Phosphorylation Of P53 Protein Research Articles

Carnosol attenuates angiotensin II-induced cardiac remodeling and inflammation via directly binding to p38 and inhibiting p38 activation

Chronic inflammation is a significant contributor to hypertensive heart failure. Carnosol (Car), primarily derived from the sage plant (Salvia carnosa), exhibits anti-inflammatory properties in a range of systems. Nevertheless, the influence of angiotensin II (Ang II) on cardiac remodeling remains uncharted. Car was shown to protect mice's hearts against Ang II-induced heart damage at dosages of 20 and 40 mg/kg/d. This protection was evident in a concentration-related decrease in the remodeling of the heart and dysfunction. Examination of the transcriptome revealed that the pivotal roles in mediating the protective effects of Car involved inhibiting Ang II-induced inflammation and the activation of the mitogen-activated protein kinase (MAPK) pathway. Furthermore, Car was found to inhibit p38 phosphorylation, therefore reducing the level of inflammation in cultured cardiomyocytes and mouse hearts. This effect was attributed to the direct binding to p38 and inhibition of p38 protein phosphorylation by Car both in vitro and in vivo. In addition, the effects of Car on inflammation were neutralized when p38 was blocked in cardiomyocytes.

Beauvericin, produced by Fusarium oxysporum inhibits bisphenol A-induced proliferation of human breast cancer cell line by regulating ERα/p38 pathway

Beauvericin (BEA) is a cyclic depsipeptide secondary metabolite of Fusarium species. It causes chemical hazards in food products and exists in an environment containing soil and various food types. On the other hand, the purified BEA has various biological activities and is regarded as a potential candidate for pharmaceutical research. This study was performed to assess the anti-proliferation activity of BEA against human breast cancer cells by regulating the estrogen receptor-alpha (ERα)/p38 pathway. TA and BA assays verified that BEA is a completed ER antagonist. Additionally, BEA suppressed cell proliferation in the anti-proliferation assay involving ER-positive human breast cancer cells co-treated with BPA and BEA. In respect to an anti-proliferation activity, the BPA-induced phosphorylation of p38 protein was inhibited in the presence of BEA. These results suggested that BEA exerts inhibitory potentials on endocrine disrupting effect and possibly acts as a natural therapeutic material for human estrogen hormonal health.

UHRF1-Mediated Epigenetic Reprogramming Regulates Glycolysis to Promote Progression of B-Cell Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is a hematologic malignant disease with high heterogeneity, including B-ALL and T-ALL. The 5-year overall survival rate of children ALL has been significantly improved, which is over 90%. However, the prognosis of adult patients is not satisfactory, and the 5-year OS is only 30%-50%. Among them, the prognosis of patients with relapsed/refractory B-ALL is very poor. Exploring the molecular mechanism of disease progression is of great significance to improve the outcome. Methods Application of 10× Genomics chromium sequencer reveals the single-cell transcriptome profiling of peripheral blood or bone marrow MNC cells, which were from 6 patients with B-ALL（3 new diagnosed patients and 3 relapsed patients） and 3 healthy donors. The expression of key genes in 84 clinical blood or bone marrow samples was detected, and the prognosis was analyzed in TARGET database by bioinformatics technology. The molecular mechanism was investigated by RNAseq, 850k methylation microarray, qRT-PCR, Western blot, BSP, CHIP-qPCR and flow cytometry. Additionally, we identified a UHRF1 inhibitor using virtual compounds screening, and demonstrated its therapeutic efficacy both in vitro and vivo. Results Using Seurat method to cluster and dimension reduction of scRNA-seq data, 29 subclusters were obtained from 9 clinical samples. It mainly includes eight progenitor B cells, one B cell, six T cells, two NK cells, three monocytes, two neutrophils and three primordial erythrocytes. Nine samples were divided into three groups: Newly Diagnosis group (ND), Relapsed/Refractory group (RR) and Healthy Donors group (HD). The proportion of three subpopulations (cluster 2,5,16) in progenitor B cell group showed a decreasing trend of RR group > ND group > HD group. The marker genes of these three subgroups were enriched by GO and KEGG using ClusterProfiler R package. Cluster 2 was enriched into cancer signal pathway, cluster 5 was enriched into DNA replication process, and the marker genes of cluster 16 was mainly involved in metabolic pathway. The differential expression of these three subgroups between RR group and ND group was analyzed by FindMarkers function. The results showed that cluster 16 was the main differential cluster. The intergroup differential genes of cluster 16 mainly involve metabolic pathway, cancer pathway, cell cycle and p53 signaling pathway (Figure 1). The three groups were compared in pairs. After the intersection of differential genes in cluster 16 and all progenitor B cell groups, four common differential genes (HSPB1, UHRF1, H1F0 and IGFBP2) were screened. The results showed that the expression of UHRF1 gene was up-regulated in acute lymphoblastic leukemia patients, especially in patients with relapsed/refractory. Moreover, UHRF1 overexpression is an independent prognostic risk factor for B-ALL patients in our clinical data, and it is also a molecular marker of poor prognosis in the TARGET dataset. In vitro experiments, knockout of gene UHRF1 promoted the cycle arrest and apoptosis of B-ALL tumor cell line (Reh and nalm6), inhibited the proliferation of cells, and improved the drug sensitivity of vindesine. RNAseq and methylation microarray showed that the gene SFRP5 was demethylated and the expression of mRNA was overexpressed in UHRF1 knockout cell lines. This was verified by subsequent qRT-PCR, Western Blot and Bisulfite sequencing. Further studies showed that UHRF1 was involved in glycolysis by regulating the SFRP5-p38 MAPK-HK2 signal axis. Subsequently, through overexpressing SFRP5, we confirmed that upregulating SFRP5 can inhibit the P38 MAPK-HK2 signaling pathway, thereby suppressing glycolytic levels. Moreover, virtual compound screening identified UM164 as an inhibitor of UHRF1, which significantly inhibits P38 protein phosphorylation, downregulates HK2 expression, and reduces the production of glycolysis-related metabolites. Furthermore, UM164 demonstrated the ability to inhibit the proliferation of B-ALL cells both in vitro and in vivo, leading to prolonged survival in a B-ALL mouse model. In conclusion, we have unraveled the molecular mechanisms of epigenetic and metabolic rearrangements in relapsed/refractory B-ALL, and have provided potential targeted therapeutic strategies to improve its unfavorable prognosis.

