MMP-9 Gene Research Articles

Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4h after exposure, and then decreased at 6h in most groups. Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Association Analysis of MMP-13 (rs2252070) Gene Polymorphism and the Susceptibility to Chronic Periodontitis.

Chronic periodontitis is a multifactorial inflammatory condition influenced by genetic factors. Matrix metalloproteinase(MMP)-13, serving as a crucial enzyme involved in extracellular matrix remodeling, is associated with the degradation of periodontal tissues. Therefore, this study assesses the genetic link between the MMP-13 (rs2252070) genetic variation and chronic periodontitis in a Southern Indian demographic. The study was conducted at Saveetha Dental Collegein Chennai, India. It involved a total of 100 subjects,50 individualsaffected with periodontitis (classified as stage II and above, American Association of Periodontology 2018 criteria) and 50 individuals who were periodontally healthy or were diagnosed as having mild gingivitis. We isolated DNAfrom the blood samples obtained from the participants. Specific primers that flank the BsrI region of the MMP-13 receptor gene were used in the process of DNA amplification. Subsequently, a restriction fragment length analysis using the BsrI enzyme was carried out for genotyping of the amplicon. Based on the restriction fragment length polymorphism pattern, we obtained certain genotypes. These were further recorded and followed by statistical analysis. We conducted a chi-square test to draw a comparison in terms of their genotype and allele frequencies. We calculated the odds ratio, along with 95% confidence intervals. The frequency of genotypes and distribution of MMP-13 polymorphism did not exhibit a statistically significant difference at χ2 degrees of freedom (P = 0.913). We inferred from our study that there was no significant difference between the groups concerning homozygous and heterozygous mutant genotypes (AA vs. AG + GG), with a P-value of 0.6871. The observed frequencies of GG (47% vs. 43%) and AG+AA (41% vs. 42%) genotypes did not indicate a significant difference between the groups. Similarly, there was no noteworthydistinction between the A allele (62% vs. 65%) and G allele (38% vs. 35%) in the case and control groups. Thefindings of the study reveal that there is no correlation between MMP-13 (rs2252070)gene polymorphism and periodontitis.

Restoration of miR-451a-5p/miR-34a-5p could suppress the proliferation and migration of human breast cancer cells through Wnt/β- catenin and ERK/P-ERK signaling pathways.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a length of 21-25 nucleotides and play an essential role in the regulation of cancer initiation, development and progression. Breast cancer (BC) is the most commonly detected malignancy in women and one of the leading causes of death worldwide. In this study, the effects of transfection of microRNA-451a-5p and miR-34a-5p (tumor suppressors), individually and in combination on apoptosis, proliferation and migration of breast cancer cells in vitro were investigated. For this study, malignant breast cancer cells (MDA-MB-231) were transfected with the miR-451a-5p and miR-34a-5p mimics. Subsequently cytotoxicity, apoptosis, proliferation, migration protein and gene expression of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc of the cancer cells were analyzed by MTT, flow cytometry, q-RT-PCR (expression level of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc genes), wound healing and Western blot assays. The results showed that miR-34a-5p and miR-451a-5p could additionally induce apoptosis and cell cycle arrest in the sub-G1phase, suppress proliferation and migration in breast cancer cells, and also decrease the expression of β- catenin and ERK/P-ERK proteins . The present data document that restoration of the tumor suppressor miR-451/miR-34 strongly induces programmed cell death in vitro and apparently inhibits cell proliferation and migration in human breast cancer cells. In summary, miR-451a and miR-34a play an important role in breast cancer cell proliferation and migration via the Wnt/β-catenin and ERK/P-ERK signaling pathways. Therefore, the simultaneous restoration of the presented tumor suppressor miRNAs can be proposed as a valuable and potential therapeutic strategy in the treatment of breast cancer. However, further studies should be useful.

Retraction Note.

