MMP-9 mRNA Levels Research Articles

Altered choline level in atherosclerotic lesions: Upregulation of choline transporter-like protein 1 in human coronary unstable plaque.

Inflammatory activity and hypoxia in atherosclerotic plaques are associated with plaque instability and thrombotic complications. Recent studies show that vascular cell metabolism affects atherogenesis and thrombogenicity. This study aimed to identify the metabolites in macrophage-rich unstable plaques that modulate atherogenesis and serve as potential markers of plaque instability. Atherosclerotic plaques were induced by balloon injury in the iliofemoral arteries of rabbits fed on a conventional or 0.5% cholesterol diet. At 3 months post-balloon injury, the arteries and cardiac tissues were subjected to histological, quantitative real-time polymerase chain reaction, and metabolomic analyses. The identified metabolite-related proteins were immunohistochemically analyzed in stable and unstable plaques from human coronary arteries. The factors modulating the identified metabolites were examined in macrophages derived from human peripheral blood mononuclear cells. Metabolomic analysis revealed that choline and guanine levels in macrophage-rich arteries were upregulated compared with those in non-injured arteries and cardiac tissues. Vascular choline levels, but not guanine levels, were positively correlated with the areas immunopositive for macrophages and tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP) 9 mRNA levels in injured arteries. In human coronary arteries, choline transporter-like protein (CTL) 1 was mainly localized to macrophages within plaques. The area that was immunopositive for CTL1 in unstable plaques was significantly higher than that in stable plaques. Intracellular choline levels were upregulated upon stimulation with TNF-α but were downregulated under hypoxia in cultured macrophages. Administration of choline upregulated the expression of TNF-α and CTL1 mRNA in cultured macrophages. The transfection of CTL1 small interfering RNA decreased CTL1, TNF-α, and MMP9 mRNA levels in cultured macrophages. These results suggest that choline metabolism is altered in macrophage-rich atherosclerotic lesions and unstable plaques. Thus, CTL1 may be potential markers of plaque instability.

Rebalancing SMAD7/SMAD3 Signaling Reduces Adhesion Formation during Flexor Tendon Healing.

Transforming growth factor-β is a key factor in regulating adhesion formation during tendon healing. We investigated the effectiveness of SMAD family members, SMAD7 and SMAD3, in the TGF-β/Smad signaling during flexor tendon repair. Mouse flexor toe deep tendon rupture anastomosis models were made. On days 3, 7, 14, 21, and 28, the expressions of smad7 and smad3 in flexor tendon tissues were detected by RT-qPCR and western blot. Furthermore, postoperative intraperitoneal injections of SMAD7 agonists or SMAD3 antagonists were given. The degree of tendon healing was evaluated by adhesion testing and biomechanical experiments. Hematoxylin and eosin (HE) staining was used to observe the pathological changes. Immunohistochemistry was used to evaluate the expressions of collagen III, SMAD3, and SMAD7. The mRNA levels of matrix metalloproteinases, Mmp2 and Mmp9, and scleraxis (SCX) in flexor tendon tissue were detected by RT-qPCR. Smad3 expression increased and Smad7 expression decreased in flexor tendon tissue after injury. In addition, the SMAD7 agonist blocked SMAD3 phosphorylation. SMAD7 agonist and SMAD3 antagonist both improved adhesion formation during flexor tendon healing, and decreased the expressions of collagen III, Mmp9, and SCX, while increasing Mmp2 expression. This study provides a possible theoretical basis for the SMAD7-SMAD3 signal cascade during flexor tendon adhesion healing.

Analyses of Transcriptomics upon IL-1β-Stimulated Mouse Chondrocytes and the Protective Effect of Catalpol through the NOD2/NF-κB/MAPK Signaling Pathway

The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1β (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 μM catalpol 2 h prior to treatment with IL-1β (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 μM and 100 μM) could significantly reduce NO, IL-6, IL-1β, and TNF-α in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1β. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-α mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKBα protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-κB and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1β. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of IκBα, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.

Cinnamaldehyde modulates host immunoinflammatory responses in rat ligature-induced periodontitis and peripheral blood mononuclear cell models.

