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This research aimed to investigate the effects of dietary fructooligosaccharides (FOS) on attenuating the Aeromonas hydrophila (A. hydrophila)-induced oxidative stress and apoptosis in blunt snout bream Megalobrama amblycephala. Fish were divided into three groups as follows: C1 (Control), T1 (A. hydrophila), and T2 (A. hydrophila + 4 g/kg FOS). The results showed that the activities of antioxidant enzymes increased, the liver morphology had disorderly arrangement, and extensive cell necrosis occurred because of A. hydrophila-infection. While the dietary FOS improved the above-mentioned liver damage. Additionaly, FOS elevated mRNA levels of pro-apoptotic molecules, including caspase-8 and 9, and down-regulated mRNA levels of the anti-apoptotic molecule Bcl-2, which is triggered by A. hydrophila-infection. The transcriptome analysis showed that the oxidative stress-related DEGs pathways were activated in intestine of blunt snout bream by A. hydrophila-infection. The FOS-added group led to the enrichment of more pathways to health. Further WGCNA co-expression network analysis showed that the screened single genes were clustered into 49 modules. The two modules with the highest association to the five traits (10 hub genes) were chosen to build the network by combining the physiological and biochemical characteristic. In summary, this research offers a foundation for the exploring of A. hydrophila-restoration genes in dietary FOS, and also lays a theoretical foundation for aquaculture in the future., (© 2024. The Author(s).)

Avermectin is a highly effective insecticide that has been widely used in agriculture since the 1990s. In recent years, the safety of avermectin for non-target organisms has received much attention. The vasculature is important organs in the body and participate in the composition of other organs. However, studies on the vascular safety of avermectin are lacking. The vasculature of zebrafish larvae is characterized by ease of observation and it is a commonly used model for vascular studies. Therefore, zebrafish larvae were used to explore the potential risk of avermectin on the vasculature. The results showed that avermectin induced vascular damage throughout the body of zebrafish larvae, including the head, eyes, intestine, somite, tail and other vasculature. The main forms of damage are reduction in vascular diameter, vascular area and vascular abundance. Meanwhile, avermectin induced a decrease in the number of endothelial cells and apoptosis within the vasculature. In addition, vascular damage may be related to impairment of mitochondrial function and mitochondria-mediated apoptosis. Finally, exploration of the molecular mechanisms revealed abnormal alterations in the expression of genes related to the VEGF/Notch signaling pathway. Therefore, the VEGF/Notch signaling pathway may be an important mechanism for avermectin-induced vascular damage in zebrafish larvae. This study demonstrates the vascular toxicity of avermectin in zebrafish larvae and reveals the possible molecular mechanism, which would hopefully draw more attention to the safety of avermectin in non-target organisms., 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 Elsevier B.V. All rights reserved.)

Melatonin, as an endocrine neurotransmitter, can promote the development of the ovary. Meanwhile, it also has protective effect on the ovary as an antioxidant. Thyroid hormone (TH) is essential for normal human reproductive function. Many studies have shown that 3,5,3'-triiodothyronine (T 3 ) regulates the development of ovarian granulosa cells. However, little is known about the specific mechanisms by which melatonin combines with T 3 to regulate granulosa cell development. The aim of present study was to investigate the effects and the possible mechanisms of melatonin and T 3 on ovarian granulosa cell development. In the present study, cell development and apoptosis were detected by CCK8, EdU and TUNEL, respectively. The levels of related proteins were analyzed by Western blotting. The results showed that oxidative stress (OS) and reactive oxygen species (ROS) were induced by H 2 O 2 in granulosa cells, and cell apoptosis was also increased accompanied with the decreased cellular proliferation and viability. Melatonin protects granulosa cells from H 2 O 2 -induced apoptosis and OS by downregulating ROS levels, especially in the presence of T 3 . Co-treatment of cell with melatonin and T 3 also promotes the expression of GRP78 and AMH, while inhibiting CHOP, Caspase-3, and P16. It was demonstrated that melatonin alone or in combination with T 3 had positive effect on the development of granulosa cells. In addition, the AMPK/SIRT1 signaling pathway is involved in the process of melatonin/T 3 promoting granulosa cell development.

