MMP-9 Gene Expression Research Articles

5-aza-2\u2032-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/\u03b2-catenin pathway in human uterine leiomyomas

BackgroundUterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2′-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option.MethodsProspective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR.ResultsDNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577).Conclusions5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

The Effect of B9 and B12 Vitamins Deficiency on Hypomethylation of MMP-9 gene Promoter Among Women With Preterm Parturition.

Preterm birth is one of the problems that pregnant women may encounter during pregnancy. Over-expression of Matrix metalloproteinases (MMPs) has the main role in the untimely remodeling of the extracellular matrix (ECM) and preterm delivery, but its exact mechanism is unknown. This study was undertaken to examine the methylation and expression patterns of this gene in women delivering preterm and also to analyze the correlation of serum B9 and B12 vitamins levels with expression and methylation of MMP-9 gene. The MMP-9 RNA and protein levels in 50 women delivering preterm and 50 women delivering at term were determined by Real-time PCR and enzyme-linked immunosorbent assay (ELISA) methods. Methylation pattern of this gene was evaluated by Epitect Methyl-II PCR assay and methylation-sensitive restriction enzymes combined with PCR amplification method; then, their correlation with serum B9 and B12 vitamins was studied. Placental MMP-9 RNA and protein levels were higher (P < 0.03, P < 0.01) in women delivering preterm as compared to those delivering at term. We also observed significant hypomethylation of promoter of this gene in preterm samples (P < 0.012). Moreover, significant negative correlation between B9 and B12 vitamins concentration and hypomethylation of MMP-9 gene was seen (r = - 0.68 P < 0.001). Our study suggests an impressive role of deficiency of B9 and B12 vitamins concentration on the hypomethylation and overexpression of MMP-9 gene which may lead to preterm parturition of pregnant women.

Quercetin synergistically potentiates the anti-metastatic effect of 5-fluorouracil on the MDA-MB-231 breast cancer cell line.

Breast cancer (BC) cells' ability to metastasize to other tissues increases mortality. The Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) facilitate cancer cell migration. 5-fluorouracil is a frequently applied chemotherapeutic agent in cancer treatment with destructive side effects on normal tissues. Hence, researchers have focused on finding a way to reduce the dose of chemotherapeutic drugs. Quercetin, a natural polyphenolic compound, has inhibitory effects on proliferation and migration of tumor cells. This study evaluated the effect of the combination of Quercetin and 5-fluorouracil on migration of the MDA-MB-231 breast cancer cell line. The effect of Quercetin, 5-fluorouracil , and their combination on MDA-MB-231 breast cancer cell proliferation was investigated through MTT assay. Inhibition of tumor cell migration was examined by wound healing assay. Finally, the effect of treatments on gene expression of MMP-2 and MMP-9 was evaluated by quantitative real-time PCR. The IC50 values for Quercetin and 5-fluorouracil after 48 hr treatment were 295 μM and 525 μM, respectively. The combination index (CI) for Quercetin and 5-fluorouracil was <1, indicating synergy between them. The combination of Quercetin plus 5-fluorouracil resulted in a significant reduction in migration rate and MMP-2 and MMP-9 gene expressions of MDA-MB-231 cancer cells compared with the individual application of 5-FU. Quercetin enhances the suppressory effect of 5-fluorouracil on migration of BC cells. The combination of Quercetin and 5-fluorouracil can be an attractive field for future studies.

Abstract 1139: Potent SMARCA2 targeted degraders induce genetic synthetic lethality in SMARCA4 deleted cancer