Cinnamaldehyde is a natural product with anti-inflammatory and immune-modulatory properties, known to regulate host responses to bacterial stimuli. This study aimed to investigate the effects of cinnamaldehyde on ligature-induced periodontitis in rats, and its impact on the modulation of human peripheral blood mononuclear cells (PBMC). Male Wistar rats were assigned into three groups:i) control: no ligature+vehicle; ii) ligature: ligature+vehicle; and iii) ligature+cinnamaldehyde (50mg/kg); all treatments by daily oral gavage. After 14days of induced periodontitis, the hemimandibles were collected for bone loss evaluation. The gingival levels of IL-1β, MMP-9 and iNOS mRNA were evaluated. Nitric oxide (NO) was measured in both rat saliva and plasma. PBMC were stimulated with Aggregatibacter actinomycetemcomitans (Aa) in the presence or absence of cinnamaldehyde (5, 20 e 40µM), and cytokine production was quantified in cell supernatant. Proliferating lymphocytes were taken for flow cytometer reading, while culture supernatants were used for IFN-γ and IL-10 assessment. The ligature group had both increased alveolar bone loss and gingival expression of IL-1β, MMP-9 and iNOS compared to the control group. All parameters were attenuated by cinnamaldehyde treatment. Lower salivary but not plasma NO was detected in the cinnamaldehyde compared to the ligature group. Aa-stimulated PBMCs treated with cinnamaldehyde produced less IL-1β; the compound also attenuated lymphocyte proliferation in a dose-dependent manner, as well as cell IL-10 production. Cinnamaldehyde treatment reduced periodontal bone loss, and downregulated key inflammatory mediators and human PBMC responses, pointing to novel potential therapeutic effects of this compound.

YB-1 regulates mesothelioma cell migration via snail but not EGFR, MMP1, EPHA5 or PARK2.

Pleural mesothelioma (PM) is characterized by rapid growth, local invasion, and limited therapeutic options. The multifunctional oncoprotein Y-box-binding protein-1 (YB-1) is frequently overexpressed in cancer and its inhibition reduces aggressive behavior in multiple tumor types. Here, we investigated the effects of YB-1 on target gene regulation and PM cell behavior. Whereas siRNA-mediated YB-1 knockdown reduced cell motility, YB-1 overexpression resulted in scattering, increased migration, and intravasation in vitro. Furthermore, YB-1 stimulated PM cell spreading in zebrafish. Combined knockdown and inducible overexpression of YB-1 allowed bidirectional control and rescue of cell migration, the pattern of which was closely followed by the mRNA and protein levels of EGFR and the protein level of snail, whereas the mRNA levels of MMP1, EPHA5, and PARK2 showed partial regulation by YB-1. Finally, we identified snail as a critical regulator of YB-1-mediated cell motility in PM. This study provides insights into the mechanism underlying the aggressive nature of PM and highlights the important role of YB-1 in this cancer. In this context, we found that YB-1 closely regulates EGFR and snail, and, moreover, that YB-1-induced cell migration depends on snail.

The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels.

Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.

N-Acetylcysteine Administration Improves the Redox and Functional Gene Expression Levels in Spleen, Mesenteric Lymph Node and Gastrocnemius Muscle in Piglets Infected with Porcine Epidemic Diarrhea Virus.

Our previous study reported that N-acetylcysteine (NAC) administration improved the function of intestinal absorption in piglets infected with porcine epidemic diarrhea virus (PEDV). However, the effects of NAC administration on the functions of other tissues and organs in PEDV-infected piglets have not been reported. In this study, the effects of NAC on the liver, spleen, lung, lymph node, and gastrocnemius muscle in PEDV-infected piglets were investigated. Thirty-two 7-day-old piglets with similar body weights were randomly divided into one of four groups: Control group, NAC group, PEDV group, and PEDV+NAC group (eight replicates per group and one pig per replicate). The trial had a 2 × 2 factorial design consisting of oral administration of 0 or 25 mg/kg body weight NAC and oral administration of 0 or 1.0 × 104.5 TCID50 PEDV. The trial lasted 12 days. All piglets were fed a milk replacer. On days 5-9 of the trial, piglets in the NAC and PEDV + NAC groups were orally administered NAC once a day; piglets in the control and PEDV groups were orally administered the same volume of saline. On day 9 of trial, piglets in the PEDV and PEDV+NAC groups were orally administrated 1.0 × 104.5 TCID50 PEDV, and the piglets in the control and NAC groups were orally administrated the same volume of saline. On day 12 of trial, samples, including of the liver, spleen, lung, lymph node, and gastrocnemius muscle, were collected. PEDV infection significantly increased catalase activity but significantly decreased the mRNA levels of Keap1, Nrf2, HMOX2, IFN-α, MX1, IL-10, TNF-α, S100A12, MMP3, MMP13, TGF-β, and GJA1 in the spleens of piglets. NAC administration ameliorated abnormal changes in measured variables in the spleens of PEDV-infected piglets. In addition, NAC administration also enhanced the antioxidant capacity of the mesenteric lymph nodes and gastrocnemius muscles in PEDV-infected piglets. Collectively, these novel results revealed that NAC administration improved the redox and functional gene expression levels in the spleen, mesenteric lymph nodes, and gastrocnemius muscle in PEDV-infected piglets.