Pyraclostrobin is a highly effective and broad-spectrum strobilurin fungicide. With the widespread use of pyraclostrobin to prevent and control crop diseases, its environmental pressure and potential safety risks to humans have attracted much attention. Herein, the toxicological risks of pyraclostrobin toward HepG2 cells and the mechanisms of intoxication in vitro were investigated. The liver toxicity of pyraclostrobin in zebrafish larvae was also evaluated. It was found that pyraclostrobin induced DNA damage and reactive oxygen species generation in HepG2 cells, indicating the potential genotoxicity of pyraclostrobin. The results of fluorescent staining experiments and the expression of cytochrome c, Bcl-2 and Bax demonstrated that pyraclostrobin induced mitochondrial dysfunction, resulting in cell apoptosis. Monodansylcadaverine staining and autophagy marker-related proteins LC3, p62, Beclin-1 protein expression showed that pyraclostrobin promoted cell autophagy. Furthermore, immunoblotting analysis suggested that pyraclostrobin induced autophagy accompanied with activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/mTOR signaling pathway. Visualization of zebrafish liver and oil red staining indicated that pyraclostrobin could induce liver degeneration and liver steatosis in zebrafish. Collectively, these results help to better understand the hepatotoxicity of pyraclostrobin and provide a scientific basis for its safe applications and risk control., 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 © 2023 Elsevier Ltd. All rights reserved.)

Background: Cuproptosis, an emerging form of programmed cell death, has recently been identified. However, the association between cuproptosis-related long non-coding RNA (lncRNA) signature and the prognosis in prostate carcinoma remains elusive. This study aims to develop the novel cuproptosis-related lncRNA signature in prostate cancer and explore its latent molecular function., Methods: RNA-seq data and clinical information were downloaded from the TCGA datasets. Then, cuproptosis-related gene was identified from the previous literature and further applied to screen the cuproptosis-related differentially expressed lncRNAs. Patients were randomly assigned to the training cohort or the validation cohort with a 1:1 ratio. Subsequently, the machine learning algorithms (Lasso and stepwise Cox (direction = both)) were used to construct a novel prognostic signature in the training cohorts, which was validated by the validation and the entire TCGA cohorts. The nomogram base on the lncRNA signature and several clinicopathological traits were constructed to predict the prognosis. Functional enrichment and immune analysis were performed to evaluate its potential mechanism. Furthermore, differences in the landscape of gene mutation, tumour mutational burden (TMB), microsatellite instability (MSI), drug sensitivity between both risk groups were also assessed to explicit their relationships., Results: The cuproptosis-related lncRNA signature was constructed based on the differentially expressed cuproptosis-related lncRNAs, including AC005790.1, AC011472.4, AC099791.2, AC144450.1, LIPE-AS1, and STPG3-AS1. Kaplan-Meier survival and ROC curves demonstrate that the prognosis signature as an independent risk indicator had excellent potential to predict the prognosis in prostate cancer. The signature was closely associated with age, T stage, N stage, and the Gleason score. Immune analysis shows that the high-risk group was in an immunosuppressive microenvironment. Additionally, the significant difference in landscape of gene mutation, tumour mutational burden, microsatellite instability, and drug sensitivity between both risk groups was observed., Conclusions: A novel cuproptosis-related lncRNA signature was constructed using machine learning algorithms to predict the prognosis of prostate cancer. It was closely with associated with several common clinical traits, immune cell infiltration, immune-related functions, immune checkpoints, gene mutation, TMB, MSI, and the drug sensitivity, which may be useful to improve the clinical outcome., (© 2023. The Author(s).)

