Intracellular Protein Levels Research Articles

Nuclear Factor-κB Signaling Regulates the Nociceptin Receptor but Not Nociceptin Itself.

The nociceptin receptor (NOP) and nociceptin are involved in the pathways of pain and inflammation. The potent role of nuclear factor-κB (NFκB) in the modulation of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β on the nociceptin system in human THP-1 cells under inflammatory conditions were investigated. Cells were stimulated without/with phorbol-myristate-acetate (PMA), TNF-α, IL-1β, or PMA combined with individual cytokines. To examine NFκB's contribution to the regulation of the nociceptin system, PMA-stimulated cells were treated with NFκB inhibitor BAY 11-7082, JSH-23, or anacardic acid before culturing with TNF-α or IL-1β. NOP and prepronociceptin (ppNOC) mRNA were quantified by RT-qPCR; cell membrane NOP and intracellular nociceptin protein levels were measured by flow cytometry. Phosphorylation and localization of NFκB/p65 were determined using ImageStream. PMA + TNF-α decreased NOP mRNA compared to stimulation with PMA alone, while PMA + IL-1β did not. BAY 11-7082 and JSH-23 reversed the repression of NOP by PMA + TNF-α. TNF-α and IL-1β attenuated PMA's upregulating effects on ppNOC. None of the inhibitors preserved the upregulation of ppNOC in PMA + TNF-α and PMA + IL-1β cultures. TNF-α strongly mediated the nuclear translocation of NFκB/p65 in PMA-treated cells, while IL-1β did not. Proinflammatory cytokines suppressed NOP and ppNOC mRNA in PMA-induced human THP-1 cells. NFκB signaling seems to be an important regulator controlling the transcription of NOP. These findings suggest that the nociceptin system may play an anti-inflammatory role during immune responses.

PGRN protects against serum deprivation-induced cell death by promoting the ROS scavenger system in cervical cancer

Progranulin (PGRN), an autocrine growth factor with tumorigenic roles in a variety of tumors, is a putative survival factor for normal and cancer cells in vitro. However, the fundamental mechanism of PGRN-mediated survival of cancer cells suffering from various types of microenvironmental stresses, such as serum deprivation, remains unknown. We show here that serum deprivation decreases intracellular PGRN protein levels in cervical cancer cells. PGRN protects cervical cancer cells against serum deprivation-induced apoptosis, limits reactive oxygen species (ROS) levels, maintains mitochondria integrity, and reduces oxidative damage of protein, lipid and DNA. PGRN enhances the ROS scavenger system, as evidenced by increased superoxide dismutase (SOD), catalase protein expression and activity, elevated GSH and NADPH levels and increased phase II detoxification enzyme expression in cervical cancer cells after serum withdrawal. The role of PGRN in ROS clearance is mediated by the PGRN-stimulated nuclear factor erythroid-derived 2-like 2 (NFE2L2)-antioxidant response element (ARE) pathway. Our study reveals an antioxidant role of PGRN in supporting the survival of cervical cancer cells under oxidative stress. This insight provides a new perspective on the how cervical cancer cells adapt to microenvironmental stress, contributing to cell viability and other malignant characteristics.

The neuroprotective effect of 1,25-dyhydroxyvitamin D3 (calcitriol) and probiotics on the rotenone-induced neurotoxicity model in SH-SY5Y cells

