TIMP1 Research Articles

Fibroblasts repair blood-brain barrier damage and hemorrhagic brain injury via TIMP2.

The function of fibroblasts in intracerebral hemorrhage (ICH) remains elusive. By targeting Col1α1, a fibroblast-specific marker, we generate mice with ablated Col1α1+ fibroblasts. These mutants show exacerbated blood-brain barrier (BBB) damage, enlarged injury volume, and worse neurological function, highlighting a beneficial role of Col1α1+ fibroblasts in ICH. Echoing these findings, fibroblasts significantly decrease endothelial permeability in an invitro ICH model. Next, we demonstrate that fibroblasts promote BBB integrity in ICH mainly via up-regulating tight junction proteins without affecting transcytosis-associated proteins, indicating a paracellular rather than transcellular mechanism. A subsequent mechanistic study reveals that the BBB-protective effect of fibroblasts is partially mediated by TIMP metallopeptidase inhibitor 2 (TIMP2). Furthermore, we find that exogenous TIMP2 attenuates BBB disruption in these mutants after ICH. These results suggest that Col1α1+ fibroblasts repair BBB damage in ICH via the paracellular pathway in a TIMP2-dependent manner, and that Col1α1+ fibroblasts and TIMP2 may be targeted in ICH treatment.

DZNep, a Histone Modification Inhibitor, Inhibits HIF1α Binding to TIMP2 Gene by Reducing Open Chromatin Area

DZNep, a Histone Modification Inhibitor, Inhibits HIF1α Binding to TIMP2 Gene by Reducing Open Chromatin Area

Results of PCR diagnostics after gingiva soft tissue augmentation

Aim. To demonstrate the results of polymerase chain reaction in real time.Materials and methods. The gum fragment was placed in 1 ml of RNA-leiter ("QIAGEN", Germany), incubated for a day at +4C, samples were stored at -70 °C.To obtain cDNA from the RNA matrix, a ready-made set of reagents MMLV RT Kit ("Eurogen", Russia) was used, the reaction was carried out according to the attached instructions. 2 µl of random decanucleotide primer (Random dN) and 1 µl of sterile RNase-free water were added to 6 µl of RNA, heated at +70C in a “Termit” thermostat (“DNA-Technology”, Russia) for 2 min to melt secondary RNA structures, then stored on ice (+4C). 4 ml of 5X buffer was added to the reaction mixture for the synthesis of the first chain (280 mM Tris-HCl, 375 mM KCl, 30 mM MgCl2, pH 8,7), 2 ml of dNTP mixture, 2 ml of DTT and 2 ml of sterile water free of RNase. Immediately before the reaction, 1 ml of MMLV revertase (reverse transcriptase of mouse leukemia virus) was added to the mixture and added to the RNA. The test tubes were heated in the Gnome thermostat (“DNA- Technology”, Russia) at +39C for 60 minutes, then at +70C for 10 minutes in the “Termit” thermostat (“DNA-Technology”, Russia).Results. The expression of IL-4, IL-10, IL-6, IL-12b, IL-1ß, MMP9 mRNA was not detected in the studied samples. The results of the polymerase chain reaction in real time showed that the expression levels of the proinflammatory cytokine TNFa and TIMP1 genes did not differ in both the autograft group and the collagen membrane group. The expression of MMP 2 and TIMP 2 was higher in the group using a collagen membrane, which is probably associated with tissue regeneration processes.Conclusions. Fibro-guide collagen matrix shows no less effective clinical results in comparison with autografts

Complement complex 1 subunit q-mediated hepatic stellate cell activation with connective tissue growth factor elevation is a prognostic factor for survival in rat and human chronic liver diseases.