Difenoconazole is one of the most typical triazole fungicides. Difenoconazole is widely used in the field of agricultural production, and its health and safety problems need to be further studied. The main purpose of this paper is to verify the neurotoxicity of Difenoconazole at the cellular level. In this study, SH-SY5Y cell line of human neuroblastoma was used to evaluate its potentially toxic effects and molecular mechanism in vitro. The research indicated that Difenoconazole could reduce cell viability and inhibit cell proliferation, induce DNA damage and accelerate programmed cell death. Further studies showed that Difenoconazole induced DNA double-strand breaks, intracellular generation of ROS, cleaved PARP, mitochondrial membrane potential collapse, induced Cyt c release, and Bax/Bcl-2 ratio increase in SH-SY5Y cells. In conclusion, the cytotoxicity of Difenoconazole revealed its toxic effect on SH-SY5Y cells, and the IC 50 value was 55.41 μM after 24 h exposure. Meanwhile, the genetic toxicity of Difenoconazole has revealed that it can induce DNA damage and apoptosis of SH-SY5Y cells. Through this study, the toxic effects of Difenoconazole on SH-SY5Y cells are further understood, which provides a more scientific basis for its safe use and risk control., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Preeclampsia is a gestational hypertensive disease; however, preeclampsia remains poorly understood. Bioinformatics analysis was applied to find novel genes involved in the pathogenesis of preeclampsia and identified CLDN1 as one of the most differentially expressed genes when comparing patients with preeclampsia and healthy controls. The results of the qRT-PCR, Western blotting and immunohistochemistry experiments demonstrated that CLDN1 was significantly downregulated in the chorionic villi in samples from patients with preeclampsia. Furthermore, knockdown of CLDN1 in HTR-8/SVneo cells resulted in the inhibition of proliferation and induction of apoptosis, and overexpression of CLDN1 reversed these effects. In addition, RNA-seq assays demonstrated that the gene BIRC3 is potentially downstream of CLDN1 and is involved in the regulation of apoptosis. Knockdown of CLDN1 confirmed that the expression level of BIRC3 was obviously decreased and was associated with a significant increase in cleaved PARP. Interestingly, the apoptotic effect in CLDN1 knockdown cells was rescued after BIRC3 overexpression. Overall, these results indicate that a decrease in CLDN1 inhibits BIRC3 expression and increases cleaved PARP levels thus participating in the pathogenesis of preeclampsia.

Follicle development is a complicated process regulated by thyroid hormone (TH). TH dysregulation is associated with reproductive disorders; however, the mechanism underlying these relationships remains unclear. Glucose-related protein 78 (GRP78) is a well-characterised endoplasmic reticulum stress protein related to ovarian cell apoptosis. To clarify whether GRP78 is regulated by TH and the involvement of GRP78 in follicle development, we established rat models of hypothyroidism and hyperthyroidism and investigated the effects of TH dysregulation on levels of GRP78, C/EBP homologous protein (CHOP) and cleaved caspase-3. TH dysregulation decreased levels of GRP78 and increased those of CHOP and cleaved caspase-3 in both rat models. However, treatment with equine chorionic gonadotrophin reversed these effects, as well as granulosa cell apoptosis induced by TH dysregulation. Together, these results provide evidence that TH dysregulation alters the GRP78 expression profile, triggering the apoptotic signalling pathway, and suggest that GRP78 is a novel mediator of TH in follicle development.

Protein disulfide isomerase 3 (PDIA3) is a chaperone protein that modulates the folding of newly synthesized glycoproteins, has isomerase and redox activity, and has been implicated in the pathogenesis of many diseases. However, the role of PDIA3 in pregnancy-associated diseases remains largely unknown. Our present study reveals a key role for PDIA3 in the biology of placental trophoblasts from women with preeclampsia (PE). Immunohistochemistry and Western blot analysis revealed that PDIA3 expression was decreased in villous trophoblasts from women with PE compared to normotensive pregnancies. Further, using a Cell Counting Kit-8 assay, flow cytometry, and 5-ethynyl-2'-deoxyuridine (EdU) staining, we found that siRNA-mediated PDIA3 knockdown significantly promoted apoptosis and inhibited proliferation in the HTR8/SVneo cell line, while overexpression of PDIA3 reversed these effects. Furthermore, RNA sequencing and Western blot analysis demonstrated that knockdown of PDIA3 inhibited MDM2 protein expression in HTR8 cells, concurrent with marked elevation of p53 and p21 expression. Conversely, overexpression of PDIA3 had the opposite effects. Immunohistochemistry and Western blot further revealed that MDM2 protein expression was downregulated and p21 was increased in trophoblasts of women with PE compared to women with normotensive pregnancies. Our findings indicate that PDIA3 expression is decreased in the trophoblasts of women with PE, and decreased PDIA3 induces trophoblast apoptosis and represses trophoblast proliferation through regulating the MDM2/p53/p21 pathway.