Protective Effects of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells on Human Retinal Endothelial Cells in an In Vitro Model of Diabetic Retinopathy: Evidence for Autologous Cell Therapy.

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood-retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1β, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-β1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-β axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR.

PATTERN OF MMP2 AND MMP9 EXPRESSION DEPENDS ON BREAST CANCER PATIENTS' AGE.

Despite the large number of studies devoted to the study of the features of tumor microenvironment in breast cancer (BCa), presently there is no consensus on the features of MMP-2 and MMP-9 expression in the tumor tissue of BCa patients depending on the age. The aim of the study was to investigate the relationship between MMP-2 and -9 expression at the protein and mRNA levels in BCa tissues and the clinical and pathological features of BCapatientsin different age groups. The expression level of MMP-2 and -9in the BCa tissue of patients of two age groups (< 45 years and > 45 years) was studied using the bioinformatics method (UALCAN database), immunohistochemical method, and real-time PCR. It was established that a characteristic feature of BCa in young patients is the low level of MMP2 mRNA against the background of increased expression of this gelatinase at the protein level, as well as decreased expression of MMP9 at both the mRNA and protein levels. When analyzing the correlation of the gelatinase expression indices in BCa tissue of young patients, depending on the clinical and pathological features, a significantly lower level of MMP-2 expression was recorded in BCa cases of stage II compared to the indices of stage I cases. High expression of MMP-2 and -9 was recorded in BCa tissue in node-positive cases and the basal molecular BCa subtype. The identified relationship between the expression of the studied gelatinases and such indices of BCa malignancy as its stage, positive regional lymph node status, and the molecular BCa subtype in young patients indicates the need for further research of the features of the tumor microenvironment to predict the cancer aggressiveness.

Gene Expression Linked to Reepithelialization of Human Skin Wounds.

Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change (MMP1, MMP3, IL6, CXCL8, SERPINE1, IL1B, PTGS2, HBEGF, CXCL5, CXCL2, TIMP1, CYR61, CXCL1, MMP12, MMP9, HGF, CTGF, ITGB3, MT2A, FGF7, COL4A1 and PLAUR). The expression of the most upregulated gene, MMP1, correlated strongly with MMP3 followed by IL6 and IL1B. rhIL-1β, but not rhIL-6, exposure of cultured normal human epidermal keratinocytes and normal human dermal fibroblasts increased both MMP1 mRNA and MMP-1 protein levels, as well as TIMP1 mRNA levels. The increased TIMP1 in wounds was validated by immunohistochemistry. The six downregulated DEGs (COL7A1, MMP28, SLC39A2, FLG1, KRT10 and FLG2) were associated with epidermal maturation. KLK8 showed the strongest correlation with MKI67 mRNA levels and is a potential biomarker for keratinocyte proliferation. The observed gene expression changes correlate well with the current knowledge of physiological reepithelialization. Thus, the gene expression panel described in this paper could be used in patients with impaired healing to identify possible therapeutic targets.

Hypoxia inducible factor-1α mediates the mechanism of the Hedgehog pathway in tendinopathy repair by Asperosaponin VI.

