MMP-9 mRNA Levels Research Articles

Steroid receptor coactivator 1 promotes human hepatocellular carcinoma invasiveness through enhancing MMP-9.

SRC-1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC-1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC-1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta-catenin signalling. In this study, we uncover a previously unknown role of SRC-1 in HCC metastasis. Our findings reveal that SRC-1 promotes HCC metastasis through the augmentation of MMP-9 expression. The knockdown of SRC-1 effectively mitigated HCC cell metastasis both invitro and invivo by suppressing MMP-9 expression. Furthermore, we observed a positive correlation between SRC-1 mRNA levels and MMP-9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC-1 operates as a coactivator for NF-κB and AP-1, enhancing MMP-9 promoter activity in HCC cells. Higher levels of SRC-1 and MMP-9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC-1 expression, significantly decreased MMP-9 expression and inhibited HCC metastasis in both invitro and invivo settings. Our results demonstrated the pivotal role of SRC-1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.

Pharmacological Stimulation of Soluble Guanylate Cyclase Counteracts the Profibrotic Activation of Human Conjunctival Fibroblasts.

Conjunctival fibrosis is a serious clinical concern implicated in a wide spectrum of eye diseases, including outcomes of surgery for pterygium and glaucoma. It is mainly driven by chronic inflammation that stimulates conjunctival fibroblasts to differentiate into myofibroblasts over time, leading to abnormal wound healing and scar formation. Soluble guanylate cyclase (sGC) stimulation was found to suppress transforming growth factor β (TGFβ)-induced myofibroblastic differentiation in various stromal cells such as skin and pulmonary fibroblasts, as well as corneal keratocytes. Here, we evaluated the in vitro effects of stimulation of the sGC enzyme with the cell-permeable pyrazolopyridinylpyrimidine compound BAY 41-2272 in modulating the TGFβ1-mediated profibrotic activation of human conjunctival fibroblasts. Cells were pretreated with the sGC stimulator before challenging with recombinant human TGFβ1, and subsequently assayed for viability, proliferation, migration, invasiveness, myofibroblast marker expression, and contractile properties. Stimulation of sGC significantly counteracted TGFβ1-induced cell proliferation, migration, invasiveness, and acquisition of a myofibroblast-like phenotype, as shown by a significant downregulation of FAP, ACTA2, COL1A1, COL1A2, FN1, MMP2, TIMP1, and TIMP2 mRNA levels, as well as by a significant reduction in α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein expression. In addition, pretreatment with the sGC stimulator was capable of significantly dampening TGFβ1-induced acquisition of a contractile phenotype by conjunctival fibroblasts, as well as phosphorylation of Smad3 and release of the proinflammatory cytokines IL-1β and IL-6. Taken together, our findings are the first to demonstrate the effectiveness of pharmacological sGC stimulation in counteracting conjunctival fibroblast-to-myofibroblast transition, thus providing a promising scientific background to further explore the feasibility of sGC stimulators as potential new adjuvant therapeutic compounds to treat conjunctival fibrotic conditions.

8-Oxoguanine DNA glycosylase protects cells from senescence via the p53-p21 pathway.

Cellular senescence is an important factor leading to pulmonary fibrosis. Deficiency of 8-oxoguanine DNA glycosylase (OGG1) in mice leads to alleviation of bleomycin (BLM)-induced mouse pulmonary fibrosis, and inhibition of the OGG1 enzyme reduces the epithelial mesenchymal transition (EMT) in lung cells. In the present study, we find decreased expression of OGG1 in aged mice and BLM-induced cell senescence. In addition, a decrease in OGG1 expression results in cell senescence, such as increases in the percentage of SA-β-gal-positive cells, and in the p21 and p-H2AX protein levels in response to BLM in lung cells. Furthermore, OGG1 promotes cell transformation in A549 cells in the presence of BLM. We also find that OGG1 siRNA impedes cell cycle progression and inhibits the levels of telomerase reverse transcriptase (TERT) and LaminB1 in BLM-treated lung cells. The increase in OGG1 expression results in the opposite phenomenon. The mRNA levels of senescence-associated secretory phenotype (SASP) components, including IL-1α, IL-1β, IL-6, IL-8, CXCL1/CXCL2, and MMP-3, in the absence of OGG1 are obviously increased in A549 cells treated with BLM. Interestingly, we demonstrate that OGG1 binds to p53 to inhibit the activation of p53 and that silencing of p53 reverses the inhibition of OGG1 on senescence in lung cells. Additionally, the augmented cell senescence is shown in vivo in OGG1-deficient mice. Overall, we provide direct evidence in vivo and in vitro that OGG1 plays an important role in protecting tissue cells against aging associated with the p53 pathway.