PF-04449913 Inhibits Proliferation and Metastasis of Colorectal Cancer Cells by Down-regulating MMP9 Expression through the ERK/p65 Pathway.

Colorectal cancer remains a life-threatening malignancy with increasing morbidity and mortality worldwide. Therefore, new and effective anti-colorectal cancer therapeutics are urgently needed. In this study, we have studied the anti-tumor properties and potential mechanisms of PF-04449913. Colorectal cancer cell viability was reduced by PF-04449913 in a dose-dependent manner. The migration and invasion ability of malignant colon cells were attenuated by the drug, as demonstrated by the Transwell test. Moreover, PF-04449913 repressed the phosphorylation levels of ERK and other proteins, and the expression levels of MMP9. The anti-tumor effects of the drug in vivo were demonstrated in BALB/c-nude mice models, and PF-04449913 inhibited the malignant phenotype of colorectal cancer cells, including reduction of tumor size and promotion of apoptosis. At the molecular level, PF-04449913 induced a significant decrease in ERK and p65 protein phosphorylation levels and inhibited MMP9 protein expression. Both in vivo and in vitro results showed PF-04449913 to demonstrate antitumor effects, which have been proposed to be mediated through blockade of the ERK/p65 signaling pathway, and subsequent repression of MMP9 expression. Our study provides a new perspective on the potential clinical application of PF-04449913 in the treatment of colorectal cancer.

Rabdosichuanin C inhibits productions of pro-inflammatory mediators regulated by NF-κB signaling in LPS-stimulated RAW264.7 cells.

Chronic pharyngitis (CP) is an inflammatory disease of the pharyngeal mucosa and its lymphatic tissues that is difficult to treat clinically. However, research on the exact therapeutic agents and molecular mechanisms of CP is still unclear. In this study, we investigated Rabdosichuanin C (RC) to attenuate lipopolysaccharide (LPS)-induced inflammatory damage in RAW264.7 cells by a combination of targeted virtual screening and in vitro activity assay and further clarified its molecular mechanism of action centering on the IκB/nuclear factor kappa B (NF-κB) pathway. Molecular docking and pharmacophore simulation methods were used to screen compounds with IκB inhibitory effects. Expression of genes and proteins related to the IκB/NF-κB signaling pathway by RC in LPS-induced inflammatory injury model of RAW264.7 cells was detected by PCR, enzyme-linked immunosorbent assay, and Western blot. The docking of RC with IκB protein showed good binding energy, and pharmacophore simulations further confirmed the active effect of RC in inhibiting IκB protein. RC intervention in LPS-induced RAW264.7 cells significantly reduced the expression levels of inflammatory factors tumor necrosis factor-α, interleukins-6, iNOS, and CD-86 at the messenger RNA and protein levels, downregulated IκB, p65 protein phosphorylation levels, and significantly inhibited IκB/NF-κB signaling pathway activation. Virtual screening provided us with an effective method to rapidly identify compounds RC that target inhibit the action of IκB, and the activity results showed that RC inhibits NF-κB signaling pathway activation. It is suggested that RC may play a role in the treatment of CP by inhibiting the IκB/NF-κB signaling pathway.