Background/aims: Obesity induces hippocampal neuronal apoptosis and leads to cognitive function deficits. Sonic hedgehog (SHH) signaling is crucial during nervous system development and is neuroprotective in many neurologic diseases. This study assessed the role of SHH signaling in the cognitive deficits in high-fat diet (HFD)-induced obese mice., Methods: Flow cytometry assay was used to examine cell apoptosis. Tissue pathology was evaluated by Nissl staining. Immunofluorescent staining and western blotting were used to detect SHH signaling molecules and apoptosis-related proteins. The Morris water maze test was performed to evaluate mouse spatial learning and memory., Results: After HFD feeding for 24 weeks, the expression of SHH signaling molecules was downregulated in the mouse hippocampus. In vitro, GANT61 inhibited SHH signaling and induced apoptosis in HT22 mouse hippocampal cells. Smoothened agonist (SAG), an agonist of SHH signaling, reduced apoptosis in GANT61-treated HT22 cells by regulating caspase-9 and caspase-3 activation. In vivo, 12-week SAG treatment also inhibited the apoptosis of hippocampal neurons in HFD-fed mice by increasing mitofusin 2 (Mfn2) and B-cell lymphoma 2 (Bcl-2) levels and decreasing dynamin-related protein 1 (Drp1) and Bcl-2 homologous antagonist/killer (Bak) levels. Behavioral testing showed that SAG administration ameliorated the cognitive impairment in HFD-fed mice., Conclusion: Downregulation of hippocampal SHH signaling leads to neuronal apoptosis and cognitive deficits in HFD-fed mice. These findings provide useful information for the identification of potential targets for research and therapeutic interventions for cognitive impairment in obesity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Resveratrol has been shown to prevent high ambient temperature (HT)-induced spleen dysplasia, but the mechanisms of action are not clear. This study aims to examine the hypothesis that HT-induced spleen dysplasia may be associated with HT-induced oxidative stress and apoptosis, and resveratrol may activate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, thus reducing oxidative stress and apoptosis., Results: Results showed that HT caused spleen dysplasia in broilers, reflecting the lower relative weight of the spleen (P < 0.05). Compared with birds in a normal ambient temperature group, birds in the HT group exhibited higher (P < 0.05) malondialdehyde (MDA), protein carbonyl (PC), 8-hydroxydeoxyguanosine (8-OHdG) and Bcl-2 associated X protein (Bax) content, higher Bax, caspase-3 and caspase-9 mRNA levels, and caspase-3 and caspase-9 activity, and a higher Bax/B-cell lympoma/leukemia-2 (Bcl-2) ratio, but they exhibited lower (P < 0.05) glutathione (GSH) and Bcl-2 content, and lower Nrf2, glutathione peroxidase (Gpx), MnSOD, heme oxygenase 1, glutathione reductase (GR) and Bcl-2 mRNA levels, and lower total antioxidant capacity (T-AOC), T-SOD and catalase and maganese superoixide dismutase (CAT) activity, indicating HT-induced oxidative stress and apoptosis. Compared with birds in the HT group, birds in the HT + Res group exhibited higher (P < 0.05) GSH and Bcl-2 content, higher Nrf2, CAT, MnSOD, GR and Bcl-2 mRNA levels, and higher T-AOC, T-SOD and CAT activity, but lower (P < 0.05) MDA content, and Bax and caspase-3 mRNA levels, lower caspase-3 and caspase-9 activities, and Bax/Bcl-2 ratio, indicating that resveratrol activated the Nrf2 signaling pathway and decreased apoptosis in the spleen., Conclusion: Resveratrol was effective in ameliorating HT-induced spleen dysplasia in broilers through the activation of the Nrf2 signaling pathway, thereby decreasing apoptosis, suggesting that resveratrol may offer a potential nutritional strategy to protect against some HT-induced detriments. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