The publisher and Editor in Chief have withdrawn four articles published between 2016 and 2018. These articles were discovered to contain problems, such as image manipulation, absence of ethics approval, and inconsistencies in data presented. This retraction notice pertains to the following articles, with detailed explanations below: - "The effects of MCA-MAO on cAMP pathway in rats with cerebral hemorrhage" by Y. Jiang, X.-F. Wang, Y.-L. Niu, J.-H. Xia-Eur Rev Med Pharmacol Sci 2016; 20 (9): 1834-1838-PMID: 27212177. Readers have pointed out on PubPeer that Figures 1B and D appear to contain some overlaps. Moreover, there is no ethical approval statement for this study. The editorial team has contacted the authors but received no reply. The journal investigation revealed a figure duplication. Therefore, the Editor in Chief mistrusts the results and decided to retract the manuscript. Although the authors were notified of the retraction decision, they remained unresponsive. - "Research on the role of GLP-2 in the central nervous system EPK signal transduction pathway of mice with vascular dementia" by C.-L. Chi, S.-A. Zhang, Z. Liu, M.-X. Chang, H. Wang, Y. Huang-Eur Rev Med Pharmacol Sci 2017; 21 (1): 131-137-PMID: 28121365. Readers have pointed out on PubPeer that Figure 3 appears to contain some overlaps. Moreover, there is no ethical approval statement for this study. The editorial team has contacted the authors but received no reply. The journal investigation revealed a figure duplication. Therefore, the Editor in Chief mistrusts the results and decided to retract the manuscript. Although the authors were notified of the retraction decision, they remained unresponsive. - "Studies on the relationship between P13K/AKT signal pathway-mediated MMP-9 gene and lung cancer" by H. Zheng, J.-F. Liu-Eur Rev Med Pharmacol Sci 2017; 21 (4): 753-759-PMID: 28272723. Readers on PubPeer have expressed concerns about Western blots, as shown in Figure 2. Moreover, there is no ethical approval statement for this study. The editorial team has contacted the authors but received no reply. The journal investigation did not detect figure manipulation; however, since the lack of ethics statement and the lack of the original data, the Editor in Chief decided to retract the manuscript. Although the authors were notified of the retraction decision, they remained unresponsive. - "LncRNA LINC01116 competes with miR-145 for the regulation of ESR1 expression in breast cancer" by H.-B. Hu, Q. Chen, S.-Q. Ding-Eur Rev Med Pharmacol Sci 2018; 22 (7): 1987-1993-DOI: 10.26355/eurrev_201804_14726-PMID: 29687853. Readers on PubPeer have expressed concerns regarding inconsistencies between data presented in Tables and figures, deficiencies in the description of sample collection, and a lack of ethics approval. The editorial team has contacted the authors to request the raw data of the manuscript and a reply to those concerns but has not received any response. Throughout the journal investigation, numerous flaws and inconsistencies were discovered in the presented results. Therefore, the editors deem the conclusions of this article to be invalid and decided to retract the manuscript. The corresponding author did not respond to journal correspondence about the investigation and retraction of this article. These articles have been retracted. The Publisher apologizes for any inconvenience this may cause.

Application of Network Pharmacology to Explore the Anti-Aging Molecular Mechanism of Radix notoginseng

Objective: To study the anti-aging effects of Radix Notoginseng and to explore its molecular network mechanism. Methods: Aging and Radix notoginseng gene targets were searched and downloaded from the Genecards website, then Venn intersection analysis was performed to find common genes for diseases and drugs to explore candidate targets for Radix notoginseng in the treatment of aging. Bioinformatics was then used to analyze the biological processes, cellular components, molecular functions, and KEGG signaling pathways of the shared target network. Protein molecular network construction was carried out to find the core molecular network genes of the drug Radix notoginseng for the treatment of aging. A final PubMed literature comparison was performed to assess the value of the potential role of core network genes. Results: The keywords “Aging” and “Radix notoginseng” were queried in Genecards and 25,000 aging-related targets were obtained, 17 for Radix notoginseng. GO and KEGG analysis of the intersecting genes obtained from the Venn intersection analysis then showed that the BP with the highest potential to be associated with disease and drugs is positive regulation of protein phosphorylation, CC is macromolecular complex and MF is identical protein binding. The KEGG with the higher correlation is lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, and proteoglycans in cancer. A total of 10 hub genes were identified in the PPI network construction, including EGFR, MMP9, TNF, VEGFA, RHOA, CDKN1A, CASP3, CCND1, AKT1, and IL1B. Among these, it found that a large number of MMP9 and TNF genes were reported in the literature, with the remaining hub genes less frequently reported in the literature. Conclusion: This study uses bioinformatics and network pharmacology to explain the core network mechanisms of the drug Radix notoginseng in the treatment of aging using the latest databases. The results show that hub genes such as CDKN1A, EGFR, and AKT1 are involved in the core biological processes of aging. The results of the study provide an important reference for resolving the core molecular network mechanism of anti-aging properties and provide a validation basis for future experimental validation.

GEO dataset mining analysis reveals novel Staphylococcus aureus virulence gene regulatory networks and diagnostic targets in mice.

Staphylococcus (S.) aureus infection is a serious, worldwide health concern, particularly in many communities and hospitals. Understanding the S. aureus pathogenetic regulatory network will provide significant insights into diagnostic target screening to improve clinical treatment of diseases caused by S. aureus. We screened differentially expressed genes between normal mice and S. aureus-infected mice. We used the Gene Expression Omnibus (GEO) DataSets database for functional analysis (GO-analysis) and the DAVID and KEGG databases for signaling pathway analyses. We next integrated the gene and pathway analyses with Transcriptional Regulatory Element Database (TRED) to build an antimicrobial resistance gene regulatory network of S. aureus. We performed association analysis of network genes and diseases using DAVID online annotation tools. We identified a total of 437 virulence genes and 15 transcription factors (TFs), as well as 444 corresponding target genes, in the S. aureus TF regulatory network. We screened seven key network nodes (Met, Mmp13, Il12b, Il4, Tnf, Ptgs2, and Ctsl), four key transcription factors (Jun, C3, Spil, and Il6) and an important signaling pathway (TNF). We hypothesized that the cytokine activity and growth factor activity of S. aureus are combinatorically cross-regulated by Met, Mmp13, Il12b, Il4, Tnf, Ptgs2, and Ctsl genes, the TFs Jun, C3, Spi1, and Il6, as well as the immune response, cellular response to lipopolysaccharide, and inflammatory response. Our study provides information and reference values for the molecular understanding of the S. aureus pathogenetic gene regulatory network.