Novel Pathways between Invasiveness Modulators in Breast Cancer Single Cells.

Individual cells are known to behave differently than their whole populations of cells. The present work focused on proteins that control cancer invasiveness. Individual Dicer siRNA knockdown of HER4, CDC42, and E-cadherin decreased MMP1 mRNA levels in SCP2, a cancer single-cell progeny that is highly metastatic to bone and adrenal gland. Individual knockdown of β-catenin, CDC42, HER3, and the γ catalytic subunit of PI3K raised MMP1 mRNA levels in SCP21, a single-cell progeny of the same tumor and patient, with low metastasis to bone and adrenal.

The CircHAS2/RPL23/MMP9 Axis Facilitates Brain Tumor Metastasis

Abstract Background: Circular RNAs (circRNAs) regulate tumor development by interacting with microRNAs. However, limited research has been conducted on the roles of circRNAs in gliomas. Therefore, we sought to demonstrate the function and molecular mechanism of circHAS2 in gliomas. Methods: CircHAS2, hsa-miR-508-3p, RPL23, and MMP9 mRNA levels were assessed with qRT-PCR. RPL23 and MMP9 protein levels were determined with western blotting and immunohistochemical staining. Glioma cell migration and invasion were assessed with Transwell assays. The interaction between hsa-miR-508-3p and circHAS2 or RPL23 was predicted with RNAhybrid and miRanda, and confirmed through luciferase reporter assays. The effects of circHAS2 on glioma cells were demonstrated in a nude mouse orthotopic xenograft glioma model. Results: We computationally analyzed the differentially expressed circRNAs in glioma tissues by using the GEO database. The screening indicated that circHAS2 was located primarily in the cytoplasm. Functionally, silencing of circHAS2 inhibited glioma migration and invasion. Mechanically, hsa-miR-508-3p was identified as a downstream target of circHAS2. CircHAS2 was found to regulate RPL23 and influence MMP9 via hsa-miR-508-3p, thereby promoting glioma migration and invasion. Moreover, inhibition of circHAS2 impeded the progression of U87 glioma cells in vivo. Conclusion: CircHAS2 regulates RPL23 and subsequent MMP9 expression by sponging hsa-miR508-3p in glioma cells.

Expression Profiles of Matrix Metalloproteinases and Their Inhibitors in Nasal Polyps.

Nasal polyp (NP) is characterized by inflammation of the sinonasal mucosa with predominant inflammatory cell infiltration. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are recognized to play an important role in leukocyte migration in airway inflammation. Herein, efforts were made to confirm the expression levels of MMPs/TIMPs and study the relationship between the infiltration of inflammatory cells and local expression levels of MMPs/TIMPs in NPs. NP tissues were obtained from 42 Chinese patients with bilateral nasal polyps during the endoscopic sinus surgery. Inferior turbinate (IT) tissues from 19 patients with septal deviation were taken during the rhinoplasty surgery as controls. mRNA and protein levels of MMP1, MMP9, MMP10, MMP12, TIMP1 and TIMP3 were assessed by quantitative PCR and immunohistochemistry. Eosinophilia (72%, 23/32 samples), neutrophilia (41%, 13/32 samples), and increase in macrophages (38%, 12/32 samples) were found in NP tissues. mRNA expression of MMP1 (10.9-fold), MMP9 (4.1-fold), MMP10 (6.7-fold) and MMP12 (3.5-fold) were significantly up-regulated, while TIMP1 (1.5-fold) and TIMP3 (6.0-fold) were significantly down-regulated in NPs (n=42) as compared to the controls (n=19). The immunostaining levels of all 4 MMPs and two TIMPs were higher in NPs than those in controls. The co-localization of MMP1/MMP10/MMP12 and macrophages were identified in NPs. MMP9 was mainly expressed in neutrophils, while TIMP1 or TIMP3 were mostly found in eosinophils in NPs. The results of our study indicate that tissue remodeling is significant in NPs, where MMPs/TIMPs play important roles in both tissue remodeling and inflammatory cells infiltration.

