Phosphorylation Of P53 Protein Research Articles

Betulinic Acid Alleviates Spleen Oxidative Damage Induced by Acute Intraperitoneal Exposure to T-2 Toxin by Activating Nrf2 and Inhibiting MAPK Signaling Pathways.

T-2 toxin, which is mainly produced by specific strains of Fusarium in nature, can induce immunotoxicity and oxidative stress, resulting in immune organ dysfunction and apoptosis. Betulinic acid (BA), a pentacyclic triterpenoids from nature plants, has been demonstrated to possess immunomodulating and antioxidative bioactivities. The purpose of the study was to explore the effect of BA on T-2 toxin-challenged spleen oxidative damage and further elucidate the underlying mechanism. We found that BA not only ameliorated the contents of serum total cholesterol (TC) and triglyceride (TG) but also restored the number of lymphocytes in T-2 toxin-induced mice. BA dose-dependently reduced the accumulation of reactive oxygen species (ROS), enhanced superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content, as well as increased the total antioxidant capacity (T-AOC) in the spleen of T-2-toxin-exposed mice. Moreover, BA reduced inflammatory cell infiltration in the spleen, improved the morphology of mitochondria and enriched the number of organelles in splenocytes, and dramatically attenuated T-2 toxin-triggered splenocyte apoptosis. Furthermore, administration of BA alleviated the protein phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERK); decreased the protein expression of kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein1 (Keap1); and increased the protein expression of nuclear factor erythroid 2 [NF-E2]-related factor (Nrf2) and heme oxygenase-1 (HO-1) in the spleen. These findings demonstrate that BA defends against spleen oxidative damage associated with T-2 toxin injection by decreasing ROS accumulation and activating the Nrf2 signaling pathway, as well as inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway.

Lidocaine attenuates CFA-induced inflammatory pain in rats by regulating the MAPK/ERK/NF-κB signaling pathway.

Lidocaine is a commonly used local anesthetic that also confers analgesic effects, resistance to hyperalgesia and anti-inflammatory properties. The present study aimed to explore the effects of lidocaine on complete Freund's adjuvant (CFA)-induced inflammatory pain. In the present study, rats were subcutaneously injected with CFA to investigate the molecular mechanisms associated with lidocaine in an inflammation-induced pain model. Firstly, CFA was subcutaneously injected into the paws of Sprague-Dawley rats, following which lidocaine or saline and the ERK agonist recombinant human epidermal growth factor (rh-EGF) were injected via the tail vein. Rat behavior was then assessed at 0 and 4 h, 1, 4, 7 and 14 days after CFA treatment. Proinflammatory cytokine levels in the serum were measured using ELISA. Western blotting was performed to detect the protein levels of phosphorylated (p)-ERK1/2, ERK1/2 and NF-κB subunits p-p65 and p65. Reverse transcription-quantitative PCR was used to measure the mRNA expression of ERK1/2 and p65 in rat spinal cord tissues. The results showed that injection of CFA significantly reduced the mechanical withdrawal threshold, thermal withdrawal latency and the frequency of responses to cold stimulation in rats, whilst promoting tumor necrosis factor-α, interleukin (IL)-1β, IL-6 levels in addition to ERK1/2, p65 protein phosphorylation. These effects were alleviated by lidocaine treatment. Furthermore, treatment with rh-EGF reversed the protective effects of lidocaine on inflammatory pain caused by CFA. In conclusion, lidocaine inhibits the inflammatory response and pain through the MAPK/ERK/NF-κB pathway in a rat model of pain induced by CFA.

Bupleurum chinense Polysaccharide Improves LPS-Induced Senescence of RAW264.7 Cells by Regulating the NF-κB Signaling Pathway.

Macrophages are important inflammatory cells that play a vital role in inflamm-aging. Bupleurum chinense polysaccharide (BCP), an effective component of the Bupleurum chinense herb, exerts multiple beneficial pharmacological effects, such as improving immunity and antioxidant activity. However, the effects of BCP on macrophage-aging and inflamm-aging are yet to be established. In this study, we examined the effects of BCP on proliferation, inflammatory cytokines, β-galactosidase (SA-β-gal), senescence-associated heterochromatin foci (SAHF), reactive oxygen species (ROS), mitochondrial membrane potential, p53, p16, and p65/NF-κB signaling proteins in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BCP significantly inhibited production of interleukin-1α (IL-1α), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), reduced the expression of SA-β-gal and formation of SAHF, as well as ROS level, and stabilized the mitochondrial membrane potential in RAW264.7 cells stimulated with LPS. Furthermore, BCP inhibited the expression of aging-related genes, p53 and p16, suppressed phosphorylation of p65 protein, and enhanced the expression of I-κBα protein through the NF-κB signaling pathway in LPS-stimulated RAW264.7 cells. Accordingly, we conclude that BCP effectively suppresses inflamm-aging by reducing inflammatory cytokine levels and oxidative stress production following activation of the NF-κB signaling pathway in RAW264.7 cells stimulated with LPS. Our collective findings support the utility of BCP as a novel pharmaceutical agent with potential anti-inflamm-aging effects.