Abstract SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core subunits of the SWI/SNF complexes which contain ATPase domain and DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to damaging mutations (e.g. nonsense, frameshift deletion, splice site mutations) and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers through the use of selective SMARCA2 degraders induces synthetic lethality. We designed and synthesized a variety of novel SMARCA2 degraders and tested them for degradation potency and selectivity by high-throughput in-cell western blot and probed for both SMARCA2 and SMARCA4 proteins in SMARCA4 WT expressing non-small cell lung cancer (NSCLC) NCI-H520 cells. We identified a unique series of potent SMARCA2 degraders with 20-40 fold greater selectivity for SMARCA2 vs SMARCA4. Global proteomics analysis confirmed that these degraders are highly selective for SMARCA2, and demonstrate minimal degradation of any other protein except PBRM1, another bromodomain VIII family protein. Our data suggest that the greater degradation selectivity observed is a result of preferential SMARCA2 ternary complex formation and/or poly-ubiquitination processing. Global RNA expression analysis shows that treatment with SMARCA2 degraders downregulates gene signatures associated with the cell cycle, cell proliferation, apoptosis and cell adhesion in SMARCA4-deleted NSCLC NCI-H1693 cells. Specifically, we found a significant reduction of gene expression of AXL, SMAD3, MMP2, and KRT80, all of which are important factors for tumor cell signaling and invasion. Additionally, protein set enrichment analysis (PSEA) shows that SMARCA2 degrader-treated NCI-H1693 cells downregulate expression of cell cycle and DNA replication related protein signatures as well as upregulated antigen processing and presentation pathway. A cell line panel analysis for cell viability and clonogenicity demonstrated that our selective SMARCA2 degraders effectively inhibit proliferation of SMARCA4-deleted cancer cells with nanomolar potencies, but not SMARCA4 WT or SMARCA2/4 null cancer cells. Our SMARCA2 degraders also significantly suppressed the growth of patient derived SMARCA4-deleted NSCLC tumor cells in primary 3D culture assay. In summary, consistent with previous studies and genomic perturbation analyses, our potent and selective SMARCA2 degraders induce synthetic lethality in SMARCA4-deleted cancers in vitro. Citation Format: Koichi Ito, Anjana Agarwal, Philip Pitis, Min Wang, Jack Carter, Miles Cowart, Chaofeng Dai, William Gowen-MacDonald, Eliza Elliot, Brian Vidal, Jacob Spruance, Hsin-Yao Tang, Bruce Ruggeri, Liang Lu, Andrew Combs, Hong Lin, Peggy Scherle, Kris Vaddi. Potent SMARCA2 targeted degraders induce genetic synthetic lethality in SMARCA4 deleted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1139.

GSK-3 Inhibition Modulates Metalloproteases in a Model of Lung Inflammation and Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is mainly characterized by aberrant extracellular matrix deposition, consequent to epithelial lung injury and myofibroblast activation, and inflammatory response. Glycogen synthase kinase 3 (GSK-3) is a serine–threonine kinase involved in several pathways, and its inhibition has been already suggested as a therapeutic strategy for IPF patients. There is evidence that GSK-3 is able to induce matrix metalloproteinase (MMP) expression and that its inhibition modulates MMP expression in the tissues. The aim of our study was to investigate the role of GSK-3 and its inhibition in the modulation of MMP-9 and -2 in an in vivo mouse model of lung fibrosis and in vitro using different cell lines exposed to pro-inflammatory or pro-fibrotic stimuli. We found that GSK-3 inhibition down-modulates gene expression and protein levels of MMP-9, MMP-2, and their inhibitors TIMP-1 and TIMP-2 in inflammatory cells harvested from bronchoalveolar lavage fluid (BALF) of mice treated with bleomycin as well as in interstitial alveolar macrophages and cuboidalized epithelial alveolar cells. To the same extent, GSK-3 inhibition blunted the increased MMP-9 and MMP-2 activity induced by pro-fibrotic stimuli in a human lung fibroblast cell line. Moreover, the αSMA protein level, a marker of fibroblast-to-myofibroblast transition involved in fibrosis, was decreased in primary fibroblasts treated with TGFβ following GSK-3 inhibition. Our results confirm the implication of GSK-3 in lung inflammation and fibrosis, suggesting that it might play its role by modulating MMP expression and activity but also pushing fibroblasts toward a myofibroblast phenotype and therefore enhancing extracellular matrix deposition. Thus, its inhibition could represent a possible therapeutic strategy.