Complement complex 1 subunit q (C1q) has multiple functions, including cell migration, in addition to its traditional complement-activating effect. Research shows C1q is a ligand for frizzled receptors (FZDs). FZD-induced yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) alternate Wnt signaling activation induces connective tissue growth factor (CTGF) production and hepatic stellate cell (HSC) activation. However, no study exists in which C1q directly induces CTGF in HSCs. Here, we investigated the role of C1q in HSC activation. Human HSCs (LX2) were incubated with C1q to assess HSC activation. C1q and fibrotic markers were assessed using immunohistochemistry, immunoblotting, and quantitative reverse-transcription polymerase chain reaction in cirrhotic rats administered CCl4 for 21 weeks. Serum C1q, liver function, and fibrosis score were measured in 91 patients with chronic liver disease. The correlations between serum C1q and liver function, fibrosis score, and survival prognosis were examined. C1q-activated LX2s showed morphologic changes, up-regulation of CTGF, tissue inhibitors of metalloproteinases (TIMP-1),and alternate Wnt signal genes FZD2, TAZ, and cysteine-rich angiogenic inducer 61 (Cyr61). Cirrhotic rat liver C1q expression correlated with the Azan-positive area and expression of CTGF, TIMP-1, hyaluronan synthase (HAS)1, HAS3, and CD44. Expression of C1q protein and C1q, CTGF, and TIMP-1 genes were higher in deceased cirrhotic rat livers compared to surviving rats. Human serum C1q levels increased in liver cirrhosis compared to chronic hepatitis and correlated with liver fibrosis and functional markers. Ten patients suffered liver-related death over a 66-month observation period. The C1q cut-off value (11 mg/dl) showed patients with serum values < 11 mg/dl had longer rates of survival compared to C1q ≥ 11 mg/dl. Conclusion: C1q-mediated HSC activation in liver fibrosis is associated with CTGF elevation. Additionally, serum C1q may be diagnostic for survival in human chronic liver diseases.

Viral protein revives host cell miRNA function by dampening the circular RNA sponge

In this study, Liu et al. have investigated the inhibitory effects of hepatitis B viral X gene-coded protein (HBx) on circular RNA circSFMBT2 expression in host cells in connection to hepatocellular carcinoma (HCC) cell metastasis. The authors have delineated a novel mechanism to show how HBx lowers circSFMBT2 expression to upregulate miR-665 activity linked with miR-665 target gene TIMP3 downregulation and enhanced HCC metastasis.1 They also found that circSFMBT2 RNA acts as sponge for miR-665 in hepatic cells.

Effect of Irisin on Human Nucleus Pulposus Cells: New Insights into the Biological Cross-talk Between Muscle and Intervertebral Disk.

In vitro study. To investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Physical exercise (PE) favours weight loss and ameliorates function in patients with low back pain. Although there is no biological evidence that the intervertebral disk (IVD) can respond to PE, recent studies have shown that running is associated with increased IVD hydration and hypertrophy. Irisin, a myokine released upon muscle contraction, has demonstrated anabolic effects on different cell types, including chondrocytes. hNPCs were exposed to 5, 10, and 25ng/mL irisin. Cell proliferation, glycosaminoglycan (GAG) content, metabolic activity, gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-3, aggrecan (ACAN), interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 were assessed. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1, and IL-1β gene expression were evaluated following incubation with irisin for 24 hours and subsequent culture with 10ng/mL IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with irisin). Irisin increased hNPC proliferation, metabolic activity, and GAG content, as well as COL2, ACAN, TIMP-1 and TIMP-3 gene expression, while decreasing MMP-13 and IL-1β mRNA levels. Irisin pretreatment of hNPCs cultured in proinflammatory conditions resulted in a rescue of metabolic activity and a decrease of IL-1β levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to irisin led to an increment of metabolic activity, COL2 gene expression, and a reduction of IL-1β and ADAMTS-5 levels. Irisin increases hNPC proliferation, GAG content, metabolic activity, and promotes anabolic gene expression while reducing catabolic markers. Irisin may be one of the mediators by which PE and muscle tissues modulate IVD metabolism, suggesting the existence of a biological cross-talk between the muscle and IVD.