Endothelial progenitor cells promote osteogenic differentiation in co-cultured with mesenchymal stem cells via the MAPK-dependent pathway

BackgroundThe role of bone tissue engineering is to regenerate tissue using biomaterials and stem cell-based approaches. Combination of two or more cell types is one of the strategies to promote bone formation. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of mesenchymal stem cells (MSCs) and promote bone healing; this study aimed to investigate the possible mechanisms of EPCs on promoting osteogenic differentiation of MSCs.MethodsMSCs and EPCs were isolated and co-cultured in Transwell chambers, the effects of EPCs on the regulation of MSC biological properties were investigated. Real-time PCR array, and western blotting were performed to explore possible signaling pathways involved in osteogenesis. The expression of osteogenesis markers and calcium nodule formation was quantified by qRT-PCR, western blotting, and Alizarin Red staining.ResultsResults showed that MSCs exhibited greater alkaline phosphatase (ALP) activity and increased calcium mineral deposition significantly when co-cultured with EPCs. The mitogen-activated protein kinase (MAPK) signaling pathway was involved in this process. p38 gene expression and p38 protein phosphorylation levels showed significant upregulation in co-cultured MSCs. Silencing expression of p38 in co-cultured MSCs reduced osteogenic gene expression, protein synthesis, ALP activity, and calcium nodule formation.ConclusionsThese data suggest paracrine signaling from EPCs influences the biological function and promotes MSCs osteogenic differentiation. Activation of the p38MAPK pathway may be the key to enhancing MSCs osteogenic differentiation via indirect interactions with EPCs.

Association of MicroRNA-21 with p53 at Mutant Sites R175H and R248Q, Clinicopathological Features, and Prognosis of NSCLC

This study aimed to investigate the association of miRNA-21 with mutant p53 expression, prognosis, interaction, and clinicopathological features of non-small cell lung cancer (NSCLC). Tissue specimens from 200 NSCLC patients were collected for qRT-PCR analysis of miR-21 and p53 expression, and p53 mutations were analyzed by Sanger sequencing. NSCLC cell lines were used to determine the effects of miR-21 knockdown on cell viability, cell cycle distribution, and p53 expression. We found that miR-21 expression was upregulated in NSCLC tissues, which was associated with an increase in p53 mRNA levels and with advanced tumor-node-metastasis (TNM) stages and lymph node metastasis. The most common mutant sites of p53 in NSCLC were R175H and R248Q. Moreover, elevated miR-21 and p53 expression levels were associated with shorter overall survival. Knockdown of miR-21 reduced NSCLC cell viability, arrested NSCLC cells at the G0-to-G1 phase of the cell cycle, and downregulated mutant p53 mRNA levels and phosphorylated p53 protein expression in A549 and H1650 cells compared to control cells. miR-21 is associated p53 at mutant sites R175H and R248Q, which seems not to be oncogenic, as it is being reported, since in a normal cell, without a mutated p53, it will probably have a protective role.

Extracellular signal-regulated kinases, P38 and c-Jun N-terminal kinases phosphorylation changes in PC12 cells with diabetic disturbances and comorbid excitotoxicity - a preliminary report.