Our previous study found that asperosaponin VI (ASA VI) has a positive effect on the repair of tendinopathy. However, its molecular biological mechanism is unclear. To investigate the role of hypoxia inducible factor-1α (HIF-1α) in mediating the hedgehog (Hh) pathway in tendinopathy repair by ASA VI. A total of 36 2-month-old female SD rats were classified into the normal group (NG, n=10) and tendinopathy model group (n=26). The tendinopathy model group was further divided into the model group (MG), ASA VI group (AG), and triamcinolone acetonide+lidocaine group (TG). Compared with those in the MG group, IL-1 mRNA was significantly downregulated and IL-4 and IL-10 were increased in the AG group (P<0.01). The mRNA expression levels of MMP3, TIMP3, VEGF-A, KDR, and VWF mRNA decreased (P<0.01). Immunofluorescence staining revealed that CD31/endomucin levels were significantly attenuated. Scx, Mkx, EYA1, EYA2, COL1, COL3, and TNC mRNA levels showed significant differences (P<0.01). Immunofluorescence staining suggested the upregulation of Scx and the downregulation of Sox9. Shh, Ptch1, Smo, Gli1, Cyc-D1, Cyc-E1, and c-Myc mRNA levels were downregulated (P<0.01). The protein expression levels of Gli 1, Shh, and Ptch1 decreased significantly (P<0.01). The immunofluorescence staining levels of Shh, Ptch, and Gli 1 significantly decreased. ASA VI inhibits local vascular hyperproliferation and downregulates the HIF-1α/Hh pathway to promote the tendinous differentiation of tendon stem/progenitor cells and the repair of tendinopathy. The effect of ASA VI on HIF-1α levels may be an effective target in the treatment of tendinopathy.

Santamarine Isolated from Artemisia scoparia Inhibits UVB-induced Matrix Metalloproteinase Expression via Repression of MAPK/AP-1 Pathway in Human Keratinocytes

Background: Artemisia scoparia has a widespread distribution and can be found commonly in Eurasia. In traditional Korean medicine, A. scoparia leaves and flowers are used against urethral complications, phlogistic problems, and in the treatment of hepatitis.BRMethods and Results: In the present study, the protective effect of santamarine isolated from A. scoparia was evaluated in Ultraviolet B (UVB)-damaged keratinocytes. Results showed that santamarine suppressed the production of reactive oxygen species in a concentration-dependent manner. Treating cells with santamarine decreased the generation of Matrix Metalloproteinase (MMP)-1 compared to that in cells treated with UVB alone. Additionally, the mRNA levels of MMP-1 and MMP-3 were remarkably lower in cells treated with santamarine than those in cells with UVB irradiation. Furthermore, upregulated protein levels of MMP-1, MMP-2, MMP-3, and MMP-9 following UVB exposure were ameliorated by the introduction of santamarine. Investigation of the mechanisms underlying the photoprotective effect of santamarine showed enhanced inhibition of MAPK/AP-1 signaling following santamarine treatment. The presence of santamarine also recovered the UVB-induced decrease in collagen amount.BRConclusions: Overall, these results demonstrated that santamarine has a potential protective effect against UVB-induced skin photoaging in keratinocytes in vitro. The mechanism behind this effect was suggested to be via suppression of MAPK/AP-1 signaling.

VEGF regulates the blood‑brain barrier through MMP‑9 in a rat model of traumatic brain injury.

Blood-brain barrier (BBB) damage is closely related to morbidity and mortality in patients with traumatic brain injury (TBI). Inhibition of VEGF effectively protects BBB integrity in clinical ischemic stroke. Protecting BBB integrity, reducing brain edema and alleviating post-TBI secondary brain injury are key to a favorable patient prognosis. MMP-9 affects BBB integrity by destroying the tight junction of vascular endothelial cells and inhibiting the transport and enzymatic systems. The present study aimed to examine the possible interplay between VEGF and MMP-9 in TBI. A TBI model was established in 87 male Sprague-Dawley rats. Reverse transcription-quantitative PCR, western blotting, wet-dry brain edema assessment, TUNEL and Fluoro-Jade C staining were performed to analyze the brain tissue samples of the rats. The results showed that compared with in the Sham group rats, the mRNA and protein expression levels of VEGF and MMP-9 were increased at 24 h post-TBI. After bevacizumab treatment, BBB permeability and nerve cell apoptosis were markedly reduced. In conclusion, the present study revealed a potential role for TBI-associated VEGF and MMP-9 upregulation in BBB disruption and nerve damage post-TBI.

The Cytotoxicity and Anticancer Effects of Propolis against the Oral Squamous Cell Carcinoma: In Vitro Study

Background and aim: A wide range of therapeutic properties, including anti-cancer properties, are attributed to propolis, a resinous product obtained from several plants that possess a variety of medicinal properties. A study on honeybee-produced propolis showed that in mice, it showed a significant reduction in the progression of squamous cell carcinoma in the head and neck, but in humans, its role in HNSCC remains unclear.&#x0D; Method and materials: Propolis was sampled from two types of Iranian. Extraction was done using ethanolic extracts of propolis. The cll viability was evaluated by MTT assay. Cancer cell lines were assessed for gene expression, such as mmp-2, mmp-9, bax, and bcl-2.&#x0D; Results: Increased sample concentrations reduced cell viability but did not cause significant cytotoxicity. A RT-PCR indicated that the Khalkhal sample produced more effects among the two samples, and the level of bax mRNA gene expression in the Khalkhal sample was increased. With an increasing concentration of Khalkhal samples, the expression increased. Increasing Khalkhal sample concentration also reduced mRNA levels of bcl-2, mmp-2, and mmp-9.&#x0D; Conclusion: Khalkhal’s propolis can be considered a suitable sample for the study of antiapoptotic and proapoptotic gene expression. Additionally, it can be used as a potential candidate for inhibiting the proliferation and spread of oral cancer cells.