This study aimed to investigate the neuroprotective role of probiotics and 1,25-dyhydroxyvitamin D3 (calcitriol) against neurotoxicity on rotenone-induced human neuroblastoma cell line SH-SY5Y. Rotenone was administered to induce neurotoxic effects in SH-SY5Y cells. Calcitriol and probiotics were administered at different concentrations as pre- and post-treatment. The thiazolyl blue tetrazolium bromide (MTT) assay was performed to measure cell viability. Intracellular protein levels of antioxidant enzymes (protein tyrosine kinase (PTK), superoxide dismutase (SOD), glutathione peroxidase (GSH), glutathione reductase (GSR), and catalase (CAT)) were determined by the enzyme-linked immunosorbent assay (ELISA). Rotenone (150 nM) reduced (p < 0.001) cell viability compared to control cells. Single and combined pretreatments with probiotics (0.01 mg/ml, 0.05 mg/ml, and 0.1 mg/ml) and calcitriol (1.25 µM, 2.5 µM, and 5 µM) increased (p < 0.05) cell viability compared to rotenone group. In the pre- and post-treatment design, all treatment groups increased the SOD and GSH levels and decreased the GSR levels compared to rotenone. None of the pretreatments reversed the PTK levels (except probiotics: 0.01 mg/ml). Calcitriol (2.5 µM) increased the CAT levels in pretreatment design, and probiotics (0.05 mg/ml and 0.1 mg/ml) increased CAT levels in post-treatment design compared to rotenone group. Calcitriol and probiotics protect against rotenone-induced neurotoxicity in SH-SY5Y cells by decreasing reactive oxygen species (ROS) and increasing antioxidant enzyme parameters. These neuroprotective effects of calcitriol and probiotics against rotenone-induced dopaminergic neurotoxicity provide an experimental basis for their potential clinical use in the treatment of Parkinson’s disease (PD).

Bidirectional roles of extracellular and intracellular HMGB2 in the development of atherosclerosis

Abstract Rationale: HMGB family plays an important role in homeostasis of immunity and regulation of inflammation. However, the role of HMGB2 in atherosclerosis remains uncharacterized. Objective The current study aimed to ascertain the relation of HMGB2 levels in macrophages of atherosclerotic plaques and in circulatory monocytes/macrophages with coronary artery disease (CAD), and to investigate the influence of HMGB2 on atherogenesis in mice and inflammatory reaction in vitro. Methods/Results We evaluated HMGB2 levels in macrophages of atherosclerotic plaques in coronary endarterectomy tissue from patients with angiographically documented severe coronary artery disease (CAD)(n=23), and in coronary artery specimens from autopsy cases with no or mild atherosclerosis (n=11) by immunohistochemistry. HMGB2 levels were significantly decreased in macrophages of progressive atherosclerotic plaques, as compared with those from mild plaques. Moreover, we also analyzed HMGB2 levels in peripheral blood monocytes from patients with CAD (n=44) or age- and sex-matched non-CAD controls (n=44). The results showed that decreased intracellular HMGB2 protein levels in monocytes were associated with CAD. In experiments, HMGB2 deficiency significantly promoted atherosclerosis in ApoE-/- mice, whereas such increment of atherosclerosis was markedly attenuated after transplantation of bone marrow derived macrophages from ApoE-/-mice. Mechanistically, intracellular HMGB2 repressed IL-1 transcription and induced autophagic degradation of NLRP3, thus suppressing NLRP3 inflammasome priming and activation. Extracellular HMGB2 levels increased in progressive atherosclerotic plaques with macrophage apoptosis. Recombinant HMGB2 protein promoted inflammation in vitro via RAGE and intraperitoneal administration of this protein exacerbated atherosclerosis in mice. Conclusion This study indicates that decreased HMGB2 level in macrophage is associated with inflammation and atherosclerosis in patients with CAD. Intracellular HMGB2 inhibits NLRP3 inflammasome and atherosclerosis in mice, whereas released HMGB2 promotes inflammation and atherosclerosis in mice.

Histone deacetylase inhibition disrupts the molecular signature of the glioblastoma secretome related to extracellular vesicle-associated proteins and targets RAB7a and RAB14 in vitro

Glioblastoma (GBM) is the most aggressive brain tumor with a poor prognosis. While Histone Deacetylase inhibitors have shown promising results in inhibiting cancer cell invasion and promoting apoptosis, their effects on GBM secretion, specifically focusing on extracellular vesicles (EVs) secretion, remain largely unexplored. Using label-free NANOLC-MS/MS methodology, we identified significant changes in the abundance of membrane traffic regulatory proteins in the secretome of U87MG cells after the treatment with the HDAC inhibitor Trichostatin A (TSA). In silico analysis showed that TSA treatment disrupted the secretion pattern of EVs-associated proteins and cellular signaling pathways, both qualitatively and quantitatively. Notably, RAB14/RAB7a interaction was only observed in the secretome of cells treated with TSA. In vitro assays revealed that TSA treatment of glioma cells increased EVs secretion and intracellular protein levels of RAB7a and RAB14 without affecting gene expression, suggesting a role of these two EVs-associated proteins in grade IV glioma cells. Additionally, an integrative approach using clinical data highlighted a correlation between DNA mutations affecting vesicle traffic coding-genes and clinical and phenotypic outcomes in glioma patients. These findings provide insights into the interplay between epigenetics and GBM intracellular trafficking, potentially leading to improved strategies for targeting and modifying the complex signaling network established between GBM cells and the tumor cell microenvironment.

