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Refreezing the remaining genetic resources after in vitro fertilization (IVF) can conserve genetic materials. However, the precise damage inflicted by repeated freezing and thawing on bovine sperm and its underlying mechanism remain largely unexplored. Thus, this study investigates the impact of repeated freeze-thaw cycles on sperm. Our findings indicate that such cycles significantly reduce sperm viability and motility. Furthermore, the integrity of the sperm plasma membrane and acrosome is compromised during this process, exacerbating the advanced apoptosis triggered by oxidative stress. Additionally, transmission electron microscopy exposed severe damage to the plasma membranes of both the sperm head and tail. Notably, the "9 + 2" structure of the tail was disrupted, along with a significant decrease in the level of the axonemal protein DNAH10, leading to reduced sperm motility. IVF outcomes revealed that repeated freeze-thaw cycles considerably impair sperm fertilization capability, ultimately reducing the blastocyst rate. In summary, our research demonstrates that repeated freeze-thaw cycles lead to a decline in sperm viability and motility, attributed to oxidative stress-induced apoptosis and DNAH10-related dynamic deficiency. As a result, the utility of semen is compromised after repeated freezing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Background: Liver hepatocellular carcinoma (HCC) is a prevalent cancer that lacks a sufficiently efficient approach to guide immunotherapy. Additionally, cuproptosis is a recently identified regulated cell death program that is triggered by copper ionophores. However, its possible significance in tumor immune cell infiltration is still unclear., Methods: Cuproptosis subtypes in HCC were identified using unsupervised consensus cluster analysis based on 10 cuproptosis regulators expressions, and a cuproptosis-related risk signature was generated using univariate and LASSO Cox regression and validated using the ICGC data. Moreover, the relationship between signature and tumor immune microenvironment (TME) was studied through tumor immunotherapy responsiveness, immune cell infiltration, and tumor stem cell analysis. Finally, clinical specimens were analyzed using immunohistochemistry to verify the expression of the three genes in the signature., Results: Two subtypes of cuproptosis regulation were observed in HCC, with different immune cell infiltration features. Genes expressed differentially between the two cuproptosis clusters in the TCGA were determined and used to construct a risk signature that was validated using the ICGC cohort. Greater immune and stromal cell infiltration were observed in the high-risk group and were associated with unfavorable prognosis. Elevated risk scores were linked with higher RNA stemness scores (RNAss) and tumor mutational burden (TMB), together with a greater likelihood of benefitting from immunotherapy., Conclusion: It was found that cuproptosis regulatory patterns may play important roles in the heterogeneity of immune cell infiltration. The risk signature associated with cuproptosis can assess each patient's risk score, leading to more individualized and effective immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Xiao, Zhao, Sun, Guo, Li, Zhang, Bian, Qiu and Wang.)

As a member of the long non‑coding (lnc)RNA family, lncRNA maternally expressed 8, small nucleolar RNA host gene ( MEG8 ), has been reported to serve an oncogenic role in several types of malignancies, including hepatocellular carcinoma, non‑small cell lung cancer and pancreatic cancer. The current study aimed to investigate the effect of the knockdown of MEG8 on human hemangioma endothelial cell (HemEC) proliferation, apoptosis and invasion, in addition to determining the underlying molecular mechanism. The knockdown of lncRNA MEG8 was achieved by transfecting lncRNA MEG8 small interfering (si)RNA into HemECs, while the combined knockdown of lncRNA MEG8 knockdown and microRNA (miR)‑203 was established by co‑transfecting lncRNA MEG8 siRNA and a miR‑203 inhibitor into HemECs. The cell proliferation, apoptosis and invasion and the expression levels of miR‑34a, miR‑200b , miR‑200b and Notch signaling pathway‑related factors were detected via CCK‑8 Kit, flow cytometry, Transwell, reverse transcription‑quantitative PCR and western blot assay, respectively. The knockdown of lncRNA MEG8 significantly inhibited proliferation (P<0.05) and invasion (P<0.05), but promoted apoptosis (P<0.01) in HemECs. Furthermore, lncRNA MEG8 knockdown upregulated miR‑203 (P<0.01) expression, but did not alter miR‑34a or miR‑200b expression (both P>0.05). Subsequent experiments revealed that miR‑203 silencing exerted no significant effect on the expression levels of lncRNA MEG8 (P>0.05) in HemECs. In addition, miR‑203 silencing increased cell proliferation (P<0.05) and invasion (P<0.01), but suppressed apoptosis (P<0.05). miR‑203 silencing also reversed the effect of lncRNA MEG8 knockdown on the proliferation (P<0.05), apoptosis (P<0.001) and invasion (P<0.01) of HemECs. Moreover, lncRNA MEG8 knockdown downregulated jagged canonical notch ligand 1 (JAG1; P<0.05) and Notch1 (P<0.05) expression levels, while miR‑203 silencing upregulated JAG1 (P<0.01) and Notch1 (P<0.01) expression levels and reversed the effects of lncRNA MEG8 knockdown on JAG1 (P<0.01) and Notch1 (P<0.01) expression in HemECs. In conclusion, the findings of the present study suggested that lncRNA MEG8 knockdown may inhibit cell proliferation and invasion, but promote cell apoptosis in hemangioma via miR‑203 ‑induced mediation of the Notch signaling pathway.

Non-coding RNAs participate in many cardiac pathophysiological processes, including myocardial infarction (MI). Here we showed the interplay between long non-coding RNA taurine-upregulated gene 1 (lncR-TUG1), miR-9a-5p (miR-9) and Krüppel-like factor 5 (KLF5). LncR-TUG1 was upregulated in ischemic heart and in cultured cardiomyocytes exposed to H 2 O 2 . Knockdown of lncR-TUG1 markedly ameliorated impaired cardiac function of MI mice. Further study showed that lncR-TUG1 acted as a competitive endogenous RNA of miR-9, and silencing of lncR-TUG1 inhibited cardiomyocyte apoptosis by upregulating miR-9 expression. Furthermore, the miR-9 overexpression obviously prevented ischemia injury and significantly inhibited H 2 O 2 -induced cardiomyocyte apoptosis via inhibition of mitochondrial apoptotic pathway. KLF5, as a target gene of miR-9 by dual-luciferase reporter assay, was involved in the process of miR-9 in regulating cardiomyocyte apoptosis. Our data identified the KLF5 was downregulated by miR-9 overexpression and knockdown of KLF5 inhibited cardiomyocyte apoptosis induced by H 2 O 2 . MiR-9 exerts anti-cardiomyocyte apoptotic affects by targeting KLF5. Collectively, our data identify a novel function of lncR-TUG1/miR-9/KLF5 axis in regulating cardiomyocyte apoptosis that affects myocardial infarction progression.

Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100 μM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100 μM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species (ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.