3D HeLa spheroids as a model for investigating the anticancer activity of Biginelli-hybrids

In previous study, we examined the anticancer effects of novel Biginelli-hybrids against HeLa cell line on 2D monolayer culture. The five most effective compounds were chosen for further analysis of their anticancer activity against HeLa spheroids. Using the 3D models implies the possible differences in anticancer effects and mechanisms of activity of tested compounds. The compounds 4c and 4d exerted the strongest activity against 3D HeLa spheroids and induced to some extent loosened cell-cell contacts in spheroids, leading to the largest reduction in the diameter of the spheroids. Additionally, the highest accumulation of the cells in the subG1 phase of the cell cycle was observed after the treatment with compounds 4d and 4c, while the compound 4f led to the G2/M arrest. The invasion potential of treated HeLa cells in spheroids was monitored by imaging of spheroids embedded in a matrix made of matrigel and collagen and by determination of MMP2, MMP9, and VEGF gene expression levels. The compound 4l did not show invasion-suppressive activity, while the compounds 4c and 4d exerted the strongest anti-invasive activity.

Effect on physiological and production parameters upon supplementation of fermented yeast culture to Nicobari chickens during and post summer.

Nicobari is an indigenous bird reared for meat and eggs. This study evaluated the effect of heat stress on plasma levels of leptin, growth hormone and their receptors, liver AMP kinase, plasma cholesterol and lipid peroxide (MDA). The laying period coincided with the post summer period. The birds were equally divided into three groups, control group was offered ad libitum feed and treatment groups were supplemented with fermented yeast culture at 700mg (T1) and 1.4g/kg (T2) of feed/day. The levels of plasma Leptin and GH hormones were higher (p<0.05) in the control group when compared to the treatment groups. The expression of the hormone receptors was higher in the brain, and MMP3 gene expression in the magnum was lower in the treatment group. Plasma cholesterol, MDA and AMP kinase were significantly higher (p<0.05) in the control group. Fermented yeast culture supplementation decreased feed intake and increased egg production parameters, which indicates a greater efficiency of supplementation. Supplementation reduced the severity of necrosis of villi in the jejunum when compared to control. In conclusion, higher ambient temperature during summer had negative effect on production parameters through modulation of physiological parameters which could be ameliorated by supplementation of FYC.

Doxorubicin-paclitaxel sequential treatment: insights of DNA methylation and gene expression changes of luminal A and triple negative breast cancer cell lines.

Breast cancer is one of the significant causes of death among women diagnosed with cancer worldwide. Even though several chemotherapy combinations are still the primary treatment of breast cancer, unsuccessful treatments, and poor prognostic outcomes are still being reported. DNA methylation and gene expression changes among two breast cancer cell lines representing luminal A (MCF-7) and triple-negative (MDA-MB-231) cancers were determined after sequential combination treatment of doxorubicin and paclitaxel and analyzed using Ingenuity Pathway Analysis. Promoter methylation changes were seen in different treated MCF-7 cells and accompanied by changes in the gene expression of CCNA1 and PTGS2. In MDA-MB-231 cells, the hypomethylation of ESR1 was not accompanied by an increase in its gene expression in any treated cells. The hypomethylation of GSTP1 and MGMT was accompanied by an increase in gene expression levels in the group treated with doxorubicin only. Also, significant downregulation of several genes like MUC1 and MKI67 in MCF-7 cells treated with doxorubicin showed much lower gene expression (- 37.63, - 10.88 folds) when compared with cells treated with paclitaxel (- 2.47, - 2.05 folds) or the combination treatment (- 18.99, - 2.81 folds), respectively. On the other hand, a synergistic effect on MMP9 gene expression was significantly seen in MDA-MB-231 cells treated with the combination (- 9.99 folds) in comparison with the cells treated with doxorubicin (- 3.62 folds) or paclitaxel (1.75 folds) alone. Chemotherapy combinations do not always augment the molecular changes seen in each drug alone, and these changes could be utilized as treatment response markers.

Celecoxib treatment dampens LPS-induced periapical bone resorption in a mouse model.