Anti-aging formula protects skin from oxidative stress-induced senescence through the inhibition of CXCR2 expression

Ethnopharmacological relevanceThe skin is affected by endogenous and exogenous factors, which are the intuitive consequence expression of aging. Aging not only affects the aesthetics of the skin but also causes the decline of skin functions, leading to many skin diseases and even skin cancer. Anti-aging formula (AAF) has various biological effects such as antioxidants, regulation of intestinal flora metabolism, anti-aging, and memory improvement. However, it is not clarified whether it could be anti-aging of the skin and the anti-aging mechanism. Aim of the studyThis study aimed to investigate whether AAF could prevent skin from oxidative stress-induced senescence and explore the underlying molecular mechanisms. Materials and methodsA mouse skin oxidative stress aging model was established based on ultraviolet (UV) irradiation, and parameters such as skin water content, melanogenesis, wrinkle production, pathological changes, and aging marker proteins were measured to elucidate whether AAF has an anti-aging effect on the skin. Subsequently, transcriptome sequencing (RNA-Seq) was used to identify target genes. An in vitro cellular senescence model was established to assess the role of AAF against cellular oxidative stress senescence by detecting senescence-related markers, while the specific mechanism of action of AAF in delaying skin senescence was elucidated by silencing or overexpression of targets. ResultsIn vivo experiments demonstrated that AAF significantly increased skin water content, reduced skin sensitivity and melanin content, slowed wrinkles, improved UV-induced epidermal thickening, increased collagen fiber content, improved elastic fiber morphology, and reduced the expression of senescence proteins P21 and P16 in skin tissues. The RNA-Seq results identified chemokine receptor 2 (CXCR2) as one of the potential targets for delaying skin senescence. In vitro experiments showed that AAF markedly improved the aging phenotype, and knockdown or overexpression experiments verified the essential role of CXCR2 in the skin senescence process. Mechanistic studies suggested that AAF inhibited the P38/P53 pathway by reducing CXCR2 expression, which improved the aging phenotype, reduced oxidative damage, and ultimately delayed cellular senescence. ConclusionThe results reveal that AAF protects skin from oxidative stress-induced senescence by regulating the expression of critical target CXCR2, reducing P38 protein phosphorylation, and inhibiting P53 pathway activation. These discoveries implicate the potential of AAF in the protection of skin aging disease.

Cell cycle-specific phosphorylation of p53 protein in A549 cells exposed to cisplatin and standardized air pollutants.

Exposure to particulate matter is associated with DNA damage and the risk of lung cancer. Protein p53 is activated by multi-site phosphorylation in the early stages of DNA damage and affects cell outcome. Our study aimed to assess the effect of (100µg/mL-1/24h) standardized air pollutants: carbon black (CB), urban dust (UD), and nanoparticle carbon black (NPCB) on cell cycle, DNA damage and 53 phosphorylation at Ser 9, Ser 20, Ser 46, and Ser 392 in proliferating and quiescent A549 cells and in cells that survived cisplatin (CisPT) exposure. Phosphorylated p53 was quantified in cell subpopulations by flow cytometry using specific fluorochrome-tagged monoclonal antibodies and analysis of bivariate fluorescence distribution scatterplots. CisPT, UD and NPCB increased site-specific p53 phosphorylation producing unique patterns. NPCB activated all sites irrespectively on the cell cycle, while the UD was more selective. p53 Ser 9-P and p53 Ser 20-P positively correlated with the numbers of CisPT-treated cells at G0/G1, and NPCB and NPCB + CisPT produced a similar effect. A positive correlation and integrated response were also found between Ser 20-P and Ser 392-P in resting A549 cells treated with NPCB and CisPT but not UD. Interdependence between the expression of p53 phosphorylated at Ser 20, and Ser 392 and cell cycle arrest show that posttranslational alterations are related to functional activation. Our data suggest that p53 protein phosphorylation in response to specific DNA damage is driven by multiple independent and integrated pathways to produce functional activation critical in cancer prevention and treatment.