Regenerative capacity of morselized cartilage in vitro and in an osteochondral defect model

IntroductionNumerous treatment modalities for focal articular cartilage defects exist, including the use of chondrons from minced cartilage morsels. However, the use of consistent and reproducible morsel sizes to heal these lesions, especially with a recently characterized alginate-Nanocellulose (NC) hydrogel scaffold, has not been investigated. MethodsCartilage was harvested and morselized into 0.5mm, 1.0mm, and 5.0mm diameters, and cultured. Cell viability and chondrogenic gene expression was analyzed. 0.5mm diameter minced cartilage morsels were harvested and used to produce the implant paste. The NC paste was filled into a rabbit articular cartilage defect, and histology was used to assess cartilage repair. ResultsChondrocyte viability of the morsels was highly maintained. Chondrogenic and proliferative genes like ACAN, COL2, COMP, PCNA, SOX9, and PRG4 all increased expression to a varying degree as morsel size decreased. 0.5mm morsels exhibited the highest chondrogenic potential. Also, NC paste containing 0.5mm morsels resulted in complete defect closure unlike marrow stimulation alone. Safranin-O staining demonstrated superior cartilage formation in the defect area. DiscussionSmaller consistent morsel size was found to be linked to increased chondrogenic potential, as hypothesized. However, since PRG4 expression was highest in 1mm morsels and MMP-13 gene expression was significantly decreased in only 0.5mm morsels, this demonstrates the need to adjust morsel size to optimize gene expression of interest. NC paste mixed with 0.5mm morsels produced the greatest regenerative capacity. Exploring the regenerative potential of unique hydrogels and chondron morsel sizes is warranted to provide insight on optimizing clinical implants to improve patient recovery.

Nephroprotective effects of silymarin and its fabricated nanoparticles against aluminum-induced oxidative stress, hyperlipidemia, and genotoxicity.

Aluminum (Al) is a ubiquitous element with proven nephrotoxicity. Silymarin (SM) is a mixture of polyphenolic components extracted from Silybum marianum and exhibited protective influences. However, SM bioactivity can be enhanced by its incorporation in chitosan (CS) through the use of nanotechnology. This work proposed to assess the protective influence of SM and its loaded chitosan nanoparticles (SM-CS-NPs) on aluminum chloride (AlCl3)-induced nephrotoxicity. Six groups were created randomly from 42 male Wistar rats and each one contains 7 rats (n = 7). Group I, acted as a control and received water. Group II received SM (15 mg/kg/day) and group III administered with SM-CS-NPs (15 mg/kg/day). Group IV received AlCl3 (34 mg/kg) and groups V and VI were treated with SM and SM-CS-NPs with AlCl3 respectively for 30 days. AlCl3 administration significantly elevated TBARS, H2O2, and kidney function levels besides LDH activity. Whereas GSH, CAT, SOD, GPx, GST, and GR values were all substantially reduced along with protein content, and ALP activity. Additionally, significant alterations in lipid profile, hematological parameters, and renal architecture were observed. Moreover, TNF-α, TGF-β, and MMP9 gene expression were upregulated in kidney tissues. The administration of SM or its nanoparticles followed by AlCl3 intoxication attenuated renal dysfunction replenished the antioxidant system, and downregulated TNF-α, TGF-β, and MMP9 gene expression in renal tissues compared to the AlCl3 group. SM-CS-NPs have more pronounced appreciated protective effects than SM and have the proficiency to balance oxidant/antioxidant systems in addition to their anti-inflammatory effect against AlCl3 toxicity.

Abstract 6150: MMP14 and MKLN1 as colorectal cancer susceptibility genes and a drug repurposing candidate from genome-wide association study in South Korea

Abstract Background: One of the major topics about colorectal cancer (CRC) is to find susceptible genes. Furthermore, functional interpretation of post-GWAS analysis gains attention and various approaches to find drug-repurposing candidates are used in cancer research. Our study aims to identify susceptible genes for CRC and provide drug-repurposing candidates. Methods: CRC patients in Seoul National University Hospital with prospective follow-up were recruited. From blood-derived DNA, genome-wide SNPs were genotyped using the Korea Biobank Array and 409,063 SNPs were extracted. SNP-based logistic regression model was fit for the event of CRC estimating beta values. Furthermore, post-GWAS analysis was conducted using regulatory function, multiple annotation and gene expression database. Drug-repurposing candidate was identified through the pre-trained deep neural network with susceptible genes. Results: We used 500 participants of CRC cases and 1,500 participants of healthy controls. For GWAS, statistically significant associations were mapped to MKLN1, MMP14, PTPN14 and MKL2 genes. For post-GWAS analysis, strong associations were found for MKLN1 (rs75170436, 7q32.3, Beta=-0.90, P-value=5.90×10-13) and MMP14 (rs3751489, 14q11.2, Beta=-1.91, P-value=2.31×10-12). From the drug-repurposing analysis, PPARδ/β agonist (GW0742; Binding score=3.79(MKLN1), 12.06(MMP14)) was identified for both genes with the highest score. Conclusions: We revealed MKLN1 and MMP14 genes as susceptible genes associated with CRC development. Additionally, we identified PPARδ/β agonist (GW0742) as a drug-repurposing candidate for the treatment of CRC. These findings can promote the understanding of CRC development and provide a novel therapeutic candidate for CRC patients in the future. Citation Format: Dabin Yun, Jung-Ho Yang, Sun-Seong Kweon, Jin-ah Sim, Minjung Kim, Ji Won Park, Seung Yong Jeong, Aesun Shin, Nan Song. MMP14 and MKLN1 as colorectal cancer susceptibility genes and a drug repurposing candidate from genome-wide association study in South Korea [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6150.