Phosphorylation of amino acid residues of extracellular signal-regulated kinases (ERK), p38 and c-Jun N-terminal kinases (JNK) contributes to the initiation of complex pathways of intracellular signal transduction, which play a role in the development of excitotoxicity, which is important in pathogenesis both in diabetes and neurodegeneration. Due to reports on the relationship between these two diseases, it is important to explore pathways in the coexistence of both of them. This study investigated ERK, p38 and JNK protein kinases phosphorylation changes in diabetic in vitro conditions with accompanying excitotoxicity reflected by high L-glutamate concentrations. An InstantOne ELISA test in cell lysates was performed to evaluate the intensity of phosphorylation of ERK, p38 and JNK occurring as a result of the incubation of undifferentiated PC12 cells with solutions of glucose (G1,G2), insulin (I1,I2) and L-glutamate (L1,L2). We observed increased phosphorylation of JNK (Thr183/Tyr185) and p38 (Thr180/Tyr182) kinases. For both these kinases, we have shown an increase in phosphorylation in case of double combinations for the following reagents: G1I1, G1I2, G2I1, G2I2, G2L1, I2L2 and the triple ones: G1I2L1 and G2I1L2. The research based on the analysis of selected protein kinases under diabetic conditions with accompanying excitotoxicity, represents an important cognitive issue for research on neurodegenerative disorders resulting from long-term type 2 diabetes. The confirmed changes in protein phosphorylation of p38 and JNK kinases suggests changes in the conformation and activity of these proteins under conditions of increased excitotoxicity resulting from diabetes.

MicroRNA miR-155-5p knockdown attenuates Angiostrongylus cantonensis-induced eosinophilic meningitis by downregulating MMP9 and TSLP proteins

Angiostrongylus cantonensis infection is a major cause of eosinophilic meningitis (EM). Severe cases or cases that involve infants and children present poor prognoses. MicroRNAs (miRNAs), which are important regulators of gene expression in many biological processes, were recently found to be regulators of the host response to infection by parasites; however, their roles in brain inflammation caused by A. cantonensis are still unclear. The current study confirmed that miR-155-5p peaked at 21 days after A. cantonensis infection, and its expression was positively correlated with the concentration of excretory and secretory products (ESPs). We found that miR-155-5p knockdown lentivirus successfully ameliorated brain injury and downregulated the expression of major basic protein (MBP) in vivo, and the number of eosinophils in CSF (and the percentage of eosinophils in peripheral blood were also decreased in the miR-155-5p knockdown group. Moreover, the expression of several eosinophilic inflammation cytokines such as CCL6/C10, ICAM-1, and MMP9, declined after the miR-155-5p knockdown. SOCS1 protein, which is an important negative regulator of inflammation activation, was identified as a direct miR-155-5p target. We further detected the effect of miR-155-5p knockdown on phosphorylated-STAT3 and phosphorylated-p65 proteins, which were found to be negatively regulated by SOCS1 and play an important role in regulating the inflammatory response. We found that miR-155-5p knockdown decreased the activity of p-STAT3 and p-p65, thereby leading to lower expression of MMP9 and TSLP proteins, which were closely related to the chemotaxis and infiltration of eosinophils. Interestingly, the inhibition of p-STAT3 or p-p65 was found to induce the downregulation of miR-155-5p in an opposite manner. These observations suggest that a positive feedback loop was formed between miR-155-5p, STAT3, and NF-κB in A. cantonensis infection and that miR-155-5p inhibition might provide a novel strategy to attenuate eosinophilic meningitis.

Urban particulate matter regulates tight junction proteins by inducing oxidative stress via the Akt signal pathway in human nasal epithelial cells

Recent studies have revealed that increased reactive oxidative stress (ROS) induced by particulate matter (PM) affects tight junction (TJ) functions; however, the molecular mechanisms underlying this effect have not been evaluated fully. Cultured human epithelial cells obtained from inferior turbinate tissues were exposed to an urban PM (UPM) standard reference material (SRM 1648a). Intracellular ROS level and expression of proinflammatory cytokines and TJ proteins were examined. Expression level of phosphorylated (p)-Akt, p38, p65 were compared between exposed and unexposed cells. Cells were pretreated with the ROS scavenger N-acetylcysteine (NAC) or Akt inhibitor MK-2206 before exposure to determine whether the changes in cellular ROS and TJ protein expression could be reversed. Exposure to UPM significantly increased ROS levels and inflammatory cytokine expression levels, and decreased expression of TJ proteins zonula occludins (ZO)-1, occludin, claudin-1, and E-cadherin. UPM exposure increased p-Akt, p-p38, and p65 expression levels, and NAC pretreatment reversed these effects. Akt inhibition decreased UPM-induced ROS formation and p38 and p65 protein phosphorylation, and restored the decreased ZO-1 and E-cadherin expression. Akt inhibition and ROS scavenging may provide targets for maintaining epithelial integrity by restoring decreased TJ protein expression during exposure to UPM.

