Cell Biochemistry and Biophysics最新文献

Icariside II exerts protective effects against various diseases; however, its specific effects on osteoarthritis (OA) remain unclear. Therefore, in this study, we aimed to investigate the effects of icariside II in an in vitro model of OA and analyze its action mechanisms. We established an in vitro OA model by treating a human chondrocyte cell line (CHON-001) with interleukin (IL)-1β, followed by treatment with different concentrations of icariside II. Cell viability was measured using the methyl thiazolyl tetrazolium assay, and the level of lactate dehydrogenase (LDH) released from cells was determined using the appropriate kit. Tumor necrosis factor (TNF)-α, IL-6, and IL-8 levels were determined via enzyme-linked immunosorbent assay. Flow cytometry was used to assess apoptosis. Apoptosisrelated protein expression levels and TNFAIP3-interacting protein 2 (TNIP2)/nuclear factor (NF)-κB signaling pathway were analyzed via reverse transcription-quantitative polymerase chain reaction and western blotting. Furthermore, TNIP2-small interfering RNA (siRNA) was used to determine whether the TNIP2/NF-κB pathway influences the effects of icariside II on OA. Results indicated that Icariside II did not exert any significant toxic effects on CHON-001 cells. It inhibited IL-1β-induced apoptosis and increase in LDH levels and enhanced the inflammatory response. Additionally, icariside II reversed the IL-1β-induced decrease in TNIP2 levels and increase in NF-κB phosphorylation. TNIP2-siRNA revealed that the TNIP2/NF-κB signaling pathway influenced the alleviating effects of icariside II on OA. In conclusion, our results revealed that icariside II attenuated IL-1β-induced inflammatory injury in chondrocytes by increasing TNIP2 expression and inhibiting NF-κB pathway activation, highlighting its therapeutic potential for OA.

In the contemporary era of drug discovery, herbal treatments have demonstrated an unparalleled ability to produce anticancer drugs. An important part of the therapy of cancer is the use of plants and their by-products via analogues, which alter the tumor microenvironment and several signaling pathways. The objective of the current investigation was to conclude the rate at which the herbal medications quercetin (QT) and sulforaphane (SFN) repressed the growth of breast carcinoma cells in MDA-MB-231 by preventing the ERK/MAPK signaling systems. The cells were assessed for several studies after being subjected to different concentrations (0-70 µM) of QT and SFN (QT + SFN) for duration of 24 h. We investigated the combination that QT + SFN generated cytotoxicity using the MTT assay. The DCFH-DA staining technique was utilized to assess ROS. The protein spectra of survival of cells, cell cycle progression, and apoptosis were evaluated employing flow cytometry and western blotting. The consequences illustrated that the relative cytotoxicity of QT and SFN was roughly 28.74 μM and 39.87 μM for MDA-MB-231 cells, respectively. Following the 24-h incubation period, MDA-MB-231 cells exhibit considerable cytotoxicity when QT and SFN are combined, with IC₅₀ values of 19.48 μM. Moreover, MCF-7 and MDA-MB-231 cells treated with QT and SFN concurrently showed substantial production of ROS and increased apoptotic signals. Consequently, because QT + SFN inhibit the production of ERK/MAPK/JNK/p38-based control of proliferation and cell cycle-regulating proteins, it has been considered a chemotherapeutic medication. To determine the extent to which the co-treatment induces apoptosis, more in vivo study will be required before they can be used commercially.