Aloe-emodin (AE) is an anthraquinone derived from rhubarb and has a variety of pharmacological actions. However, the role of AE in regulating ischemic heart diseases is still unclear. The present study investigated the effect of AE on cardiac injuries induced by myocardial infarction (MI) in vivo and oxidative insults in vitro and explored the mechanisms involved. TUNEL and Flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect expression of Bcl-2, Bax and Caspase-3 proteins. Real-time PCR was used to quantify the microRNAs levels. Our data showed that AE protected neonatal rat ventricular myocytes (NRVMs) from hydrogen peroxide (H 2 O 2 ) induced apoptosis and significantly inhibited H 2 O 2 -induced reactive oxygen species (ROS) elevation. Furthermore, AE treatment significantly reversed H 2 O 2 -induced upregulation of Bax/Bcl-2 and the loss of mitochondrial membrane potential. In vivo, AE treatment significantly reduced infarct size, ameliorated impaired cardiac function and obviously decreased cardiac apoptosis and oxidative stress in MI mice heart. Meanwhile, AE restored H 2 O 2 -induced downregulation of miR-133, and transfection with miR-133 inhibitor abolished the anti-apoptotic and anti-oxidative effects of AE. Moreover, AE prevented H 2 O 2 -induced increase in caspase-3 activity, which was diminished by application of miR-133 inhibitor. Our results indicate that AE protectes against myocardial infarction via the upregulation of miR-133, inhibition of ROS production and suppression of caspase-3 apoptotic signaling pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Smad ubiquitylation regulatory factor 1 (Smurf1) has been identified to play a critical role in bone homeostasis, development, cell cycle regulation and tumorigenesis. However, the role of Smurf1 in macrophage proliferation, apoptosis and migration is still unclear. Here, we show that Smurf1 expression was elevated in LPS-induced RAW264.7 macrophage and mouse embryonic fibroblasts (MEFs). And we found that knockdown of Smurf1 suppresses macrophage proliferation but promotes apoptosis and migration. Furthermore, JNK and p38 MAPK signaling were upregulated in Smurf1-depleted cells. And inhibition of JNK and p38 MAPK signaling in Smurf1 knockdown cells rescue the phenotypes of macrophage proliferation, apoptosis and migration. Therefore, our study suggests that Smurf1 is a new positive regulator for macrophage proliferation and apoptosis, but a negative regulator for macrophage migration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long-chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR-associated protein 9 (Cas9) system. Cas9 and single-guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS-positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X-box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR-like ER kinase (PERK), and an increase in the mRNA expression of the ER stress-regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca 2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction., (© 2017 Wiley Periodicals, Inc.)

This study aims to design and synthesize a novel harmine derivative N-(4-(hydroxycarbamoyl) benzyl)-1-(4-methoxyphenyl)-9H-pyrido [3,4-b]indole-3-carboxamide (HBC) as histone deacetylase (HDAC) inhibitor, and evaluate its antitumor activities and anti-metastasis mechanism. HBC not only exerted significant ant-proliferation activity against five human cancer cell lines, especially for HepG2 cell with an IC 50 value of 2.21 μM, which is nearly three-fold lower than SAHA (IC 50 = 6.26 µM), but also showed selective HDAC1/6 inhibitory effects in vitro. However, HBC had little effect on normal hepatic cells LO2. Furthermore, HBC simultaneously increased the acetylation of histone H3, H4, and α-tubulin, induced hypochromism by electrostatical interaction with CT-DNA, triggered more significant cancer cell apoptosis and cell cycle arrest at G2/M than SAHA by inhibition of both CDK1 and cyclin B in a concentration dependent manner. In addition, scratch and invasion assay showed that HBC also dose-dependently suppressed migration and invasion capacities of highly metastatic HCC HepG2 cells through down-regulated the expression of tumor metastasis related proteins MMP-2 and MMP-9, significantly better than SAHA. Finally, HBC showed low acute toxicity to mice and significant growth inhibition of the hepatoma tumor in vivo. These results demonstrate that novel harmine-based HDAC inhibitor HBC not only exhibited selective HDAC1/6 inhibitory activity and significant in vitro and in vivo antitumor activity, but also possessed DNA binding effect, apoptosis induction, cell cycle arrest effects, and potent anti-metastasis mechanisms, which may hold great promise as therapeutic agent targeting HDAC1/6 for the intervention of human cancers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background/aims: MicroRNAs play an important role in regulating myocardial infarction (MI)-induced cardiac injury. MicroRNA-124 (miR-124) plays a vital role in regulating cellular proliferation, differentiation and apoptosis. Although the alteration of miR-124 was confirmed in peripheral blood of MI patients, little is known regarding the biological functions of miR-124 in cardiomyocytes. This study was designed to explore the role of miR-124 in MI and its underlying mechanisms., Methods: Real-time PCR was used to quantify the microRNAs levels. TUNEL and Flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect expression of Bcl-2, Bax, Caspase-3 and STAT3 proteins., Results: We revealed that miR-124 was significantly up-regulated in a mice model of MI and in neonatal rat ventricular myocytes (NRVMs) with H2O2 treatment. H2O2 treatment induced cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death, whereas silencing expression of miR-124 by AMO-124 (antisense inhibitor oligodeoxyribonucleotides) alleviated these deleterious changes. AMO-124 decreased the expression of Bax and cleaved-caspase-3 and upregulated the expression of Bcl-2 in H2O2-treated NRVMs. Besides, AMO-124 improved mitochondrial dysfunction of NRVMs induced by H2O2 treatment. Moreover, antagomir-124 markedly decreased the infarct area and apoptotic cardiomyocytes and improved cardiac function in MI mice. Furthermore, we identified STAT3 as a direct target of miR-124, and downregulation of miR-124 ameliorated the diminished levels of STAT3 and p-STAT3 (Tyr705) in response to H2O2 or MI. STAT3 inhibitor, stattic, was shown to attenuate the elevation of p-STAT3 in NRVMs with AMO-124 transfection. Inhibiting of STAT3 activity by stattic abrogated protective effects of AMO-124 on H2O2-induced cardiomyocytes apoptosis., Conclusion: Taken together, our data demonstrate that downregulation of miR-124 inhibits MI-induced apoptosis through upregulating STAT3, which suggests the therapeutic potential of miR-124 for myocardial infarction., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

A series of bis-pyrazole derivatives were designed and synthesized, and their antitumor effects in vitro and in vivo were investigated. Several compounds displayed good antiproliferative activity with IC 50 values in low-micromolar range against three human cancer cell lines in vitro, superior to 5-FU. The most potent compound 10M selectively inhibited human hepatocellular carcinoma cells but not non-tumor liver cell proliferation in vitro, and significantly triggered SMMC-7721 cell apoptosis by cleavage of both PARP and caspase-3 in a concentration-dependent manner. Further study revealed that the potent activity in the cell growth inhibition and apoptosis induction effects of 10M were related to DNA damage and activation of the p53 signaling pathway. Moreover, 10M showed low acute toxicity to mice and significant growth inhibition of the hepatoma tumor in vivo., (Copyright © 2017. Published by Elsevier Masson SAS.)