Abstract 2958: Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype

Abstract Multiple myeloma (MM) is an incurable plasma cell (PC) cancer in which all patients are expected to relapse. Cytotoxic agents such as high-dose melphalan (HDM), which is used as a myeloablative agent prior to autologous stem cell transplant (ASCT) in MM, drive therapy-induced senescence (TIS) in non-cancerous tissues. We hypothesized that HDM similarly activates TIS pathways in surviving MM cells and this response may correlate with patient outcomes post-ASCT. To test this, we developed an in vitro model in which 5TGM1 mouse MM cells are cultured with 10uM melphalan (HDM) or vehicle (Veh) for 6 hours, followed by co-culture in normal media with primary mouse bone marrow stromal cells. Cultures were maintained for 10 days and imaged to quantify cumulative population doublings (CPDs). 5TGM1 were then isolated by FACS to measure telomere-associated DNA damage foci (TAFs), sensitivity to senolytic drugs, and senescence gene expression (RT-qPCR and scRNA-seq). To translate these studies, scRNA-seq was used to analyze CD138+ PCs from MM patient bone marrow post-ASCT. Additionally, longitudinal bone biopsies from MM patients at diagnosis and post-ASCT were stained for CD138+ PCs; PC burden was quantified using artificial intelligence assisted histology (HALO). HDM-5TGM1 exhibited stable growth arrest (decreased CPDs) and sustained DNA damage (increased TAFs) compared to Veh-5TGM1. HDM-5TGM1 were also significantly more sensitive to senolytic ablation (Dasatinib+Quercetin or Fisetin). HDM-5TGM1 showed increased senescence (Cdkn1a, Cdkn1c, Glb1), anti-apoptosis (Bcl2l1), and senescence associated secretory phenotype (Ccl5, Icam1, Mmp13) genes, as well as myeloid markers found to be increased in MM dormancy (Axl, Fcer1g, Mpeg1). Analysis by scRNA-seq revealed that HDM-5TGM1 were G0/G1 arrested, and gene set enrichment analysis (GSEA) confirmed upregulation of senescence pathways with downregulation of DNA replication and cell cycle pathways. Consistent with RT-qPCR results, these cells exhibited myeloid gene expression. Analysis of patient CD138+ PCs post-ASCT by scRNA-seq also revealed a myeloid-like cluster. Of interest, of the 491 genes upregulated in this cluster, 268 were also upregulated in HDM-5TGM1. CD138+ PCs could also be detected in patient bone biopsies post-ASCT, although PC burden was significantly reduced post-ASCT. Surprisingly, the percent reduction in PC burden post-ASCT was greater in relapsed (≤3 years) vs non-relapsed patients. Further, in relapsed patients, PC burden post-ASCT positively correlated with time in remission. Altogether, these findings demonstrate that HDM can induce senescence features and a myeloid-dormancy signature in surviving MM cells. This phenotype is associated with longer durable response, suggesting that activation of TIS may be a strategy to extend PFS. Further, senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent relapse. Citation Format: Angelo Jose Guilatco, Marta Diaz-del-Castillo, Gabriel Alvares Borges, Neal I. Sannuli, Megan L. Ritting, Syed Mohammed Aalam, Nagarajan Kannan, Tamar Tchkonia, James L. Kirkland, Yi Lin, Taxiarchis Kourelis, Matthew T. Drake, Thomas Levin Andersen, Megan Weivoda. Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2958.