Telmisartan inhibits microglia-induced neurotoxic A1 astrocyte conversion via PPARγ-mediated NF-κB/p65 degradation

Astrocytes are crucially involved in neuroinflammation. Activated astrocytes exhibit at least two phenotypes, A1 (neurotoxic) and A2 (neuroprotective). The A1 phenotype is the major reactive astrocyte phenotype involved in aging and neurodegenerative diseases. Telmisartan, which is an antihypertensive agent, is a promising neuroprotective agent. This study aimed to investigate the effects of telmisartan on the phenotype of reactive astrocytes. Astrocytes were activated by culturing with the conditioned medium derived from lipopolysaccharide-stimulated microglia. This conditioned medium induced early, transient A2 astrocyte conversion (within 24 h) and late, sustained A1 conversion (beginning at 24 h and lasting up to 7 days), with a concomitant increase in the production of pro-inflammatory cytokines (interleukin [IL]-1β, tumor necrosis factor [TNF]α, and IL-6) and phosphorylation of nuclear factor-κB (NF-κB)/p65. Telmisartan treatment promoted and inhibited A2 and A1 conversion, respectively. Telmisartan reduced total and phosphorylated p65 protein levels. Losartan, a specific angiotensin II type-1 receptor (AT1R) blocker, did not influence the reactive state of astrocytes. Additionally, AT1R activation by angiotensin II did not induce the expression of pro-inflammatory cytokines and A1/A2 markers, indicating that the AT1R signaling pathway is not involved in the astrocyte-mediated inflammatory response. A peroxisome proliferator-activated receptor γ (PPARγ) antagonist reversed the effects of telmisartan. Moreover, telmisartan-induced p65 downregulation was reversed by the proteasome inhibitor MG132. These results indicate that telmisartan suppresses activated microglia-induced neurotoxic A1 astrocyte conversion through p65 degradation. Our findings contribute towards the elucidation of the anti-inflammatory activity of telmisartan in brain disorders.

PKC-δ Promotes IL-1β-Induced Apoptosis of Rat Chondrocytes and Via Activating JNK and P38 MAPK Pathways.

Protein kinase C-delta (PKC-δ) is involved in apoptosis. This study aimed to establish whether PKC-δ can further promote IL-1β-induced chondrocyte apoptosis by mediating the phosphorylation of the JNK and p38 mitogen-activated protein kinase (MAPK) signaling pathways In osteoarthritis (OA). We employed chondrocyte staining to determine the extent of cartilage degeneration. PKC-δ and p38 signal expressions were used in the immunohistochemical (IHC) test and apoptosis was assayed at the TUNEL test in human osteoarthritic and controls. We stimulated rat cartilage cells using IL-1β (10 ng/ml)/rottlerin (10 μM) or lentivirus. To determine the apoptosis rate, we employed flow cytometry. The mRNA of both BCL2-related X (BAX) and cysteine aspartate protease 3 (caspase-3) could be measured via qRT-PCR. Western blot measured the protein levels of BAX, caspase-3, PKC-δ, p-JNK/JNK and p-p38/p38. The positive rate of PKC-δ and the apoptotic rate of chondrocytes in OA were higher than controls. The manifestation of PKC-δ was positively related to the degree of cartilage degeneration, p38 protein expression, and apoptosis rate. IL-1β exposure upregulated PKC-δ expression in chondrocytes in a dose-dependent manner. Decreasing PKC-δ expression and its phosphorylation in OA can inhibit MAPK signaling pathway activation (phosphorylation) by downregulating JNK and p38 protein phosphorylation and expression. This inhibition decreases caspase-3 and BAX levels, consequently lowering the apoptosis rate in chondrocytes. PKC-δ activation by IL-1β in OA promotes chondrocyte apoptosis via activation of the JNK and p38 MAPK signal pathways, thereby promoting the OA progression.

Downregulation of XBP1s aggravates lipopolysaccharide-induced inflammation by promoting NF-κB and NLRP3 pathways’ activation in goat endometrial epithelial cells