Associations of Prenatal Metals Exposure with Kidney Injury Biomarkers in Children

Background and Aim: Prenatal exposure to toxic metal(loid)s, including arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) during development may have nephrotoxic effects in children. We assessed associations between prenatal metal exposure and urinary kidney injury biomarkers in children. Methods: Analyses included 418 children participating in the PROGRESS birth cohort study in Mexico City. Concentrations of As, Cd, Pb, and Hg (urine only) were quantified in maternal second trimester (2T) blood and urine. Kidney parameters were assessed at 8-10 years of age. We examined associations between individual metals and 1) kidney injury biomarkers using linear regression and 2) a multi-protein kidney index using weighted quantile sum (WQS) regression. Associations of separate urine and blood metal mixtures with individual kidney injury biomarkers were assessed via WQS. Covariates included child’s age, sex, body mass index z-score, urine creatinine, 2T socioeconomic status, and 2T smoking exposure. Results: In single metal analyses, doubling of urine Cd was associated with higher urinary TIMP metallopeptidase inhibitor 1 (TIMP1), alpha-1-microglobulin (A1M), clusterin, and cystatin C. Within the multi-protein index, the association with increased urinary As was predominated by albumin, fatty acid binding protein 1 (FABP1), and interferon gamma-induced protein 10 (IP10), and the association with increased urinary Cd was predominated by albumin, A1M, TIMP1, lipocalin, and FABP1. Decile increases in urine metal mixture was associated with increased urinary albumin, IP10, and cystatin C. Urinary As and Cd comprised 60-96% of the metal mixture weights contributing to the associations. Conclusions: Prenatal urinary metals, individually and as a mixture were associated with increased urinary kidney injury biomarkers in healthy children. Prenatal metals exposure may lead to subclinical glomerular or tubular injury in school age children. Further research is required to ascertain resolution or worsening of subclinical kidney injury at later life stages. Keywords: Heavy metals; Renal health; Mixtures

Study the expression rate and methylation of TIMP-3 gene in the blood samples of people suffering from gastric cancer

TIMPs inhibit the necrosis factor converting enzyme of alpha tumor and induce planned cellular death. Methylation in TIMP3 promoter would cause tumor progress in cancer. The present research seeks to study the expression rate and methylation of the TIMP3 gene in the blood of individuals who have gastric cancer. The present research uses MS PCR and Real-time PCR to study the expression rate and methylation of the TIMP3 gene, which has recently attracted much attention in gastric cancer. In this research, we have compared the blood samples of those with gastric cancer and ordinary people among Iranian patients. The expression rate of TIMP3 in the blood samples obtained from those who have gastric cancer has decreased significantly compared to the blood samples obtained from ordinary people. A significant correlation was also observed between the expression rate of this gene, age and methylation in gastric cancer. Methylation and expression levels of the TIMP3 gene were statistically significant in patient samples compared to healthy subjects, and decreased methylation and increased expression of this gene in healthy individuals indicated the role of altered gene expression in gastric cancer. Keywords. Gastric cancer; TIMPs; Methylation; Real-time PCR.

Differential gene expression of ADAMTS-1, ADAMTS-9 and TIMP-3 in periodontitis

ABSTRACT A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are metalloproteinases that bind to components of the extracellular matrix (ECM) to regulate tissue remodeling and homeostasis. ADAMTS can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). Expression of ADAMTS increases under inflammatory conditions. We investigated the mRNA expression of ADAMTS-1, ADAMTS-9 and TIMP-3 genes in both healthy gingival tissues and periodontitis. Clinical periodontal measurements were conducted and gingival biopsies were obtained from stage IIIgrade C generalized periodontitis and healthy (control) groups. mRNA expression was evaluated using real-time quantitative polymerase chain reaction (RTqPCR). All clinical periodontal parameters were significantly higher in the periodontitis group than for the control group. ADAMTS-1 levels were significantly higher in the periodontitis group and were significantly correlated with clinical attachment level and probing pocket depth. Differences in ADAMTS-9 and TIMP-3 mRNA in the periodontitis group compared to the control group were not statistically significant. Increased ADAMTS-1 mRNA expression in periodontitis indicates that members of the ADAMTS family of metalloproteinases are associated with pathogenesis and progression of periodontal disease. Maintaining balance between ADAMTS and TIMP is important for limiting ECM catabolism and preventing tissue damage.