To evaluate the efficacy of selective and nonselective inhibitors of cyclooxygenase-2 enzymes in the treatment of experimental apical periodontitis induced by bacterial lipopolysaccharide (LPS) in vivo in a mouse model. Thirty-six C57BL/6 mice were used. After access cavity preparation, a solution containing E.coli LPS (1.0µgµL-1 ) was inoculated into the root canals of the mandibular and maxillary right first molars (n=72) After 30days, apical periodontitis was established and the animals were systemically treated with celecoxib, a selective COX-2 inhibitor (15mgkg-1 ), or indomethacin, a nonselective COX-2 inhibitor (5mgkg-1 ), for 7 and 14days. Blocks containing teeth and bone were removed for histopathological and histometric analyses (haematoxylin and eosin), evaluation of osteoclasts numbers (tartrate-resistant acid phosphatase enzyme - TRAP) and immunohistochemistry for RANK, RANKL and OPG. Gene expression was performed using reverse transcription and real-time polymerase chain reaction (qRT-PCR) for RANK, RANKL, OPG, TRAP, MMP-9, cathepsin K and calcitonin receptor. Histopathological, histometric, TRAP, immunohistochemistry and qRT-PCR data were evaluated using Kruskal-Wallis followed by Dunn's test (α=0.05). Systemic administration of celecoxib for 7 and 14days prevented periapical bone resorption (P<0.0001), differently from indomethacin that exacerbated bone resorption at 7days (P<0.0001) or exerted no effect at 14days (P=0.8488). Celecoxib treatment reduced osteoclast formation in apical periodontitis, regardless of the period of treatment (P<0.0001 for 7days and P=0.026 for 14days). Administration of celecoxib or indomethacin differentially modulated the expression of genes involved in bone resorption. At 7days, celecoxib and indomethacin treatment significantly inhibited expression of mRNA for cathepsin K (P=0.0005 and P=0.016, respectively) without changing TRAP, MMP-9 and calcitonin receptor gene expression. At 14days, celecoxib significantly inhibited expression of mRNA for MMP-9 (P<0.0001) and calcitonin receptor (P=0.004), whilst indomethacin exerted no effect on MMP-9 (P=0.216) and calcitonin receptor (P=0.971) but significantly augmented cathepsin K gene expression (P=0.001). The selective COX-2 inhibitor celecoxib reduced osteoclastogenic signalling and activity that dampened bone resorption in LPS-induced apical periodontitis in mice, with greater efficacy than the nonselective inhibitor indomethacin.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1β), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1β. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1β. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

Effect of Endurance Training and Stem Cell on Fgf2 and Mmp13 Gene Expression in Knee Tissue of Rats with Osteoarthritis

Introduction: The aim of this study was to investigate the effect of endurance training and stem cell injection on FGF2 and MMP13 gene expression in knee tissue of rats with knee osteoarthritis. Methods: The type of study was an experimental one.Male Wistar rats were randomly divided into 5 groups: control - healthy, control - patient, patient - stem cell, patient - training, and patient - training - stem cell. The training program consisted of 30 minutes of running on a treadmill with no slope at 16 m / min for the first week, with 1 m / min added weekly. Rats received MSCs through intracellular injection of 1*106 cells / kg. Expression of FGF2 and MMP13 genes was measured by Real Time PCR. One way ANOVA and if there was a significant difference, Tukey post hoc test were used to determine the difference between groups. All statistical analyzes were performed using SPSS version 20. Results: Data analysis showed that training and stem cell therapy have significantly increased in genes expression of FGF2 and MMP-13 in mice with osteoarthritis (p &lt;0.000). Moreover, Tukey post hoc test showed a significant difference in the level of FGF2 changes in the training-stem cell groups compared to the training group and the saline group (p=0.000‌). There was also a significant difference between the MMP-13 follow-up test with the control group with the saline group, the training group and the stem cell with the train-stem cell, and the saline group with the training group9(P=0.000‌). Conclusion: The results showed that FGF2 levels and MMP13 in the cartilage tissue of mice with osteoarthritis have increased and endurance training and stem cells therapy caused a decrease in the level of factors.

