Identification and validation of matrix metalloproteinase hub genes as potential biomarkers for Skin Cutaneous Melanoma.

Objectives: The role of matrix metalloproteinases (MMPs) in Skin Cutaneous Melanoma (SKCM) development and progression is unclear so far. This comprehensive study delved into the intricate role of MMPs in SKCM development and progression. Methods: RT-qPCR, bisulfite sequencing, and WES analyzed MMP gene expression, promoter methylation, and mutations in SKCM cell lines. TCGA datasets validated findings. DrugBank and molecular docking identified potential regulatory drugs, and cell line experiments confirmed the role of key MMP genes in tumorigenesis. Results: Our findings unveiled significant up-regulation of MMP9, MMP12, MMP14, and MMP16, coupled with hypomethylation of their promoters in SKCM cell lines, implicating their involvement in disease progression. Mutational analysis highlighted a low frequency of mutations in these genes, indicating less involvement of mutations in the expression regulatory mechanisms. Prognostic assessments showcased a significant correlation between elevated expression of these genes and poor overall survival (OS) in SKCM patients. Additionally, functional experiments involving gene silencing revealed a potential impact on cellular proliferation, further emphasizing the significance of MMP9, MMP12, MMP14, and MMP16 in SKCM pathobiology. Conclusion: This study identifies Estradiol and Calcitriol as potential drugs for modulating MMP expression in SKCM, highlighting MMP9, MMP12, MMP14, and MMP16 as key diagnostic and prognostic biomarkers.

Astragalus polysaccharide restores insulin secretion impaired by lipopolysaccharides through the protein kinase B /mammalian target of rapamycin/glucose transporter 2 pathway.

BACKGROUND: Lipopolysaccharide (LPS)-induced dysfunction of pancreatic ß-cells leads to impaired insulin (INS) secretion. Astragalus polysaccharide (APS) is a bioactive heteropolysaccharide extracted from Astragalus membranaceus and is a popular Chinese herbal medicine. This study aimed to elucidate the mechanisms by which APS affects INS secretion from ß-cells under LPS stress. METHODS: Rat insulinoma (INS-1) cells were treated with LPS at a low, medium, or high concentration of APS. Glucose-stimulated insulin secretion (GSIS) was evaluated using an enzyme-linked immunosorbent assay (ELISA). Transcriptome sequencing was used to assess genome-wide gene expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to determine the signaling pathways affected by APS. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to evaluate the gene expression of glucose transporter 2 (GLUT2), glucokinase (GCK), pancreatic duodenal homeobox-1 (PDX-1), and INS. Western blot analysis was used to detect the protein expression of phosphorylated protein kinase B (p-Akt), total Akt (t-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), total mTOR (t-mTOR), and GLUT2. RESULTS: LPS decreased GLUT2, GCK, PDX-1, and INS expression and reduced GSIS. These LPS-induced decreases in gene expression and GSIS were restored by APS treatment. In addition, transcriptome sequencing in combination with KEGG enrichment analysis revealed changes in the INS signaling pathway following APS treatment. LPS decreased p-Akt and p-mTOR expression, which was restored by APS treatment. The restorative effects of APS on GSIS as well as on the expression of GLUT2, GCK, PDX-1, and INS were abolished by treatment with the Akt inhibitor MK2206 or the mTOR inhibitor rapamycin (RPM). CONCLUSIONS: APS restored GSIS in LPS-stimulated pancreatic ß-cells by activating the Akt/mTOR/GLUT2 signaling pathway.

Rapid Treatment with Intramuscular Magnesium Sulfate During Cardiopulmonary Resuscitation Does Not Provide Neuroprotection Following Cardiac Arrest.