Hiding in plain sight: Optimizing topoisomerase IIα inhibitors into Hsp90β selective binders

Due to their impact on several oncogenic client proteins, the Hsp90 family of chaperones has been widely studied for the development of potential anticancer agents. Although several Hsp90 inhibitors have entered clinical trials, most were unsuccessful because they induced a heat shock response (HSR). This issue can be circumvented by using isoform-selective inhibitors, but the high similarity in the ATP-binding sites between the isoforms presents a challenge. Given that Hsp90 shares a conserved Bergerat fold with bacterial DNA gyrase B and human topoisomerase IIα, we repurposed our ATP-competitive inhibitors of these two proteins for Hsp90 inhibition. We virtually screened a library of in-house inhibitors and identified eleven hits for evaluation of Hsp90 binding. Among these, compound 11 displayed low micromolar affinity for Hsp90 and demonstrated a 12-fold selectivity for Hsp90β over its closest isoform, Hsp90α. Out of 29 prepared analogs, 16 showed a preference for Hsp90β over Hsp90α. Furthermore, eleven of these compounds inhibited the growth of several cancer cell lines in vitro. Notably, compound 24e reduced intracellular levels of Hsp90 client proteins in MCF-7 cells, leading to cell cycle arrest in the G0/G1 phase without inducing HSR. This inhibitor exhibited at least a 27-fold preference for Hsp90β and was selective against topoisomerase IIα, a panel of 22 representative protein kinases, and proved to be non-toxic in a zebrafish larvae toxicology model. Finally, molecular modeling, corroborated by STD NMR studies, and the binding of 24e to the S52A mutant of Hsp90α confirmed that the serine to alanine switch drives the selectivity between the two cytoplasmic isoforms.

Sm(Ⅲ), Gd(Ⅲ), and Eu(Ⅲ) complexes with 8-hydroxyquinoline derivatives as potential anticancer agents via inhibiting cell proliferation, blocking cell cycle, and inducing apoptosis in NCI-H460 cells.

Four lanthanide complexes with 8-hydroxyquinoline-2-aldehyde-2-hydrazinopyridine (H-L1), 8-hydroxyquinoline-2-aldehyde-2-hydrazimidazole (H-L2): [Sm(L1)2][Sm(L1)(NO3)3]·CHCl3·2CH3OH (1), [Gd(L1)2][Gd(L1)(NO3)3]·CHCl3·2CH3OH (2), [Sm(L2)(NO3)2]2·CH3OH (3), and [Eu(L2)(NO3)2]2·CH3OH (4) were synthesized and characterized. In vitro cytotoxicity evaluation showed that the ligands and four lanthanide complexes exhibited cytotoxicity to the five tested tumor cell lines. Among them, complex 1 showed the best antiproliferative activity against NCI-H460 tumor cells. Mechanistic studies demonstrated that complex 1 arrested the cell cycle of NCI-H460 cells in G1 phase and induced mitochondria-mediated apoptosis, which resulted in the loss of mitochondrial membrane potential, enhanced intracellular Ca2+ levels and reactive oxygen species generation. In addition, complex 1 affected the expression levels of intracellular apoptosis-related proteins and activated the caspase-3/9 in NCI-H460 cells. Therefore, complex 1 is a potential anticancer agent.