The impact of hypoestrogenism and occlusal function on MMP1, MMP8 and MMP13 expression in the odontogenic region in rats

Background: The impact of estrogen deficiency and occlusion in the matrix metalloproteinases (MMPs) expression in dental tissues has not yet been elucidated. Objective: To evaluate the influence of estrogen deficiency and occlusal hypofunction and hyperfunction on the gene expression of MMP1, MMP8 and MMP13 in the odontogenic region of teeth in continuous growth, in the murine model. Material and methods: Rats (Wistar Hannover lineage) were divided into two groups according to the intervention received: Hypoestrogenism Group - ovariectomy surgery and Control Group - fictitious surgery. Occlusal hypofunction and hyperfunction conditions were also established in all animals (each animal presented both conditions). After euthanasia, the hemimandibles were removed to evaluate the gene expression through real time PCR. T-test was used to compare the mean differences between groups (P&lt;0.05). Results: There was no statistically significant difference of the relative gene expression of MMP1, MMP8 and MMP13 between the hypoestrogenism and control groups (P&gt;0.05). A statistically significant difference of the relative MMP13 expression between the occlusal hypofunction and hyperfunction tooth was observed (P=0.03). In the hypoestrogenism group, MMP13 was overexpressed in hypofunction tooth (P=0.045). Conclusion: Occlusal function affects MMP13 expression in the odontogenic region, in murine model.

Importance of Altered Gene Expression of Metalloproteinases 2, 9, and 16 in Acute Myeloid Leukemia: Preliminary Study.

Acute myeloid leukemia is a group of hematological neoplasms characterized by a heterogeneous course and high mortality. The important factor in the neoplastic process is metalloproteinases, proteolytic enzymes capable of degrading various components of the extracellular matrix, which take an active part in modifying the functioning of the cell, including transformation to cancer cell. They interact with numerous signaling pathways responsible for the process of cell growth, proliferation, or apoptosis. In the present study, changes in the expression of MMP2, MMP9, and MMP16 genes between patients with AML and people without cancer were examined. The impact of cytogenetic changes in neoplastic cells on the expression level of MMP2, MMP9, and MMP16 was also assessed, as well as the impact of the altered expression on the effectiveness of the first cycle of remission-inducing therapy. To evaluate the expression of all studied genes MMP2, MMP9, and MMP16, SYBR Green-based real-time PCR method was used; the reference gene was GAPDH. For two investigated genes MMP2 and MMP16, the lower expression level was observed in patients with AML when compared to healthy people. The MMP9 gene expression level did not differ between patients with AML and healthy individuals which may indicate a different regulation of gene expression in acute myeloid leukemia. However, no correlation was observed between the genes' expression of all tested metalloproteinases and the result of cytoreductive treatment or the presence of cytogenetic changes. The obtained results show that the expression of MMP2 and MMP16 genes is reduced while the expression of MMP9 is unchanged in patients with acute myeloid leukemia. This may indicate a different regulation of the expression of these genes, and possible disruptions in gene transcription or posttranscriptional mechanisms in the MMP2 and MMP16 genes, however, do not affect the level of MMP9 expression. Obtained results in AML patients are in contrary to various types of solid tumors where increased expression is usually observed.

Loss of microRNA-147 function alleviates synovial inflammation through ZNF148 in rheumatoid and experimental arthritis.

MicroRNA-147 (miR-147) had been previously found induced in synoviocytes by inflammatory stimuli derived from Tcells in experimental arthritis. This study was designed to verify whether loss of its function might alleviate inflammatory events in joints of experimental and rheumatoid arthritis (RA). Dark Agouti (DA) rats were injected intradermally with pristane to induce arthritis, and rno-miR-147 antagomir was locally administrated into individual ankle compared with negative control or rno-miR-155-5p antagomir (potential positive control). Arthritis onset, macroscopic severity, and pathological changes were monitored. While in vitro, gain or loss function of hsa-miR-147b-3p/hsa-miR-155-5p and ZNF148 was achieved in human synovial fibroblast cell line SW982 and RA synovial fibroblasts (RASF). The expression of miRNAs and mRNAs was detected by using RT-quantitative PCR, and protein expression was detected by using Western blotting. Anti-miR-147 therapy could alleviate the severity, especially for the synovitis and joint destruction in experimental arthritis. Gain of hsa-miR-147b-3p/hsa-miR-155-5p function in TNF-α stimulated SW982 and RASF cells could upregulate, in contrast, loss of hsa-miR-147b-3p/hsa-miR-155-5p function could downregulate the gene expression of TNF-α, IL-6, MMP3, and MMP13. Hence, such alteration could participate in synovial inflammation and joint destruction. RNAi of ZNF148, a miR-147's target, increased gene expression of TNF-α, IL-6, MMP3, and MMP13 in SW982 and RASF cells. Also, mRNA sequencing data showed that hsa-miR-147b-3p mimic and ZNF148 siRNA commonly regulated the gene expression of CCL3 and DEPTOR as well as some arthritis and inflammation-related pathways. Taken together, miR-147b-3p contributes to synovial inflammation through repressing ZNF148 in RA and experimental arthritis.