Metallopeptidades 2 and 9 genes epigenetically modulate equine endometrial fibrosis.

Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.

Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes.

BackgroundIn most cell types, replicative senescence (RS) is supposed to be a principle causative factor for aging. Atrial fibrosis, pathologically characterized by proliferation of atrial fibroblasts (AFs) and excessive accumulation of extracellular matrix proteins, is the most common substrate of atrial fibrillation (Afib) in the elderly. However, whether AFs’ RS develops in the aged and fibrotic left atrium (LA) and, if yes, what is the key regulator for the pathogenesis of AFs’ RS remain largely unknown.MethodsWe obtained the left atrial tissues from young (6–8 weeks old) and aged (24 months old) C57BL/6 male mice. Screening and validation of differential genes were performed using comparative analysis of RNA-seq results. Replicative senescence was examined in primary AFs after cell passage. Further gain-of-function and loss-of-function experiments were performed to explore the regulation of the AFs’ RS progression.ResultsIn the present study, we demonstrated that there was a considerable extent of AFs’ RS in the aged and fibrotic LA. Transcriptome screening showed that Ezh2 (Enhancer of zeste homolog 2) was significantly downregulated in the LA tissue of aged mice. Ezh2 is a histone methyltransferase that catalyzes H3K27me3 and mediates transcriptional silencing. We confirmed that Ezh2 was downregulated in the isolated pure senescent AFs. Knockdown of Ezh2 by siRNA or inhibition of Ezh2ʹs methyltransferase activities by GSK-126 and GSK-343 accelerated RS in the early passage of AFs, while its overexpression deaccelerated RS in the late passage of AFs. Mechanistically, Ezh2 suppressed CDKN2a (p16, p19) and Timp4 gene transcription by forming canonical H3K27me3 modifications in their promoter regions. Furthermore, the functional balance between Timp4 and MMP8 in AFs could be collapsed by changes in Ezh2 expression.ConclusionThese results thus indicate that Ezh2 is a key regulator of AFs’ RS and this work may provide a basis for future treatments for atrial fibrosis in the elderly.

Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells-Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing.

Limbus-derived stromal/mesenchymal stem cells (LMSCs) are vital for corneal homeostasis and wound healing. However, despite multiple pre-clinical and clinical studies reporting the potency of LMSCs in avoiding inflammation and scarring during corneal wound healing, the molecular basis for the ability of LMSCs remains unknown. This study aimed to uncover the factors and pathways involved in LMSC-mediated corneal wound healing by employing RNA-Sequencing (RNA-Seq) in human LMSCs for the first time. We characterized the cultured LMSCs at the stages of initiation (LMSC−P0) and pure population (LMSC−P3) and subjected them to RNA-Seq to identify the differentially expressed genes (DEGs) in comparison to native limbus and cornea, and scleral tissues. Of the 28,000 genes detected, 7800 DEGs were subjected to pathway-specific enrichment Gene Ontology (GO) analysis. These DEGs were involved in Wnt, TGF-β signaling pathways, and 16 other biological processes, including apoptosis, cell motility, tissue remodeling, and stem cell maintenance, etc. Two hundred fifty-four genes were related to wound healing pathways. COL5A1 (11.81 ± 0.48) and TIMP1 (20.44 ± 0.94) genes were exclusively up-regulated in LMSC−P3. Our findings provide new insights involved in LMSC-mediated corneal wound healing.

Reveal the Mechanisms of Yi-Fei-Jian-Pi-Tang on Covid-19 through Network Pharmacology Approach.

