LuARF11 contained up to 4, 6, and 7 elements associated with gibberellin, MeJA, and abscisic acid, respectively. Additionally, three regulatory elements related to environmental stress, the MBS (drought inducibility), LTR (low temperature responsive), and TC-rich repeats (defense- and stress-responsive), were identified in the LuARF promoter; 17, 21, and 14 LuARF genes contained MBS, LTR, and TC-rich repeats, respectively. Among the 21 genes, LuARF11 had the most LTR elements.The database . The anaAAT-box) . FurtherLuARF genes, we first analyzed their expression patterns in different tissues (stem and capsule) and cultivars (Longya-10 and Heiya-14) using the previously published transcriptome data  were highly expressed in capsules, while nine  had higher expression levels in stems. Further, we investigated these genes’ expression patterns in flax stem and seeds by qRT-PCR. As shown in LuARF4/13 exhibited differences in the expression patterns between the two cultivars, Longya-14 and Zhangya-2, with different oil content. Subsequent analysis of the correlation between oil content and the expression levels of LuARF4/13 in seeds at different developmental stages indicated that the expression level in 10 d seeds was positively related to the oil content in Longya-14, while the expression level in 25 d and 40 d seeds was positively associated with the oil content in Zhangya-2 , these genes exhibited higher expression in the whole stem than in the stem peel, while the opposite was found at point A. Below the snap point (D to H), LuARF8 and LuARF32/33 showed lower expression at position G than C in the stem peel, while the other genes showed obviously higher expression levels at all positions than that at C. The expression patterns of these genes in the whole stem were opposite to that in the stem peel, and the levels diminished from C to D. In general, the nine LuARFs exhibited significantly higher expression in the stem peel than in the whole stem at points D to H, with the expression patterns of LuARF8 and 32/33 at point G and LuARF21 at point E as the exceptions. LuARF8 expression at point G was lower in the stem peel than in the whole stem, and LuARF32/33 expression at point E was lower in the stem peel than in the whole stem.To further confirm the importance of the nine genes explicitly expressed in the stem, their expression levels were tested in both whole stems and stem peels at nine positions along the Longya-10 stem c. The niLuARF genes in response to phytohormones and abiotic stresses, we investigated their expression levels in flax seedlings under IAA, NAA, and GA3 treatments and salinity, drought, cold, and heat stresses. The analysis showed that all LuARFs were significantly induced 4 h after exposure to cold and heat stress, and the response of all genes, except LuARF6/26, to cold stress was more pronounced than to heat stress  after exposure to NAA  [O. sativa (24%) [M. truncatula (37.5%) [Arabidopsis AtARF3 [Domain characteristics are crucial for predicting the functions of transcription factors . The pre17.39%) , O. sativa (24%) , and M.  (37.5%) . General.39% , O.s AtARF3 . Therefos AtARF3 ,49,50. Ts AtARF3 ,46. Thess AtARF3 .LuARF genes (2 gene pairs) experienced TD events, while 27  experienced SD events. Studies have demonstrated the significance of SD/WGD and TD in gene expansion and functional differentiation of multiple gene families [s analysis implied that the most recent WGD event was the leading cause of the expansion of the LuARF gene family [S. italica, the WGD event caused the expansion of the ARF gene family [LuARF genes might have contributed to function specificity or redundancy. Additionally, the Ka/Ks ratios of all the duplicated gene pairs were less than 0.4, indicating a purifying selection force on LuARF genes  contained ARFs from both dicots and monocots, suggesting relatively conserved ARF functions among the species. However, clade IIIb and IVb were dicot-specific subgroups and clade IVa only had one monocot ARF member, suggesting specific roles for these ARFs. Our study also found that the LuARFs within the same clade had similar exon–intron structures and domain composition, similar to those of other species ,36,46, ifamilies ,52. Genefamilies . Subseque family . In S. iRF genes . Researc species . In the us avium , probablLuARF genes  in seeds and stems at different development stages. LuARF4 showed higher expression in capsules and exhibited different expression patterns in cultivars with different oil content . The seeds collected from Longya-14 and Zhangya-2 plants at 5, 10, 15, 20, 25, 30, 40, and 50 days post anthesis and the stem tissues collected from Longya-10 plants (52~54 cm) at about 5 weeks post-emergence were used to determine the expression patterns of Deyholos , the whoLuARF genes under various phytohormone and abiotic stress treatments, seeds of Longya-15 were allowed to germinate in Petri dishes at 25 °C under 16 h:8 h light/dark conditions in a climate chamber. After 10 days of growth, uniformly grown seedlings were selected, transferred to 1/2 MS liquid medium, and cultivated for 3 days under the same conditions. Then, the medium was replaced with fresh medium containing 150 mM NaCl and 20% PEG to induce salinity and drought treatment, respectively, and 15 µM indole-3-acetic acid (IAA) or 5 µM naphthalene