Potential effects of parietin on apoptosis and cell cycle related genes in SH-SY5Y neuroblastoma cells

Background: Ingredients obtained from natural products have been used in cancer treatments for years. High diversity and non-toxicity compared to chemotherapeutic agents are the main reasons for their preference. Lichens having potential for treatment of cancer consist of fungus and 1-2 species of algae. Under the name of lichen substances, many of them have also been synthesized as specific substances. The secondary metabolites in lichens are generally insoluble in water, and have many biological activities such as antiviral, antitumor, antibacterial, and antioxidant; they store in the fungal cell or on the surface of the hyphae and can only be extracted with organic solvents.
 Parietin extracted from lichen species such as xanthoria parietina is an anthraquinone pigment and a secondary metabolite. 
 In our study, the effects of parietin on cytotoxicity, gene expression, migration, invasion, and colony formation in neuroblastoma cells treated with parietin were investigated. The SH-SY5Y neuroblastoma (NB), which is not treated with parietin, was administered as a control group.
 Methods: The IC50 value of the parietin was determined using XTT assay. The total RNA extractions were performed from the cells using the Tri-Reagent kit. The expressions of BCL-XL, BCL-2, MMP2, MMP9, P21, CYCLIN D1, CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA genes were investigated by Lightcycler 480 (Roche) using SYBR Green dye. Migration analysis of the control and the dose group cells were performed in accordance with the Wound-healing assay protocol. Invasion activities were determined using the “Invasion Chamber” (BD Biosciences) protocol. The colony assay was treated with crystal violet and the cells were observed under the light microscope.
 Results: The IC50 value of the parietin used for 48-hour treatment on the cells was determined as 35 µM. It was found that the expression levels of BCL-XL, BCL-2, MMP2, MMP9, P21, and CYCLIN D1 mRNA were downregulated, and it was also shown the expression levels of CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA to be upregulated. It was determined that parietin suppressed both cell invasion and migration, and colony formation in the neuroblastoma cells. 
 Conclusions: Thus, it can be possible parietin to be used as an alternative, complementary, and supportive agent together with the other drugs in the treatment of neuroblastoma. However, more comprehensive studies supporting these significant effects of parietin will increase its potential in the application.

Exploring the Role of Lycium barbarum Polysaccharide in Corneal Injury Repair and Investigating the Relevant Mechanisms through In Vivo and In Vitro Experiments.