Objectives The Traditional Chinese Medicine (TCM) formula Yi-Fei-Jian-Pi-Tang (YFJPT) has been demonstrated effective against Corona Virus Disease 2019 (Covid-19). The aim of this article is to make a thorough inquiry about its active constituent as well as mechanisms against Covid-19 via TCM network pharmacology. Methods All the ingredients of YFJPT are obtained from the pharmacology database of the TCM system. The genes which are associated with the targets are obtained by utilizing UniProt. The herb-target network is built up by utilizing Cytoscape. The target protein-protein interaction network is built by utilizing the STRING database and Cytoscape. The critical targets of YFJPT are explored by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Results The outcomes show that YFJPT might has 33 therapeutic targets on Covid-19, namely, interleukin 2 (IL2), heme oxygenase 1 (HMOX1), interleukin 4 (IL4), interferon gamma (FNG), α nuclear factor of kappa light polypeptide gene enhancer in Bcells inhibitor, alpha (NFKBIA), nuclear factor-k-gene binding (NFKB), nitric oxide synthase 3 (NOS3), intercellular adhesion molecule 1 (ICAM1), hypoxia inducible factor 1 subunit alpha (HIF1A), mitogen-activated protein kinase 3 (MAPK3), epidermal growth factor receptor (EGFR), interleukin 10 (IL10), jun proto-oncogene (JUN), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 8 (CXCL8), tumor protein p53 (TP53), interleukin 1 beta (IL1B), AKT serine/threonine kinase 1 (AKT1), tumor necrosis factor (TNF), interleukin 6 (IL6), erb-b2 receptor tyrosine kinase 2 (ERBB2), RELA proto-oncogene (RELA), NF-κB subunit, caspase 8 (CASP8), peroxisome proliferator activated receptor alpha (PPARA), TIMP metallopeptidase inhibitor 1 (TIMP1), transforming growth factor beta 1 (TGFB1), interleukin 1 alpha (IL1A), signal transducer and activator of transcription 1 (STAT1), mitogen-activated protein kinase 8 (MAPK8), myeloperoxidase (MPO), matrix metallopeptidase 3 (MMP3), matrix metallopeptidase 1 (MMP1), and NFE2 like bZIP transcription factor 2 (NFE2L2). The gene enrichment analysis prompts that YFJPT most likely contributes to patients related to Covid-19 by regulating the pathways of cancers. Conclusions That will lay a foundation for the clinical rational application and further experimental research of YFJPT.

Fibroblast morphology, growth rate and gene expression in facial melasma

BackgroundIn addition to melanocytic hyperfunction, changes are observed in the upper dermis of melasma, and fibroblasts play a central role in collagen synthesis and pigmentation induction. ObjectiveTo explore the morphology, growth rate, and gene expression profile of fibroblasts from the skin with melasma in comparison to fibroblasts from the adjacent healthy skin. MethodsTen women with facial melasma were biopsied (lesion and adjacent healthy skin), and the fragments were processed for fibroblast culture. Samples from five participants were seeded to evaluate growth (days 2, 5 and 8) and senescence (SA-β-gal) curves. The samples from the other participants were submitted to real-time PCR to comparatively evaluation of the expression of 39 genes. ResultsCultured fibroblasts from melasma skin were morphologically less fusiform in appearance and on average a 34% (95% CI 4%‒63%) greater proportion of cells labeled with SA-β-gal than the fibroblasts from the adjacent skin. The cell growth rate was lower for the melasma samples after eight days (p < 0.01). TheWNT3A, EDN3, ESR2, PTG2, MMP1, and SOD2 genes were up-regulated, whereas the COL4A1, CSF2, DKK3, COL7A1, TIMP4, CCL2, and CDH11 genes were down-regulated in melasma skin fibroblasts when compared to the ones from adjacent healthy skin. Study limitationsSmall sample size; absence of functional tests. ConclusionsFibroblasts from the skin with melasma showed a lower growth rate, less fusiform morphology and greater accumulation of SA-β-gal than those from adjacent photo exposed skin. Moreover, their gene expression profile comprised factors that may contribute to upper dermis damage and sustained melanogenesis.

Effects of TIMP1 rs4898 Gene Polymorphism on Early-Onset Preeclampsia Development and Placenta Weight.

