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Ischemia/reperfusion injury (IRI), the most common cause of acute kidney injury (AKI), is correlated with oxidative stress and subsequent inflammation. Taraxasterol, a natural product, has been shown to exert anti-oxidative and anti-inflammatory effects. However, the role of taraxasterol in renal IRI remains unknown. In this study, mice were subjected to 30 min of bilateral renal ischemia-reperfusion to induce AKI. Cellular hypoxia/reoxygenation (H/R) was used to mimic IRI in vitro. Western blotting, immunochemistry, immunofluorescence, TUNEL staining, ELISA, and flow cytometry were performed to evaluate kidney damage, oxidative stress, inflammation, and apoptosis in vivo and in vitro. Treatment with taraxasterol attenuated the following in a dose-dependent manner: tubular damage; infiltration of F4/80-positive macrophages; renal interstitial fibrosis; myeloperoxidase (MPO) activity; and expression of the inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1). Moreover, taraxasterol treatment remarkably ameliorated apoptosis in the kidney by decreasing Bax expression and conserving Bcl2. Notably, MitoSOX assay revealed that treatment with taraxasterol suppressed the production of mitochondrial reactive oxygen species. Furthermore, taraxasterol suppressed phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) signaling pathways in vivo and in vitro. In conclusion, these findings indicate that taraxasterol has a protective effect on IRI-induced AKI via inhibition of oxidative stress, inflammation, and apoptosis., (Copyright © 2020. Published by Elsevier B.V.)

Aims: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that regulates tissue and cell homeostasis and has been shown to antagonize heart fibrosis, which indicates a potential protective effect of CREG against cardiomyocyte chronic damage. However, little is known about the role of CREG in myocardial tissue acute injury, in this study, we aimed to investigate the role of CREG in myocardial ischemia/reperfusion (MI/R) injury and clarify the mechanism of action., Methods and Results: Wild-type Creg (Creg +/+ ), heterozygous Creg (Creg +/- ) mice and mice pretreated with infusion of recombinant 0.3mg/kg·d CREG protein (reCreg +/+ ) were subjected to 30min of left ascending coronary ischemia and 24h of reperfusion. Evan's Blue-triphenyl- tetrazolium chloride (TTC) solution and echocardiography analysis were used to evaluate the effects of CREG on MI/R mice. The underlying mechanisms were further determined by cultured myocardial cells in vitro. Our findings revealed that the level of CREG protein in mouse hearts was significantly decreased after mice were subjected to MI/R. Moreover, Creg +/- mice had larger infarction size 2h after reperfusion and worse cardiac function 28days after MI/R injury compared to that in Creg +/+ mice. However, reCreg +/+ mice could maintain CREG at a high level even after MI/R injury, and mitigated infarction size and improved cardiac function significantly. In Creg +/- mice, myocardial autophagy was dysfunctional characterized by accumulation of LC3A and p62, while apoptotic cell number increase was detected by cleaved caspase-3 blotting and TUNEL staining. Conversely, decreased apoptosis and activated autophagy were detected in reCreg +/+ mice. Furthermore, chloroquine, a kind of autophagy blocker, was used to demonstrate recombinant CREG protected cardiomyocytes against apoptosis mediated by activating autophagy both in vivo and in vitro. Finally, we found CREG was involved into lysosomal protein transfer and improve cellular autophagy., Conclusion: CREG protects heart against MI/R injury-induced cardiomyocytes apoptosis by activating lysosomal autophagy. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren and Megan Yingmei Zhang., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To explore the roles of mitochondrial Uncoupling Protein 2 (UCP2) in cumulus cells (CCs), human CCs were cultured in vitro, and the UCP2 was inhibited by treatment with Genipin, a special UCP inhibitor, or by RNA interference targeting UCP2. No significant differences in adenosine triphosphate levels and the ratio of ADP/ATP were observed after UCP2 inhibition. UCP2 inhibition caused a significant increase in cellular oxidative damage, which was reflected in alterations to several key parameters, including reactive oxygen species (ROS) and lipid peroxidation levels and the ratio of reduced GSH to GSSG. UCP2 blocking resulted in an obvious increase in active Caspase-3, accompanied by the decline of proactive Caspase-3 and a significant increase in the LC3-II/LC3-I ratio, suggesting that UCP2 inhibition triggered cellular apoptosis and autophagy. The mRNA and protein expression of connexin 43 (Cx43), a gap junction channel protein, were significantly reduced after treatment with Genipin or siRNA. The progesterone level in the culture medium was also significantly decreased after UCP2 inhibition. Our data indicated that UCP2 plays highly important roles in mediating ROS production and regulating apoptosis and autophagy, as well as maintaining gap junction integrity and progesterone synthesis, which suggests that UCP2 is involved in the regulation of follicle development and early embryo implantation and implies that it might serve as a potential biomarker for oocyte quality and competency., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Endothelium dysfunction plays a critical role in atherosclerosis. MicroRNAs are endogenous non-coding RNAs that suppress gene expression by binding to the 3' untranslated regions of target genes. MiR-495 can regulate the proliferation and apoptosis of cancer cells, however, the roles of miR-495 in endothelial cells (ECs) remain unclear. Therefore, this study aims to investigate the roles and mechanisms of miR-495 on ECs proliferation and apoptosis., Materials and Methods: MiR-495 and CCL2 expressions were examined using quantitative RT-PCR, ELISA assay and western blot. Bioinformatics analysis and luciferase reporter assay were used to examine the regulatory relationship between miR-495 and CCL2. CCK8 assay, BrdU incorporation assay and flow cytometry were used to analyze the roles of miR-495 and CCL2 on the proliferation of human umbilical vein endothelial cells (HUVECs). The effects of miR-495 and CCL2 on HUVECs apoptosis were examined by tunnel staining and western blot., Results: MiR-495 was down-regulated in patients with coronary artery disease compared with healthy controls. CCL2 was a novel target gene of miR-495. MiR-495 significantly promoted HUVECs proliferation by altering cell cycle distribution, and it also inhibited HUVECs apoptosis by affecting the expression of cleaved caspase 3. Effects of miR-495 on HUVECs proliferation and apoptosis were significantly reversed by overexpression of CCL2., Conclusions: MiR-495 could affect HUVECs proliferation and apoptosis by directly targeting CCL2. This is the first report to disclose the roles and mechanisms of miR-495 on HUVECs proliferation and apoptosis, which may provide a theoretical basis for clarifying the mechanisms of atherosclerosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The hepatitis B virus X protein (HBx) is the most important determinant in viral pathogenesis. HBx regulates HBV replication, cellular transcription, signal transduction, proteasome activity and cell cycle progression. In this study, HK-2 cells were transiently transfected with the HBx gene using a eukaryotic vector, pcDNA3.1/myc-HBx. Transfection with the HBx gene increased the number of apoptotic cells. In addition, cultured HK-2 cells became irregular in shape. The expression of α-SMA and E-cadherin, TLR4, MHC-II and CD40 was increased. The transfection resulted in increased IL-1, IL-6, TNF-α and IFN-γ levels in the supernatant and decreased IL-4 in the supernatant. In conclusion, overexpression of the HBx gene in renal tubular epithelial cells induces apoptosis of HK-2 cells and promotes epithelial-mesenchymal transdifferentiation. HBx transfection upregulates the expression of immune molecules in renal tubular epithelial cells and induces an imbalance in cytokine levels.