Curcumin-primed human BMSC-derived extracellular vesicles reverse IL-1\u03b2-induced catabolic responses of OA chondrocytes by upregulating miR-126-3p

BackgroundCurcumin has anti-inflammatory effects and qualifies as a potential candidate for the treatment of osteoarthritis (OA). However, curcumin has limited bioavailability. Extracellular vesicles (EVs) are released by multiple cell types and act as molecule carrier during intercellular communication. We assume that EVs can maintain bioavailability and stability of curcumin after encapsulation. Here, we evaluated modulatory effects of curcumin-primed human (h)BMSC-derived EVs (Cur-EVs) on IL-1β stimulated human osteoarthritic chondrocytes (OA-CH).MethodsCellTiter-Blue Viability- (CTB), Caspase 3/7-, and live/dead assays were used to determine range of cytotoxic curcumin concentrations for hBMSC and OA-CH. Cur-EVs and control EVs were harvested from cell culture supernatants of hBMSC by ultracentrifugation. Western blotting (WB), transmission electron microscopy, and nanoparticle tracking analysis were performed to characterize the EVs. The intracellular incorporation of EVs derived from PHK26 labeled and curcumin-primed or control hBMSC was tested by adding the labeled EVs to OA-CH cultures. OA-CH were pre-stimulated with IL-1β, followed by Cur-EV and control EV treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay). Relative expression of selected anabolic and catabolic genes was assessed with qRT-PCR. Furthermore, WB was performed to evaluate phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in OA-CH. The effect of hsa-miR-126-3p expression on IL-1β-induced OA-CH was determined using CTB-, Caspase 3/7-, live/dead assays, and WB.ResultsCur-EVs promoted viability and reduced apoptosis of IL-1β-stimulated OA-CH and attenuated IL-1β-induced inhibition of migration. Furthermore, Cur-EVs increased gene expression of BCL2, ACAN, SOX9, and COL2A1 and decreased gene expression of IL1B, IL6, MMP13, and COL10A1 in IL-1β-stimulated OA-CH. In addition, phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK, induced by IL-1β, is prevented by Cur-EVs. Cur-EVs increased IL-1β-reduced expression of hsa-miR-126-3p and hsa-miR-126-3p mimic reversed the effects of IL-1β.ConclusionCur-EVs alleviated IL-1β-induced catabolic effects on OA-CH by promoting viability and migration, reducing apoptosis and phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK thereby modulating pro-inflammatory signaling pathways. Treatment of OA-CH with Cur-EVs is followed by upregulation of expression of hsa-miR-126-3p which is involved in modulation of anabolic response of OA-CH. EVs may be considered as promising drug delivery vehicles of curcumin helping to alleviate OA.