A dual-pathway pyroptosis inducer based on Au–Cu2-xSe@ZIF-8 enhances tumor immunotherapy by disrupting the zinc ion homeostasis

The regulation of intracellular ionic homeostasis to trigger antigen-specific immune responses has attracted extensive interest in tumor therapy. In this study, we developed a dual-pathway nanoreactor, Au-Cu2-xSe@ZIF-8@P18 NPs (ACS-Z-P NPs), which targets danger-associated molecular patterns (DAMPs) and releases Zn2+ and reactive oxygen species (ROS) within the tumor microenvironment (TME). Zn2+ released from the metal–organic frameworks (MOFs) was deposited in the cytoplasm, leading to aberrant transcription levels of intracellular zinc-regulated proteins and DNA damage, thereby inducing pyroptosis and immunogenic cell death (ICD) dependent on caspase1/gasdermin D (GSDMD) pathway. Furthermore, upon laser irradiation, ACS-Z-P NPs could break through the limitations of inherent defects of immunosuppression in TME, enhance ROS generation through a Fenton-like reaction cascade, which subsequently triggered the activation of inflammatory vesicles and the release of damage-associated molecular patterns (DAMPs). This cascade effect led to the amplification of pyroptosis and immunogenic cell death (ICD), thereby remodeling the immunosuppressed TME. Consequently, this process improved dendritic cell (DC) antigen presentation and augmented anti-tumor T-cell responses, effectively initiating antigen-specific immune responses and further enhancing pyroptosis and ICD. This study explores the therapeutic properties of these mechanisms in detail. Statement of significanceThe synthesized Au-Cu2-xSe@ZIF-8@P18 nanoparticles (ACS-Z-Ps) can effectively enhance the bodyʼs immune response by regulating zinc ion levels within cells. This regulation leads to abnormal levels of zinc-regulated protein transcription and DNA damage, which induces cellular pyroptosis. As a result, antigen presentation to dendritic cells (DCs) is improved, and anti-tumor T-cell responses are enhanced.The ACS-Z-P NPs overcome the limitations of ROS deficiency and immunosuppression in the tumor microenvironment by using H2O2 in the tumor microenvironment through a Fenton-like reaction. This leads to an increased production of ROS and O2, remodeling of the immunosuppressed tumor microenvironment, and enhanced induction of cell pyroptosis and immunogenic cell death.ACS-Z-P NPs targeted B16 cells using the photosensitizer P18 in combination with PDT treatment. This approach significantly inhibited the proliferation of B16 cells and effectively inhibited tumor growth.

UTRs and Ago-2/miR-335 Complex Restricts Amylin Translation in Insulinoma and Human Pancreatic β-Cells.

Amylin promoter and transcriptional factors are well-established, inducible factors in the production of the main amyloidogenic pancreatic hormone, human islet amyloid peptide (hIAPP) or amylin. However, posttranscriptional mechanisms driving hIAPP expression in pancreas remain enigmatic, and hence were explored here. The translational assay revealed that both 5' and 3' untranslated regions (UTRs) of hIAPP restricted expression of the luciferase constructs only in constructs driven by the hIAPP promoter. Bioinformatics analysis revealed several putative seed sequences for a dozen micro RNAs (miRNAs) in hIAPP's 3' UTR. miR-182, miR-335, and miR-495 were the most downregulated miRNAs in stressed human islets exposed to endoplasmic reticulum (ER) or metabolic stressors, thapsigargin (TG) or high glucose (HG). Correspondingly, miR-335 mimics alone or in combination with miR-495 and miR-182 mimics significantly and potently (>3-fold) reduced hIAPP protein expression in HG-treated cultured human islets. siRNA-mediated silencing of Ago2 but not Ago1 significantly stimulated hIAPP expression and secretion from transfected, HG-treated human islets. Conversely, ectopic expression of Ago2 in hIAPP-expressing RIN-m5F cell line driven by CMV promoter reduced hIAPP intracellular protein levels. Collectively, the results point to a novel and synergistic role for hIAPP promoter, 5/3' UTRs and Ago-2/miR-335 complex in post-transcriptional regulation of hIAPP gene expression in normal and metabolically active β-cells.

Dichloroacetate Prevents Sepsis Associated Encephalopathy by Inhibiting Microglia Pyroptosis through PDK4/NLRP3.

Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, is often used to treat lactic acidosis and malignant tumors. Increasing studies have shown that DCA has neuroprotective effects. Here, we explored the role and mechanism of DCA in Sepsis associated encephalopathy (SAE). Single-cell analysis was used to determine the important role of PDK4 in SAE and identify the cell type. GO and GSEA analysis were used to determine the correlation between DCA and pyroptosis. Through LPS + ATP stimulation, a microglia pyroptosis model was established to observe the expression level of intracellular pyroptosis-related proteins under DCA intervention, and further detect the changes in intracellular ROS and JC-1. Additionally, a co-culture environment of microglia and neuron was simply constructed to evaluate the effect of DCA on activated microglia-mediated neuronal apoptosis. Finally, Novel object recognition test and the Morris water maze were used to explore the effect of DCA on cognitive function in mice from different groups after intervention. Based on the above experiments,this study concludes thatDCA can improve the ratio of peripheral and central M1 macrophages, inhibit NLRP3-mediated pyroptosis through ROS and mitochondrial membrane potential (MMP). DCA can reduce neuron death caused by SAE and improve cognitive function in LPS mice. In SAE, DCA may be a potential candidate drug for the treatment of microglia-mediated neuroinflammation.

Extracellular ISG15 triggers ISGylation via a type-I interferon independent non-canonical mechanism to regulate host response during virus infection.

Type-I interferons (IFN) induce cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity and regulate cellular activities associated with inflammatory and neurodegenerative diseases and cancer. Apart from ISGylation, unconjugated free ISG15 is also released from cells during various conditions, including virus infection. The role of extracellular ISG15 during virus infection was unknown. We show that extracellular ISG15 triggers ISGylation and acts as a soluble antiviral factor to restrict virus infection via an IFN-independent mechanism. Specifically, extracellular ISG15 acts post-translationally to markedly enhance the stability of basal intracellular ISG15 protein levels to support ISGylation. Furthermore, extracellular ISG15 interacts with cell surface integrin (α5β1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified extracellular ISG15 protein as a new soluble antiviral factor that confers IFN-independent non-canonical ISGylation via the integrin-FAK pathway by post-translational stabilization of intracellular ISG15 protein.

Machine Learning-Enhanced Estimation of Cellular Protein Levels from Bright-Field Images.

In this study, we aimed to develop a novel method for non-invasively determining intracellular protein levels, which is essential for understanding cellular phenomena. This understanding hinges on insights into gene expression, cell morphology, dynamics, and intercellular interactions. Traditional cell analysis techniques, such as immunostaining, live imaging, next-generation sequencing, and single-cell analysis, despite rapid advancements, face challenges in comprehensively integrating gene and protein expression data with spatiotemporal information. Leveraging advances in machine learning for image analysis, we designed a new model to estimate cellular biomarker protein levels using a blend of phase-contrast and fluorescent immunostaining images of epidermal keratinocytes. By iterating this process across various proteins, our model can estimate multiple protein levels from a single phase-contrast image. Additionally, we developed a system for analyzing multiple protein expression levels alongside spatiotemporal data through live imaging and phase-contrast methods. Our study offers valuable tools for cell-based research and presents a new avenue for addressing molecular biological challenges.

Prostaglandin E2 inhibits the differentiation of T regulatory cells by Peroxisome Proliferator-Activated Receptor-Gamma during allergic rhinitis.

Allergic rhinitis (AR) represents a significant global health concern that can give rise to numerous diseases and result in labor productivity. T regulatory (Treg) cells are pivotal players in the pathogenesis of AR, and their deficiencies are closely related to Prostaglandin E2 (PGE2). However, the downstream mechanisms of this relationship remain poorly understood. This study aims to investigate the inhibitory mechanisms through which PGE2 impacts the differentiation of Treg cells. We compared the differentiation of Treg cells from naïve CD4+ T cells of AR patients and healthy controls, with or without the presence of PGE2 by flow cytometry. Intracellular cAMP concentration, mRNA and protein levels of cyclic-AMP dependent protein kinase A (PKA), as well as their downstream target, Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) were examined in Treg cells from AR and healthy donors. AR mouse model was established by pollen administration. PGE2 suppressed the differentiation of Treg cells from human naïve CD4+ T cells through the EP4 receptor. Furthermore, in AR patients and AR mouse, the expression of EP4 receptor were observed enhanced. The PGE2-EP4 signal was carried out by activating cAMP-PKA signaling pathway. Subsequently, phospholated PKA would suppress PPAR-γ expression. Treatment of Pioglitazone, a PPAR-γ agonist, was demonstrated to rescue the differentiation of Treg and help alleviate inflammation in the AR mouse model. In AR disease, the PGE2-EP4 signaling exerts an inhibitory effect on Treg differentiation by influencing the cAMP-PKA pathway and its downstream target PPAR-γ.

