Biochemistry and Cell Biology最新文献

Intramuscular adipose tissue is associated with an increased risk for the development of metabolic syndrome. A cellular model of adipogenesis in muscular tissues would be an invaluable tool for studying regulatory factors in this important process. Cellular stress can impact the homeostasis of various metabolic pathways, including lipid metabolism. In this study, a porcine intramuscular preadipocyte cell line was established, which displayed mature adipocyte attributes such as lipid accumulation and increased expression of adipogenic gene markers. Since it is well established that endoplasmic reticulum (ER) and Golgi stress impact adipogenesis, we sought to investigate the effects of ER/Golgi stress and an associated protein, CREB3, in this cell line model. We found that this novel model maintains robust adipogenic capabilities, and that ER stress can negatively affect adipogenic markers. Overall, these findings demonstrate the strength of the new cell model for studying adipogenesis, and highlight the impact of ER stress on lipid metabolism.

Our aim is to explore the protective effect of ophiopogonin D (OPD) on cerebral ischemia/reperfusion injury (CIRI) and its relevant molecular mechanisms. OPD effect on brain injury of CIRI rats was evaluated using 2,3,5-triphenyltetrazolium chloride staining, neurological deficit score, brain water content, hematoxylin and eosin staining, Nissl staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining. Immunofluorescence, immunohistochemistry, qRT-PCR, Western blot, and relative kits were used for detecting inflammatory factors, oxidative stress related factors, and mRNA and protein levels. The Cell Counting Kit-8 assay and flow cytometry were applied for assessing the viability and apoptosis of oxygen-glucose deprivation)/reoxygenation (OGD/R)-induced PC12 cells. OPD ameliorated brain injury via inhibiting neurons apoptosis, oxidative stress, and inflammatory response in cerebral infarction rats. In addition, we found that OPD attenuated the apoptosis, oxidative stress and inflammatory response in OGD/R-induced PC12 cells. Both in CIRI rats and OGD/R-induced PC12 cells, OPD was demonstrated to inhibit signal transducer and activator of transcription 3 (STAT3) phosphorylation. Moreover, Colivelin TFA (a STAT3 activator) reversed OPD effect on the apoptosis, oxidative stress, and inflammatory response in OGD/R-induced PC12 cells. Our findings demonstrated that OPD could protect against CIRI in rats by inhibiting STAT3 phosphorylation.

Background: In eukaryotic cells, phospholipid asymmetry is actively maintained, with phosphatidylserine (PS) typically confined to the inner leaflet of the plasma membrane (PM), due to the active performance of the PS flippase ATP11/CDC50A complex. However, in the tumor microenvironment (TME), PS exposure on the outer leaflet occurs from multiple sources, including apoptotic tumor cells, necrotic tissue, viable endothelial cells, and tumor-derived exosomes. Especially, in apoptotic cells, the PS scamblase Xkr8 mediates PS externalization. This exposed PS plays a crucial role in immune suppression within the TME. PS binds to receptors on phagocytes, primarily macrophages and dendritic cells (DCs), triggering efferocytosis (the engulfment of PS-positive cells, usually apoptotic cells) and promoting anti-inflammatory responses.

Method: To understand the immune suppressive role of PS exposure on tumor cells, we deleted CDC50A, the PS flippase, from the tumor. Thus, in this tumor, PS is constantly on the outer leaflet of PM, called PS^out tumor model. On the contrary, we knocked out Xkr8 from tumor cells. Thus, even tumor cells undergo apoptosis, PS can still stay in the inner leaflet of PM, called PSⁱⁿ tumor model. Taking the advantage of the PSⁱⁿ and PS^out model, we could investigate the anti-tumor immune responses of PS externalization in TME.

Results: Using PS^out model, we found that these PS^out tumors exhibited enhanced growth, M2-polarized tumor-associated macrophages (TAMs), and reduced tumor-antigen-specific T cell infiltration. In TME, the PS receptor TIM-3 on TAMs was responsible for PS sensing. Using PSⁱⁿ model, we found that the PSⁱⁿ tumors exhibited increased anti-tumor immunity, featuring suppressed tumor progress, TAM M1 polarization, suppressed IL-10 secretion, and enhanced natural killer (NK) cell cytotoxicity. Thus, blocking PS externalization via targeting Xkr8 could serve as a promising strategy for anti-tumor immunotherapy.

Therapeutic applications: However, there is no available Xkr8 inhibitor or direct anti-PS blocking antibody for therapeutic use. Thus, we developed our unique "PS all-block" strategy leveraging an engineered protein which binds only to PS, without sending signals to immune receptors, functioning as a dominant negative. The "PS all-block" can neutralize PS molecules from all sources without triggering downstream immune suppression pathways. Our data suggested that the "PS all-block" was a more effective antitumor immunotherapy compared to our successfully developed Xkr8 inhibition, as Xkr8 targeting only neutralized apoptotic PS, while the "PS all-block" approach could neutralize PS from all sources.

Cellular aging, driven by oxidative stress, mitochondrial dysfunction, and inflammation, is exacerbated by a high-glucose and high-lipid (HGHL) diet, leading to collagen degradation and skin aging. Psilocybin, a naturally occurring compound, has shown potential in reducing symptoms of aging. This study explores the protective effects of psilocybin on BJ-5ta fibroblasts exposed to HGHL, focusing on cellular viability, apoptosis, senescence, the inflammatory responses, and wound healing. First, fibroblasts were exposed to 25 mmol/L glucose and 400 µmol/L palmitic acid to establish cell aging. Then, psilocybin effects were tested in co- and post-treatment with HGHL. Post-treatment with psilocybin at 15 µmol/L (P15) and co-treatment with psilocybin at 10 µmol/L (P10) preserved cellular viability and decreased beta-galactosidase activity. P10 was most effective in reducing apoptosis and alleviating HGHL-induced S phase arrest. P15 also reduced senescence markers and decreased the expression of inflammatory cytokines IL-1β, IL-6, and COX-2. Additionally, psilocybin promoted nonsignificant fibroblast migration, and P10 co-treated with HGHL significantly upregulated elastin gene expression. These findings suggest that psilocybin's antioxidative, anti-inflammatory, and regenerative properties make it a promising natural compound for reducing skin aging, particularly under oxidative stress conditions. Further research is needed to explore its long-term effects, optimal dosages, and clinical applications.