Anticancer, anticholinesterase and antidiabetic activities of tunceli garlic (Allium tuncelianum): determining its phytochemical content by LC–MS/MS analysis

We aimed to investigate the phytochemical content and pharmaceutical activity of methanol extracts of Allium tuncelianum (MAT). In this study, phytochemical contents of MAT were analyzed by liquid chromatography mass-spectrometry (LC–MS/MS) technique. Anticancer activity was determined on six different cell lines. By flow cytometric method, apoptotic activity was examined with annexin V in human prostate carcinoma (PC3) cell lines where the highest cytotoxic activity was observed. Real Time PCR was used to investigate the molecular genetic size of the effect. Anticholinesterase and antidiabetic activities were performed using enzyme inhibition technique. Enzyme inhibitory activity of MAT was determined using AChE, α-glucosidase enzymes. In these extracts, “μg Analyte/g MAT” as its unit; quinic acid (6458.45 ± 2.389), malic acid (4187.25 ± 0.544) and vanilic acid (349.4 ± 0.052) were found to be major components. The highest cytotoxic activity was found in PC3 cells (IC50: 105.72 µg/mL). Therefore, the mechanism of apoptotic effect in this cell line was also investigated. In a statistically meaningful proportion, MAT increased Caspase 3 and Caspase 9 gene expressions and suppressed IL6, IL8, MMP2 and MMP9 gene expressions. On the other hand, MAT demonstrated effective inhibition effects against AChE (IC50: 11.25 μg/mL), and α-glycosidase (IC50: 9.85 μg/mL) enzymes. As a result, it can be argued that Allium tuncelianum can be used as an alternative treatment for prostate cancer, diabetes and some neurodegenerative diseases.

The Effect of Two Types of Exercise Preconditioning on the Expression of TrkB, TNF-α, and MMP2 Genes in Rats with Stroke.

Despite the beneficial effects of exercise and physical activity, there is little knowledge about the effects of different types of physical activity on neural function. The present study assessed the effects of two types of selected aerobic exercises prior to stroke induction and characterized the expression of TrkB, TNF-α, and MMP2 genes in vivo. Forty male adult Wistar rats were exposed to aerobic exercises following randomization into four groups, including swimming + MCAO (Middle Cerebral Artery Occlusion) (n = 10), treadmill training + MCAO (n = 10), MCAO (n = 10), and control (n = 10). The swimming + MCAO group included swimming for 30 minutes each day, while the treadmill training + MCAO group program involved running for 30 minutes each day at an intensity of 15 m/min, for three weeks, five days a week. Neurological deficit was assessed using modified criteria at 24 h after the onset of cerebral ischemia. In the control group, the animals worked freely for three weeks without undergoing ischemia. The MCAO group also operated freely for three weeks after they underwent a stroke. Both training groups underwent ischemia after three weeks of training. TrkB, TNF-α, and MMP2 gene expressions were increased in the MCAO+ swimming training and in the MCAO + running training group compared to the control and MCAO groups, respectively. Preconditioning aerobic exercises significantly increased brain trophic support and reduced brain damage conditions in exercise groups, which support the importance of aerobic exercise in the prevention and treatment of stroke.

Expression and significance of related genes in the early stage of post-traumatic heterotopic ossification in a rat model of Achilles tenotomy.

This study aims to determine expression profiles of relevant genes in the early stages of post-traumatic heterotopic ossification (HO) in a rat model of Achilles tenotomy. A total of 80 male Sprague-Dawley rats were randomly assigned to two groups: the HO group and the control group. Tenotomy was performed in the Achilles tendon of the rats in the HO group, and no intervention was conducted in the control group. On the 3rd, 5th, 8th, and 14th days after the operation, 8 rats were taken from each group at each time point, and the Achilles tendon and surrounding tissue specimens were collected. Gene expressions of TGF-β, BMP, GDF, IL, and MMP families as well as TNF-α, HIF-1α chordin, gremlin, noggin, and NODAL were analyzed by qRT-PCR. The relevant genes that were highly expressed at different time points were screened, and immunohistochemical staining was then used to verify their expression. At the 10th week, HO formation was explored by radiographic and histological examination in the remaining 8 rats of each group. Both the radiographic and histological analyses indicated that all the rats developed HO in the HO group (100%), whereas no HO occurred in the control group. Surrounding tissues obtained from the HO group showed significantly higher gene expressions of TGF-β1, BMP-1, IL1β, HIF-1α, and MMP-2 but lower expressions of BMP-4, GDF-8, and TNF-α compared with the control group. In addition, immunohistochemical staining confirmed the higher protein expression levels of relevant genes in the HO group. TGF-β1, BMP-1, IL-1β, HIF-1α, MMP-2, BMP4, GDF-8, and TNF-α may be associated with the formation of traumatic HO; and BMP4, GDF-8, and TNF-α may play a protective role in the early stage of HO. In this study, we investigated the expression levels of the related cytokines in the early stages of traumatic HO in the Achilles tendon tenotomy rat model to better understand the pathogenesis of HO.