Chronic obstructive pulmonary disease (COPD) stands as a major contributor to mortality worldwide, with cigarette smoke being a primary causative factor. Acacetin has been reported to possess lung protective effects. However, the precise role and mechanism of Acacetin in COPD remains elusive. In this study, human bronchial epithelial cell line HBE135-E6E7 was treated with Acacetin under cigarette smoke extract (CSE) conditions. Cellular viability was assessed using CCK-8 and LDH kits. Reactive oxygen species (ROS) generation was tested with DCFH-DA staining. JC-1 staining was employed to examine the mitochondrial membrane potential (MMP). Additionally, hydroxynonenal (4-HNE) level was tested using immunofluorescence staining and mitochondrial lipid peroxidation was evaluated using MitoPeDPP staining. MitoSOX staining was used to detect mitochondrial (mito)-ROS. Fe²⁺ level was measured using FerroOrange staining and the expression of ferroptosis-related proteins was detected with western blot. Besides, the binding between Acacetin and NRF2 was analyzed by molecular docking. The sequent NRF2 overexpression or knockdown was used to explore the regulation of Acacetin on NRF2/SLC7A11/GPX4 signaling. Results indicated that CSE significantly reduced the viability, augmented ROS generation and decreased MMP in HBE135-E6E7 cells, which were blocked by Acacetin addition. Moreover, Acacetin inhibited lipid peroxidation and ferroptosis in CSE-treated HBE135-E6E7 cells. Specifically, Acacetin targeted NRF2 and activated the NRF2/SLC7A11/GPX4 signaling in CSE-induced HBE135-E6E7 cells. Furthermore, NRF2 deficiency or ML-385 treatment notably restored the influences of Acacetin on oxidative stress and ferroptosis in HBE135-E6E7 cells challenged with CSE. In conclusion, Acacetin alleviated CSE-induced injury in HBE135-E6E7 cells by activating The NRF2/SLC7A11/GPX4 signaling to inhibit ferroptosis.

O-linked N-acetylglucosamine transferase (OGT)-catalyzed O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation) is closely associated with diabetes progression. This study aims to investigate the mechanism of OGT in regulating endothelial dysfunction in gestational diabetes mellitus (GDM). Expressions of OGT, O-linked N-acetylglucosamine (O-GlcNAc), enhancer of zeste homolog 2 (EZH2), and HEK27me3 in human umbilical vein endothelial cells (HUVECs) and GDM-derived HUVECs (GDM-HUVECs) were assessed by western blot. RT-qPCR and western blot assays were used to test the OGT overexpression and EZH2 silencing levels. CCK-8, EdU, wound healing, and transwell invasion assays were used to analyze the cell proliferative, migratory, and invasive abilities. Tube formation assay was performed to evaluate angiogenesis ability of cells. Western blot assay was performed to estimate vascular endothelial growth factor (VEGF) and p-VEGFR2 levels in cells. The binding of O-GlcNAc and EZH2 after OGT overexpression was measured by Co-IP assay. The results showed that OGT, O-GlcNAc, EZH2, and HEK27me3 expressions were declined in GDM-HUVECs. OGT overexpression induced the proliferation, migration, and invasion of GDM-HUVECs, and also elevated angiogenesis and the expressions of VEGF and p-VEGFR2 in cells. O-GlcNAc, EZH2, and HEK27me3 expressions were upregulated after OGT overexpression. OGT upregulation induced the binding between O-GlcNAc and EZH2. EZH2 silencing attenuated the promotion of OGT overexpression on the proliferative, invasive, migratory, and angiogenic capacities of GDM-HUVECs. To be concluded, OGT overexpression stabilized EZH2 expression by promoting O-GlcNAcylation modification of EZH2, and further enhanced proliferation, migration, and invasion as well as angiogenesis of GDM-HUVECs. While these effects were decayed after EZH2 absenting. Overall, the modulation of OGT on endothelial dysfunction in GDM provides a novel perspective for the clinical treatment of GDM.