Lycium barbarum polysaccharide (LBP) is the main active component of Fructus Lycii, exhibiting various biological activities. This study aims to explore the protective effects of LBP on human corneal epithelial cells (HCEC) and a rat corneal injury model. Potential target points for LBP improving corneal injury repair were screened from public databases, and functional and pathway enrichment analyses of core targets were conducted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Rat corneal alkali burns and HCEC oxidative stress injury models were established, and the results were validated through slit lamp examination, HE staining, TUNEL assay, immunofluorescence, CCK-8 assay, flow cytometry, scratch assay, and qRT-PCR methods. In the context of database retrieval, identification of 10 LBP monosaccharide components and 50 corneal injury repair-related targets was achieved. KEGG pathway analysis suggested that LBP might regulate the IL-17 and TNF signaling pathways through targets such as JUN, CASP3, and MMP9, thereby improving corneal damage. In vivo and in vitro experimental results indicated that LBP could reduce the increase of inflammation index scores (p < 0.05), inflammatory cell density (p < 0.01), TUNEL-positive cells (p < 0.01), corneal opacity scores (p < 0.01), and expression of corneal stromal fibrosis-related proteins α-SMA, FN, and COL (p < 0.01) caused by chemical damage to rat corneas. LBP inhibited oxidative stress-induced decreases in cell viability (p < 0.001) and migration healing ability (p < 0.01) in HCECs, reducing apoptosis rates (p < 0.001), ROS levels (p < 0.001), and the expression of inflammatory factors TNF-α and IL-6 (p < 0.01). qRT-PCR results demonstrated that LBP intervention decreased the mRNA levels of JUN, CASP3, and MMP9 in H2O2-induced alkaline-burned corneas and HCECs (p < 0.01).The integrated results from network pharmacology and validation experiments suggest that the inhibitory effects of LBP on apoptosis, inflammation, and fibrosis after corneal injury may be achieved through the suppression of the TNF and IL-17 signaling pathways mediated by JUN, CASP3, and MMP9.

Fructose-bisphosphatase1 (FBP1) alleviates experimental osteoarthritis by regulating Protein crumbs homolog 3 (CRB3)

PurposeTo identify the role of gluconeogenesis in chondrocytes in osteoarthritis (OA).Materials and methodsCartilage samples were collected from OA patients and C57 mice and were stained with Safranin O-Fast Green to determine the severity of OA. Periodic acid Schiff staining was used to characterize the contents of polysaccharides and SA-βGal staining was used to characterize the aging of chondrocytes. Immunohistochemistry and western blotting were used to detect fructose-bisphosphatase1 (FBP1), SOX9, MMP13, P21, and P16 in cartilage or chondrocyte. The mRNA levels of fbp1, mmp13, sox9, colX, and acan were analyzed by qPCR to evaluate the role of FBP1 in chondrocytes.ResultsThe level of polysaccharides in cartilage was reduced in OA and the expression of FBP1 was also reduced. We treated the chondrocytes with IL-1β to cause OA in vitro, and then made chondrocytes overexpress FBP1 with plasma. It shows that FBP1 alleviated the degeneration and senescence of chondrocytes in vitro and that it also showed the same effects in vivo experiments. To further understand the mechanism of FBP1, we screened the downstream protein of FBP1 and found that CRB3 was significantly downregulated. And we confirmed that CRB3 suppressed the degeneration and delayed senescence of chondrocytes.ConclusionsFBP1 promoted the polysaccharide synthesis in cartilage and alleviated the degeneration of cartilage by regulating CRB3, so FBP1 is a potential target in treating OA.

Expression profiles of MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps.

Metalloproteinases (MMPs) are implicated in tissue remodeling in chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to evaluate the expression profiles of MMP-9 and the extracellular matrix metalloproteinase inducer (EMMPRIN) in nasal polyps compared to healthy mucosa. Tissue samples from 37 CRSwNP patients undergoing functional endoscopic sinus surgery and mucosal specimens from 12 healthy controls were obtained intra-operatively. MMP-9 and EMMPRIN mRNA levels were assessed by reverse transcription-polymerase chain reaction and their protein expression by Western blot analysis. MMP-9 mRNA expression levels were significantly elevated in CRSwNP compared to controls (p < 0.05). MMP-9 protein levels presented an increasing trend but with no statistical significance (p > 0.05). No statistically significant difference in EMMPRIN mRNA and protein levels was identified. Upregulation of MMP-9 in nasal polyps is evident and highlights its role in the pathogenesis of CRSwNP. The lack of concordance between MMP-9 mRNA and protein levels may be attributed to post-translational gene expression regulation. Although EMMPRIN expression was not significantly different between the two groups, its role remains to be elucidated. MMP-9 may be a valuable biomarker and treatment target in CRSwNP.

The Protective Role of L-carnitine on Psychosocial Stress-induced Changes in Gene Expression and Protein Levels of Matrix Metalloproteinases, Serum Corticosterone in a Rat Model.