Anti-Inflammatory Profile of Jungia sellowii Less. by Downregulation of Proinflammatory Mediators and Inhibition of NF-κB and p38 Pathways.

Jungia sellowii Less. (Asteraceae) is a native plant found in Southeast Brazil used traditionally to treat inflammatory diseases. This study was conducted (1) to investigate the toxicity of the crude extract (CE) and (2) to investigate the mechanism of the anti-inflammatory action of J. sellowii L. roots. The potential acute toxicity of CE was performed by administration of only different doses of CE (500, 1,000, and 2,000 i.p.) on mice for 14 days. The anti-inflammatory effect was evaluated using carrageenan-induced acute pleural cavity inflammation in a mouse model, evaluated through the following inflammatory variables: leukocyte, protein concentrations of the exudate, myeloperoxidase (MPO), adenosine deaminase (ADA), nitric oxide metabolites (NOx), and proinflammatory cytokine (tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin- (IL-) 6, and IL-12) levels in mouse pleural fluid leakage. The p65 protein phosphorylation of nuclear factor NF-kappa B (p65 NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation were analyzed in lung tissue. Our results demonstrated that the administration of CE up to 2,000 mg/kg did not present a toxic effect. In addition, the pretreatment of mice with CE; its derived fractions (aqueous fraction (AqF), butanol fraction (BuOHF), and ethyl acetate fraction (EtOAcF)); and isolated compounds (curcuhydroquinone O-β-glucose (CUR) and α and β piptizol (Pip)) reduced the following inflammatory variables: neutrophils, protein concentrations of the exudate, MPO, ADA, NOx, and proinflammatory cytokine (TNF-α, IFN-γ, IL-6, and IL-12) levels in mouse pleural fluid leakage. The compounds CUR and Pip also decreased the p65 protein phosphorylation of NF-kappa B and p38 (MAPK) in lung tissue. J. sellowii L. has important anti-inflammatory activity with potential applications in drug development against inflammatory disorders. These effects found can be attributed to the ability of the new isolated compounds CUR and Pip to suppress p65 NF-κB and p-p38 MAPK pathways.

Lipocalin 2 Induces Neuroinflammation through Liver‐Brain Axis in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is chronic liver disorder characterized by accumulation of excess fat in liver due to consumption of high fat/lipid diets. A multiple hit paradigm that include lipotoxicity, environmental and genetic causes for prolonged period often leads to nonalcoholic steatohepatitis (NASH) with scarring and inflammation in liver that consequently causes irreversible hepatic damage. Both clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with several ectopic manifestations like type 2 diabetes, cardiovascular disease, chronic kidney disease and more recently neuroinflammation and neurodegeneration. Notably, mechanisms of neuroinflammation in underlying NAFLD are poorly understood. In this report we identified adipokine Lipocalin 2 (Lcn2) induced neuroinflammation by acting through liver‐brain axis in diet induced murine NAFLD/NASH model. We found that Lcn2 is overexpressed in liver and in portal circulation that activated 24p3R (Lipocalin2 receptor) in brain and induced the release of high mobility group box 1 (HMGB1) from brain cells. Released HMGB1 acted as a preferential ligand to Toll like receptor 4 (TLR 4) and induced oxidative stress by activation of NOX‐2 signaling. NOX2 downstream signaling activated NF‐kB by phosphorylation of p65 protein. Activation of p65 and NOX‐2 led to NLRP‐3 inflammasome activation which subsequently released proinflammatory cytokines IL‐6 and IL‐1β from brain cells. Furthermore, we show that elevated Lcn2 level from neurons in underlying NAFLD caused a non disruptive blood brain barrier dysfunction by over production of TNFα, IL‐1β and MMP 9 without alteration of tight junction proteins Claudin 5 and Occludin. Based on the above, the above findings identify Lcn2 as a potent molecule inducing neuroinflammation in underlying NAFLD.Support or Funding InformationNIH#P01ES028942‐01 to Dr. Saurabh Chatterjee