Cholinergic deficiency and neuroinflammation are the two main factors of Alzheimer's disease. Recent studies have shown that water-soluble ginseng oligosaccharides (WGOS) derived from Panax ginseng roots can protect against scopolamine-induced impairments in learning and memory. However, the fundamental mechanisms remain unclear for the most part. The purpose of this study was to examine the effect of WGOS on cholinergic function and protein levels of proinflammatory cytokines in the hippocampus of mice. Mice were first pretreated with WGOS or saline, and then treated with scopolamine to establish an Alzheimer's disease model. The cognition memory of the mice was assessed through the behavioral test. The effect of WGOS on the cholinergic system was evaluated by measuring acetylcholine (ACh) neurotransmitter concentration and acetylcholinesterase (AChE) activity in the hippocampus. Using ELISA, the inflammatory cytokines IL-1β and TNF-α in the hippocampus were identified. This study found that WGOS treatment prevented the scopolamine-induced impairment of mice's recognition memory, as seen by their enhanced object recognition. In addition, WGOS prevented the scopolamine-induced decrease in ACh concentration and increase in AChE activity. Moreover, WGOS treatment inhibited scopolamine-induced upregulation of the inflammatory proteins IL-1β and TNF-α. These findings suggest that the amelioration of scopolamine-induced cognitive impairment in mice by WGOS was a consequence of the control of cholinergic function and inflammatory response in the hippocampus. Our findings suggest that WGOS should be investigated as a dietary supplement or medication for the treatment of learning and memory disorders in humans.

Silymarin, a flavonoid complex isolated from Silybum marianum, possesses various biological properties, including antioxidant, anti-inflammatory, anti-glycation, and hepatoprotective effects. In the present study, we investigated the effects of silymarin on the SPC212 human mesothelioma cell line. MTT and neutral red assays were performed to examine the cytotoxic effects of silymarin. The apoptotic effect was investigated using AO/EB and DAPI staining, and morphological changes were observed using H&E and May-Grünwald staining. Additionally, immunocytochemistry was performed to detect Bax, Bcl2, and PCNA. Our results indicated that silymarin has a dose-dependent cytotoxic effect on SPC212 cells, with an IC₅₀ value of approximately 187.5 µM. Silymarin induces apoptotic hallmarks such as apoptotic bodies, cell shrinkage, and nuclear condensation. In conclusion, silymarin demonstrated cytotoxic and apoptotic effects as well as morphological changes in SPC212 human mesothelioma cells. Further detailed studies are warranted to explore the potential of silymarin as an anti-cancer agent.

d-limonene is a type of colorless liquid hydrocarbon that falls under the category of cyclic monoterpene. It is the component found in the oil extracted from fruit peels. Isoproterenol, a synthetic β-adrenergic agonist, was administered to rats to induce myocardial injury by increasing heart rate and myocardial oxygen demand, leading to ischemia and oxidative stress. This study aims to investigate the properties of d limonene, against myocardial infarction induced by isoprenaline (ISO) in rats. Male Sprague Dawley rats were treated with d-limonene (200 & 400 mg/kg, p.o) daily for 28 days and administered ISO (85 mg/kg, s.c) on the 29th and 30th days at an interval of 24 hr to induce myocardial injury. Morphological and antioxidant parameters, biochemical markers, lipid profile, troponin-I, cardiac ATPase, heart mitochondrial, and lysosomal enzymes were assayed followed by histopathological screening. Rats treated with isoproterenol (85 mg/kg, s.c), administered twice at an interval of 24 h on 29th and 30th day showed a significant change in morphological and antioxidant parameters, biochemical markers, lipid profile, troponin-I, cardiac ATPase, heart mitochondrial, lysosomal enzymes activities and transcription factor (TNF-α/IL-6/NF-kB) expression. Pretreatment with d-limonene (200 and 400 mg/kg, p.o) for 28 days followed by ISO administration on 29^th and 30^th day significantly reversed the effects of isoproterenol-induced ischemic changes. Moreover, the biochemical results were validated by histopathological findings. The research indicates that d-limonene demonstrates cardioprotective potential against isoproterenol-induced myocardial infarction. This is attributed to its antioxidant properties, stabilization of myocardial membranes, improved scavenging of free radicals, and inhibition of membrane lipid peroxidation.