Alzheimer's disease (AD) is characterized by the loss of neurogenesis and excessive induction of apoptosis. The induction of neurogenesis and inhibition of apoptosis may be a promising therapeutic approach to combating the disease. Celecoxib (CB), a cyclooxygenase-2 specific inhibitor, could offer neuroprotection. Specifically, the CB-encapsulated erythrocyte membranes (CB-RBCMs) sustained the release of CB over a period of 72 h in vitro and exhibited high brain biodistribution efficiency following intranasal administration, which resulted in the clearance of aggregated β-amyloid proteins (Aβ) in neurons. The high accumulation of the CB-RBCMs in neurons resulted in a decrease in the neurotoxicity of CB and an increase in the migratory activity of neurons, and alleviated cognitive decline in APP/PS1 transgenic (Tg) mice. Indeed, COX-2 metabolic products including prostaglandin E2 (PGE 2 ) and PGD 2 , PGE 2 induced neurogenesis by enhancing the expression of SOD2 and 14-3-3ζ, and PGD 2 stimulated apoptosis by increasing the expression of BIK and decreasing the expression of ARRB1. To this end, the CB-RBCMs achieved better effects on concurrently increasing neurogenesis and decreasing apoptosis than the phospholipid membrane-encapsulated CB liposomes (CB-PSPD-LPs), which are critical for the development and progression of AD. Therefore, CB-RBCMs provide a rational design to treat AD by promoting the self-repairing capacity of the brain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

2-hydroxyethyl methacrylate (HEMA) is the major resin monomer that is released from incomplete polymerized dental restorative and adhesive biomaterials during dental therapy. Autophagy and apoptosis are biologically connected and the relationship between autophagy and apoptosis is complex under various circumstances. This study aimed to determine whether autophagy is activated by HEMA and further explore the function of autophagy during the HEMA-induced apoptosis of dental mesenchymal cells (DMCs). We exposed DMCs to different concentrations of HEMA. Cell viability showed a time- and concentration-dependent decrease when exposed to HEMA. We showed that HEMA exposure increased autophagic vacuoles and the expression of autophagic biomarkers (Beclin1, Atg5 and LC3). Pre-incubated with autophagy inhibitors (3-methyladenine and chloroquine) significantly prevented HEMA-induced apoptosis. Interestingly, HEMA initiated nuclear factor-κB (NF-κB) expression and nuclear translocation, whereas the NF-κB inhibitor (Bay 11-7082) markedly suppressed HEMA-induced autophagic activation and apoptosis. As is consistent with the in vitro results, HEMA treatment resulted in dental pulp tissue toxicity and activation of typical autophagic vacuoles in the tooth slice organ culture model ex vivo. In summary, we demonstrated that NF-κB signaling functioned upstream of HEMA-inducecd autophagy in DMCs and that the activation of NF-κB-autophagy axis was responsible for HEMA-induced apoptosis. Our findings provide novel insights into the mechanisms of resin monomer-mediated dental pulp damage during dental treatment, highlighting the activation of NF-κB-autophagy axis as an important mechanism of HEMA-mediated apoptosis., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Our previous studies suggested that brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) axis inhibited cardiomyocyte apoptosis in myocardial infarction (MI). However, the relationship between BDNF and microRNA (miRNA) in cardiomyocytes are unclear. The present study was performed to investigate the role of miR-195 and the interplay between BDNF and miR-195 in ischemic cardiomyocyte apoptosis., Methods: Male Wistar rats were subjected to coronary artery ligation, and primary neonatal rat ventricular myocytes were treated with hypoxia or hydrogen peroxide (H2O2). BDNF level in rat ventricles was measured by enzyme linked immunosorbent assay (ELISA). miR-195 mimic, inhibitor or negative control was transfected into the cardiomyocytes. Cell viability and apoptosis were detected by MTT assay and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Cardiac function and apoptosis were detected in MI rats intravenously injected with antagomiR-195. Luciferase assay, Western blot and Real-time RT-PCR were employed to clarify the interplay between miR-195 and BDNF., Results: miR-195 level was dynamically regulated in response to MI and significantly increased in ischemic regions 24 h post-MI as well as in hypoxic or H2O2-treated cardiomyocytes. Meanwhile, BDNF protein level was rapidly increased in MI rats and H2O2-treated cardiomyocytes. Apoptosis in both hypoxic and H2O2-treated cardiomyocytes were markedly reduced and cell viability was increased by miR-195 inhibitor. Moreover, inhibition of miR-195 significantly improved cardiac function of MI rats. Bcl-2 but not BDNF was validated as the direct target of miR-195. Furthermore, BDNF abolished the pro-apoptotic role of miR-195, which was reversed by its scavenger TrkB-Fc., Conclusion: Up-regulation of miR-195 in ischemic cardiomyocytes promotes ischemic apoptosis by targeting Bcl-2. BDNF mitigated the pro-apoptotic effect of miR-195 in rat cardiomyocytes. These findings may provide better understanding of the pro-apoptotic role of miR-195 in MI and suggest that BDNF/miR-195/Bcl-2 axis may be beneficial for limiting myocardial ischemic injury.

Platinum agents can cause DNA damage and thus induce apoptosis of cancer cells, which has made them the backbone of cancer chemotherapy regimens. However, most cancers will develop drug resistance over the course of treatment. Meanwhile, most tumors meet energy needs largely by aerobic glycolysis (glycolysis in the presence of oxygen, called the Warburg effect), which is related to their resistance to apoptosis. Therefore, we have used a biodegradable polymer carrier to conjugate with DACH-Pt and dichloroacetate, a PDK inhibitor that can reverse the Warburg effect and derepress the resistance to apoptosis, thus sensitizing cancer cells to platinum. The as-prepared polymer-drug conjugates can be assembled into nanoparticles for effective delivery and better synergism. In vitro and in vivo studies revealed that the combination of polymer-DCA and polymer-DACH-Pt are much better than the free drugs administered simultaneously, in terms of both safety and antitumor efficacy.