In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015–January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.

Mixotrophic cultivation of microalgae using agro-industrial waste: Tolerance level, scale up, perspectives and future use of biomass

Alternative culture media make large-scale microalgae biomass production more viable and cost-effective. The aim of this work was to study the addition of whey as an alternative source of organic nutrients in fed-batch mode for the cultivation of microalgae (Spirulina platensis, Chlorella homosphaera and Scenedesmus obliquus). The microalgae demonstrated acceptance of whey at low concentrations, causing the increasing in the maximum specific growth rates (μmax) and biomass concentrations ([X]max) compared with the cultivation without whey in standard medium (μmax of 0.168 d−1 versus 0.075 d−1, and [X]max of 2.274 g L−1 versus 0.970 g L−1, respectively, for S. platensis). Therefore, a cost savings in cultivation media of up to 32 %, 57 %, and 73 % were possible in the cultivation of S. obliquus, S. platensis, and C. homosphaera, respectively, considering the production of 1 kg of biomass. The addition of whey presented different effects in the levels of intracellular protein considering the species of microalgae: an increasing was observed in C. homosphaera (24.18 to 43.96 g/100 g of biomass) and a decreasing in S. platensis (73.75 to 44.06 g/100 g of biomass), comparing with the controls. Scale-up with a natural light source can affect microalgal productivity and biomass composition in a medium supplemented with whey, because of the influence of irradiation. Optimizing the large-scale production of microalgae using agro-industrial residues can lead to significant advancements in biomass production at reduced costs. These biomasses can be integrated into bioenergy production, biofertilizer manufacturing, and animal feed production. By utilizing waste as a nutritional source and optimizing cultivation processes and biomass generation, a closed-loop cycle is established, aligning with the principles of circular bioeconomy and enhancing the sustainability of the process.

Thymoquinone potentiates anti-cancer effects of cisplatin in oral squamous cell carcinoma via targeting oxidative stress.

Recent evidence has proved that thymoquinone as a natural polyphenol has great anticancer and anti-proliferative effects in cancer cells. In this study, we aimed to examine the effects of thymoquinone on increasing cisplatin-induced apoptosis human oral squamous cell carcinoma cells and its underlying molecular mechanisms. SCC-25 cancer cells treated by thymoquinone and cisplatin with different concentrations. Cell viability will determine by using MTT assay. The concentrations of reactive oxygen species (ROS) and antioxidant activities were determined using specific related kits. DNA damage, lipid, and protein oxidation were assessed. Real-time PCR and Western blot analysis will be used to determine the expression of apoptosis-related proteins including Bax, Bcl-2, and caspase-3. Combination of thymoquinone and cisplatin suppressed synergistically SCC-25 cancer cell viability and induced apoptosis in dose-depended manner. Cell treatment with combination of thymoquinone and cisplatin led to accumulation of ROS within cells and increase in the intracellular levels of DNA damage, protein and lipid peroxidation. In addition, the combination of thymoquinone and cisplatin modulated the mRNA and protein expression levels of apoptosis-related proteins including Bax, Bcl-2, and caspase-3. Thymoquinone potentiated cisplatin anti-cancer effect on OSCC by inducing oxidative stress in cells.

Effects of hindlimb unloading on the mevalonate and mechanistic target of rapamycin complex 1 signaling pathways in a fast-twitch muscle in rats.

Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.

Kinase PIM1 governs ferroptosis to reduce retinal microvascular endothelial cell dysfunction triggered by high glucose.