Brain injury is the most common cause of death for patients resuscitated from cardiac arrest. Magnesium is an attractive neuroprotective compound which protects neurons from ischemic injury by reducing neuronal calcium overload via NMDA receptor modulation and preventing calcium-induced mitochondrial permeability transition. Intramuscular (IM) delivery of MgSO4 during CPR has the potential to target these mechanisms within an early therapeutic window. We hypothesize that IM MgSO4 administrated during CPR could achieve therapeutic serum magnesium levels within 15 min after ROSC and improve neurologic outcomes in a rat model of asphyxial cardiac arrest. Male Long Evans rats were subjected to 8-min asphyxial cardiac arrest and block randomized to receive placebo, 107 mg/kg, 215 mg/kg, or 430 mg/kg MgSO4 IM at the onset of CPR. Serum magnesium concentrations increased rapidly with IM delivery during CPR, achieving twofold to fourfold increase by 15 min after ROSC in all magnesium dose groups. Rats subjected to cardiac arrest or sham surgery were block randomized to treatment groups for assessment of neurological outcomes. We found that IM MgSO4 during CPR had no effect on ROSC rate (p > 0.05). IM MgSO4 treatment had no statistically significant effect on 10-day survival with good neurologic function or hippocampal CA1 pyramidal neuron survival compared to placebo treatment. In conclusion, a single dose IM MgSO4 during CPR achieves up to fourfold baseline serum magnesium levels within 15 min after ROSC; however, this treatment strategy did not improve survival, recovery of neurologic function, or neuron survival. Future studies with repeated dosing or in combination with hypothermic targeted temperature management may be indicated.

Berberine mitigates nonalcoholic hepatic steatosis by downregulating SIRT1-FoxO1-SREBP2 pathway for cholesterol synthesis.

OBJECTIVE: To investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms. METHODS: A steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L, oleic acid:palmitic acid = 2:1), and then treated with three concentrations of BBR; cell viability was assessed with cell counting kit-8 assays. Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay. The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction. In addition, both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation. RESULTS: FFA-induced steatosis was successfully established in HepG2 cells. Lipid accumulation and TC content in BBR groups were significantly lower (P < 0.05, P < 0.01), associated with significantly higher mRNA and protein levels of SIRT1(P < 0.05, P < 0.01), significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P < 0.05, P < 0.01), as well as higher Acetyl-FoxO1 protein level (P < 0.05, P < 0.01) compared to the FFA only group. Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects. Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation, and promoted its nuclear translocation. CONCLUSION: BBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway. BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.

Impact of community respiratory viral infections in urban children with asthma.

BACKGROUND: Upper respiratory tract viral infections cause asthma exacerbations in children. However, the impact of natural colds on children with asthma in the community, particularly in the high-risk urban environment, is less well defined. OBJECTIVE: We hypothesized that children with high-symptom upper respiratory viral infections have reduced airway function and greater respiratory tract inflammation than children with virus-positive low-symptom illnesses or virus-negative upper respiratory tract symptoms. METHODS: We studied 53 children with asthma from Detroit, Michigan, during scheduled surveillance periods and self-reported respiratory illnesses for 1 year. Symptom score, spirometry, fraction of exhaled nitric oxide (FeNO), and nasal aspirate biomarkers, and viral nucleic acid and rhinovirus (RV) copy number were assessed. RESULTS: Of 658 aspirates collected, 22.9% of surveillance samples and 33.7% of respiratory illnesses were virus-positive. Compared with the virus-negative asymptomatic condition, children with severe colds (symptom score ≥5) showed reduced forced expiratory flow at 25% to 75% of the pulmonary volume (FEF25%-75%), higher nasal messenger RNA expression of C-X-C motif chemokine ligand (CXCL)-10 and melanoma differentiation-associated protein 5, and higher protein abundance of CXCL8, CXCL10 and C-C motif chemokine ligands (CCL)-2, CCL4, CCL20, and CCL24. Children with mild (symptom score, 1-4) and asymptomatic infections showed normal airway function and fewer biomarker elevations. Virus-negative cold-like illnesses demonstrated increased FeNO, minimal biomarker elevation, and normal airflow. The RV copy number was associated with nasal chemokine levels but not symptom score. CONCLUSION: Urban children with asthma with high-symptom respiratory viral infections have reduced FEF25%-75% and more elevations of nasal biomarkers than children with mild or symptomatic infections, or virus-negative illnesses.

Antioxidant properties and neuroprotective effects of isocampneoside II on hydrogen peroxide-induced oxidative injury in PC12 cells.