After delivery, bacterial contamination and uterine tissue degeneration in animals can lead to the development of uterine diseases, such as endometritis, accompanied by endoplasmic reticulum stress (ERS). Increasing evidence suggests that spliced X-box binding protein 1 (XBP1s), a critical component of ERS, is involved in several pathological processes in various organisms. However, the specific molecular mechanisms by which XBP1s mediates the inflammatory response in goat endometrial epithelial cells (gEECs) remain largely unknown. In the present study, XBP1s protein was induced into the nucleus in the lipopolysaccharide (LPS, 5 μg/mL)-induced inflammatory response of gEECs. Lipopolysaccharide-induced expression and nucleation of XBP1s were reduced by the inhibition of Toll-like receptor 4 (TLR4) using TAK-242 (1 μM; a TLR4 inhibitor). Expression and nucleation of XBP1s were similarly reduced when the activity of inositol-requiring enzyme 1α (IRE1α) was inhibited using 4μ8C (10 μM; an IRE1α inhibitor). In addition, inhibition of IRE1a increased IL-1β, TNF-α, and IL-8 levels and secretion of IL-6 induced by LPS. Notably, phosphorylation of nuclear factor kappa-B (NF-κB) P65 protein and expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) were similarly increased. Furthermore, knockdown of XBP1s in gEECs consistently promoted NF-κB P65 protein phosphorylation, NLRP3 protein expression, and inflammatory cytokine secretion. In summary, the current results suggest that in the LPS-induced inflammatory response in gEECs, LPS generates intracellular signaling cascades in gEECs via TLR4, which may promote XBP1s protein expression and nucleation by activating IRE1a. However, downregulation of XBP1s expression exacerbates inflammation by promoting activation of the NF-κB and NLRP3 inflammatory vesicle pathways. These results will potentially contribute to the treatment and prevention of endometritis in ruminants.

PQBP1 regulates the cellular inflammation induced by avian reovirus and interacts with the viral p17 protein

Avian reovirus (ARV) can commonly infect a flock and cause immunosuppressive diseases in poultry. The nonstructural protein p17 is involved in viral replication, and significant progress has been made in showing its ability to regulate cellular signaling pathways. In our previous study, to further investigate the effect of ARV p17 protein on viral replication, the host protein polyglu-tamine binding protein 1 (PQBP1) was identified to interact with p17 by a yeast two-hybrid system. In the current study, the interaction between PQBP1 and p17 protein was further confirmed by laser confocal microscopy and coimmunoprecipitation assays. In addition, the N-terminal WWD of PQBP1 was found to mediate the process of binding to the p17 protein. Interestingly, we found that ARV infection significantly inhibited PQBP1 expression. While the quantity of ARV replication was largely influenced by PQBP1, PQBP1 overexpression decreased ARV replication. In contrast, upon PQBP1 knockdown, the quantity of ARV was notably increased. ARV infection and p17 protein expression were both proven to induce PQBP1 to mediate cellular inflammation. In the current study, we revealed through qRT‒PCR, ELISA and Western blotting methods that PQBP1 plays a positive role in ARV-induced inflammation. Furthermore, the mechanism of this process was shown to involve the NFκB-dependent transcription of inflammatory genes. In addition, PQBP1 was shown to regulate the phosphorylation of p65 protein. In conclusion, this research provides clues to elucidating the function of the p17 protein and the pathogenic mechanism of ARV, especially the cause of the inflammatory response. It also provides new ideas for the study of therapeutic targets of ARV.

SALL4 correlates with proliferation, metastasis, and poor prognosis in prostate cancer by affecting MAPK pathway.

The mechanism involved in prostate cancer (PCa) metastasis is still poorly understood, and several oncogenes are known to regulate this process. However, the role of spalt-like transcription factor 4 (SALL4) in PCa metastasis remains unclear. We performed RNA-sequencing to compare the mRNA expression profiles of seven localized PCa tissues and six metastatic PCa tissues. SALL4 was then identified and compared in the localized PCa and metastatic PCa. Immunohistochemical studies, qRT-PCR, and Western blot were performed to analyze the expression of SALL4 in PCa patients and cell lines. SALL4 expression and its relevance to clinical traits and prognosis were further explored in the TCGA database and in our 68 clinical samples. Subsequently, we knocked down SALL4 in DU145 and PC3 cells and performed a series of functional assays to explore the effect of SALL4 on PCa progression. Finally, protein levels of SALL4 and core components of the MAPK pathway were measured by Western blot, and cells were treated with PD0325901 to observe proliferation and metastasis. Significantly higher expression of SALL4 was found in metastatic PCa than in localized PCa. In addition, high SALL4 expression was significantly associated with high pathological T stage, N stage, Gleason score, and poor disease-free survival in TCGA database and in our clinical samples. Functional studies indicated that knockdown of SALL4 in DU145 and PC3 inhibited proliferation, migration, and angiogenesis. Furthermore, the ERK and P38 protein phosphorylation significantly reduced after knockdown of SALL4 in DU145 and PC3, indicating the inactivation of the MAPK signaling pathway. Finally, the proliferation and migration ability of DU145 and PC3 cells were significantly decreased after PD0325901 treatment. SALL4 predicts unfavorable outcome and is closely associated with PCa progression, suggesting that SALL4 may be a promising prognostic marker and potential therapeutic target for PCa.