Radiosensitization effect of selenocystine on HeLa cells in vitro

AbstractObjectivesTo study the effect of selenocystine (Selenocystine, SeC) on the radiosensitivity of human cervical cancer Hela cells, and to provide a new target for radiosensitivity of cervical cancer.MethodsSeC were added to the cervical cancer Hela cells, and then irradiate with 200 cGy/min X‐rays. The radiosensitization effect of SeC on cervical cancer Hela cells were confirmed with the experiments of cell growth inhibition, clone formation, flow cytometry and Western blotting to detect apoptosis.ResultsSeC were added to the Hela cells at different doses of 0, 5, 10, and 20 μmol/L and incubating for 24 hours, the cell growth inhibition rates were: 0%, 22.68%, 41.71%, 74.19%, and the calculated IC50 was 14.83 μmol/L. The growth inhibition rate of Hela cells was dose‐dependent with the concentration of SeC. The results of the colony formation experiments showed that the cell colony formation rates of the single X‐ray irradiation doses of 0, 2, 4, 6, and 8 Gy were 100%, 77.56%, 46.89%, 28.67%, and 10.89%, respectively. After adding 5μmol/L SeC under the same irradiation dose, the colony formation rate was reduced to 70.98%, 59.45%, 30.67%, 14.29%, 3.72%; 5μmol/L SeC combined with radiation therapy can shift the survival curve to the left, and the D0 and Dq values were significantly smaller than those in the radiation group, P < 0.05. Hela cell apoptosis were detected by the flow cytometry showed that when the single X‐ray irradiation dose was 0, 2, 4, 8 Gy, the apoptosis scores were: 2.51 ± 0.06, 6.76 ± 0.03, 14.51 ± 0.15, 21.41 ± 0.08; When 5 μmol/L SeC was added and then combined with X‐ray irradiation, and the irradiation dose was 0, 2, 4, 8Gy, the apoptosis score of each group was: 10.89 ± 0.36, 15.18 ± 0.21, 31.19 ± 0.08, 45.53 ± 0.28, SeC combined with X‐ray had an interaction effect on Hela cells, and the apoptosis rate increased (F = 21.52, P < 0.01). When Hela cells were treated with SeC and X‐rays, the expression of PARP protein and phosphorylated p53 protein was significantly increased compared with the control group.ConclusionsSeC may have a radiosensitizing effect on Hela cells, and its mechanism may be related to promoting apoptosis. SeC will be expected to become a new sensitization target for cervical cancer radiotherapy.

Evaluation of the Genotoxic and Oxidative Damage Potential of Silver Nanoparticles in Human NCM460 and HCT116 Cells.

Nano Ag has excellent antibacterial properties and is widely used in various antibacterial materials, such as antibacterial medicine and medical devices, food packaging materials and antibacterial textiles. Despite the many benefits of nano-Ag, more and more research indicates that it may have potential biotoxic effects. Studies have shown that people who ingest nanoparticles by mouth have the highest uptake in the intestinal tract, and that the colon area is the most vulnerable to damage and causes the disease. In this study, we examined the toxic effects of different concentrations of Ag-NPs on normal human colon cells (NCM460) and human colon cancer cells (HCT116). As the concentration of nanoparticles increased, the activity of the two colon cells decreased and intracellular reactive oxygen species (ROS) increased. RT-qPCR and Western-blot analyses showed that Ag NPs can promote the increase in P38 protein phosphorylation levels in two colon cells and promote the expression of P53 and Bax. The analysis also showed that Ag NPs can promote the down-regulation of Bcl-2, leading to an increased Bax/Bcl-2 ratio and activation of P21, further accelerating cell death. This study showed that a low concentration of nano Ag has no obvious toxic effect on colon cells, while nano Ag with concentrations higher than 15 μg/mL will cause oxidative damage to colon cells.

Effects of Lentinan on Endothelial Cell Activity, Inflammatory Response, Endoplasmic Reticulum Stress, and Apoptosis in Sepsis