Secretomes of human pluripotent stem cell-derived smooth muscle cell progenitors upregulate extracellular matrix metabolism in the lower urinary tract and vagina

BackgroundAdult mesenchymal stem cells (MSCs) have been studied extensively for regenerative medicine; however, they have limited proliferation in vitro, and the long culture time induces cell senescence. MSCs also contribute to tissue repair through their paracrine function. In this study, we sought to examine the paracrine effects of human smooth muscle cell progenitors (pSMC) on the urethra and adjacent vagina of stress urinary incontinence rodents. We use human pluripotent stem cell (PSC) lines to derive pSMCs to overcome the issue of decreased proliferation in tissue culture and to obtain a homogenous cell population.MethodThree human PSC lines were differentiated into pSMCs. The conditioned medium (CM) from pSMC culture, which contain pSMC secretomes, was harvested. To examine the effect of the CM on the extracellular matrix of the lower urinary tract, human bladder smooth muscle cells (bSMCs) and vaginal fibroblasts were treated with pSMC-CM in vitro. Stress urinary incontinence (SUI) was induced in rats by surgical injury of the urethra and adjacent vagina. SUI rats were treated with pSMC-CM and monitored for 5 weeks. Urethral pressure testing was performed prior to euthanasia, and tissues were harvested for PCR, Western blot, and histological staining. Kruskal-Wallis one-way ANOVA test and Student t test were used for statistical comparisons.ResultspSMC-CM upregulated MMP-2, TIMP-2, collagen, and elastin gene expression, and MMP-9 activity in the human bladder and vaginal cells consistent with elastin metabolism modulation. pSMC-CM treatment in the SUI rat improved urethral pressure (increase in leak point pressure compared to intact controls, p < 0.05) and increased collagen and elastin expression in the urethra and the adjacent vagina.ConclusionConditioned media from smooth muscle cell progenitors derived from human pluripotent stem cells improved urethral leak point pressure and collagen and elastin content in the SUI rat. These findings suggest a novel therapeutic potential for PSC-based treatments for SUI and pelvic floor disorders where tissues are affected by collagen, elastin, and smooth muscle loss.

Differential inflammatory response to Aspergillus flavus and Candida albicans infection in human retinal pigment epithelial cells: Role in treatment of endophthalmitis

ObjectiveFungal endophthalmitis is an emerging vision-threatening complication in tropical countries and the Retinal pigment epithelial cells (RPE) are said to play a major role in the retinal pathology. The aim of this study was to compare the immune response of Retinal pigment epithelial cells (RPE) challenged with A. flavus and C. albicans strains, isolated from patients with fungal endophthalmitis. Material and MethodsRetinal Pigment epithelial cells (ARPE-19) cells were infected with A. flavus and C. albicans, and gene expression were assessed for TLRs, immune-mediators, and matrix metalloproteinases (MMPs). ResultsWe observed a time-dependent gene expression of TLRs (TLR-2,-7 and -9); IL-8 and TNF-α in RPE cells challenged with A. flavus and C. albicans. Additonally, IL-6 (3.3 fold), IL-10 (15.2 fold), and IL-17 (5.6 fold) were significantly up-regulated only in cells infected with A. flavus. Additionally, MMP-9 gene expression was up-regulated in both A.flavus as well as C.albicans infected cells, while MMP- 2 gene expression was induced only in cells infected with C.albicans. A total of 9 upregulated differential expression of genes (DEGs) in A. flavus infected cells and 7 DEGs in C. albicans infected cells were used to construct Protein-protein interaction (PPI) network. ConclusionRPE cells induced a differential innate immune response depending on fungal species involved (A.flavus and C.albicans) and may provide clues for novel treatment targets and better prognosis.