Objective: To investigate the effects of calcitriol on airway remodeling and airway epithelial cell apoptosis in a murine model of bronchial asthma., Methods: Twenty-four SPF female Balb/c mice were randomly allocated into three groups according to a random digits table with 8 mice in each group: the control group, the chronic asthma group and the calcitriol intervention group.(1) The chronic asthma group: the mice were sensitized by intraperitoneal injection of Ovalbumin (OVA, 20 μg) and aluminium hydroxide (50 μl) on days 1 and 14.From day 21, the mice were challenged by inhalation of 1% OVA solution (10 ml, 30 min/time, three times a week for consecutively 8 weeks). (2) The calcitriol intervention group: the mice were sensitized and challenged as above, and were given calcitriol 100 ng through intraperitoneal injection 30 min before every inhalation.(3) The control group: the mice were sensitized and challenged by saline instead of OVA.The mice were sacrificed 24 hours after the last challenge, and the left lung were removed, fixed with paraformaldehyde, embedded with paraffin and sectioned.HE staining, Alcian blue and Periodic acid Schiff (AB-PAS) staining, Masson staining, and α-smooth muscle actin (α-SMA) immunohistochemistry staining were conducted.Bronchial basement membrane perimeter (Pbm), total bronchial wall area and positive areas of AB-PAS staining, Masson staining, and α-SMA staining were determined with image analysis software.The results were standardized with the basement membrane perimeter.Paraffin sections of mice lung tissue were detected with terminal transferase deoxyuridine triphosphate (dUTP) nick end labeling enzyme mediated method (TUNEL) for apoptosis of airway epithelial cells and with immunohistochemistry staining for expression of B cell lymphoma/lewkmia-2 (Bcl-2) in the airway epithelium., Results: The mice in the chronic asthma group and calcitriol intervention group showed characteristic airway inflammation and airway remodeling of asthma.The ratios between the total bronchial wall area and positive areas of AB-PAS staining, Masson staining and α-SMA staining and the basement membrane perimeter in the calcitriol intervention group were (14.12±2.13), (3.72±0.57), (4.31±0.65) and (3.27±0.46) μm2/μm, respectively, all of them were significantly lower than (19.24±1.70), (5.23±0.90), (7.63±1.55) and (5.40±0.69) μm2/μm in the chronic asthma group and higher than (7.79±1.01), (0.05±0.03), (1.37±0.25) and (1.40±0.24) μm2/μm in the control group (all P<0.01). The airway epithelial cell apoptosis index in the calcitriol intervention group was significantly lower than that in the chronic asthma group and higher than the control group [(14.89±1.75)% vs (29.73±5.74)% and (0.45±0.38)%, both P<0.01]. The relative expression of Bcl-2 in the calcitriol intervention group was significantly higher than that in the chronic asthma group and lower than the control group (0.114±0.009 vs 0.091±0.023 and 0.160±0.021, both P<0.05)., Conclusions: Calcitriol attenuates airway remodeling and reduces the apoptosis of airway epithelial cells in a murine model of chronic asthma.The mechanism of calcitriol in reducing apoptosis of airway epithelial cells is by regulation of expression of the important molecule Bcl-2 protein in mitochondrial apoptotic pathway.

Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, exerted cytotoxicity against prostate cancer cell lines (PC3, DU145 and LNCaP cells) with IC₅₀ values in the range of 11.17 to 26.13 μmol/L, whereas RWPE-1 cells (a non-malignant human prostate epithelial cell line) were not significantly affected. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PI staining). After being treated with PD for 48 hours, DU145 and LNCaP cells were arrested in the G0 /G1 phase, and PC3 cells were arrested in the G2/M phase. A Western blotting analysis indicated that PD increased the expression of the FOXO3a transcription factor, decreased the expression of p-FOXO3a and MDM2 and increased the expression of FOXO-responsive genes, p21 and p27. MDM2 silencing (transiently by siRNA-MDM2) increased the PD-induced FOXO3a protein expression, while MDM2 overexpression (in cells transiently transfected with a pcDNA3-MDM2 plasmid) decreased the PD-induced expression of the FOXO3a protein. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blotting analysis of the excised xenograft tumors indicated that similar changes in protein expression also occurred in vivo. These results suggest that PD exhibits significant activity against prostate cancer in vitro and in vivo. The FOXO3a transcription factor appears to be involved in the activity of PD. Together, all of these findings provide a basis for the future development of this agent for human prostate cancer therapy.

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

Objective: Protecting the heart from myocardial ischemia and reperfusion (I/R) damage is the focus of intense research. Coptisine is an isoquinoline alkaloid isolated from Coptidis Rhizoma. The present study investigated the potential effect of coptisine on myocardial I/R damage in rats and the underlying mechanisms., Methods and Results: Electrocardiogram examination showed that the administration of coptisine 10 min before ischemia significantly decreased I/R-induced arrhythmia after 30 min ischemia followed by 3 h reperfusion. The release of cardiac markers was also limited. Echocardiography was performed before ischemia and 24 h post-I/R, separately. The M-mode records showed that the reductions of ejection fraction (EF) and fractional shortening (FS) were attenuated in coptisine-treated rats compared with the I/R rats. Similar results were obtained with Evans Blue/triphenyl tetrazolium chloride (TTC) staining, in which coptisine notably reduced infarct size. Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated coptisine suppressed myocardial apoptosis, which may be related to the upregulation of Bcl-2 protein and inhibition of caspase-3 activation. Coptisine treatment also attenuated the proinflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in heart tissue. Additionally, Western blot and immunohistochemical analysis showed that coptisine markedly reduced Rho, Rho-kinase 1 (ROCK1), and ROCK2 expression and attenuated the phosphorylation of myosin phosphatase targeting subunit-1, a downstream target of ROCK., Conclusions: Coptisine exerts pronounced cardioprotection in rats subjected to myocardial I/R likely through suppressing myocardial apoptosis and inflammation by inhibiting the Rho/ROCK pathway., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Hyperactivation of mechanistic target of rapamycin (MTOR) is a common feature of human cancers, and MTOR inhibitors, such as rapamycin, are thus becoming therapeutics in targeting certain cancers. However, rapamycin has also been found to compromise the efficacy of chemotherapeutics to cells with hyperactive MTOR. Here, we show that loss of TSC2 or PTEN enhanced etoposide-induced DNA damage and apoptosis, which was blunted by suppression of MTOR with either rapamycin or RNA interference. cAMP response element-binding protein 1 (CREB1), a nuclear transcription factor that regulates genes involved in survival and death, was positively regulated by MTOR in mouse embryonic fibroblasts (MEFs) and cancer cell lines. Silencing Creb1 expression with siRNA protected MTOR-hyperactive cells from DNA damage-induced apoptosis. Furthermore, loss of TSC2 or PTEN impaired either etoposide or nutrient starvation-induced autophagy, which in turn, leads to CREB1 hyperactivation. We further elucidated an inverse correlation between autophagy activity and CREB1 activity in the kidney tumor tissue obtained from a TSC patient and the mouse livers with hepatocyte-specific knockout of PTEN. CREB1 induced DNA damage and subsequent apoptosis in response to etoposide in autophagy-defective cells. Reactivation of CREB1 or inhibition of autophagy not only improved the efficacy of rapamycin but also alleviated MTOR inhibition-mediated chemoresistance. Therefore, autophagy suppression of CREB1 may underlie the MTOR inhibition-mediated chemoresistance. We suggest that inhibition of MTOR in combination with CREB1 activation may be used in the treatment of cancer caused by an abnormal PI3K-PTEN-AKT-TSC1/2-MTOR signaling pathway. CREB1 activators should potentiate the efficacy of chemotherapeutics in treatment of these cancers.