Identification and Verification of Potential Core Genes in Pediatric Septic Shock.

Septic shock is a frequent and costly problem among patients in the pediatric intensive care unit (PICU) and is associated with high mortality and devastating survivor morbidity. In this study, we aimed to screen candidate biomarkers and potential therapeutic targets for septic shock. GSE26440 dataset was downloaded from Gene Expression Omnibus (GEO), including 32 normal controls and 98 children with septic shock RNA samples from whole blood. The pathways and functional annotations of differentially expressed genes (DEGs) in the two types of samples were examined by GO and KEGG pathway enrichment analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. Protein-protein interactions (PPI) of the above-described DEGs were investigated using the Search Tool for the Retrieval of Interacting Genes (STRING) and Hub gene identification was performed by the plug-in cytoHubba in Cytoscape software. A total of 140 genes were identified as DEGs, of which 98 genes were up-regulated and 42 genes were down-regulated. GO function analysis showed that DEGs were significantly enriched in biological processes, including immune response, leukocyte activation involved in immune response, and so on. The top hub genes, namely MMP9, CEACAM8, ARG1, MCEMP1, LCN2, RETN, S100A12, GPR97, and TRAT1 were recognized from the protein-protein interaction (PPI) network. Furthermore, qRT-PCR results demonstrated that the mRNA level of MMP9, CEACAM8, ARG1, MCEMP1, LCN2, RETN, and S100A12 was elevated while GPR97 was decreased in involved mouse and human models. However, TRAT1 expression is species-dependent which was decreased in the mouse septic shock model but elevated in the human LPS-treated macrophages model. Taken together, the identification and validation of several novel hub genes, especially GPR97 and TRAT1, deepen our comprehension of the molecular mechanisms of septic shock progression. These genes may be therapeutic molecular targets or diagnostic biomarkers in patients with septic shock.

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

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

Alterations in p53 Influence hTERT, VEGF and MMPs Expression in Oral Cancer Patients.

Mutant p53 is the crucial molecule in the etiopathogenesis of oral cancer. Therefore, we aimed to evaluate the impact of alterations of the p53 gene and its negative feedback regulator, MDM2, on the expression of hTERT, VEGF, and MMPs; the critical genes involved in oral cancer progression. p53 and MDM2 genotyping were done by PCR-RFLP. p53 mutation analysis was performed using PCR-SSCP and sequencing. hTERT, VEGFA isoforms, MMP2, and MMP9 mRNA levels were analyzed by semi-quantitative Reverse Transcriptase PCR. Arg allele at p53 exon 4 was significantly associated with overexpression of hTERT, MMP2, and MMP9 individually. Expression of hTERT, VEGF A isoforms, MMP2 and MMP9 were significantly altered in the presence of p53 and MDM2 polymorphisms and p53 mutations in a specific combination. Mutant p53, Arg allele at p53 exon 4 locus, and G/G/or T/T genotype at MDM2revealed increased expression of hTERT, VEGF A isoforms, and MMP2/9. This study provides evidence that apart from mutant p53, naturally occurring sequence variants in p53codon 72 (Arg72Pro) (rs1042522) and MDM2 (rs2279744) significantly alter the expression of hTERT, VEGF-A isoforms, and MMP2/9 in a specific combination. The differential interaction of codon 72 variants with MDM2, hTERT, VEGF-A isoforms and MMP2/9 play a role in the aggressiveness of oral cancer. The results have important implications for oral cancer progression and should be explored for innovative treatment options.