The kinesin light chain-2, a target of mRNA stabilizing protein HuR, inhibits p53 protein phosphorylation to promote radioresistance in NSCLC.

Radioresistance hinders radiotherapy for the treatment of lung cancer. Kinesin light chain-2 (KLC2) has been found to be upregulated in lung cancer and also to be associated with poor prognosis. This study aimed to investigate the effect of KLC2 on radiosensitivity in lung cancer. The radioresistant role of KLC2 was determined by colony formation, neutral comet assay, and γH2AX immunofluorescent staining assay. We further verified the function of KLC2 in a xenograft tumor model. The downstream of KLC2 was identified through gene set enrichment analysis and validated by western blot. Finally, we analyzed clinical data from the TCGA database to reveal the upstream transcription factor of KLC2, which was validated by RNA binding protein immunoprecipitation assay. Here, we found that downregulation of KLC2 could significantly reduce colony formation, increase γH2AX level, and double-stranded DNA breaks in vitro. Meanwhile, overexpressed KLC2 significantly increased the proportion of the S phase in lung cancer cells. KLC2 knockdown could activate P53 pathway, and ultimately promoting radiosensitivity. The mRNA of KLC2 was observed to bind with Hu-antigen R (HuR). The mRNA and protein expression of KLC2 in lung cancer cells was significantly reduced when combined with siRNA-HuR. Interestingly, KLC2 overexpression significantly increased the expression of HuR in lung cancer cells. Taken together, these results indicated that HuR-KLC2 forms a positive feedback loop, which decreases the phosphorylation of p53 and thereby weaken the radiosensitivity of lung cancer cells. Our findings highlight the potential prognosis and therapeutic target value of KLC2 in lung cancer patients treated with radiotherapy.

Lipopolysaccharide Activating NF-kB Signaling by Regulates HTRA1 Expression in Human Retinal Pigment Epithelial Cells.

Inflammation and elevated expression of high temperature requirement A serine peptidase 1 (HTRA1) are known high risk factors for age-related macular degeneration (AMD). However, the specific mechanism that HTRA1 causes AMD and the relationship between HTRA1 and inflammation remains unclear. We found that lipopolysaccharide (LPS) induced inflammation enhanced the expression of HTRA1, NF-κB, and p-p65 in ARPE-19 cells. Overexpression of HTRA1 up-regulated NF-κB expression, and on the other hand knockdown of HTRA1 down-regulated the expression of NF-κB. Moreover, NF-κB siRNA has no significant effect on the expression of HTRA1, suggesting HTRA1 works upstream of NF-κB. These results demonstrated that HTRA1 plays a pivotal role in inflammation, explaining possible mechanism of overexpressed HTRA1-induced AMD. Celastrol, a very common anti-inflammatory and antioxidant drug, was found to suppress inflammation by inhibiting phosphorylation of p65 protein efficaciously in RPE cells, which may be applied to the therapy of age-related macular degeneration.

New verticillane-diterpenoid as potent NF-κB inhibitor isolated from the gum resin of Boswellia sacra

Two new verticillane-diterpenoids (1 and 2) were isolated from the gum resin Boswellia sacra. Their structures were elucidated by physiochemical and spectroscopic analysis, as well as ECD calculation. In addition, the in vitro anti-inflammatory activities of the isolated compounds were evaluated by determining the inhibitory effects on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse monocyte-macrophages. The results showed that compound 1 exhibited significant inhibitory effect on NO generation with an IC50 value of 23.3 ± 1.7 μM suggesting that it might be a candidate for an anti-inflammatory agent. Furthermore, 1 potently inhibited the release of inflammatory cytokines IL-6 and TNF-α induced by LPS in a dose-dependent manner. Using Western blot and Immunofluorescence methods, compound 1 was found to inhibit inflammation mainly by restraining the activation of NF-κB pathway. And in the MAPK signaling pathway, it was found to have inhibitory effects on the phosphorylation of JNK and ERK proteins and have no effect on the phosphorylation of p38 protein.

SiNPs induce ferroptosis in HUVECs through p38 inhibiting NrF2 pathway.