T-cell lymphomas (TCLs) are a malignancy characterized by tumor aggression and resistance to traditional chemotherapy. Disruption of the extrinsic cell death pathway is essential for resistance to chemotherapy. PIM1 serves as a crucial modulator in cancers. However, the role of PIM1 in TCLs remains unclear. In this study, we studied the roles of PIM1 in established T-lymphoma cell lines Jurkat and HUT-78. CCK-8 assay was conducted to evaluate cell survival and flow cytometry was performed to evaluate cell death of TCL cells. siRNAs were used to knockdown the expression of PIM1 and c-myc. qRT-PCR was used to evaluate the mRNA expression levels of c-myc and PIM1. Western blot analysis was used to evaluate the protein expression levels of PIM1, c-myc, STAT3, and phospho-STAT3. Doxorubicin was used to determine the effect of PIM1 on apoptosis. Our results showed that PIM1 expression was markedly enhanced and induced c-myc expression in TCL cells. Doxorubicin inhibited the expressions of c-myc and PIM1, and triggered the extrinsic cell death of TCLs by suppressing the JAK-STAT3 signaling pathway. Moreover, PIM1 silencing via siRNA suppressed c-myc expression, promoted the cell death of TCLs, and increased doxorubicin sensitivity. Conversely, PIM1 overexpression in TCL cells induced c-myc expression, suppressed TCL cell death, and promoted doxorubicin resistance. Collectively, our results demonstrate that PIM1 overexpression in TCLs participates in cancer cell protection from apoptosis and leads to doxorubicin resistance by inducing c-myc expression, indicating that PIM1 may be a promising target in TCL treatment.

The use of chemotherapeutic agents prior to treatment with infusion of cluster of differentiation (CD)19-chimeric antigen receptor (CAR)‑T cells is important for the efficacy of clinical therapies against hematological malignancies. However, the effect of chemotherapeutic agents on CD19‑CAR‑T cells and the associated underlying mechanisms remain unknown. The first aim of the present study was to determine the effect of chemotherapeutic agents on CAR‑T cells using the in vitro Cell Counting kit 8 assay. The second aim was to evaluate the abilities of fludarabine (FDR) and mafosfamide (MFA; a metabolite of cyclophosphamide) to induce apoptosis of CD19‑CAR‑T cells via the use of Annexin V/propidium iodide double staining. In addition, a JC‑1 fluorescent probe was used to detect alterations in cell membrane potential, and flow cytometry analysis was used to measure concentrations of caspase‑3/7 to identify apoptotic pathways of CD19‑CAR‑T cells. The data of the present study suggested that FDR and MFA inhibit the activities of CD19‑CAR‑T cells. Alterations to the mitochondrial membrane potential and an increase in the concentration of caspase‑3/7 indicated early apoptosis of FDR‑ and MFA‑treated CD19‑CAR‑T cells. The present study laid a theoretical foundation for the development of programs for clinical treatment.

Based on the mechanisms by which Wee1 inhibitor and cisplatin played their own role, a promising strategy of Wee1 inhibitor combined with cisplatin was proposed, which was investigated in gastric cancer (GC). Either Wee1 inhibitor AZD1775 or cisplatin alone had a certain inhibitory effect on in vitro cell proliferation; however, the inhibitory effect was more significant when AZD1775 combined with cisplatin in vitro and in vivo . The underlying mechanisms unveiled that the increased DNA damage indicated by increased γ H2AX protein, as well as augmented cell apoptosis indicated by upregulated proapoptotic proteins, was responsible for the significant inhibitory effect of AZD1775 plus cisplatin. Moreover, compared to any single drug, in vitro cell migration and invasion abilities were further attenuated by AZD1775 combined with cisplatin. There were suggestive results that the potentiated cytotoxicity between AZD1775 and cisplatin deserved a deep exploration in the future.