Introduction: Some evidence indicates that the improper trophoblast invasion of maternal spiral arteries could be caused by an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to preeclampsia (PE) development. This study aimed to assess the potential role of MMP1, MMP9, TIMP1 and TIMP2 gene polymorphisms in the pathogenesis of PE. Materials and methods: A total of 308 Polish women, 115 preeclamptic (55 with early-onset preeclampsia [EOPE], 60 with late-onset preeclampsia [LOPE]) and 193 healthy pregnant women, all of Caucasian origin, were recruited to the study. PE was diagnosed following the ACOG criteria. The polymorphic variants of the MMP-TIMP pathway (MMP1 rs1799750, MMP9 rs17576, MMP9 rs17577, TIMP1 rs4898, TIMP2 rs2277698, TIMP2 rs55743137) were genotyped by polymerase chain reaction and restriction fragment length polymorphism. Results: Analyzing all SNPs in the MMP-TIMP pathway, no significant differences in allele frequencies between preeclamptic women and controls were observed. However, comparing the EOPE and LOPE groups with each other, we observed a statistically significant difference between them for the TIMP1 rs4898 variant. In the whole group of 308 women, the mean placenta weight was the lowest in carriers of the rs4898 CC genotype. Post hoc analysis revealed significant differences between CC-CT (p = 0.0209) and CC-TT (p = 0.0469). Additionally, during allele analysis, a statistically significant difference in the mean placenta weight (for C allele 529.32 ± 157.11 g, for T allele 560.24 ± 162.24 g, p = 0.021) was also observed. Conclusion: Our findings suggest a relationship between TIMP1 rs4898 (372T > C) polymorphism and increased risk of early-onset preeclampsia in a population of pregnant Polish women. Our data suggest that the TIMP1 rs4898 C allele might be associated with increased risk for early-onset, but not for late-onset preeclampsia. To evaluate the role of the TIMP1 polymorphic variants in the etiopathology of preeclampsia, further studies with a larger sample size are needed.

P-523 rs2277698 Single nucleotides polymorphisms in tissue inhibitors of metalloproteinases 2 (TIMP2) affected clinical outcomes of women undergoing in vitro fertilization

Abstract Study question Is there any association between clinical outcomes of women undergoing in vitro fertilization (IVF) and the polymorphic variants of matrix metalloproteinases (MMPs) or tissue inhibitors of metalloproteinases (TIMPs)? Summary answer Our findings suggested that clinical outcomes of women undergoing IVF were associated with rs2277698 SNPs in TIMP2. What is known already The extracellular matrix proteolytic machinery is one of the most important mechanisms for regulating trophoblast invasion. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which are involved in the extracellular matrix proteolysis process, have the ability to regulate the degradation of the extracellular matrix and play a critical role in trophoblast invasion. Study design, size, duration A prospective study was composed of 1014 women undergoing their first fresh IVF cycle with non-donor from January 2014 to December 2015. Participants/materials, setting, methods The present study was carried out in Lee Women’s Hospital, Taiwan. DNA was extracted from the peripheral blood of all participants, and the SNPs were genotyped by real-time polymerase chain reaction. The effects of the following three single nucleotide polymorphisms (SNPs) on IVF outcomes were explored: TIMP1 (rs4898 C/T), TIMP2 (rs2277698 C/T) and MMP2 (rs243865 C/T). The SNP genotype, correlation with clinical pregnancy, embryo implantation, abortion and live birth rates of IVF were analyzed. Main results and the role of chance In the analysis of 1014 patients attempting their first cycle of IVF, the genotypes frequencies of the study population showed a statistically significant association between TIMP2 (rs2277698 C/T) and clinical outcome. For TIMP2 polymorphisms, women with wild genotype (CC) hadhigher clinical pregnancy rate (34.8% v.s 28.0%; p = 0.032), embryo implantation rate (19.4% v.s 15.4%; p = 0.009), live birth rate (29.8% v.s 22.1%; p = 0.006), and lower abortion rate (7.0% v.s 17.1%; p = 0.005) compared women with CT/TT genotype. On the other hand, the clinical outcomes in different TIMP1 or MMP2 gene polymorphisms groups were no statistically significant difference. Limitations, reasons for caution The size number of the study samples. Wider implications of the findings This study showed that the TIMP2 SNP polymorphisms CT/TT was associated with low trend of clinical outcomes. The mechanism of TIMP2 gene T allele affected the outcomes of IVF remains to be determined. Further studies should focus on the mechanism of these associations in a larger, more heterogeneous cohort. Trial registration number CS13194