The aim of this study is to explore the protective effects of lentinan on endoplasmic reticulum stress, inflammation, and apoptosis in sepsis endothelial cells. Firstly, lentinan was extracted, purified, and analyzed. When the concentration of lentinan was in the range of 0.04–4 μM, there was no obvious effect on the morphology of HUVECs. When the concentration reached 10 M, the cells were obviously contracted and necrotic. CCK-8 cell activity experiment showed that when the concentration of lentinan reached 4 μM, the cell activity decreased significantly (P<0.001), and it was in a dose-dependent manner. Then, the cells were divided into the control group (0 μM lentinan), sepsis group, sepsis + lentinan 1.2 μM group, and sepsis + lentinan 2 μM group. Enzyme-linked immunosorbent assay showed that lentinan could significantly reduce the expression of TNF-α, IL-1β, and IL-6 in sepsis endothelial cells (P<0.001). In addition, flow cytometry and TUNEL staining showed that compared with the control group, the apoptosis of cells in the sepsis group increased significantly (P<0.001), and lentinan could inhibit apoptosis (P<0.001). In terms of mechanism research, the mRNA and protein expression of endoplasmic reticulum stress-related protein in endothelial cells were detected by real-time fluorescent quantitative PCR (qPCR) and Western blotting, respectively. It was found that the expression of SIRT1, the upstream factors of endoplasmic reticulum stress in sepsis cells, was obviously inhibited (P<0.001), and the expression of CHOP, GRP78, IRE1α, and ATF6 was significantly increased (P<0.001), However, the pretreatment of lentinan could significantly reverse the above changes (P<0.001). Besides, lentinan could also reduce the expression of phosphorylated p65 protein (the activation marker of NF-κb) and iNOS. Conclusion. When sepsis occurs, lentinan can protect endothelial cells from ERS inflammation and apoptosis induced by sepsis. Thus, lentinan is expected to be a new target for the treatment of sepsis-induced endothelial damage.

The protective effects of aloperine against ox-LDL-induced endothelial dysfunction and inflammation in HUVECs

Atherosclerosis is a potentially life-threatening cardiovascular disease characterized by chronic endothelial inflammation and the formation of atherosclerotic lesions. Circulating ox-LDL is known to induce atherosclerosis by triggering oxidative stress, the expression of inflammatory mediators and adhesion molecules, as well as downregulating the atheroprotective transcriptional factor KLF2. Aloperine is an alkaloid compound isolated from the plant Sophora alopecuroides. Here, we employed various experimental methods to determine the effects of aloperine on ox-LDL-induced markers of atherosclerosis. DHE staining revealed that aloperine may restore the oxidant/antioxidant balance in HUVECs by reducing the level of ROS and rescuing the reduction in NOQ-1 and GCLC induced by ox-LDL. Aloperine treatment reduced ox-LDL-induced expression of IL-6, MCP-1, VCAM-1, and E-selectin and rescued the reduction in KLF2. Aloperine also downregulated ox-LDL-induced expression of the LOX-1. We also demonstrate that aloperine improved cell viability and inhibited the adhesion of U937 monocytes to HUVECs. Finally, we demonstrate that the effects of aloperine are mediated through the rescue of KLF2 expression via suppression of the phosphorylation of p53 protein. Together, our results implicate the potential of aloperine as a safe and effective antiatherosclerosis treatment.

LncRNA NR_120420 promotes SH-SY5Y cells apoptosis by regulating NF-κB after oxygen and glucose deprivation

Stroke has serious implications on patients and a huge impact on society. The current treatment regimens with drug for acute cerebral infarction are unsatisfactory. Here, we explore whether the two long non-coding RNA (lncRNA) candidates from preliminary research regulate apoptosis after cerebral infarction, and evaluate the underlying mechanism of action. Bioinformatics analysis of the lncRNA microarray in the preliminary research of our group was performed. Changes in the expression of candidate lncRNAs in SH-SY5Y cells were detected by quantitative polymerase chain reaction (qPCR) after treatment with seven different oxygen and glucose deprivation (OGD) methods. The changes were detected after transfection of cells with six small-interfering RNAs (siRNAs). Cell models were established by OGD after transfection with siRNAs. Cell viability was evaluated with the cell counting kit 8 (CCK8) assay, while TUNEL staining and flow cytometry analysis were performed to determine apoptosis. Changes in the expression and phosphorylation of three proteins were detected by western blotting after the knockdown of NR_120420. Changes in the expression and phosphorylation of P65 protein were detected by western blotting after this cell model was treated with PDTC. Cells were transfected with siNR_120420 and treated with and without PDTC, followed by analysis of cell viability and apoptosis. Bioinformatics analysis revealed that the differentially expressed lncRNAs after acute cerebral infarction were mainly involved in nuclear factor kappa B (NF-κB) and apoptosis. Expression of the two lncRNA candidates in SH-SY5Y cells was the maximum after incubation under the OGD condition for 8 h. The knockdown efficiency was more than 60% for four of the six siRNAs, and knockdown of NR_120420 increased the cell viability and decreased the percentage of TUNEL-positive cells and apoptotic cells. Knockdown of lnc-GCH1-2:3 resulted in none of these effects. Phosphorylation of NF-κB (P65) decreased significantly after the knockdown of NR_120420. Expression and phosphorylation of P65 was significantly reduced after it was treated with PDTC. The inhibitor of NF-κB (PDTC) could abolish the effect of NR_120420 on the regulation of apoptosis in this cell model. Both NR_120420 and lnc-GCH1-2:3 had significant changes in this cell model. Knockdown of NR_120420 inhibited the apoptosis of cells, while NR_120420 knockdown inhibited apoptosis after cerebral infarction by downregulating the phosphorylation of a subunit of NF-κB (P65). This study may provide new idea for improving drug treatment of acute cerebral infarction.