Ethnopharmacology Relevance: He-Ying-Qing-Re Formula (HF) was empirically modified from Si-Miao-Yong-An Decoction (SD), which was recorded in the literature of Divine Doctor's Secret Transmission, and has been utilized for centuries to treat vasculopathy through clearing heat and accelerating bloodstream. HF has been used as an effective holistic treatment of diabetic retinopathy (DR) for decades and experimentally reported to ameliorate retinal condition in diabetic mice., Aim of the Study: Our study aims to investigate the effect of HF in preventing sustained hyperglycemia and hyperlipidemia-associated retinal ganglion cell (RGC) cell death and its possible mechanism., Materials and Methods: Chromatographic fingerprint of HF was obtained upon the UPLC-based analytic system; Diabetic retinopathy was established in streptozotocin (STZ) injection-induced hyperglycemic mice; Alterations of retinal structure was assayed by H&E staining. Expression of PSD-95 and CHOP in retinae was assessed by immunofluorescence; RGC cell line (mRGC) was used for in vitro study. Cell death was analyzed by flow cytometry; Intracellular reactive oxygen species (ROS) was measured by 2',7'-dichlorofluorescin diacetate (DCFDA); Apoptosis-related proteins and signaling were monitored with immunoblotting and colorimetric assay., Results: Chlorogenic acid, ferulic acid, and rutin were identified in HF. HF attenuates the loss of RGCs, thinning of inner retinal layers in diabetic mice. Furthermore, expressions of Brn3a and PSD-95 were restored while CHOP level was downregulated upon HF treatment. In vitro study, HF alleviates H 2 O 2 -induced apoptosis of mRGCs and loss of postsynaptic protein via scavenging ROS and suppressing ATF4/CHOP-mediated endoplasmic reticulum stress and mitochondria-related pro-apoptotic factors, probably as cleaved-caspase-3, and phospho-p38 mitogen-activated protein kinase (MARK). Meanwhile, both pro-survival protein levels like Bcl-2/Bcl-xL and postsynaptic protein of PSD-95 were upregulated upon HF treatment., Conclusion: HF administration was a valid therapeutic approach for DR treatment, oriented at the blockade of endoplasmic reticulum- and mitochondria-dependent oxidative stress-induced retinal neurodegeneration including RGC apoptosis., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Isoliquiritigenin (ISL) has been reported to have a wide range of biological activities. This study evaluated the cytotoxic effect of ISL on norvegicus pheochromocytoma cell line (PC-12 cells) and its possible molecular mechanism. The cytotoxicity in vitro of ISL against PC-12 cells was investigated by MTT assay. The migration and invasion of PC-12 cells were performed by scratch test and transwell assay. Apoptosis was evaluated by microscopy and flow cytometry. The reactive oxygen species (ROS) and mitochondrial membrane potential were studied by fluorescent microscopy. DNA damage of PC-12 cells was analyzed by comet assay. The protein expression of caspase, Bcl-2 family member, autophagy-associated protein Beclin-1, and LC3 was detected by western blot. The autophagy of PC-12 cells was investigated by acridine orange (AO) and monodansylcadaverine (MDC) staining. The IC 50 value of ISL against PC-12 cell is 17.8 ± 1.8 μM. ISL could suppress PC-12 cell migration and invasion. AO/ethidium bromide staining and flow cytometry suggested that ISL caused apoptosis of PC-12 cells. Significant DNA damages of PC-12 cells treated with ISL were observed in a comet assay. ISL inhibited the cell growth of PC-12 cells at S phase. Exposure of PC-12 cells to ISL increased the levels of cellular reactive oxygen species (ROS) and decreased the mitochondrial membrane potential. Additionally, ISL trigged the release of cytochrome c from the mitochondria to the cytoplasm. The expression levels of caspase-9, caspase-3, caspase-7, Bax, and Bim were upregulated, whereas the expression levels of Bcl-2 and Bcl-x were downregulated. AO and monodansylcadaverine (MDC) staining assay showed that ISL caused autophagy of PC-12 cells. The upregulation of protein Beclin-1 and LC3 was observed in PC-12 cells. Therefore, the results show that ISL induces apoptosis of PC-12 cells through ROS-mediated activation of the intrinsic mitochondria-cytochrome c-caspase protease mechanism and causes the autophagy of PC-12 cells. Graphical Abstract The in vitro cytotoxicity, apoptosis, comet assay, ROS, mitochondrial membrane potential, cell cycle arrest, autophagy, and western blot induced by ISL were investigated.