Abstract 4039: Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor

Abstract Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. AhR plays a crucial role in inhibiting the activation of immune cells and promoting the growth of tumor cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy in combination with anti-PD-1. Methods: To explore the anti-tumor effects, DA-4505 was administered at a daily dose of 10 mg/kg either alone or in combination with anti-PD-1 (10 mg/kg) in a syngeneic mouse model. Tumor volume, survival rates, and metastasis were measured, and immune profiles were evaluated using mIHC, flow cytometry, and scRNAseq. Results: The synergistic anti-tumor effects of the DA-4505 and aPD-1 combination therapy were observed across four mouse tumor models (CT26, 4T1, LLC, TC1). The combination group demonstrated a significant improvement in anti-tumor efficacy compared to the monotherapy groups. Despite the moderate response of aPD-1 in the CT26 colon tumor model, the combination group displayed heightened anti-tumor effects through increase in CD8 T cell activity (P<0.05). In the LLC and TC1 lung cancer models, the combination therapy demonstrated improved anti-tumor effects and survival rates (P<0.001). Notably, the LLC model exhibited a significant increase in M1-type macrophages, while the TC1 model showed a significant augmentation in functional markers of CD3+ T cells within the combination group (P<0.05). In both models, there was a significant reduction in the ratio of immunosuppressive M-MDSC and PMN-MDSC in the combination group. Immune depletion assays elucidated that DA-4505 mediated anti-tumor effects by increase in CD8+ T cells, NK cells, and macrophages in the CT26 model. In contrast, the LLC model exhibited the contribution of macrophages and CD4+ T cells to anti-tumor effects in the combination group (P<0.05). To assess the impact on metastasis inhibition, an orthotopic model of 4T1 mouse tumor was established, revealing a significantly increased anti-tumor effects and survival rate in the combination group, accompanied by a reduced lung metastasis ratio. H&E staining confirmed diminished migration of tumor tissue to adjacent areas. scRNAseq analysis of CT26 tumor samples indicated consistent changes at the gene expression level, reflecting a decrease in metastasis-related MMP and VEGF genes in the combination group and an increasing trend in cytotoxicity-related genes following DA-4505 treatment. Conclusion: The AhR inhibitor, DA-4505, has demonstrated enhanced anti-tumor efficacy when administered in combination with anti-PD-1. Furthermore, it not only promoted tumor elimination but also demonstrated significant efficacy in inhibiting metastasis. This study highlights the potential of combining AhR inhibitors with immunotherapies for clinical treatment. Citation Format: Dong Kwon Kim, Sujeong Baek, Seung Min Yang, Young Taek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Mi hyun Kim, Heekyung Han, Kwangmin Na, Chai Young Lee, Yu Jin Han, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4039.

A machine learning-based diagnostic model for myocardial infarction patients: Analysis of neutrophil extracellular traps-related genes and eQTL Mendelian randomization.

To identify neutrophil extracellular trap (NET)-associated gene features in the blood of patients with myocardial infarction (MI) using bioinformatics and machine learning, with the aim of exploring potential diagnostic utility in atherosclerosis. The datasets GSE66360 and GSE48060 were downloaded from the Gene Expression Omnibus (GEO) public database. GSE66360 was used as the training set, and GSE48060 was used as an independent validation set. Differential genes related to NETs were screened using R software. Machine learning was performed based on the differential expression of NET-related genes across different samples. The advantages and disadvantages of 4 machine learning algorithms (Random Forest [RF], Extreme Gradient Boosting [XGBoost, XGB], Generalized Linear Models [GLM], and Support Vector Machine-Recursive Feature Elimination [SVM-RFE]) were compared, and the optimal method was used to screen feature genes and construct diagnostic models, which were then validated in the external validation dataset. Correlations between feature genes and immune cells were analyzed, and samples were reclustered based on the expression of feature genes. Differences in downstream molecular mechanisms and immune responses were explored for different clusters. Weighted Gene Co-expression Network Analysis was performed on different clusters, and disease-related NET genes were extracted, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Finally, Mendelian randomization was employed to further investigate the causal relationship between the expression of model genes and the occurrence of MI. Forty-seven NET-related differential genes were obtained, and after comparing the 4 machine learning methods, support vector machine was used to screen ATG7, MMP9, interleukin 6 (IL6), DNASE1, and PDE4B as key genes for the construction of diagnostic models. The diagnostic value of the model was validated in an independent external validation dataset. These five genes showed strong correlations with neutrophils. Different sample clusters also demonstrated differential enrichment in pathways such as nitrogen metabolism, complement and coagulation cascades, cytokine-cytokine receptor interaction, renin-angiotensin system, and steroid biosynthesis. The Mendelian randomization results demonstrate a causal relationship between the expression of ATG7 and the incidence of myocardial infarction. The feature genes ATG7, MMP9, IL6, DNASE1, and PDE4B, identified using bioinformatics, may serve as potential diagnostic biomarkers and therapeutic targets for Myocardial infarction. Specifically, the expression of ATG7 could potentially be a significant factor in the occurrence of MI.

Abstract P524: Prolonged Mild Sleep Restriction Results in Specific Alterations in Genome-Wide DNA Methylation: A Randomized Crossover Trial

Introduction: Short sleep duration is associated with increased cardiometabolic risk. DNA methylation plays a critical role in the regulation of gene expression and studies suggest that sleep deprivation may alter DNA-methylation patterns. However, most findings are from acute sleep deprivation or short duration studies. Hypothesis: Prolonged sleep restriction study that mimics real-life situations is associated with differentially methylated CpG loci (DML) in the core clock candidate genes and across the epigenome in an epigenome-wide association study (EWAS) Methods: Sixty participants (65% women) aged 21-73 y were included in randomized crossover studies with 2 phases of 6 weeks each. Phases differed in sleep duration: either adequate sleep (AS; sleep ≥7h/night) or sleep restriction (SR; -1.5h/night relative to AS). DNA was isolated from whole blood at wk 0 and wk 6 of each phase. DNA methylation (DNAm) was quantified the Illumina Infinium MethylationEPIC BeadChip array v.2.0 (>900K methylation sites) and Partek Genomic Suite v.720.0831. After quality control and normalization, several global and stratified EWASs were performed to test the impact of sleep condition on DNAm. Analyses were adjusted for week, phase, sex, age, BMI and cell type composition. We also focused on methylation changes of the selected core-clock candidate genes ( RORA, CRY1, NR1D2, TIMELESS, NPAS2, NFIL3, RORB, PER3, CSNK1E, ARNTL2, CRY2, CLOCK, ARNTL, RORC, PER2, BHLHE40, NR1D1, CSNK1D, BHLHE41 and PER1 ). Results: In the candidate gene approach, we detected several significant DML. Top-ranked DML in selected core clock genes with significant interactions between condition and time were: cg02394126 at ARNTL (p<0.001), cg23506964 at CLOCK (p=0.001), cg03701037 at NPAS2 (p=0.009), cg06606972 at NPAS2 (p=0.009), and cg13576304 at RORA (p=0.008). Hypermethylation was primarily observed after 6 wk of SR vs AS for cg02394126, cg23506964, cg03701037, and cg06606972, while hypomethylation was observed cg13576304 ( RORA ). In our EWAS, we detected some suggestive significant condition x time interactions in cg23738833 at SNHG3-RCC1 (p=1,34E-06), cg13280380 in the FAF1 gene (p=2,25E-05), cg03179866 in the MMP12 gene (p=2,78E-05), and cg13063696 (p=3,21E-05). All but cg23738833 ( SNHG3-RCC1 ) showed hypermethylation after 6 wk of SR vs AS. Conclusion: Six weeks of mild SR was associated with changes in DNAm in core-clock candidate genes and in other genes in the EWAS. However, further studies are needed to confirm these findings and determine the functional pathways.