Oxidative stress has been considered as a major cause of cell damage in various neurodegenerative disorders. One of the reasonable strategies for delaying the disease's progression is to prevent reactive oxygen species (ROS) mediated cellular injury by dietary or pharmaceutical augmentation of free radical scavengers. Isocampneoside II (ICD) is an active phenylethanoid glycoside isolated from the medicinal hardwood genus Paulownia. This study was designed to explore free radical scavenging potential of ICD in different in vitro systems and its protective role in hydrogen peroxide (H₂O₂)-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. The results showed ICD eliminated approximately 80.75% superoxide radical at the concentration of 0.1mg/ml and inhibited metal chelating by 22.07% at 8 mg/ml. Additionally, ICD showed a strong ability on reducing power and provided protection against oxidative protein damage induced by hydroxyl radicals. Pretreatment of PC12 cells with ICD prior to H₂O₂ exposure elevated cell viability, enhanced activity of superoxide dismutase and catalase, and decreased levels of malondialdehyde and intracellular ROS. Furthermore, ICD inhibited cell apoptosis and Bax/Bcl-2 ratio induced by H₂O₂. These findings suggested ICD may be considered as a potential antioxidant agent and should encourage for further research in neurodegenerative diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The thyroid transcription factor 1 (TTF-1) gene is associated with the differentiation of lung epithelial cells and has been reported to be an independent prognostic factor for lung adenocarcinoma patients. The aim of the present study was to detect the expression of TTF-1 in human lung cancer cell lines and to evaluate the association of overexpressed TTF-1 with Ki-67 and apoptosis in the A549 cell line. We also investigated the expression of TTF-1 and Ki-67 in Xuanwei lung adenocarcinoma. TTF-1 mRNA expression was evaluated in 10 non-small cell lung cancer (NSCLC) cell lines by quantitative real-time RT-PCR (qRT-PCR). Overexpression of TTF-1 in A549 cells was achieved by transient transfection. The TTF-1 and Ki-67 proteins were detected by immunohistochemistry and apoptosis was detected by flow cytometry. We also investigated immunohistochemically the expression of TTF-1 and Ki-67 in 62 resected cases of Xuanwei lung adenocarcinoma. Overall the expression of TTF-1 mRNA in the 10 cell lines was low. Overexpression of TTF-1 mRNA was found only in 3 (30%) of 10 NSCLC cell lines, including 1 (25%) of 4 adenocarcinoma cell lines. A549 cells overexpressing TTF-1 were found to have repressed expression of Ki-67 (P=0.012) and increased apoptosis (P=0.000). Immunohistochemical analysis of resected cases of Xuanwei lung adenocarcinoma (n=62) showed the expression of TTF-1 in 58 (93%) of 62 and Ki-67 in 22 (35%) of 62. Patients with strong immunohistochemical expression TTF-1 were statistically associated with well-differentiated phenotype (P=0.006) and inverse correlation with Ki-67 expression (P=0.016). These data suggest that TTF-1 may serve as a tumor suppressor gene based on its inverse correlation with Ki-67 proliferative activity and increase of cellular apoptosis.

This paper is to report the study of resveratrol-induced apoptosis and its mechanisms in MCF-7 cells. MTT assay was performed to assess the cytotoxicity of resveratrol on MCF-7 cells. Hoechst 33258 staining was used to observe cellular morphologic changes in apoptosis. Apoptosis was measured by flow cytometric analysis and the protein expression was examined by Western blotting analysis. The results indicated that resveratrol could inhibit MCF-7 cell growth in a time- and concentration-dependent manner. Remarkable morphologic changes in the cells after 60 micromol L(-1) resveratrol treatment, including cell nuclear shrinkage, DNA condensation and apoptotic bodies, were observed by Hoechst 33258 staining. Resveratrol could induce apoptosis and activate p38 and p53 in a time dependent manner in MCF-7 cells. In addition, the cell growth inhibitory ratio and the apoptotic ratio of resveratrol-treated group decreased markedly by the p38 MAPK inhibitor SB203580 or p53 inhibitor pifithrin-alpha. Further experiments confirmed that resveratrol-induced p53 activation was reduced by SB203580 whereas the activation of p38 was not affected by pifithrin-alpha. In conclusion, resveratrol induced apoptosis in MCF-7 cells could be through activating p38-p53 signal pathway.

Objective: To explore the effects of receptor interacting protein (RIP) 140 gene overexpression upon the in vitro proliferation, apoptosis, invasion and migration of microglioma cells., Methods: The BV-2 RIP140 overexpression model (BV-2-1) was constructed by Lipofection and G418 selection, then validated by real-time PCR and Western blotting. The proliferation, apoptosis, invasion and migration potencies were compared between BV-2-1 and its parents by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, flow cytometry and Transwell chamber., Results: The BV-2-1 model was successfully constructed. Compared to those of the BV-2 group, the RIP140 mRNA and protein expression levels of BV-2-1 were markedly higher than those of the BV-2 group (t = 49.794, P < 0.01). MTT assay showed that the absorbance values in the BV-2 group were 1.157 ± 0.013, 1.679 ± 0.005 and 2.609 ± 0.008 at 24, 48, and 72 hours respectively. And those were 0.929 ± 0.013, 1.188 ± 0.008 and 1.528 ± 0.012 in the BV-2-1 group respectively. The proliferation at the time points of 48 and 72 hours of the BV-2-1 group were significantly lower than that of the BV-2 group (t = 6.058 and 9.245, both P < 0.01). Annexin-V staining showed that there were significant differences in the apoptosis rates between the BV-2 and BV-2-1 cells [(5.35 ± 0.23)% vs (3.46 ± 0.45)%, t = 6.619, P = 0.003)]. Transwell assay showed that the invaded cell number of the BV-2-1 group was 166 ± 43. And it was obviously higher than that of the BV-2 group (93 ± 32, t = 3.403, P = 0.007). Transwell assay also showed that the migrated cell number of BV-2 cells was 101 ± 25. And the migration potency of the BV-2-1 group (202 ± 50) was significantly stronger than that of the BV-2 group (t = 4.104, P = 0.002)., Conclusion: RIP140 effectively inhibits the proliferation and facilitates the apoptosis of microglioma cells. And it may effectively facilitate the in vitro invasion and migration of microglioma cells.

Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer treatment. Our investigation indicates that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge, Rhabdastrella globostellata, inhibits proliferation of HL-60 cells with an IC(50) value of 0.68mug/ml, and induces apoptosis. Rhabdastrellic acid-A also induces cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and caspase-3. Pretreatment of HL-60 cells with the caspase-3 specific inhibitor, DEVD-CHO, prevents Rhabdastrellic acid-A-induced DNA fragmentation and PARP cleavage. Activated PI3K and Akt significantly decreases after treatment with Rhabdastrellic acid-A in HL-60 cells. Expression levels of protein bcl-2, bax remain unchanged in response to Rhabdastrellic acid-A treatment in HL-60 cells. These results suggest that Rhabdastrellic acid-A inhibits PI3K/Akt pathway and induces caspase-3 dependent-apoptosis in HL-60 human leukemia cells.

Background & Objective: Rhabdastrellic acid-A is an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata from South China Sea. Our previous study indicated that rhabdastrellic acid-A can inhibit the proliferation of many types of tumor cells with minor toxicity. This study was to investigate the apoptosis of human leukemia HL-60 cells induced by rhabdastrellic acid-A and its possible mechanisms., Methods: Inhibitory effect of rhabdastrellic acid-A on the proliferation of HL-60 cells was evaluated by MTT assay. DNA fragmentation was analyzed by agarose electrophoresis. Cell morphology was observed under fluorescent microscope. The protein levels of Caspase-3, poly(ADP-ribose) polymerase (PARP), P73, Bcl-2 and Bax were analyzed by Western blot. The expression profile of apoptosis-related genes was analyzed by gene microarray. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to confirm some altered genes identified by gene microarray., Results: Rhabdastrellic acid-A inhibited the proliferation of HL-60 cells and the 50% inhibition concentration (IC50) was (0.64+/-0.21) microg/ml. When treated with 1 microg/ml rhabdastrellic acid-A for 36 h, condensation of nuclear chromatin of HL-60 cells was observed under fluorescent microscope and DNA fragmentation was observed by agarose electrophoresis. Also, rhabdastrellic acid-A induced cleavage of PARP and Caspase-3. The mRNA levels of 44 genes, including p73, JunD, TNFAIP3 and GADD45A, were up-regulated and the mRNA levels of 16 genes, including MAP2K5 and IGF2R, were down-regulated. The results were further confirmed by RT-PCR. The protein level of P73 was up-regulated after rhabdastrellic acid-A treatment., Conclusion: Rhabdastrellic acid-A could induce the apoptosis of HL-60 cells which may be related to the up-regulation of apoptosis-related genes such as p73 and JunD, and the down-regulation of MAP2K5 and IGF2R.

The study was purposed to investigate the effect of phosphorylated-chk1 on cell cycle and apoptosis of human erythroleukemic cell line K562 and K562/A02, and to explore the mechanism of chk1 in regulation of drug-resistance of leukemia cells. After treatment with adrimycin for six hours, the cell cycle distribution was detected by flow cytometry; the Chk1mRNA expression was detected by RT-PCR and the Chk1 phosphorylation level was detected by Western blot. Under the condition of down-regulation of Chk1mRNA expression in cells transfected with Chk1 short hairpin RNA, the cell apoptosis rates were detected by flow-cytometry following adrimycin. The results indicated that the proportion of K562/A02 cell line in G2/M phase was (54.12 +/- 0.57)% at 6 hours after drug treatment, significantly higher than that of K562 cell line (36.99 +/- 1.28)%. No evident difference of the Chk1mRNA expression was observed between K562 and K562/A02 cell lines, while elevated Chk1 phosphorylation following DNA damage induced by adriamycin was observed in the K562/A02 cell line (0.79 +/- 0.56), significantly higher than that in K562 cell line (0.27 +/- 1.47). The cell apoptosis rate of the Chk1 shRNA group in K562/A02 cell line was 3.84-fold of blank vector group, but that in K562 cell line was 1.30-fold of blank vector group. It is concluded that the increased chk1 activity that delay the progress of cell cycle are associated with cellular resistance to adrimycin in the K562/A02 cell line.

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells. They function downstream of sensors/receptors and regulate cellular responses to external and endogenous stimuli. Recent studies demonstrated that SIPK and WIPK, two tobacco (Nicotiana spp.) MAPKs, are involved in signaling plant defense responses to various pathogens. Ntf4, another tobacco MAPK that shares 93.6% and 72.3% identity with SIPK and WIPK, respectively, was reported to be developmentally regulated and function in pollen germination. We found that Ntf4 is also expressed in leaves and suspension-cultured cells. Genomic analysis excluded the possibility that Ntf4 and SIPK are orthologs from the two parental lines of the amphidiploid common tobacco. In vitro and in vivo phosphorylation and activation assays revealed that Ntf4 shares the same upstream MAPK kinase, NtMEK2, with SIPK and WIPK. Similar to SIPK and WIPK, Ntf4 is also stress responsive and can be activated by cryptogein, a proteinaceous elicitin from oomycetic pathogen Phytophthora cryptogea. Tobacco recognition of cryptogein induces rapid hypersensitive response (HR) cell death in tobacco. Transgenic Ntf4 plants with elevated levels of Ntf4 protein showed accelerated HR cell death when treated with cryptogein. In addition, conditional overexpression of Ntf4, which results in high cellular Ntf4 activity, is sufficient to induce HR-like cell death. Based on these results, we concluded that Ntf4 is multifunctional. In addition to its role in pollen germination, Ntf4 is also a component downstream of NtMEK2 in the MAPK cascade that regulates pathogen-induced HR cell death in tobacco.

Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

Background & Objective: Proteasome inhibitor is a kind of potential anti-tumor drug,it can induce apoptosis in various tumor cells. This study was designed to investigate the molecular mechanism of apoptosis and G(2)/M arrest in leukemia cell line HL-60 induced by proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO)., Methods: Apoptosis in HL-60 cells was observed under fluorescent microscope, flow cytometry and immunoblot were used to analyze cell apoptosis, cell cycle arrest, and the mechanisms., Results: MG132 (2 micromol/L)induced apoptosis in HL-60 cells after 24-h treatment. Meanwhile, HL-60 cells were arrested at G(2)/M phase before apoptosis after induced by MG132. The percentage of G(2)/M phase in MG132-treated HL-60 cells at 12 h was 63.42+/-2.02,while that in untreated cells was 7.29+/-3.01 (P< 0.01). The percentage of apoptosis in MG132-treated HL-60 cells at 24 h was 16.67+/-1.48, while untreated cells had no death (P< 0.01). Compared to the treatment with MG132 only, caffeine (2 mmol/L) exposure can reduce G(2)/M arrest and apoptosis in MG132-treated HL-60 cells. Expression of cyclin-dependent kinase inhibitor p21waf/cip1 up-regulated after treated with MG132 for 3 h, but no p53 or p27 detected., Conclusions: Proteasome inhibitor MG132 can induce G2/M arrest before the apoptosis appeared in HL-60 cells. The obvious up-regulation of p21 indicated that it is p21(waf/cip1), but not p53 or p53-related proteins,that involved in the regulation of G(2)/M arrest and subsequent apoptosis induced by MG132 in HL-60 cells.