acetic acid (NAA) or 5 µM gibberellic acid (GA3) for the phytohormone treatment. The seedlings were subjected to 42 °C and 4 °C to induce heat and cold stress, respectively. The plants used for treatments were all from a single sowing. For each treatment, three biological replicates were set up. The leaves were collected from seedlings under heat and cold stress after 3 h of exposure and from drought, salinity, and phytohormone treatment at 3, 6, 9, 12, 24, 48, and 72 h under stress and 48 h after subsequent growth in 1/2 MS liquid medium without supplement. The leaves from the control seedlings were collected at 0 h and all other time points. For each treatment, samples were collected from three plants per replicate at each time point. All samples were frozen in liquid nitrogen immediately after collection and stored at −80 °C for RNA extraction.Then, to investigate the expression patterns of L. usitatissimum (Longya-10 and Heiya-14) and Linum bienne  were obtained from NCBI , and their annotation files were downloaded from Figshare . The ARF protein sequences of G. hirsutum, A. thaliana, G. max, V. vinifera, E. grandis, M.truncatula, O. sativa, S. bicolor, and B. distachyon were obtained from Phytozome v13  and used as queries to identify the ARF genes in Longya-10, Heiya-14, and pale flax using BLASTP analysis  [http://pfam-legacy.xfam.org/search#tabview=tab1, accessed on 15 December 2022) and the NCBI Conserved Domain Databases . Finally, the non-redundant LuARF genes were obtained using one gene model per locus. The MW and pI of all LuARF proteins were computed using the ExPASy ProtoParam  tool [http://www.csbio.sjtu.edu.cn/ bioinf/plant-multi/, accessed on 18 December 2022) [http://alternate.meme-suite.org/tools/meme, accessed on 24 December 2022) [The genome sequences of  × 10−5) . All can22) tool  and the er 2022) . MEME prer 2022) .LuARF genes was obtained from the annotation files downloaded from Figshare, and the chromosome distribution map was drawn using Map Gene 2 Chromosome . The MCScanX package was adopted to analyze the LuARF gene duplication events using default parameters [ARFs [a and Ks substitution rates per site of the duplicated gene pairs were calculated using DnaSP software (version 6.0) [a/Ks ratio was used to assess the mode and strength of the selective pressure [The chromosomal location of rameters , and thers [ARFs . The Ka ion 6.0) , and thepressure . For eacpressure :Mya = KsLuARF gene family members was determined based on the genome and coding sequences and visualized using the GSDS  [LuARF start codon was extracted from the Longya-10 genome, and the cis-acting elements were determined using the PlantCARE database  [The exon–intron organization of the ry 2023) . The 200ry 2023) .L. usitatissimum, V. vinifera, G. max, G. hirsutum, A. thaliana, E. grandis, M. truncatula, S. bicolor, B. distachyon, and O. sativa were used to construct a phylogenetic tree. First, a multiple sequence alignment was performed with ClustalX in MEGA (6.0) using default parameters. Then, a phylogenetic tree was built using the alignment files based on the neighbor-joining (NJ) method with 1000 bootstrap replicates [The full-length amino acid sequences of the ARFs of plicates .LuARFs in different tissues and genetic backgrounds [LuARF genes with high expression levels in the capsule or stem were further analyzed using qRT-PCR. The expression profiles of LuARFs under various treatments were also determined using qRT-PCR. Total RNA was extracted by using EZgeneTM Plant Easy Spin RNA Miniprep Kit following the manufacturer’ s instructions , and cDNA synthesis was performed by using PrimeScriptTM RT reagent Kit with gDNA Eraser . The specific primer pairs used for qRT-PCR were designed by Primer Premier 5.0  and listed in TM Premix Ex TaqTM II  and conducted on Eco Real-Time PCR System (Illumine). The reaction condition was as follows: 2 min at 50 °C, 10 min at 95 °C, with 40 cycles of 15 s at 95 °C, 15 s at 60 °C, and 15 s at 72 °C, followed by 10 s at 95 °C, 1 min at 65 °C, and 1 s at 97 °C. Each sample had three replicates. GAPDH was used as an internal reference gene [LuARF genes were calculated following the 2−ΔΔCt method [LuARF1/14, LuARF2/12, LuARF3/27, LuARF4/13, LuARF5/25, LuARF6/26, LuARF7/16, LuARF9/17, LuARF11/23, LuARF15/20, LuARF19/29/30, LuARF24/31, and LuARF32/33 gene pairs were extremely similar, and therefore, it was impossible to design primers specific for each gene of the pair. Thus, only one primer pair was designed to amplify the genes of each pair.Previously published transcriptome data on the capsule and stem of Longya-10 and Heiya-14 PRJNA505721) were initially used to determine the expression patterns of kgrounds , and the1 were int method . The codLuARF genes in Longya-10 (oil-use flax variety), and their gene structure, protein features, phylogeny, promoter elements, and expression patterns were analyzed. The results showed that LuARFs can function as either transcriptional activators or repressors, and the evolution and expansion of LuARFs were mainly driven by WGD events. Moreover, a positive association between LuARF gene expression and oil content was observed. All these results revealed the important roles of LuARFs in evolution, organ development, and responses to stress and phytohormone.In conclusion, the present study identified 33