Psychosocial stress (STS) is a common stress in modern societies. Chronic STS is associated with the impairment of a broad range of cognitive functions in humans and with prolonged abnormalities in memory. Matrix metalloproteinases (MMPs), a protein family of zinccontaining endopeptidases, are essential in neuro-inflammation and involved in neurodegenerative diseases. L-Car possessed neuroprotective, antioxidant, and anti-inflammatory properties and was shown to modulate MMPs. The current study aimed to examine the protective effect of L-Carnitine (L-CAR) on STSinduced changes in serum corticosterone levels, MMP-2, -9, and -12 protein and mRNA expression in the hippocampus as a possible mechanism for L-CAR protective effect on STS-induced memory impairment. The chronic STS and L-CAR (300 mg/kg/day, i.p) were simultaneously administered for 6 weeks to adult male Wistar rats. Serum corticosterone and protein levels of MMP-2, -9 and -12 were evaluated using ELISA. Real-Time PCR techniques were used to determine the mRNA levels of MMP-2, -9 and -12 in the hippocampus. The findings showed that serum corticosterone levels and MMP-2 and -9 protein levels were significantly increased (p<0.05) in the STS group compared to the control. Similarly, RT-PCR findings showed that the mRNA of those proteinases significantly increased (p<0.05) following the intruder method. On the other hand, the administration of L-CAR restored the alterations in corticosterone levels and MMPs gene and protein expression induced by chronic STS. STS induced elevations in corticosterone and MMP-2 and -9 levels in the hippocampus. L-CAR, on the other hand, exhibited protective effects against the STS-induced changes in MMP-2 and -9.

Relation of STAT3 rs1053005 Variation and miR-452-3p with Osteoarthritis Susceptibility and Severity and the Clinical Response to High-Molecular-Weight Hyaluronic Acid Injection in Osteoarthritis Patients

Polymorphisms in the 3′ untranslated region of STAT3 mRNA can derange STAT3 gene expression via modifying the microRNA-binding site. This study aimed to examine the impact of STAT3 rs1053005 variation and miR-452-3p expression on osteoarthritis (OA) susceptibility and severity and the efficacy of intra-articular high-molecular-weight hyaluronic acid (HMW-HA) injection as a therapy option for knee OA. Two hundred and fifty-eight OA patients and 200 healthy controls were enrolled in the study. STAT3 genotyping and STAT3 and miR-452-3p expression were carried out using allelic-discrimination PCR and quantitative real-time PCR. Functional assessment and pain evaluation were performed for all patients. Eighty-three patients received HMW-HA injections, and multiple follow-up visits were performed. STAT3 mRNA was upregulated, and expression was positively associated with plasmin, TNF-α, MMP-3, and STAT3 serum levels, whereas miR-452-3p was downregulated and negatively associated with the previously mentioned parameters in OA patients. Osteoarthritis patients had a lower prevalence of the minor allele of the rs1053005 variant (p &lt; 0.001). Plasmin, TNF, MMP-3, and STAT3 mRNA and protein levels were significantly decreased, and miR-452-3p expression was significantly increased in the GG genotype compared to AG and AA genotypes. HMW-HA injection improved OA patients’ clinical scores with concomitant decreased STAT3 levels and enhanced expression of miR-452-3p. More efficient improvement was observed in rs1053005 AG + GG genotype carriers vs. AA genotype carriers. The G allele of STAT3 rs1053005 (A/G) polymorphism was associated with decreased OA susceptibility and severity and enhanced clinical response to HMW-HA injection, possibly via enhancing miR-452-3p binding and a subsequent decrease in STAT3 expression.

Oral Administration of Rhamnan Sulfate from Monostroma nitidum Suppresses Atherosclerosis in ApoE-Deficient Mice Fed a High-Fat Diet.