Objective To explore the effect of lncRNA FGD5-AS1 on oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cell damage and its regulatory effect on miR-106a-5p. Methods Ox-LDL was used to induce human umbilical vein endothelial cells(HUVECs) to establish a cellular injury model. pcDNA-FGD5-AS1, anti-miR-106a-5p and its negative control were transfected into HUVECs and then added to ox-LDL-treated cells. pcDNA-FGD5-AS1 was co-transfected with miR-NC and miR-106a-5p mimics to HUVECs, then added ox-LDL to treat cells. RT-qPCR was used to detect the expression of FGD5-AS1 and miR-106a-5p. The level of MDA, SOD, and CAT was tested with commercially available kit. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter gene experiment was used to detect the targeting relationship between FGD5-AS1 and miR-106a-5p. Western blot was used to detect the protein expression of cleaved caspase-3 and cleaved caspase-9. Results The expression of FGD5-AS1 mRNA in HUVECs induced by ox-LDL decreased (P＜0.05) while the expression of miR-106a-5p mRNA increased (P＜0.05). Both transfection of pcDNA-FGD5-AS1 and transfection of anti-miR-106a-5p reduced the level of MDA, apoptosis rate and protein level of cleaved caspase-3, cleaved caspase-9 in HUVECs induced by ox-LDL(P＜0.05),enhanced the activity of SOD and CAT (P＜0.05). FGD5-AS1 could target at miR-106a-5p. Co-transfection of pcDNA-FGD5-AS1 and miR-106a-5p mimics could reverse the effect of pcDNA-FGD5-AS1 on ox-LDL-induced HUVECs apoptosis and oxidative stress. Conclusions Over-expression of FGD5-AS1 may inhibit oxidative stress and apoptosis through targeted regulation of miR-106a-5p, thereby reducing ox-LDL-induced vascular endothelial cell damage.

Discovering new treatments for melanoma will benefit human health. The mechanism by which deoxyhypusine synthase (DHPS) promotes melanoma development remains elucidated. Multi‐omics studies have revealed that DHPS regulates m6A modification and maintains mRNA stability in melanoma cells. Mechanistically, DHPS activates the hypusination of eukaryotic translation initiation factor 5A (eIF5A) to assist METTL3 localizing on its mRNA for m6A modification, then promoting METTL3 expression. Structure‐based design, synthesis, and activity screening yielded the hit compound GL‐1 as a DHPS inhibitor. Notably, GL‐1 directly inhibits DHPS binding to eIF5A, whereas GC‐7 cannot. Based on the clarification of the mode of action of GL‐1 on DHPS, it is found that GL‐1 can promote the accumulation of intracellular Cu2+ to induce apoptosis, and antibody microarray analysis shows that GL‐1 inhibits the expression of several cytokines. GL‐1 shows promising antitumor activity with good bioavailability in a xenograft tumor model. These findings clarify the molecular mechanisms by which DHPS regulates melanoma proliferation and demonstrate the potential of GL‐1 for clinical melanoma therapy. [ABSTRACT FROM AUTHOR]

Cell death regulation is essential for tissue homeostasis and its dysregulation often underlies cancer development. Understanding the different pathways of cell death can provide novel therapeutic strategies for battling cancer. This review explores several key cell death mechanisms of apoptosis, necroptosis, autophagic cell death, ferroptosis, and pyroptosis. The research gap addressed involves a thorough analysis of how these cell death pathways can be precisely targeted for cancer therapy, considering tumor heterogeneity and adaptation. It delves into genetic and epigenetic factors and signaling cascades like the phosphatidylinositol 3‐kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) pathways, which are critical for the regulation of cell death. Additionally, the interaction of the microenvironment with tumor cells, and particularly the influence of hypoxia, nutrient deprivation, and immune cellular interactions, are explored. Emphasizing therapeutic strategies, this review highlights emerging modulators and inducers such as B cell lymphoma 2 (BCL2) homology domain 3 (BH3) mimetics, tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL), chloroquine, and innovative approaches to induce ferroptosis and pyroptosis. This review provides insights into cancer therapy's future direction, focusing on multifaceted approaches to influence cell death pathways and circumvent drug resistance. This examination of evolving strategies underlines the considerable clinical potential and the continuous necessity for in‐depth exploration within this scientific domain. [ABSTRACT FROM AUTHOR]

Ginsenoside Rg1 (Rg1) is one of the major bioactive ingredients in Panax ginseng, and it attenuates inflammation and apoptosis. The aims of our study were to explore the potential of Rg1 for the treatment of intestinal I/R injury and to determine whether the protective effects of Rg1 were exerted through the Wnt/β-catenin signaling pathway. In this study, Rg1 treatment ameliorated inflammatory factors, ROS and apoptosis that were induced by intestinal I/R injury. Cell viability was increased and cell apoptosis was decreased with Rg1 pretreatment following hypoxia/reoxygenation (H/R) in the in vitro study. Rg1 activated the Wnt/β-catenin signaling pathway in both the in vivo and in vitro models, and in the in vitro study, the activation was blocked by DKK1. Our study provides evidence that pretreatment with Rg1 significantly reduces ROS and apoptosis induced by intestinal I/R injury via activation of the Wnt/β-catenin pathway. Taken together, our results suggest that Rg1 could exert its therapeutic effects on intestinal I/R injury through the Wnt/β-catenin signaling pathway and provide a novel treatment modality for intestinal I/R injury.

Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Trilineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress, and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair.