Oxidative stress has been considered as a major cause of cell damage in various neurodegenerative disorders. One of the reasonable strategies for delaying the disease's progression is to prevent reactive oxygen species (ROS) mediated cellular injury by dietary or pharmaceutical augmentation of free radical scavengers. Isocampneoside II (ICD) is an active phenylethanoid glycoside isolated from the medicinal hardwood genus Paulownia. This study was designed to explore free radical scavenging potential of ICD in different in vitro systems and its protective role in hydrogen peroxide (H2O2)-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. The results showed ICD eliminated approximately 80.75% superoxide radical at the concentration of 0.1mg/ml and inhibited metal chelating by 22.07% at 8 mg/ml. Additionally, ICD showed a strong ability on reducing power and provided protection against oxidative protein damage induced by hydroxyl radicals. Pretreatment of PC12 cells with ICD prior to H2O2 exposure elevated cell viability, enhanced activity of superoxide dismutase and catalase, and decreased levels of malondialdehyde and intracellular ROS. Furthermore, ICD inhibited cell apoptosis and Bax/Bcl-2 ratio induced by H2O2. These findings suggested ICD may be considered as a potential antioxidant agent and should encourage for further research in neurodegenerative diseases.

An activin receptor-like kinase 5 inhibitor reduces collagen deposition in a rat dermal incision wound healing model.

BACKGROUND: Excessive dermal scarring is characterized by an overabundant deposition of extracellular matrix caused by fibrosis. The purpose of this study was to modify a rodent model of cutaneous healing for use in the development of compounds to minimize scarring, and to test the model with a small molecule inhibitor of transforming growth factor-ß type I receptor, activin receptor-like kinase 5, because this class of inhibitors has been demonstrated to be effective in minimizing fibrosis in other organs. METHODS: The rodent model of cutaneous healing consists of uniform full-thickness incisional dermal wounds in rats. Wounds were allowed to heal by secondary intention, generally over a 14-day period. The usefulness of the model was tested by the application of an activin receptor-like kinase 5 inhibitor, CP-639180. Activin receptor-like kinase 5 inhibition antagonizes the transforming growth factor-ß pathway, and was used to determine whether there was an effect on collagen deposition in wounds. The compound was applied once per day for 7 days starting at postwounding day 0 or 7 (early or late treatment regimens). Wounds were analyzed histologically for collagen deposition and biochemically for quantification of collagen changes. RESULTS: Early and late treatment regimens with the activin receptor-like kinase 5 inhibitor significantly reduced collagen deposition without impairing wound healing. CONCLUSIONS: Application of a small molecular inhibitor of activin receptor-like kinase 5 appears to significantly reduce collagen deposition in rat dermal wounds as reported here for the first time. Activin receptor-like kinase 5 inhibition may offer a novel approach to reducing proliferative scars in humans because collagen accumulation is a core event in scarring.

IL-22 is involved in liver regeneration after hepatectomy.

Hepatocyte proliferation following partial hepatectomy is an important component of liver regeneration, and recent in vitro studies have shown that IL-22 is involved in cellular proliferation in a variety of cell types, including hepatocytes. IL-22 functions through IL-10Rbeta and IL-22Ralpha. The goal of this study was to investigate the potential role of IL-22 in liver regeneration after 70% hepatectomy. Following 70% hepatectomy, done under general anesthesia in mice, serum IL-22 and hepatic IL-22Ralpha mRNA were significantly increased. Although administration of exogenous IL-22 prior to hepatectomy did not increase hepatocyte proliferation, administration of anti-IL-22 antibody before hepatectomy did significantly decrease hepatocyte proliferation. Furthermore, IL-22 treatment prior to 70% hepatectomy induced stat-3 activation; no significant changes were seen in ERK1/2 activation, stat-1 activation, or stat-5 activation. IL-22 pretreatment also significantly increased hepatic and serum IL-6 levels. In addition, animals treated with anti-IL-22 antibody also expressed less TGF-alpha. In conclusion, these data suggest that IL-22 is involved in liver regeneration and this may be due to interaction with IL-6 and TGF-alpha cascades.

Stem cell factor and its receptor, c-kit, are important for hepatocyte proliferation in wild-type and tumor necrosis factor receptor-1 knockout mice after 70% hepatectomy.