Oral administration of rhamnan sulfate (RS), derived from the seaweed Monostroma nitidum, markedly suppresses inflammatory damage in the vascular endothelium and organs of lipopolysaccharide-treated mice. This study aimed to analyze whether orally administered RS inhibits the development of atherosclerosis, a chronic inflammation of the arteries. ApoE-deficient female mice were fed a normal or high-fat diet (HFD) with or without RS for 12 weeks. Immunohistochemical and mRNA analyses of atherosclerosis-related genes were performed. The effect of RS on the migration of RAW264.7 cells was also examined in vitro. RS administration suppressed the increase in blood total cholesterol and triglyceride levels. In the aorta of HFD-fed mice, RS reduced vascular smooth muscle cell proliferation, macrophage accumulation, and elevation of VCAM-1 and inhibited the reduction of Robo4. Increased mRNA levels of Vcam1, Mmp9, and Srebp1 in atherosclerotic areas of HFD-fed mice were also suppressed with RS. Moreover, RS directly inhibited the migration of RAW264.7 cells in vitro. Thus, in HFD-fed ApoE-deficient mice, oral administration of RS ameliorated abnormal lipid metabolism and reduced vascular endothelial inflammation and hyperpermeability, macrophage infiltration and accumulation, and smooth muscle cell proliferation in the arteries leading to atherosclerosis. These results suggest that RS is an effective functional food for the prevention of atherosclerosis.

Metformin attenuates inflammation and boosts autophagy in the liver and intestine of chronologically aged rats

BackgroundOur previous studies found that autophagy levels in liver and intestinal segments of naturally aging rats were downregulated, and the expression of pro-inflammatory factors was increased. The increased expression of pro-inflammatory factors might be related to the downregulation of autophagy. AMPK is the most critical upstream targeting and regulating molecule of autophagy, and Metformin, as an agonist of AMPK, has the effects of anti-inflammation and anti-aging. We pretreated 29-month-old naturally aging rats with Metformin for a short period and observed the changes in autophagy levels and pro-inflammatory factors in the liver, ileum, and colon after 31 days of intervention and preliminarily investigated the mechanism of its action. Methods29-month-old SPF male Wistar rats were divided into three groups: The control group, the Metformin 100 mg/kg intervention group, and the Metformin 250 mg/kg intervention group, with eight rats in each group. At 29 months, different concentrations of Metformin (100 mg/kg, 250 mg/kg) were given by gavage once a day until 30 months, and the control group was kept generally until 30 months. Western Blot was used to assess the expression levels of AMPK, P-AMPK, LC3, and P62 proteins in the liver and intestinal tissues. Intestinal and liver tissues were immunofluorescence labeled for LC3 and P62 proteins. Moreover, RT-qPCR was conducted to detect the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and MMP-9 mRNA in liver and intestinal tissues. ResultsShort-term Metformin pretreatment (31 days) in naturally aging rats (29 months old) increased autophagy levels and down-regulated the expression of various pro-inflammatory cytokines (IL-1β, TNF-α, MMP-9, and IL-6) in various intestinal segments and the liver—the expression of LC3II protein enriched with the increase of Metformin concentration. The level of P62 protein decreased with the accumulation of Metformin concentration. And a higher concentration of Metformin was associated with increased expression of P-AMPK protein. ConclusionsMetformin intervention can boost the autophagy level in the liver and intestine and reduce the expression of aging-related inflammatory factors in aged rats, and these effects may be related to the increase of the AMPK phosphorylation level.

Empagliflozin treatment prevented oxidative stress, macrophage infiltration, pro-inflammatory and pro-fibrotic responses in the left ventricle of isoprenaline-induced Takotsubo-like syndrome in rats