Activating transcription factor 3 protects alveolar epithelial type II cells from Mycobacterium tuberculosis infection-induced inflammation

Activating transcription factor 3 (ATF3) is a stress-inducible gene reported with anti-inflammatory response effects against bacterial infections. This study focuses on the function of ATF3 in alveolar epithelial type II cells (A549) following Mycobacterium tuberculosis (MTB) infection. First, RT-qPCR results detected reduced ATF3 expression in broncho-alveolar lavage fluid (BALF) of MTB-infected patients, whereas the ATF3 level was upregulated in A549 cells at early stages after MTB infection but decreased later. The binding relationship between ATF3 and TIMP metallopeptidase inhibitor 2 (TIMP2) promoter was predicted via bioinformatic prediction and validated by ChIP and luciferase assays. ATF3 bound to TIMP2 promoter for transcriptional activation. Overexpression of ATF3 or TIMP2 enhanced autophagy activity, elevated p62 levels and the LC3BII/LC3BI ratio, and decreased IL-6 and TNF-α levels in A549 cells. The ATF3/TIMP2 axis suppressed the NF-κB pathway to alleviate inflammatory responses in A549 cells. Mice were exposed to MTB aerosol for in vivo experiments. Increased ATF3 expression was correlated with increased autophagy activity, clearance of bacteria as well as inflammation resolution in mouse lung tissues. In conclusion, this study demonstrates that ATF3 promotes cell autophagy and suppresses inflammatory response in MTB-infected A549 cells via TIMP2 activation and NF-κB suppression.

Action mechanism and active components of Fuzheng Huayu Recipe against liver fibrosis via regulating macrophage with network pharmacology and transcriptomics methods

We have demonstrated that Fuzheng Huayu Recipe(FZHY) plays an anti-liver fibrosis role by regulating the polarization of intrahepatic macrophages, while the key targets in macrophages and the effective components of FZHY remain unclear. In this study, we obtained the potential anti-liver fibrosis target set of FZHY through network pharmacological analysis, and the differentially expressed gene set of FZHY for the prevention and treatment of mouse liver fibrosis through RNA-Seq of the liver tissue. The potential core targets of FZHY against liver fibrosis were obtained by degree value analysis of the common target proteins between the above two sets. Then, through the retrieval of PubMed database, we identified the potential key targets in macrophages. After that, the effective components in FZHY corresponding to key targets were obtained by reverse pharmacological analysis. Finally, we verified the regulatory effects of these effective components on the expression of key target genes by using the lipopolysaccharide-induced M1 macrophages derived from THP-1 cells. The RNA-Seq data combined with network pharmacological analysis showed that FZHY might alleviate liver fibrosis by regulating the expression of CCL2, TIMP1, and MMP2 genes in macrophages. The results of in vivo experiments showed that FZHY significantly inhibited the expression of CCL2 and TIMP1 genes and promoted the expression of MMP2 genes in liver tissues of liver fibrosis mice. The results of in vitro experiments demonstrated that FZHY and its four effective components(luteolin, ursolic acid, quercetin, and danshensu) significantly inhibited the expression of CCL2 and TIMP1 genes in M1 macrophages derived from THP-1 cells. In addition, the expression of MMP2 gene was up-regulated by luteolin, ursolic acid, and quercetin, not affected by FZHY, and down-regulated by danshensu. FZHY could inhibit the expression of CCL2 and TIMP1 genes in M1 macrophages by the four effective components to achieve the anti-inflammatory and anti-liver fibrosis effects.