BACKGROUND: Stem cell factor (SCF) has well-known proliferative effects on hematopoietic cells. SCF also has effects on differentiation and proliferation in other cell types. Interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha have proliferative effects in the liver. Recent studies in our laboratory have linked SCF's hepatoproliferative actions to those of IL-6, demonstrating that IL-6-induced hepatocyte proliferation depends, at least in part, on SCF. We now hypothesize that TNF-alpha's hepatoproliferative effects are also dependent on SCF. METHODS AND RESULTS: In vitro studies using primary mouse hepatocytes show that SCF is induced by TNF-alpha; anti-SCF antibody treatment in this system inhibits TNF-alpha-induced hepatocyte proliferation, suggesting that TNF-alpha-induced hepatocyte proliferation is also SCF dependent. Additional in vivo experiments were performed in which wild type and/or TNF-alpha receptor-1 knockout mice (TNFR1(-/-)) were subjected to 70% hepatectomy or sham laparotomy. TNFR1(-/-) mice are known to have delayed hepatic regeneration after partial hepatectomy. Initial experiments demonstrated that the SCF receptor, c-kit, is upregulated after partial hepatectomy in wild-type mice, further emphasizing the importance of this system in the restoration of hepatic mass. SCF administration to TNFR1(-/-) mice in the context of partial hepatectomy restores hepatocyte proliferation to normal. Further, SCF administration to TNFR1(-/-) mice before hepatectomy increases phosphotyrosine signal transducer and activator (p-stat-3) levels, suggesting that SCF-induced increases in hepatocyte proliferation may also be stat-3 mediated. CONCLUSIONS: These data suggest that TNF-alpha-induced hepatocyte proliferation depends, at least in part, on SCF and that SCF and its receptor, c-kit, are important for the liver's regenerative processes.

Stem cell factor restores hepatocyte proliferation in IL-6 knockout mice following 70% hepatectomy.

Stem cell factor (SCF) is a molecule with known proliferative effects on hematopoietic cells. More recent studies suggest that this molecule may also have effects on cellular differentiation and proliferation in other types of cells. The current investigations demonstrate that there is a large reservoir of SCF in the liver, that hepatic SCF levels change dramatically following partial hepatectomy in mice, and that SCF blockade, either by administration of anti-SCF antibodies or by using genetically altered, SCF-deficient mice, inhibits hepatocyte proliferation after partial hepatectomy; if SCF is replaced in the genetically SCF-deficient mice after partial hepatectomy, hepatocyte proliferation is restored to that seen in WT animals. Furthermore, SCF administration to IL-6 knockout mice also restores hepatocyte proliferation to normal. In vitro studies using primary mouse hepatocytes demonstrate that SCF causes hepatocyte proliferation and is induced by IL-6 and that treatment with anti-SCF antibodies inhibits IL-6-induced hepatocyte proliferation. Further in vivo studies in IL-6 knockout mice demonstrate that SCF administration to these animals increases p-stat3 levels, suggesting that the SCF-induced increase in hepatocyte proliferation in this system is stat3-mediated.

CXC chemokine expression after stimulation with interferon-gamma in primary rat hepatocytes in culture.

Monokine-induced by gamma interferon (MIG) and gamma-interferon-inducible protein (IP-10) are members of the CXC chemokine family that have been shown to be induced by interferon-gamma (IFNy) in some cell types. The purpose of this investigation was to determine whether IFNgamma influences CXC chemokine production, particularly MIG and IP-10, in primary rat hepatocytes in culture. Previous experiments in our laboratory have demonstrated that pharmacologic doses of IFNgamma in an in vivo model of hepatic ischemia/reperfusion-induced liver injury resulted in increased hepatic levels of IP-10 and MIG and decreased hepatic levels of macrophage inflammatory protein-2, Kupffer cells, and epithelial neutrophil-activating protein, with a concomitant decrease in neutrophil-mediated hepatic injury. In the current investigation, MIG and IP-10 mRNA and protein were up-regulated in primary rat hepatocytes in vitro in response to IFNgamma. Although MIG and IP-10 mRNA were both somewhat increased at early time points, larger increases in these chemokines were seen at later time points, specifically at 24, 48, and 72 h of incubation as compared to controls. Levels of Kupffer cells and macrophage inflammatory protein-2 mRNA after IFNgamma were negligible and similar to those seen in controls. These findings were confirmed by enzyme-linked immunosorbent assay analysis. These studies demonstrate that IFNgamma in vitro up-regulates the production of MIG and IP-10, at both the mRNA and protein levels.