Previous studies have implicated targeting Pim-1 proto-oncogene, serine/threonine kinase (PIM1) as a preventive measure against high glucose-induced cellular stress and apoptosis. This study aimed to reveal the potential role and regulatory mechanism of PIM1 in diabetic retinopathy. Human retinal microvascular endothelial cells (hRMECs) underwent high glucose induction, and fluctuations in PIM1 levels were assessed. By overexpressing PIM1, its effects on the levels of inflammatory factors, oxidative stress indicators, migration and tube formation abilities, tight junction protein expression levels, and ferroptosis in hRMECs were identified. Afterwards, hRMECs were treated with the ferroptosis-inducing agent erastin, and the effect of erastin on the above PIM1 regulatory functions was focused on. PIM1 was downregulated upon high glucose, and its overexpression inhibited the inflammatory response, oxidative stress, cell migration, and tube formation potential in hRMECs, whereas elevated tight junction protein levels. Furthermore, PIM1 overexpression reduced intracellular iron ion levels, lipid peroxidation, and levels of proteins actively involved in ferroptosis. Erastin treatment reversed the impacts of PIM1 on hRMECs, suggesting the mediation of ferroptosis in PIM1 regulation. The current study has yielded critical insights into the role of PIM1 in ameliorating high glucose-induced hRMEC dysfunction through the inhibition of ferroptosis.

Effect of Short-Chain Fatty Acids on Inflammatory and Metabolic Function in an Obese Skeletal Muscle Cell Culture Model.

The fermentation of non-digestible carbohydrates produces short-chain fatty acids (SCFAs), which have been shown to impact both skeletal muscle metabolic and inflammatory function; however, their effects within the obese skeletal muscle microenvironment are unknown. In this study, we developed a skeletal muscle in vitro model to mimic the critical features of the obese skeletal muscle microenvironment using L6 myotubes co-treated with 10 ng/mL lipopolysaccharide (LPS) and 500 µM palmitic acid (PA) for 24 h ± individual SCFAs, namely acetate, propionate and butyrate at 0.5 mM and 2.5 mM. At the lower SCFA concentration (0.5 mM), all three SCFA reduced the secreted protein level of RANTES, and only butyrate reduced IL-6 protein secretion and the intracellular protein levels of activated (i.e., ratio of phosphorylated-total) NFκB p65 and STAT3 (p < 0.05). Conversely, at the higher SCFA concentration (2.5 mM), individual SCFAs exerted different effects on inflammatory mediator secretion. Specifically, butyrate reduced IL-6, MCP-1 and RANTES secretion, propionate reduced IL-6 and RANTES, and acetate only reduced RANTES secretion (p < 0.05). All three SCFAs reduced intracellular protein levels of activated NFκB p65 and STAT3 (p < 0.05). Importantly, only the 2.5 mM SCFA concentration resulted in all three SCFAs increasing insulin-stimulated glucose uptake compared to control L6 myotube cultures (p < 0.05). Therefore, SCFAs exert differential effects on inflammatory mediator secretion in a cell culture model, recapitulating the obese skeletal muscle microenvironment; however, all three SCFAs exerted a beneficial metabolic effect only at a higher concentration via increasing insulin-stimulated glucose uptake, collectively exerting differing degrees of a beneficial effect on obesity-associated skeletal muscle dysfunction.

Effects of α7 nicotinic acetylcholine receptor agonist against α-synuclein-induced neurotoxicity

The α7 neuronal nicotinic acetylcholine receptor (α7 nAChR) is a potential target for the development of Parkinson’s disease (PD) therapeutics. α-Synuclein (α-Syn), a principal component of Lewy bodies (cytoplasmic inclusions), is a major contributor to PD pathophysiology. Previous studies have demonstrated that activating α7 nAChR protects against nigrostriatal dopamine degeneration in acute and chronic PD animal models induced by 6-hydroxydopamine and rotenone, respectively. In the present study, we investigated the effects of PNU282987, a selective α7 nAChR agonist, against α-Syn-induced neurotoxicity in α-SynWT-, α-SynA30P-, and α-SynE46K-N2a cells. PNU282987 exhibited substantial neuroprotection against both wild-type and mutant-type α-Syn-induced toxicity. Furthermore, PNU282987 promoted transcription factor EB activity and reduced intracellular α-Syn protein levels through autophagy induction. These results highlight the therapeutic potential of α7 nAChR activation in diseases characterized by α-Syn aggregation, such as PD.