Abstract Introduction Takotsubo Syndrome (TTS) is an acute heart failure (HF) syndrome induced by both emotional and physical stressors involving a surge of catecholamines and apical ballooning. Currently, there is no specific treatment for TTS. Sodium-glucose cotransporter2 inhibitors (SGLT2i) showed major cardiovascular protective effects in patients with HF independent of glycemic control. SGLT2i have also been shown to have anti-inflammatory and anti-oxidant effects, and to improve the endothelial function in experimental models of cardiac disease. However, a possible protective role of SGLT2i in TTS is unclear. Thus, this study evaluated the effect of SGLT2i on isoprenaline-induced TTS-like syndrome in rats, and determined the underlying mechanism. Methods 32 female Sprague-Dawley rats (10 weeks old) were divided into 4 groups: control, empagliflozin (30 mg/kg/day provided in the diet for 2 weeks), isoprenaline (Iso, 100 mg/kg, i.p.) to induce a TTS-like syndrome, and Iso plus empagliflozin (IE group). After 6 h of Iso injection, echocardiography was performed to determine left ventricular (LV) ejection fraction (EF) by area and heart rate (HR). At day 3, rats were sacrificed, main mesenteric artery and microvessels were collected for vascular reactivity studies, and LV was divided into base and apex to study mRNA expression levels using RT-qPCR, macrophage infiltration by in situ CD68 immunofluorescence staining, and oxidative stress using dihydroethidium. Results The injection of Iso to rats caused at 6 h an increased LVEF and HR in the Iso and the IE groups as compared to their corresponding controls. The HR response was significantly smaller in the IE group compared to Iso group whereas LVEF was similar. In addition, in mesenteric microvessels but not the main mesenteric artery, Iso group showed significant decreased relaxations to acetylcholine and increased contractile responses to phenylephrine in intact rings both of which were not observed in the IE group. Hearts showed significantly increased mRNA levels of IL-1β, TNF-α, NOX2, VCAM-1, MMP-9, TGF-β1 and collagen-1, oxidative stress levels and CD68 staining in the apex compared to the base in the ISO group, all of which were significantly reduced in IE group. Conclusions The present findings indicate that ISO injection caused in the LV elevated levels of oxidative stress, macrophage infiltration, pro-inflammatory, pro-remodeling and pro-fibrosis responses that were more pronounced in the apex compared to the base, and also endothelial dysfunction in the systemic microcirculation, all of which were significantly prevented by the empagliflozin treatment. They further suggest that SGLT2 appears as an interesting target in TTS.

Decreased intracellular chloride enhances cell migration and invasion via activation of the ERK1/2 signaling pathway in DU145 human prostate carcinoma cells

Our previous study revealed that changes of the intracellular Cl− concentration ([Cl−]i) affected cell proliferation in cancer cells. However, the role of Cl− on cell migration and invasion in cancer cells remains unanalyzed. Therefore, the aim of the present study is to investigate whether changes of [Cl−]i affects cell migration and invasion of cancer cells. In human prostate cancer DU145 cells, cell migration and invasion were enhanced by culturing in the low Cl− medium (replacement of Cl− by NO3−). We also found that DU145 cells in the low Cl− condition caused significant transient ERK1/2 activation followed by an increase of MMP-1 mRNA levels. Inhibition of ERK1/2 activation in the low Cl− condition reduced enhancement of MMP-1 mRNA levels and decreased cell migration and invasion. These observations indicate that [Cl−]i plays important roles in metastatic function by regulating the ERK1/2 signaling pathway in human prostate cancer cells, and intracellular Cl− would be one of the key targets for anti-cancer therapy.

Post-COVID-19 Cardiovascular Disorders and the Molecular Mechanism of NET Formation

Objective: The post-COVID-19 process is not completely understood, as it affects COVID-19 survivors at all levels of disease severity, not all of whom are hospitalized. One of the long-lasting COVID-19 symptom categories, cardiovascular disorders (including acute heart failure, palpitations, hypotension, venous thromboembolic diseases, arrhythmias, myocarditis, and increased heart rate), may derive from a systemic inflammatory response to the viral infection. NETs (neutrophil extracellular traps) that fight invading viruses in extracellular cardiac spaces accumulate due to COVID-19, hyperinflammation and cytokine storms. Our study focuses on cardiovascular disorders as COVID-19 sequelae. To determine the role of NETs in these disorders, we aimed to measure levels of PAD4, MPO, MMP-9, and H3Cit. Methods: In this study, forty people with long-term cardiac complications associated with a history of COVID-19 were recruited along with forty healthy people. Results: We found significant differences in PAD4, H3Cit, and MPO plasma levels between the post-COVID-19 and control groups (p values &lt; 0.05). The expression levels of PAD4 mRNA were lower and MMP-9 mRNA levels was higher in the post-COVID-19 group compared with the control subjects. Conclusion: These findings suggest that PAD4, MPO, MMP-9, and H3Cit are potential biomarkers of NET dysregulation and may cause post-COVID-19 symptoms, especially cardiovascular disorders.

Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture. Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions. Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA. Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged. Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

N-acetyltransferase 10 promotes the progression of oral squamous cell carcinoma through N4-acetylcytidine RNA acetylation of MMP1 mRNA.

The pathogenesis of oral squamous cell carcinoma (OSCC) remains unclear. Therefore, clarifying its pathogenesis and molecular-level development mechanism has become the focus of OSCC research. N-acetyltransferase 10 (NAT10) is a crucial enzyme involved in mRNA acetylation, regulating target gene expression and biological functions of various diseases through mediating N4-acetylcytidine (ac4C) acetylation. However, its role in OSCC progression is not well understood. In this study, we showed that NAT10 was significantly upregulated in OSCC tissues compared to normal oral tissues. Moreover, lentivirus-mediated NAT10 knockdown markedly suppressed cell proliferation, migration, and invasion in two OSCC cell lines (SCC-9 and SCC-15). Interestingly, MMP1 was found to be significantly upregulated in OSCC tissues and was a potential target of NAT10. N-acetyltransferase 10 knockdown significantly reduced both the total and ac4C acetylated levels of MMP1 mRNA and decreased its mRNA stability. Xenograft experiments further confirmed the inhibitory effect of NAT10 knockdown on the tumorigenesis and metastasis ability of OSCC cells and decreased MMP1 expression in vivo. Additionally, NAT10 knockdown impaired the proliferation, migration, and invasion abilities in OSCC cell lines in an MMP1-dependent manner. Our results suggest that NAT10 acts as an oncogene in OSCC, and targeting ac4C acetylation could be a promising therapeutic strategy for OSCC treatment.

TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/ERK/NF-kB signaling pathways

Studies have established the association between increased plasma levels of matrix metalloproteinase (MMP)-9 and adipose tissue inflammation. Tumor necrosis factor α (TNFα) was elevated in obesity and is involved in the induction of MMP-9 in monocytic cells. However, the underlying molecular mechanism was incompletely understood. As per our recent report, TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we further investigated the role of ACSL1 in TNFα-mediated MMP-9 secretion in monocytic cells. THP-1 cells and primary monocytes were used to study MMP-9 expression. mRNA and protein levels of MMP-9 were determined by qRT-PCR and ELISA, respectively. Signaling pathways were studied using Western blotting, inhibitors, and NF-kB/AP1 reporter cells. We found that THP-1 cells and primary human monocytes displayed increased MMP-9 mRNA expression and protein secretion after incubation with TNFα. ACSL1 inhibition using triacsin C significantly reduced the expression of MMP-9 in the THP-1 cells. However, the inhibition of β-oxidation and ceramide biosynthesis did not affect the TNFα-induced MMP-9 production. Using small interfering RNA-mediated ACSL1 knockdown, we further confirmed that TNFα-induced MMP-9 expression/secretion was significantly reduced in ACSL1-deficient cells. TNFα-mediated MMP-9 expression was also significantly reduced by the inhibition of ERK1/ERK2, JNK, and NF-kB. We further observed that TNFα induced phosphorylation of SAPK/JNK (p54/46), ERK1/2 (p44/42 MAPK), and NF-kB p65. ACSL1 inhibition reduced the TNFα-mediated phosphorylation of SAPK/JNK, c-Jun, ERK1/2, and NF-kB. In addition, increased NF-κB/AP-1 activity was inhibited in triacsin C treated cells. Altogether, our findings suggest that ACSL1/JNK/ERK/NF-kB axis plays an important role in the regulation of MMP-9 induced by TNFα in monocytic THP-1 cells.