Despite of growing evidence linking silica nanoparticles (SiNPs), one of the global-top-three-produced and -used nanoparticle (NP), to human health risks, there remain many knowledge gaps over the adverse effects of SiNPs exposure on cardiovascular system and the underlying molecular mechanisms. In this study, the ferroptotic effects of SiNPs (20 nm; 0, 25, 50, and 100 μg/mL) on human umbilical vein endothelial cells (HUVECs) and the possible molecular mechanism were studied with the corresponding biochemical and molecular biology assays. The results showed that at the tested concentrations, SiNPs could decrease HUVEC viability, but the deferoxamine mesylate (an iron ion chelator) might rescue this reduction of cell viability. Also, increased levels of intracellular reactive oxygen species and enhanced mRNA expression of lipid oxidation enzymes (ACSL4 and LPCAT3) with increase in lipid peroxidation (malondialdehyde), but decreased ratios of intracellular GSH/total-GSH and mitochondrial membrane potential as well as reduced enzymatic activities of anti-oxidative enzymes (CAT, SOD, and GSH-PX), were found in the SiNPs-treated HUVECs. Meanwhile, increase in p38 protein phosphorylation and decrease in NrF2 protein phosphorylation with reduced mRNA expressions of downstream anti-oxidative enzyme genes (CAT, SOD1, GSH-PX, and GPX4) was identified in the SiNPs-exposed HUVECs. These data indicated that SiNPs exposure might induce ferroptosis in HUVECs via p38 inhibiting NrF2 pathway. Ferroptosis of HUVECs will become a useful biomarker for assessing the cardiovascular health risks of environmental contaminants.

Inhibition of mycobacteria proliferation in macrophages by low cisplatin concentration through phosphorylated p53-related apoptosis pathway.

Drug resistance is a prominent problem in the treatment of tuberculosis, so it is urgent to develop new anti- tuberculosis drugs. Here, we investigated the effects and mechanisms of cisplatin (DDP) on intracellular Mycobacterium smegmatis to tap the therapeutic potential of DDP in mycobacterial infection. Macrophages infected with Mycobacterium smegmatis were treated with DDP alone or combined with isoniazid or rifampicin. The results showed that the bacterial count in macrophages decreased significantly after DDP (≤ 6 μg/mL) treatment. When isoniazid or rifampicin was combined with DDP, the number of intracellular mycobacteria was also significantly lower than that of isoniazid or rifampicin alone. Apoptosis of infected cells increased after 24 h of DDP treatment, as shown by flow cytometry and transmission electron microscopy detection. Transcriptome sequencing showed that there were 1161 upregulated and 645 downregulated differentially expressed genes (DEGs) between the control group and DDP treatment group. A Trp53-centered protein interaction network was found based on the top 100 significant DEGs through STRING and Cytoscape software. The expression of phosphorylated p53, Bax, JAK, p38 MAPK and PI3K increased after DDP treatment, as shown by Western blot analysis. Inhibitors of JAK, PI3K or p38 MAPK inhibited the increase in cell apoptosis and the reduction in the intracellular bacterial count induced by DDP. The p53 promoter Kevetrin hydrochloride scavenges intracellular mycobacteria. If combined with DDP, Kevetrin hydrochloride could increase the effect of DDP on the elimination of intracellular mycobacteria. In conclusion, DDP at low concentrations could activate the JAK, p38 MAPK and PI3K pathways in infected macrophages, promote the phosphorylation of p53 protein, and increase the ratio of Bax to Bcl-2, leading to cell apoptosis, thus eliminating intracellular bacteria and reducing the spread of mycobacteria. DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.

NF-κB Inhibitor Myrislignan Induces Ferroptosis of Glioblastoma Cells via Regulating Epithelial-Mesenchymal Transformation in a Slug-Dependent Manner.

Glioblastoma (GBM) is the most common malignant tumor of the adult central nervous system. Aberrant regulation of cell death is an important feature of GBM, and investigating the regulatory mechanisms of cell death in GBM may provide insights into development of new therapeutic strategies. We demonstrated that myrislignan has ferroptosis-promoting activity. Myrislignan is a lignan isolated from Myristica fragrans Houtt and an inhibitor of NF-κB signaling pathway. Ferroptosis is an iron-dependent form of programmed cell death characterized by the accumulation of intracellular lipid peroxidation products. Interestingly, ferroptosis was associated with other biological processes in tumor cells such as autophagy and necroptosis. Recently, the crosstalk between epithelial-mesenchymal transition (EMT) and ferroptosis has also been reported, but the mechanisms underlying the crosstalk have not been identified. Our results indicated that myrislignan suppressed growth of GBM through EMT-mediated ferroptosis in a Slug-dependent manner. Myrislignan inhibited the activation of NF-κB signaling by blocking the phosphorylation of p65 protein and induced ferroptosis through the Slug-SLC7A11 signaling pathway in GBM cells. In addition, myrislignan suppressed the progression of GBM in xenograft mouse model. Hence, our findings contribute to the understanding of EMT-induced ferroptosis and provide targets for the development of targeted therapy against GBM.