Acute lung injury (ALI), a severe pulmonary disorder that poses a significant threat to life, is closely associated with macrophage ferroptosis and polarization. Lipocalin 2 (LCN2) has been previously reported to be implicated in the pathogenesis of ALI. However, the specific role of LCN2 in macrophage ferroptosis and polarization remains undetermined. Lipopolysaccharide (LPS) was used to establish a mouse model of ALI and also to stimulate mouse RAW264.7 cells. H&E staining was used for histopathologic evaluation, and immunohistochemistry analysis was used to determine the 4-HNE-positive cells. The secretion levels of TNF-α, IL-6, and IL-1β were assessed by ELISA. Gene and protein expression assays were performed using quantitative PCR and immunoblotting. The levels of MDA, GSH, ROS, and lipid ROS were detected to evaluate the alteration in ferroptosis. CD86⁺ and CD206⁺ cells were quantified by flow cytometry. The relationship between LCN2 and interferon regulatory factor 7 (IRF7) was confirmed by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. LCN2 expression was upregulated in the lungs of LPS-induced ALI mice and LPS-stimulated RAW264.7 cells. In LPS-induced ALI mice, the depletion of LCN2 alleviated lung injury and ferroptosis, and also inhibited inflammation and macrophage M1 polarization. In LPS-stimulated RAW264.7 cells, the depletion of LCN2 suppressed ferroptosis, inflammation, and M1 polarization. Mechanistically, IRF7 enhanced LCN2 transcription in RAW264.7 cells by binding to its promoter region. More importantly, the silencing of IRF7 inhibited ferroptosis and M1 polarization in LPS-stimulated RAW264.7 cells by downregulating LCN2. Taken together, the IRF7/LCN2 cascade enhances the ferroptosis and M1 polarization of LPS-stimulated macrophages, thereby exacerbating ALI. Anti-IRF7 and anti-LCN2 therapies might potentially be exploited for the prevention and treatment in ALI.

Sudachitin, which is a polymethoxy flavonoid derived from the peer of Citrus sudachi, has several biological properties. However, the effect of sudachitin on human dental pulp cells (HDPCs) remains unclear. The aim of this study was to investigate whether sudachitin could decrease the expression of inflammatory mediators such as cytokines and prostaglandin in HDPCs stimulated with Pam3CSK4, a ligand for toll-like receptor (TLR) 2. HDPCs were pre-incubated with different concentrations of sudachitin (6.25, 12.5, 25, or 50 μM) and stimulated with Pam3CSK4 (100 ng/mL). The quantification of inflammatory cytokines (interleukin (IL)-6, IL-8, and C-X-C motif chemokine ligand (CXCL) 10) and prostaglandin E₂ (PGE₂) were performed by enzyme-linked immunosorbent assay (ELISA). The expression of cyclooxygenase (COX)-2, a key enzyme for PGE₂ formation, was analyzed by western blot. Moreover, the activations of cell signal pathways were examined by western blot analysis. Sudachitin suppressed IL-6, IL-8, CXCL10, and PGE₂ production and COX-2 protein expression in Pam3CSK4-stimulated HDPCs. In addition, we revealed that nuclear factor-kappa B (NF-κB) and protein kinase B (Akt) pathways in the Pam3CSK4-stimulated HDPCs were inhibited by sudachitin treatment. These findings suggest that sudachitin can reduce inflammatory mediator production in HDPCs stimulated with TLR2 ligand by inhibiting NF-κB and Akt activations.

Cell-extracellular matrix (ECM) interactions play multiple roles in developmental, physiological, and pathological processes. ECM stiffness substantially affects cellular morphology, migration, and function. In this study, we investigated the effect of ECM comprising gelatin methacryloyl (GelMA) on the activation of rat basophilic leukemia (RBL-2H3) cells, a model mast cell line. Maintenance of intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation and subsequent degranulation, evoked by crosslinking the high-affinity IgE receptors (FcεRI), were significantly suppressed in RBL-2H3 cells on collagen-coated GelMA hydrogel than those on collagen-coated glass dishes and plastic wells. Thapsigargin and phorbol myristate acetate caused sustained [Ca²⁺]_i increase and degranulation to a similar extent in cells on both GelMA hydrogel and plastic wells/glass dishes. F-actin was clearly accumulated along the periphery of RBL-2H3 cells in plane attached to glass, but not GelMA hydrogel, suggesting that the loose actin cytoskeleton of RBL-2H3 cells on GelMA hydrogel caused suppressive degranulation through unstable FcεRI aggregation.