A study on the anti-senescent effects of flavones derived from Prinsepia utilis Royle seed residue

Ethnopharmacological relevancePrinsepia utilis Royle, also known as the Anas fruit, is a unique perennial woody oil plant from Yunnan Province, China. In the ancient texts of Dongba sutras and Yunnan Southern Materia Medica, it has been documented that the local Naxi, Tibetan, and Mosuo communities extensively utilize the root and leaf fruits of green thorns for various purposes. These include treating mild-to-moderate specific dermatitis, moisturising the skin, providing protection against UV damage, aiding childbirth in pregnant women, safeguarding stomach health, reducing the risk of arteriosclerosis, and delaying aging. Aim of the studyIn this study, leftover residues from oil extraction were efficiently reused, and flavonoids were identified during subsequent extraction and separation processes. The anti-senescent effects of flavonoids in P. utilis Royle have not been systematically studied. Therefore, the objective of this study was to explore the anti-senescent properties of the flavonoids obtained from P. utilis Royle. MethodsFirst, HPLC and other analytical techniques were used to identify the components of the P. utilis Royle flavonoid (PURF). Next, DPPH, hydroxyl radicals, superoxide anion O2−, collagenase, and elastase were initially detected using in vitro biochemical assays. To examine its antioxidant properties, a zebrafish model was used, and to confirm its anti-senescent effects, a d-galactose-induced mouse aging model was employed. The anti-senescent mechanism of PURF was examined using a natural senescence HFF model. Furthermore, the anti-senescent target was confirmed using a 3D full T-Skin™ model. ResultsIn vitro biochemical assays demonstrated that flavones exhibited potent antioxidant activity and anti-senescent potential by inhibiting DPPH, hydroxyl radicals, superoxide anion O2−, collagenase, and elastase. It significantly enhanced the antioxidant effect on zebrafish while suppressing ROS and inflammatory injury, up-regulating COL1A1, COL3A1, AMPK, and mTOR gene expression and down-regulating MMP-9, TGF-β, p21, and p16 gene expression suggesting its potential anti-senescent ability. Findings from the D-galactose-induced aging mouse model showed that PURF greatly increased SOD levels, while simultaneously decreasing HYP and MDA levels. In addition, when PURF was given to the HFF cell and 3D full T-Skin™ model, consistent trends were observed in gene and protein expression, with up-regulation of COL1A1, COL3A1, AMPK, and mTOR genes and down-regulation of TGF-β, MMP-1, MMP-9, p21, and p16 genes. Therefore, these preliminary findings indicate that flavones can modulate AMPK/mTOR/TGF-β signalling pathways to exert its influence. ConclusionThe kernel residue of natural P. utilis Royle oil extracted from Yunnan province was previously considered agricultural waste, but we successfully extracted and isolated its flavonoid components. Our preliminary studies demonstrated its potential as an environmentally friendly anti-senescent raw material.

Identification of Hedyotisdiffusa Willd-specific mRNA–miRNA–lncRNA network in rheumatoid arthritis based on network pharmacology, bioinformatics analysis, and experimental verification