Tumor growth and metastasis are angiogenesis dependent. Angiogenic growth involves endothelial cell proliferation, migration, and invasion. Ephrin-B2 is a ligand for Eph receptor tyrosine kinases and is an important mediator in vascular endothelial growth factor-mediated angiogenesis. However, research offer controversial information regarding effects of ephrin-B2 on vascular endothelial cells. In this paper, proteome analyses showed that ephrin-B2/Fc significantly activates multiple signaling pathways related to cell proliferation, survival, and migration and suppresses apoptosis and cell death. Cytological experiments further confirm that ephrin-B2/Fc stimulates endothelial cell proliferation, triggers dose-dependent migration, and suppresses cell apoptosis. Results demonstrate that soluble dose-dependent ephrinB2 can promote proliferation and migration and inhibit apoptosis of human umbilical vein endothelial cells. These results also suggest that ephrinB2 prevents ischemic disease and can potentially be a new therapeutic target for treating angiogenesis-related diseases and tumors.

A new ligand BTCP and its iridium(III) complex [Ir(ppy) 2 (BTCP)]PF 6 (Ir-1) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1 H NMR and 13 C NMR. The cytotoxic activity in vitro of the ligand and its complex against SGC-7901, HeLa, HOS, PC-12, BEL-7402, MG-63, SiHa, A549, HepG2 and normal cell LO2 were evaluated by MTT method [MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The reactive oxygen species (ROS), mitochondrial membrane potential, autophagy and cell invasion were studied under fluorescent microscope. The expression of caspases and Bcl-2 family proteins were investigated by western blot. The IC 50 values of complex toward SGC-7901, BEL-7402 and MG-63 cells are 3.9 ± 0.5, 5.4 ± 1.2 and 4.2 ± 0.6 µM. The complex can increase the levels of ROS, and induce a decrease in the mitochondrial membrane potential. Ir-1 inhibits the cell growth at G0/G1 phase in SGC-7901 cells, and the complex can induce both autophagy and apoptosis and inhibit the cell invasion. And the complex induces apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5±1.2μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(dhbn)](ClO4)2 (N-N = dmb: 4,4'-dimethyl-2,2'-bipyridine 1; bpy = 2,2'-bipyridine 2; phen = 1,10-phenanthroline 3; dmp = 2,9-dimethyl-1,10-phenanthroline 4) were synthesized and characterized. The cytotoxicity in vitro of the ligand and complexes toward HepG-2, HeLa, MG-63 and A549 were assayed by MTT method. The IC50 values of the complexes against the above cells range from 17.7 ± 1.1 to 45.1 ± 2.8 μM. The cytotoxic activity of the complexes against HepG-2 cells follows the order of 4 > 2 > 3 > 1. Ligand shows no cytotoxic activity against the selected cell lines. Cellular uptake, apoptosis, comet assay, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest, and the expression of proteins involved in apoptosis pathway induced by the complexes were investigated. The results indicate that complexes 1-4 induce apoptosis in HepG-2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Endocrine disruptor 2,2',4,4'-tetrabromodiphenylether (PBDE-47) can harm the female reproductive system. Recent studies showed that PBDE-47 neurotoxicity is associated with endoplasmic reticulum stress (ERS); however, the role of ERS in PBDE-47-induced ovarian injury is unclear. New-born female Sprague-Dawley rats were orally exposed to PBDE-47 (1, 5, or 10mg/kg bw) on postnatal day 10. An additional 10mg/kg bw PBDE-47 group was given the ERS inhibitor 4-PBA intraperitoneally for three weeks beginning on postnatal day 8. At 2 months of age, PBDE-47 exposure significantly reduced the ovarian coefficients, increased the expression of ERS and autophagy markers, including GRP78, IRE1, Caspase-12, Beclin1, LC3 and P62. In the 10mg/kg bw PBDE-47 group, PARP and Caspase-3 were markedly activated, indicative of apoptosis. These were accompanied by histopathological damage. Intriguingly, 4-PBA attenuated all these effects. Thus, these results suggest that ERS plays a vital role in PBDE-47-induced ovarian injury by regulating autophagy and apoptosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fluoride, a ubiquitous environmental contaminant, is known to impair testicular functions and fertility; however the underlying mechanisms remain obscure. In this study, we used a rat model to mimic human exposure and sought to investigate the roles of apoptosis and autophagy in testicular toxicity of fluoride. Sprague-Dawley rats were developmentally exposed to 25, 50, or 100 mg/L sodium fluoride (NaF) via drinking water from pre-pregnancy to post-puberty, and then the testes of offspring were excised on postnatal day 56. Our results demonstrated that developmental NaF exposure induced an enhanced testicular apoptosis, as manifested by a series of hallmarks such as caspase-3 activation, chromatin condensation and DNA fragmentation. Further study revealed that fluoride exposure elicited significant elevations in the levels of cell surface death receptor Fas with a parallel increase in cytoplasmic cytochrome c, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. Intriguingly, fluoride treatment also simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II but not Beclin1. Unexpectedly, the expression of p62, a substrate that is degraded by autophagy, was also significantly elevated, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation rather than increased formation. Importantly, these were associated with marked histopathological lesions including spermatogenic failure and germ cell loss, along with severe ultrastructural abnormalities in testes. Taken together, our findings provide deeper insights into roles of excessive apoptosis and defective autophagy in the aggravation of testicular damage, which could contribute to a better understanding of fluoride-induced male reproductive toxicity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Connexin 43 (Cx43) induced apoptosis has been reported in solid tumors, but the effect of Cx43 expressed by bone marrow stromal cells (BMSC) in leukemia has not been fully investigated. Manipulating Cx43 expression could be a potential therapeutic strategy for leukemia. Here, we investigate the effect of Cx43 expressed by BMSCs (human Umbilical Cord Stem Cells over-expressed CX43, Cx43-hUCSC) on leukemia cells. When co-cultured with Cx43-hUCSC, leukemia cells show significant lower growth rate with increasing apoptosis activity, and more leukemia cells enter S phase. Functional assays of fluorescence recovery after photo bleaching (FRAP) showed improved gap junctional intercellular communication (GJIC) on leukemia cells when co-cultured with Cx43-hUCSC (p < 0.01). In a mouse minimal disease model, the mean survival time and mortality rate were significantly improved in mice transplanted with Cx43-hUCSC. Our results indicate that Cx43 expressed by BMSC induces apoptosis on leukemia cells. Small molecules or other pharmaceutical approaches for modulating Cx43 expression in BMSCs could be used for delaying relapse of leukemia.