Background: Mufangji tang (MFJT) is composed of Ramulus Cinnamomi, Radix Ginseng, Cocculus orbiculatus (Linn.) DC., and Gypsum. In clinical settings, MFJT has been effectively employed in addressing a range of respiratory disorders, notably including pulmonary arterial hypertension (PAH). However, the mechanism of action of MFJT on PAH remains unknown. Methods: In this study, a monocrotaline-induced PAH rat model was established and treated with MFJT. The therapeutic effects of MFJT on PAH rat model were evaluated. Network pharmacology was conducted to screen the possible targets for MFJT on PAH, and the molecular docking between the main active components and the core targets was carried out. The key targets identified from network pharmacology were tested. Results: Results showed significant therapeutic effects of MFJT on PAH rat model. Analysis of network pharmacology revealed several potential targets related to apoptosis, inflammation, oxidative stress, and vascular remodeling. Molecular docking showed that the key components were well docked with the core targets. Further experimental validation results that MFJT treatment induced apoptosis (downregulated Bcl-2 levels and upregulated Bax levels in lung tissue), inhibited inflammatory response and oxdative stress (decreased the levels of IL-1β, TNF-α, inducible NOS, and malondialdehyde, and increased the levels of endothelial nitric oxide synthase, nitric oxide, glutathione and superoxide dismutase), reduced the proliferation of pulmonary arterial smooth muscle cells (downregulated ET-1 and β-catenin levels and ERK1/2 phosphorylation, increased GSK3β levels). Conclusion: Our study revealed MFJT treatment could alleviate PAH in rats via induction of apoptosis, inhibition of inflammation and oxidative stress, and the prevention of vascular remodeling. [ABSTRACT FROM AUTHOR]

As a novel nuclear factor E2‐related factor 2 (NRF2) activator, the itaconate has shown significant therapeutic potential for oxidative stress diseases. However, its role in Vohwinkel syndrome in relation to the gap junction protein beta 2 (GJB2) mutation is still unclear. This study aimed at investigating the effect of 4‐octyl itaconate (OI) on HaCaT and D66H cells and clarify its potential mechanism in vitro. The optimal concentration and treatment time of OI on HaCaT cells and D66H cells were determined by CCK‐8 and LDH experiments. The effect of OI on cell proliferation was detected by EdU staining and FACS analysis of PI, while the apoptosis was evaluated by TUNEL staining and FACS analysis of Annexin V. The ROS staining was performed, and the levels of SOD, MDA, GSH and GSH/GSSG were detected to evaluate the effect of OI on oxidative damage induced by D66H‐type mutation. CO‐IP, Western blot, immunofluorescence and qPCR analyses were employed to detect the activation of KEAP1‐NRF2‐GCLC/HO‐1 pathway by OI. Finally, sh‐NRF2 was used to confirm the activation of this pathway by OI. Results showed that OI could improve the cell viability decreased by GJB2 gene mutation by regulating the balance between cell growth and apoptosis induced by oxidative damage. Furthermore, this alleviation process was regulated by the KEAP1‐NRF2‐HO‐1/GCLC pathway. In conclusion, OI could improve the viability of HaCaT and D66H cells via regulating the KEAP1‐NRF2‐GCLC/HO‐1 pathway, which provided a wide spectrum of potential targets for effective therapeutic treatments of Vohwinkel syndrome in the clinic. [ABSTRACT FROM AUTHOR]

A colon tumor, one of the digestive tract malignant tumors, is harmful to human health. A potential new treatment still deserves attention. The development of a new drug needs more resources, including time and expense. Therefore, the old drug with new targets has become a current research hotspot. Fluvoxamine, as an antidepressant, could play an effect on inhibiting 5‐hydroxytryptamine reuptake. In the present research, the antitumor effects and possible mechanisms of fluvoxamine are validated. The results showed that fluvoxamine significantly suppressed the migration and proliferation of tumor cells, and increased the apoptosis in vitro. Additionally, fluvoxamine significantly delays tumor development, and prompts the apoptosis in tumor tissues of mice‐burdened colon tumors in vivo. The tumor suppression might be related with that fluvoxamine inhibits the expression of phosphorylated signal transducer and activator of transcription 3, matrix metalloproteinase 2, and cleaved‐caspase 3. Importantly, fluvoxamine significantly reduces the expression level of programmed cell death ligand 1. This could be a possible reason that treatment with fluvoxamine drives the infiltration of T lymphocytes and M1‐type macrophages in tumor tissues. Taken together, this research suggests that fluvoxamine might be a promising drug to treat colon cancer by inhibiting the proliferation and migration, inducing apoptosis, and even increasing the immune response of antitumor. [ABSTRACT FROM AUTHOR]

Rotenone is a commonly used insecticidal chemical in agriculture and it is an inhibitor of mitochondrial complex Ⅰ. Previous studies have found that rotenone induces the production of reactive oxygen species (ROS) by inhibiting electron transport in the mitochondria of somatic and germ cells. However, there is little precise information on the effects of rotenone exposure in porcine oocytes during in vitro maturation, and the mechanisms underlying these effects have not been determined. The Cumulus-oocyte complexes were supplemented with different concentrations of rotenone to elucidate the effects of rotenone exposure on the meiotic maturation of porcine oocytes during in vitro maturation for about 48 hours. First, we found that the maturation rate and expansion of cumulus cells were significantly reduced in the 3 and 5 μM rotenone-treated groups. Subsequently, the concentration of rotenone was determined to be 3 μM. Also, immunofluorescence, western blotting, and image quantification analyses were performed to test the rotenone exposure on the meiotic maturation, total and mitochondrial ROS, mitochondrial function and biogenesis, mitophagy and apoptosis in porcine oocytes. Further experiments showed that rotenone treatment induced mitochondrial dysfunction and failure of mitochondrial biogenesis by repressing the level of SIRT1 during in vitro maturation of porcine oocytes. In addition, rotenone treatment reduced the ratio of active mitochondria to total mitochondria, increased ROS production, and decreased ATP production. The levels of LC3 and active-caspase 3 were significantly increased by rotenone treatment, indicating that mitochondrial dysfunction induced by rotenone increased mitophagy but eventually led to apoptosis. Collectively, these results suggest that rotenone interferes with porcine oocyte maturation by inhibiting mitochondrial function. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O2−) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m6A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m6A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m6A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m6A modification. [ABSTRACT FROM AUTHOR]

Background: Cumulative evidence suggests that neuronal death including autophagy, apoptosis, and necrosis is closely related to the occurrence and development of cerebral ischemia–reperfusion (I/R) injury. Moreover, vagal nerve stimulation (VNS) is involved in many different neuroprotective and neuroplasticity pathways. Thus, VNS may be a novel approach for treating various neurodegenerative diseases. The present study aims to determine whether VNS protects against cerebral I/R injury in rats by inhibiting autophagy and apoptosis. Methods: Cerebral I/R injury is induced by middle cerebral artery occlusion (MCAO) and VNS is carried out. Infarct volume, neurological deficit, autophagy, and apoptosis are examined 24 h after reperfusion. Results: Vagal nerve stimulation decreases infarct volume and suppresses neurological deficit. Moreover, obvious autophagy and apoptosis are detected in rats that have undergone I/R, and VNS inhibits autophagy and apoptosis. Conclusion: Vagal nerve stimulation exerts neuroprotective effects following I/R injury by inhibiting autophagy and apoptosis. [ABSTRACT FROM AUTHOR]

Background: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance.Methods: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance.Conclusions: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance. [ABSTRACT FROM AUTHOR]