1333-P: Therapeutic Potential of DA-1726, a Novel Oxyntomodulin Analogue, in a Diet-Induced NASH Mouse Model

DA-1726 is a novel balanced long-acting GLP-1/glucagon receptor dual agonist, which is under preclinical development for the treatment of obesity. DA-1726 represented a superior efficacy to semaglutide (SEMA) in weight loss in various animal studies. In this study, we evaluated the therapeutic potential of DA-1726 for the treatment of nonalcoholic steatohepatitis (NASH) . To induce NASH, male mice were given a diet containing 40% total fat, 20% fructose, and 2% cholesterol for 30-week. After then DA-1726 and SEMA were subcutaneously injected every three days for 8-week. At the end of treatment, vehicle control, DA-1726 (100 or 200 nmol/kg) and SEMA (250 nmol/kg) significantly reduced body weight compared to baseline (+10.4%, -8.9%, -17.2%, and -12.9%, respectively) . Plasma clinical chemistry parameters (ALT, AST, ALP and T-BIL) and hepatic fat accumulation were significantly decreased by DA-1726. The reduction of plasma glucose and total cholesterol induced by DA-1726 200 nmol/kg was superior to that of SEMA. In a histopathological analysis of steatosis, lobular inflammation, and ballooning in the liver, DA-1726 showed an excellent improvement in NAFLD Activity Score compared to SEMA (NAS: 6.75 for vehicle control, 3.88 and 3.63 for DA-1726, and 5.00 for SEMA) . Liver fibrosis was assessed by Masson Trichrome stain. Improvement of hepatic fibrosis by DA-1726 was also observed (fibrosis score: 1.88 for vehicle control, 1.25 and 1.13 for DA-1726, and 1.50 for SEMA) . In the liver tissue, the expression of inflammatory (Tnfa, Il-1β, and Ccl2) and fibrotic (Acta2, Timp1, Col1a1, Col3a1, and Mmp9) genes were significantly decreased by DA-1726 treatment. In particular, the DA-1726 low-dose group showed a higher efficacy despite lower weight loss compared to SEMA. This is thought to be a result of the dual actions of DA-1726 to GLP-1 and glucagon receptors. Taken together, our findings suggest that DA-1726 has a therapeutic potential for NASH in addition to obesity and type 2 diabetes. Disclosure I.Jung: None. T.Kim: None. M.Goo: None. M.Kim: None. Y.Chae: None.

1291-P: Bioinformatics Analysis of Gene Networks Associated with Glucose Variability and Inflammatory Pathways in Diabetes

Aim: To identify key genes linking glucose variability (GV) and inflammatory pathways in diabetes. Methods: Text mining-based reconstruction and analysis of the gene networks associated with GV and low-grade inflammation in diabetes were performed by the ANDSystem (www-bionet.sscc.ru/and/cell/) . Key elements of the networks were identified by the highest value of cross-talk centrality (CTC) and cross-talk specificity (CTS) . A comparative analysis of two networks was applied to identify genes that are up- or down-regulated by glucose fluctuations and involve in the inflammatory pathways. Results: We revealed sixty one genes related to GV and inflammation (Table) . Based on CTC values, TP53, TNF, NFKB1, IL1B, IL6, CASP3, MMP2, MMP9, TGFB1, and VEGFA were identified as the principal hubs in the gene network of inflammation that are regulated by GV. The UCP2, GPX1, SOD2, ANGPT2, GLP1R, HGF, SPP1, MAPK9, TIMP1, and MMP1 genes demonstrated the highest CTS values. Conclusions: The obtained results improve the understanding of the molecular mechanisms of the proinflammatory effect of GV in diabetes. The role of the identified molecules as key drivers of GV-induced inflammation needs further experimental verification. Disclosure O.V.Saik: None. V.Klimontov: None. Funding Russian Science Foundation (20-15-00057)