Hedyotisdiffusa Willd (HDW) possesses heat-clearing, detoxification, anti-cancer, and anti-inflammatory properties. However, its effects on rheumatoid arthritis (RA) remain under-researched. In this study, we identified potential targets of HDW and collected differentially expressed genes of RA from the GEO dataset GSE77298, leading to the construction of a drug-component-target-disease regulatory network. The intersecting genes underwent GO and KEGG analysis. A PPI protein interaction network was established in the STRING database. Through LASSO, RF, and SVM-RFE algorithms, we identified the core gene MMP9. Subsequent analyses, including ROC, GSEA enrichment, and immune cell infiltration, correlated core genes with RA. mRNA–miRNA–lncRNA regulatory networks were predicted using databases like TargetScan, miRTarBase, miRWalk, starBase, lncBase, and the GEO dataset GSE122616. Experimental verification in RA-FLS cells confirmed HDW’s regulatory impact on core genes and their ceRNA expression. We obtained 11 main active ingredients of HDW and 180 corresponding targets, 2150 RA-related genes, and 36 drug-disease intersection targets. The PPI network diagram and three machine learning methods screened to obtain MMP9, and further analysis showed that MMP9 had high diagnostic significance and was significantly correlated with the main infiltrated immune cells, and the molecular docking verification also showed that MMP9 and the main active components of HDW were well combined. Next, we predicted 6 miRNAs and 314 lncRNAs acting on MMP9, and two ceRNA regulatory axes were obtained according to the screening. Cellular assays indicated HDW inhibits RA-FLS cell proliferation and MMP9 protein expression dose-dependently, suggesting HDW might influence RA’s progression by regulating the MMP9/miR-204-5p/MIAT axis. This innovative analytical thinking provides guidance and reference for the future research on the ceRNA mechanism of traditional Chinese medicine in the treatment of RA.

Effects of Viscum coloratum (Kom.) Nakai on collagen-induced rheumatoid arthritis

Ethnopharmacological relevanceViscum coloratum (Kom.) Nakai has been traditionally used in China for nearly a thousand years to treat rheumatic diseases. However, its efficacy and mechanisms in treating rheumatoid arthritis (RA) have not been demonstrated. Aim of the studyTo investigate the anti-arthritic effects and molecular mechanisms of Viscum coloratum (Kom.) Nakai on collagen-induced arthritic mice through network pharmacology technology and experimental validation. Materials and methodsFirst, the main ingredients of the extract of Viscum coloratum (Kom.) Nakai (EVC) were identified through chemical composition characterization using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS). Then, the collagen-induced arthritis (CIA) model was established in DBA/1 J mice and the ameliorative effects of EVC on the progression of CIA mice were evaluated by oral treatment with different doses of the EVC for 28 days. After that, cytokine antibody microarray assay was used to detect the levels of multiple inflammation-related cytokines and chemokines in each group, and performed Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis. Subsequently, the potential target for the effective chemical components of EVC in treating RA was identified using various databases. Additionally, a drug-disease target protein-protein interaction network (PPI) was conducted using Cytoscape for visualization and clustering, while GO and KEGG enrichment analyses were performed with the Metascape database. Finally, identified phenotypes and targets by network pharmacology analysis were experimentally validated in vivo. ResultsTreatment with EVC significantly suppressed the severity of CIA with a dramatic reduction of paw swelling, arthritis index, levels of IgGs (IgG, IgG1, IgG2a, and IgG2b), multi-inflammation-related cytokines and chemokines on the progression of CIA. Histopathological examinations showed EVC could markedly inhibit inflammatory cell infiltration, tartrate-resistant acid phosphatase (TRAP) activity of osteoclast, and bone destruction. Furthermore, GO and KEGG enrichment analyses revealed that EVC could ameliorate RA by inhibiting osteoclast differentiation and regulating multiple signaling pathways including Osteoclast differentiation, IL-17, and TNF. PPI network analysis demonstrated that AKT1, MMP9, MAPK3, and other genes were highly related to EVC in treating RA. Finally, we proved that EVC could inhibit the expression of NFTAc1, MMP9, Cathepsin K, and AKT which were closely related to osteoclast activity. ConclusionsEVC could treat RA through multiple components, multiple targets, and multiple pathways. The present study demonstrated the therapeutic efficacy of EVC and its molecular mechanisms in treating RA, indicating that it would be a potent candidate as a novel botanical drug for further investigation.

The Effect of Leukocyte Removal and Matrix Metalloproteinase Inhibition on Platelet Storage Lesions.

The reasons for unfavorable changes in platelet concentrate (PC) quality during storage are not fully understood yet. We aimed to evaluate whether leukocytes and matrix metalloproteinases (MMPs) lead to a decrease in the quality of PCs and examine whether MMP inhibition will slow down the platelets' aging. Nine PCs were divided into three parts: (1) leukocyte-depleted (F) PCs, (2) PCs with no additional procedures (NF), and (3) PCs with the addition of an MMP inhibitor-doxycycline (D). Each PC was stored for 144 h, and a sample for testing was separated from each part on the day of preparation and after 24, 48, 72 and 144 h of storage. Blood morphological analysis, platelet aggregation, and the expression of activation markers were evaluated. MMP-2 and MMP-9 concentration, activity, and gene expression were assessed. Platelet aggregation decreased, and platelet activation marker expression increased during the storage. D concentrates showed the lowest level of platelet activation. In turn, leukocyte-depleted PCs showed the highest level of platelet activation in general. MMP-9 platelet activity was higher in leukocyte-containing concentrates at the end of the storage period. We concluded that the filtration process leads to a higher platelet activation level. The presence of doxycycline in PCs reduces the expression of the activation markers as compared to leukocyte-depleted concentrates.