Cisplatin effect on head and neck squamous cell carcinoma cells is modulated by ERK1/2 protein kinases.

The extracellular signal-regulated kinases (ERKs) are key transducers of the extracellular signals into intracellular responses and represent major molecular players in tumorigenesis. The aim of this study was to determine how curcumin (CRM) used as an adjuvant supports the apoptotic process induced by a single chemical agent treatment (cisplatin-CisPT) on two head and neck squamous cell carcinoma cell lines (FaDu and PE/CA-PJ49) and the involvement of ERK1/2 and/or p53 activation in this process. Data have shown that the CisPt effect is potentiated by CRM. CRM induced an increase of p53 protein phosphorylation in both cell lines. CisPt decreased p53 protein phosphorylation in FaDu cells, but increased it in PE/CA-PJ49 cells. Data showed that the constitutive expression of activated ERK1/2 protein-kinase was different in the two analyzed tumor cell lines. ERK1/2 activation status was essential for both cell processes, proliferation and apoptosis induced by CisPt and/or CRM treatment on squamous cell carcinoma cells. Our data suggest that p53 phosphorylation in the apoptotic process induced by CRM treatment might require the involvement of ERK1/2. In this regard the CisPt treatment suggested that p53 phosphorylation is ERK1/2 independent in FaDu cells having a p53 gene deletion and ERK1/2 dependent in PE/CA-PJ49 cells having a p53 gene amplification. Moreover, in both tumor cell lines our results support the involvement of p53 phosphorylation-ERK1/2 activation-dependent in the apoptosis induced by combined treatments (CisPt and CRM). The use of CRM as adjuvant could increase the efficiency of chemotherapy by modulating cellular activation processes of ERK1/2 signaling pathways. In conclusion, the particular mode of intervention by which ERK1/2 might influence cell proliferation and/or apoptosis processes depends on the type of therapeutic agent, the cells' particularities, and the activation status of the ERK1/2.

Melatonin Rescued Reactive Oxygen Species-Impaired Osteogenesis of Human Bone Marrow Mesenchymal Stem Cells in the Presence of Tumor Necrosis Factor-Alpha.

Accumulation of reactive oxygen species (ROS), which can be induced by inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This process can contribute to the imbalance of bone remodeling, which ultimately leads to osteoporosis. Therefore, reducing the ROS generation during osteogenesis of BMSCs may be an effective way to reverse the impairment of osteogenesis. Melatonin (MLT) has been reported to act as an antioxidant during cell proliferation and differentiation, but its antioxidant effect and mechanism of action during osteogenesis of MSCs in the inflammatory microenvironment, especially in the presence of TNF-α, remain unknown and need further study. In our study, we demonstrate that melatonin can counteract the generation of ROS and the inhibitory osteogenesis of BMSCs induced by TNF-α, by upregulating the expression of antioxidases and downregulating the expression of oxidases. Meanwhile, MLT can inhibit the phosphorylation of p65 protein and block the degradation of IκBα protein, thus decreasing the activity of the NF-κB pathway. This study confirmed that melatonin can inhibit the generation of ROS during osteogenic differentiation of BMSCs and reverse the inhibition of osteogenic differentiation of BMSCs in vitro, suggesting that melatonin can antagonize TNF-α-induced ROS generation and promote the great effect of osteogenic differentiation of BMSCs. Accordingly, these findings provide more evidence that melatonin can be used as a candidate drug for the treatment of osteoporosis.

Tobramycin Promotes Melanogenesis by Upregulating p38 MAPK Protein Phosphorylation in B16F10 Melanoma Cells.