Objective: To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with HBx gene and elucidate its role in cell apoptosis., Methods: The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells and shRNA technique employed for silencing Notch1. HK-2 cells were divided into 7 groups of normal culture, pcDNA3.1/myc, HBx, HBx + pcDNA3.1/myc, HBx + Notch1 gene, HBx + shRNA and HBx + Notch1 shRNA. Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of HBx and detect the expression of Notch1 receptor. Methyl thiazolyl tetrazolium (MTT) assay was used to observe the proliferation rate of HK-2 cells and flow cytometry to detect the apoptosis of HK-2 cells., Results: HBx and Notch1 receptor were successfully expressed in HK-2 cells post-transfection. The transfection efficiency of shRNA was 70%. The expression of Notch1 receptor in pcDNA3.1/myc-HBx group was higher than that in the normal culture and pcDNA3.1/myc groups. And the apoptotic ratio was also higher than that of normal culture and pcDNA3.1/myc group. The difference was significant (14.94% ± 0.32% vs 13.86% ± 0.18%, 13.67% ± 0.54%, both P < 0.05). The apoptotic ratio in the HBx + Notch1 gene group was lower than that in control group (10.10% ± 0.26% vs 14.79% ± 0.02%, P < 0.05). And the growth inhibition ratio of cells was also lower. But the apoptotic ratio and growth inhibition ratio were both higher in HBx + Notch1 shRNA group than those in HBx + shRNA group (both P < 0.05)., Conclusions: HBx gene is successfully transfected into HK-2 cells. And its over-expression may induce apoptosis of HK-2 cells and growth inhibition by regulating Notch1 signal.

In recent years, immunotherapy has emerged as a promising strategy for treating solid tumors, although its efficacy remains limited to a subset of patients. Transforming non-responsive "cold" tumor types into immuno-responsive "hot" ones is critical to enhance the efficacy of immune-based cancer treatments. Pyroptosis, a programmed cell death mechanism, not only effectively eliminates tumor cells but also triggers a potent inflammatory response to initiate anti-tumor immune activities. This sheds light on the potential of pyroptosis to sensitize tumors to immune therapy. Hence, it is urgent to explore and develop novel treatments (e.g., nanomedicines) which are capable of inducing pyroptosis. In this study, we constructed tumor-targeting nanoparticles (CS-HAP@ATO NPs) by loading atorvastatin (ATO) onto chondroitin sulfate (CS) modified hydroxyapatite (HAP) nanoparticles (CS-HAP). CS was strategically employed to target tumor cells, while HAP exhibited the capacity to release calcium ions (Ca2+) in response to the tumor microenvironment. Moreover, ATO disrupted the mitochondrial function, leading to intracellular energy depletion and consequential changes in mitochondrial membrane permeability, followed by the influx of Ca2+ into the cytoplasm and mitochondria. CS and HAP synergetically augmented mitochondrial calcium overload, inciting the production of substantial amount of reactive oxygen species (ROS) and the subsequent liberation of oxidized mitochondrial DNA (OX-mitoDNA). This intricate activation process promoted the assembly of inflammasomes, most notably the NLRP3 inflammasome, followed by triggering caspase-1 activation. The activated caspase-1 was able to induce gasderminD (GSDMD) protein cleavage and present the GSDM-N domain, which interacted with phospholipids in the cell membrane. Then, the cell membrane permeability was raised, cellular swelling was observed, and abundant cell contents and inflammatory mediators were released. Ultimately, this orchestrated sequence of events served to enhance the anti-tumor immunoresponse within the organism. [ABSTRACT FROM AUTHOR]