Regulation of MMP9 transcription by ETS1 in immortalized salivary gland epithelial cells of patients with salivary hypofunction and primary Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) patients exhibit enhanced degradation of the salivary epithelium initially through MMP9 overexpression. We assessed the expression of MMP9 and an associated transcription factor, ETS1, in primary salivary gland epithelial cells (SGECs) and investigated potential regulatory mechanism(s) in immortalized SGECs. SGECs and iSGECs were derived from pSS and/or xerostomic “sicca” patients. siRNA knockdown of ETS1 in iSGECs was performed to determine MMP9 mRNA (qRT-PCR) and protein expression (ELISA). ETS1 binding to MMP9 promoter was assessed by luciferase activity and binding confirmed by mutagenesis and ChIP. Effects of ETS1 overexpression on progenitor and Epithelial-Mesenchymal transition (EMT) associated markers were determined by Western blot. Expression of ETS1 and its phosphorylated form in iSGECs was determined by immunofluorescence microscopy. ETS1 and MMP9 were overexpressed in SGECs of pSS and non-pSS sicca patients with salivary gland lymphocytic infiltration compared to non-pSS sicca patients without infiltration. ETS1 siRNA knockdown reduced both MMP9 mRNA and protein levels. ETS1 overexpression affected the expression of EMT and progenitor cell markers. Lastly, ETS1 bound the MMP9 promoter within the DNA region of −296 bp to −339 bp. ETS1 may impair salivary function through direct transcriptional control of the MMP9 promoter. ETS1 upregulation may also affect other factors involved in repair of the dysfunctional pSS salivary epithelium.

Abstract P3137: Drp1-dependent Mitochondrial Fission Induces Vascular Smooth Muscle Cell Dysfunction Through Kruppel-like Factor 4 Upregulation

Introduction: Vascular smooth muscle cell (VSMC) phenotypic switching and subsequent VSMC proliferation and migration are major events that are closely associated with progression of cardiovascular diseases. Mitochondria play key roles in regulation of cell function. Recent findings have revealed that mitochondrial fission promotes proliferation and migration of VSMCs. Hypothesis: This study aimed to explore the role of a new drug candidate, ginsenoside Rh1, in Angiotensin II (Ang II)-induced VSMC migration and proliferation and its underlying mechanisms. Methods: Sulforhodamine B and wound-healing assay were used to evaluate effects on cell proliferation and migration. Molecular mechanisms were investigated using western blotting, qRT-PCR analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured by dihydroethidium and MitoSOX staining. Results: We found that pretreatment with 50 μM Rh1 significantly suppressed Ang II-induced cell proliferation (% of decrease vs. Ang II group: 17.41 ± 2.38%, p &lt; 0.01, n = 5) and migration (% of decrease vs. Ang II group: 198.8 ± 2.67%, p &lt; 0.001, n = 5). Concomitantly, Ang II-increased both protein and mRNA levels of MMP2, MMP9, PCNA, and cyclin D1 were reduced by Rh1 pretreatment. Rh1 incubation also reversed the changes in both protein and mRNA expressions of α-SMA, vimentin, and osteopontin induced by Ang II. Besides, Ang II enhanced protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, inhibitory effects of Rh1 were found to be associated with inhibition of ERK1/2 signaling pathway and ROS generation. In addition, deactivation of ERK1/2 suppressed phosphorylation of DRP1 at Ser 616. Notably, inhibition of DRP1-dependent mitochondrial fission by Mdivi-1 inhibited KLF4 expression and transcriptional activity induced by Ang II. Conclusions: In conclusion, mitochondrial fission involves Ang II-induced VSMC proliferation and migration through KLF4 upregulation, and that Rh1 inhibits VSMC dysfunction by suppressing ROS-mediated ERK1/2/DRP1/KLF4 signaling pathway.

Two dimers generated by lithospermic decarboxylation coupling from Danshen

To further reveal the active ingredients of Danshen, we systematically studied its chemical components and obtained two new lithospermic acid derivatives (compounds 1 and 2) together with five known phenylpropionic acids (compounds 3–7) from the dried rhizomes of Salvia miltiorrhiza. The structures of the two new compounds were determined by multiple spectral analyses (UV, IR, HR-ESI-MS, NMR, and ECD). In addition, the absolute configurations were established by chiral analysis and calculated and experimental circular dichroism spectra. Biological research indicated that compound 1 could significantly inhibit the proliferation of isoproterenol (ISO)-treated cardiac fibroblasts (AC16 cells), and MMP9 was found to be the most likely target of compound 1. The protein expression and mRNA levels of MMP9 were increased in ISO-induced AC16 cells, which could be reversed by treatment with compound 1. Furthermore, this treatment could alleviate the migration and activation of ISO-induced cardiac fibroblasts.