Tobramycin is an aminoglycoside-based natural antibiotic derived from Streptomyces tenebrarius, which is primarily used for Gram-negative bacterial infection treatment. Although tobramycin has been utilized in clinical practice for a long time, it has exhibited several side effects, leading to the introduction of more effective antibiotics. Therefore, we conducted our experiments focusing on new possibilities for the clinical use of tobramycin. How tobramycin affects skin melanin formation is unknown. This study used B16F10 melanoma cells to assess the effect of tobramycin on melanin production. After cytotoxicity was assessed by MTT assay, melanin content and tyrosinase activity analyses revealed that tobramycin induces melanin synthesis in B16F10 cells. Next, Western blot analyses were performed to elucidate the mechanism by which tobramycin increases melanin production; phosphorylated p38 protein expression was upregulated. Protein inhibitors have been used to elucidate the mechanism of tobramycin. Kanamycin A and B are structurally similar to tobramycin, and 2-DOS represents the central structure of these antibiotics. The effects of these substances on melanogenesis were evaluated. Kanamycin A reduced melanin production, whereas kanamycin B and 2-DOS had no effect. Overall, our data indicated that tobramycin increases melanin production by promoting p38 protein phosphorylation in B16F10 melanoma cells.

Immunomodulatory Effects of Colistin on Macrophages in Rats by Activating the p38/MAPK Pathway

Objectives: Colistin has been identified in a Caenorhabditis elegans chemical screening as an immunostimulatory agent that activates the conserved p38/PMK-1 pathway and provides protection against pathogens. Here we aimed to extend those findings to a mammalian model and evaluate the immunomodulatory effects of colistin on rat macrophages. Methods: Macrophages were isolated from Sprague-Dawley (SD) rat. The effects of colistin on the cytokine secretion, phagocytic activity and protein expression were determined by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blotting analysis, respectively. The relative microRNA expression was determined by microarray, and Kyoto Encylopedia of Genes and Genomes (KEGG) was used to identify potential signaling pathways. Results: Our data showed that 5, 10, and 20 µg/ml colistin significantly increased the secretion of TNF-α, while 20 and 5 µg/ml colistin significantly increased the levels of IL-1β and IL-6, respectively. Flow cytometry results showed that the relative mean fluorescence intensity and percentage of phagocytosis in colistin treatment groups were significantly higher compared with the control group, while the increased phagocytosis phenomenon can be blocked by p38 inhibitor. The phagocytic ability of macrophages against Staphylococcus aureus was significantly increased after colistin treatment. Microarray and KEGG pathway analyses revealed that mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (mTOR), chemokine, and B cell receptor were the main pathways involved in the colistin stimulation process. Western blotting analysis demonstrated that the phosphorylated p38 protein level of colistin treatment groups was increased in a dose dependent manner. Conclusions: Present study is the first to demonstrate that colistin had immunomodulatory effects on macrophages in mammals, and the p38/MAPK pathway was involved in such colistin-induced immunomodulatory effect.

Treatment of Type II Collagen-Induced Rat Rheumatoid Arthritis Model by Interleukin 10 (IL10)-Mesenchymal Stem Cells (BMSCs).

BackgroundRheumatoid arthritis model (CIA) rats were treated by tail vein injection of IL-10-modified bone marrow mesenchymal stem cells (BMSCs) to investigate its feasibility and intrinsic molecular mechanism.Material/MethodsThe CIA rat model was established by induction type II collagen, and IL-10-modified BMSCs was established by transfecting BMSCs with adenovirus. IL-10-modified BMSCs were used to treat the CIA rats. The therapeutic effect was evaluated by measuring the changes in body weight, ankle swelling, and forced swimming time, as well as observation of synovial hyperplasia and cartilage tissue repair by HE staining. Western blot analysis and ELISA were used to detect gene expression.ResultsAfter 4 weeks and 8 weeks of treatment with IL10-BMSCs, the body weight, swelling value, resting time, and forced swimming struggle time of CIA rats were significantly higher than those of BMSCs-treated and -untreated CIA rats (P<0.05). Compared to BMSCs-treated CIA model rats, after treatment with IL10-BMSCs, the repair rate of osteoarticular cartilage was higher and the inhibition of synovial proliferation was better, and serum IL-17, IL-1β, and TNF-α levels were lower. We found that the protein level of SIRT1 in peripheral blood mononuclear cells was lower, the protein level in spleen was higher, and phosphorylation of p65 protein in peripheral blood mononuclear cells was reduced.ConclusionsThe efficacy of tail vein injection of IL-10-modified BMSCs in treatment of CIA rats was superior to that of BMSCs alone, which may be related to the more pronounced suppression of IL-10-modified BMSCs in peripheral blood inflammation and spleen immune response.