Chemotherapy is a crucial adjuvant method of treating breast cancer, but it fails to achieve preferable prognosis as the malignant cells eventually develop drug-resistance. Reduced chemotherapeutic sensitivity is primarily caused by endogenous and exogenous resistance during the treatment. Cuproptosis is a newly discovered immunogenic cell death (ICD), characterized by accumulation of copper (Cu) ions inside tumor cells. However, intracellular Cu ion concentrations can hardly reach desired levels to induce cuproptosis due to limited import via existing Cu ionophores and synchronous export via ATP-Cu pump. Herein, our research developed an elesclomol (ES) loaded cuprous oxide (Cu 2 O) nanocomposite, called PEG@Cu 2 O-ES, to solve this dilemma. Our designed PEG@Cu 2 O-ES could efficiently enter the breast cancer cells, release encapsulated ES and Cu 2 O, while the photothermal (PTT) effect of Cu 2 O induced by near-infrared II region (NIR-II) radiation could in turn accelerate the releasing process. Cu 2 O discharged substantial amounts of Cu in cytoplasm, which directly engaged in the tricarboxylic acid (TCA) cycle in mitochondria, resulting in cuproptosis to some extent. As a result of PTT-enhanced Fenton-like reactions, Cu 2 O generated a substantial quantity of reactive oxygen species (ROS) that attacked the ATP-Cu pump on cancer cells' membranes, thereby reducing the outflow of Cu ions and aggravating cuproptosis. Additionally, free ES further promoted Cu from the tumor microenvironment (TME) to enter breast cancer cells, and exacerbated cuproptosis with higher efficiency. In vivo , PEG@Cu 2 O-ES nanocomposites showed strong anti-tumor effect by inducing cuproptosis, as well as the ability to reprogram the TME and increase the response-sensitivity to programmed cell death protein-1 antibody (αPD-1). In conclusion, this study provides a promising strategy by combining a nano-drug with αPD-1 for sensitizing immunotherapy of chemotherapy-resistant breast cancer. [Display omitted] • PEG@Cu 2 O-ES discharged substantial amounts of Cu in cytoplasm, which directly engaged in TCA cycle in mitochondria to induce cuproptosis. • PEG@Cu 2 O-ES with PTT and CDT effects could generate ROS to attack ATP-Cu pump, thereby reducing the outflow of Cu ions and aggravating cuproptosis. • Released ES from PEG@Cu 2 O-ES further promoted Cu from TME to enter breast cancer cells, and exacerbated cuproptosis with higher efficiency. • PEG@Cu 2 O-ES showed strong anti-tumor effect by inducing cuproptosis, reprogramming TME and thus increasing the response-sensitivity to αPD-1. [ABSTRACT FROM AUTHOR]

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H 2 O 2 (200 μM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 μM. Furthermore, in the H 2 O 2 -treated group, agarose gel electrophoresis displayed a “DNA ladder”. Whereas in the 10 μM crocin-pretreated group, cells remained intact and no “DNA ladder” was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 μM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H 2 O 2 , which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension). [ABSTRACT FROM AUTHOR]

Trying to develop potent and selective anticancer agents, two series of novel 1,2,4-triazolo[3,4-a]phthalazine derivatives were designed and synthesized. Their antitumor activities were evaluated by MTT method against four selected human cancer cell lines (MGC-803, EC-9706, HeLa and MCF-7). Our results showed that compound 11h exhibited good anticancer activities compared to 5-fluorouracil against the four tested cell lines, with IC 50 values ranging from 2.0 to 4.5 μM. Flow cytometry analysis indicated that compound 11h induced the cellular early apoptosis and cell cycle arrest at G2/M phase in EC-9706. [ABSTRACT FROM AUTHOR]

Abstract: KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G2 growth. We found that KYKZL-1 inhibited the growth of Hep G2 cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE2 and LTB4 production in Hep G2 cells. Accordingly, exogenous addition of PGE2 or LTB4 reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S–G2 checkpoint via the activation of p21CIP1 protein and down-regulation of cyclin A expression. These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest. Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile. [Copyright &y& Elsevier]