Human and mouse TLR2 results in different activation of p38 and JNK signal pathway in HaCaT infected by Trichophyton rubrum and Microsporum canis.

It has long been recognized that inflammation to dermatophyte infection is different among various hosts, but the mechanism underlying is still not well understood. Toll-like receptor (TLR2), mediates the innate immune response against dermatophyte infection and is very important to trigger the inflammatory response to dermatophytes. Considering the different amino acid sequences and structures of TLR2, we speculated that TLR2 from different hosts will activate the downstream signal pathways to varying degrees, resulting in different inflammatory responses to dermatophytes. In this study, we constructed the mice-human fusion TLR2 expressed HaCaT (mhTLR2-HaCaT) by replacing the extracellular ligand recognition region of human TLR2 with that of the mouse. Then hTLR2-HaCaT cells and mhTLR2-HaCaT cells were infected with T. rubrum and M. canis for 24 h followed by immunoblotting to asses associated proteins of p38 and JNK signal pathway. Compared with that of human TLR2 expressed HaCaT (hTLR2-HaCaT), levels of phosphorylated p38 protein were increased in mhTLR2-HaCaT cells stimulated by T. rubrum for 24 h, and levels of phosphorylatedJNK and c-Jun protein were increased in mhTLR2-HaCaT cells whenstimulated with M. canis for 24 h. Compared with hTLR2-HaCaT cells, p38 and JNK signal pathwayswere activated in mhTLR2-HaCaT after being infected by Trichophyton rubrumand Microsporum canis, respectively. Since p38 and JNK are the mainpathways that transduce the signal for host recognition of dermatophytes andmediate the downstream inflammatory response, it suggested that theinterspecific difference of TLR2 ectodomain may be one of the reasons for thedifferent inflammatory manifestations between humans and mice infected bythese two dermatophytes. Quite especially, the mouse-derived TLR2extracellular recognition region is more effective in recognizing T. rubrum andM. canis to activate the downstream signal pathways, resulting in a tenserinflammatory response against these two dermatophytes.

Low-Dose Metformin Treatment Reduces In Vitro Growth of the LL/2 Non-small Cell Lung Cancer Cell Line.

Lung cancer maintains a relatively small survival rate (~19%) over a 5-year period and up to 80-85% of all lung cancer diagnoses are Non-Small Cell Lung Cancer (NSCLC). To determine whether metformin reduces non-small cell lung cancer (NSCLC) LL/2 cell growth, cells were grown in vitro and treated with metformin for 48 h. qPCR was used to assess genes related to cell cycle regulation and pro-apoptotic markers, namely Cyclin D, CDK4, p27, p21, and HES1. Treatment with 10 mM metformin significantly reduced HES1 expression (p = 0.011). Furthermore, 10 mM metformin treatment significantly decreased REDD1 (p = 0.0082) and increased p-mTOR Ser2448 (p = 0.003) protein expression. Control cells showed significant reductions in phosphorylated p53 protein expression (p = 0.0367), whereas metformin treated cells exhibited reduced total p53 protein expression (p = 0.0078). There were no significant reductions in AMPK, PKB/AKT, or STAT3. In addition, NSCLC cells were treated for 48 h. with 10 mM metformin, 4 µM gamma-secretase inhibitor (GSI), or the combination of metformin (10 mM) and GSI (4 µM) to determine the contribution of respective signaling pathways. Metformin treatment significantly reduced total nucleus expression of the proliferation maker Ki-67 with an above 65% reduction in Ki-67 expression between control and metformin-treated cells (p = 0.0021). GSI (4 µM) treatment significantly reduced Ki-67 expression by ~20% over 48 h (p = 0.0028). Combination treatment (10 mM metformin and 4 µM GSI) significantly reduced Ki-67 expression by more than 50% over 48 h (p = 0.0245). As such, direct administration of metformin (10 mM for 48 h) proved to be an effective pharmaceutical agent in reducing the proliferation of cultured non-small cell cancer cells. These intriguing in vitro results, therefore, support the further study of metformin in appropriate in vivo models as an anti-oncogenic agent and/or an adjunctive therapy.