Determination of IC₅₀ values at a fixed substrate concentration ([S]) is frequently used to rank the potency of enzyme inhibitors and estimate inhibitor concentrations ([I]) to use in full inhibition analyses, particularly for structure-activity studies wherein the mode of inhibition is often known. Assays at an [S] yielding the greatest difference between the initial rates observed in the absence (v_o) and in the presence (v_i) of an inhibitor (i.e., v_o - v_i) will increase the sensitivity for the detection of enzyme inhibition. For noncompetitive and uncompetitive inhibitors of single-substrate enzymes, v_o - v_i increases with increasing [S]; however, for competitive and linear mixed-type (LMT) inhibitors, v_o - v_i obtains a maximum at a specific "optimal" substrate concentration ([S]_opt). Equations are derived describing the dependence of [S]_opt on [I], the dissociation constant for the inhibitor (K_i), and the Michaelis constant for the substrate (K_m). For example, for competitive inhibition, [S]_opt = K_m $\sqrt{1+\left(\left[I\right]/K_i\right)}$ . For [I]/K_i values typically employed for inhibition studies (e.g., 0.5 ≤ [I]/K_i ≤ 4), [S] ≈ 2K_m or 3K_m will generally maximize the v_o - v_i difference for competitive or LMT (α ≥ 7) inhibitors, respectively. For competitive inhibition of bireactant enzymes, the "optimal" substrate concentrations depend on the Michaelis constants for both substrates, [I]/K_i, and the concentration of the second substrate.

Ferroptosis, a recently discovered form of cell death, plays an important role in cancer progression. It has been reported that ferroptosis plays a complex regulatory role in the biological processes of glioma. In glioma, the long noncoding RNA, HOXD cluster antisense RNA 1 (HOXD-AS1), functions as an oncogene, contributing to glioma progression. However, its potential functions in ferroptosis are unclear. Herein, we aimed to clarify the biological function and detailed molecular mechanism of HOXD-AS1 in regulating ferroptosis in glioma. Firstly, cell viability, reactive oxygen species (ROS), and malondialdehyde (MDA) content assays were detected. The mechanisms of HOXD1-AS1's effect on ferroptosis were evaluated by detecting glutathione, Cys, and solute carrier family 7 member 11 (SLC7A11) levels. RNA immunoprecipitation and RNA pull-down techniques were employed to explore whether HOXD-AS1 can directly bind AMP-activated protein kinase (AMPK). Our findings indicated that HOXD-AS1 levels were augmented significantly in glioma tissue. HOXD-AS1 knockdown induced MDA and ROS accumulation, subsequently resulting in ferroptosis. Further molecular analysis showed that the binding between HOXD-AS1 and AMPK regulated the mechanistic target of rapamycin kinase pathway, inhibit the transport of Cys, and decrease the production of glutathione, eventually resulting in ferroptosis. Our study revealed that HOXD-AS1 regulates cell ferroptosis, thus its downregulation might be an effective strategy to suppress glioma.

Doxorubicin is a chemotherapeutic drug for cancer that intercalates into nucleosome-free regions at promoters. Doxorubicin was reported to result in loss of histone H3 trimethylated lysine 4 (H3K4me3). To further explore doxorubicin's mechanism of action, we determined the genomic location of the binding sites of doxorubicin in leukemic cells. The effect of doxorubicin intercalation into the chromatin of leukemic cells on histone modifications was also determined. We show that doxorubicin binding sites were present in the nucleosome-free regions associated with regulatory regions (promoters, enhancers, and super-enhancers) and in the gene body (introns). Doxorubicin treatment did not alter the levels of H3K4me3 and many other histone modifications but significantly lowered H2B ubiquitinated at lysine 120 (H2BK120ub), an elongation-dependent modification. Lastly, we demonstrate that doxorubicin results in the degradation of the largest subunit (RPB1) of RNA polymerase II.

G protein-coupled receptor (GPCR) signaling regulates a wide range of pathophysiological cell functions via G protein α and βγ subunits. Small molecules targeting the subunits of Gα and Gβγ have been developed as cancer therapeutics. We have previously reported that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC) HuH7 cells through the activation of AKT, p38 mitogen-activated protein kinase (MAPK), Rho-kinase, and c-Jun N-terminal kinase (JNK). This study aims to determine whether Gβγ subunits regulate the TGF-α-induced migration of HCC HuH7 cells using gallein, a Gβγ subunits inhibitor. The Janus family of tyrosine kinase/signal transducer and activator of transcription 3 (STAT3) signaling pathway was also involved in the regulation of the migration. Gallein significantly reduced the TGF-α-induced cell migration. In contrast, fluorescein, a gallein-related compound that has no effect on Gβγ subunits, failed to affect the cell migration. Gallein suppressed the TGF-α-stimulated phosphorylation of JNK without affecting the phosphorylation of epidermal growth factor receptor, AKT, p38 MAPK, target protein of Rho-kinase, and STAT3. Conversely, fluorescein did not attenuate the phosphorylation of JNK. These results strongly suggest that Gβγ subunits act as positive regulators in TGF-α-induced migration of HCC cells via the JNK signaling pathway.