Modulating the Wnt Signaling Pathway in Wound Healing: The Potential of L-Carnosine, Zinc Sulfate, and B-Complex Vitamins Cocktail

Background: The process of wound healing is a dynamic event that starts with inflammation, proliferation, and migration of various types of fibroblast cells and aims to restore the integrity and functionality of damaged tissue. One crucial signaling pathway involved in wound healing is the Wnt signaling pathway. This study aimed to investigate the effects of a cocktail consisting of L-carnosine, zinc sulfate, and B-complex vitamins on the modulation of the Wnt signaling pathway during wound healing. Methods: The optimal dosages of L-carnosine, zinc sulfate, and B-complex were used to prepare the cocktail. Then, the cocktail-mediated wound healing was measured through scratch assay on human dermal fibroblast (HDF) cells. Finally, the effects of the cocktail on the expression of SFRP1, SFRP2, SFRP4, MMP7, and RSPO2 genes involved in the Wnt signaling pathway were measured during the wound closure phenomenon by real-time quantitative polymerase chain reaction (RT-qPCR). Results: The scratch assay demonstrated the wound-healing effects of the prepared cocktail on HDF cells. Additionally, the RT-qPCR results showed that the cocktail can activate the Wnt signaling pathway by down-regulating the expression of SFRP1, SFRP2, SFRP4, and MMP7 and up-regulating the expression of RSPO2. Conclusions: The data suggest that the L-carnosine, zinc sulfate, and B-complex vitamin cocktail exhibits wound healing properties by increasing fibroblast proliferation via the activation of the Wnt pathway.

Inflammation Factors and Genistein Supplementation in Cancer-Preliminary Research.

The purpose of this study was to evaluate the effect of genistein in nano, micro, and macro forms on the intensity of the DMBA-induced tumor process in rats and to understand the mechanisms of this action. The effect of genistein supplementation on the content of selected eicosanoids (HETEs, HODE, and HEPE) in the serum of rats was evaluated. The levels and expression of genes encoding various pro-inflammatory cytokines (IL-1, IL-6) and MMP-9 in the blood of rats were also investigated. The biological material for the study was blood obtained from female rats of the Sprague Dawley strain (n = 32). The animals were randomly divided into four groups: animals without supplementation, and animals supplemented at a dose of 0.2 mg/kg b.w. (0.1 mg/mL) with macro, micro (587 ± 83 nm), or nano (92 ± 41 nm) genistein. To induce mammary neoplasia (adenocarcinoma), rats were given 7,12-dimethyl-1,2-benz[a]anthracene (DMBA). The content of selected eicosanoids was determined by liquid chromatography with UV detection. An immunoenzymatic method was used to determine the content of cytokines and MMP-9. The expression of the IL-6, IL-1beta, and MMP-9 genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes. Based on the study, it was shown that supplementation of animals with genistein in macro, micro, and nano forms increased the intensity of the tumor process in rats. It was shown that the content of 12-HEPE, HODE, and 12-HETE in the serum of genistein-supplemented rats was statistically significantly lower with respect to the content of the aforementioned markers in the serum of rats receiving only a standard diet, devoid of supplementation. It was found that animals supplemented with nano-, micro-, and macrogenistein had higher levels of metalloproteinase-9, MMP-9, compared to animals without supplementation. There was a significant increase in MMP-9 gene expression in the blood of macrogenistein-supplemented animals, relative to the other groups of rats. On the basis of the study, it was shown that supplementation of animals with nano-, micro-, and macrogenistein had an effect on the development of the tumor process. Dietary supplementation with genistein significantly decreased the level of selected eicosanoids, which may have significant impacts on cancer development and progression.

Co-delivery of epirubicin and letrozole using a metal-organic framework nanoparticle in breast cancer therapy

Combination chemotherapy is a viable approach for cancer treatment in clinics when dealing with multidrug-resistant cancer. The difficulty of concentrating medications in drug-resistant cancers remains a substantial issue, making it challenging to get sufficient multi-drug delivery into tumour cells to enhance the synergetic therapeutic effect. To enhance combination therapy, we fabricated metal-organic framework (MOF) nanoparticles (NPs) that co-deliver letrozole (Let) and epirubicin (Epi). We created UIO-66@NH2-based MOFs to achieve desired encapsulation efficiencies of 82.93 ± 2.13 for Let and 66.84 ± 1.25 for Epi. The drug release profile revealed that the release rate of Let and Epi from the nanoparticles was pH-dependent, with a significant increase in acidic environments. This indicated the adaptive release capability of UIO-66-Let/Epi@NH2 in the breast cancer milieu. The nanoparticle size and entrapment efficiency were more stable at 4 °C as compared to 25 °C. Additionally, cellular assays demonstrated that a MOF loaded with Let and Epi increased the rate of apoptosis in MCF-7 cells compared to Let, Epi, and their combination (Let + Epi). The UIO-66-Let/Epi@NH2 group showed increased expression levels of Caspase3, Caspase9, and Mfn1 genes while the expression levels of MMP-3 and MMP-9 genes decreased. The effectiveness of the formulation in inducing apoptosis was confirmed by the results of DAPI staining microscopy and flow cytometry analysis. The higher apoptotic rate and toxicity of the Let/Epi-loaded UIO-66@NH2 MOF may be due to its greater delivery effectiveness into cancer cells. This article describes a novel MOF nanocarrier for the co-delivery of Let and Epi to treat breast cancer. The results demonstrate the potential of MOF nanocarriers as stimuli-responsive co-delivery systems for various drugs.