Purpose: To evaluate the cytotoxic and apoptotic effects of bishydroxycoumarin (BHC) against human glioma cells and to study their mode of action., Methods: Three cells were used in the experiments. MTT and LDH assays were used to assess the cytotoxic effects of BHC while an in vitro wound healing assay was used to study the effect of BHC on cell migration. Fluorescence microscopy and annexin V-FITC assay were used to study the cellular morphology and apoptotic effects while flow cytometry in combination with propidium iodide (PI) were used to study cell cycle arrest induced by BHC., Results: BHC induced substantial and dose-dependent cytotoxic effects against all three cell lines with U87MG cell line being most susceptible. BHC also inhibited cell migration and induced characteristic morphological changes including chromatin condensation, nuclear shrinkage which increased with increasing dose of BHC. The apoptotic cell population (both early and late apoptotic cells) increased with increasing dose of BHC which also induced substantial G0/G1 cell cycle growth arrest in U87MG cells., Conclusion: This study provides help to elucidate the translational potential of the in vitro results to be used for further in vivo studies on human glioma using animal or human subjects. The mechanistic pathway studied in this report could be helpful in explaining the mode of action of these classes of natural products.

A new ruthenium(II) polypyridyl complex [Ru(dmp)2(pddppn)](ClO4)2Ru1 was synthesized and characterized. The cytotoxic activity in vitro of the complex was evaluated by MTT method. Ru1 shows high effect on the inhibition of the cell growth against BEL-7402, HeLa, MG-63 and A549 cells with low IC50 values of 1.6±0.4, 9.0±0.8, 1.5±0.2 and 1.5±0.3 μM, respectively. The cellular uptake indicates that Ru1 can enter into the cytoplasm and accumulate in the cell nuclei. Ru1 can induce apoptosis in A549 cells and enhance the levels of reactive oxygen species (ROS) and induce the decrease of mitochondrial membrane potential. In addition, Ru1 can down-regulate the levels of Bcl-2, Bcl-x, Bak, and Bim expression and up-regulate the expression of Bag-1 and Bad. The complex induces apoptosis of A549 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of caspases and Bcl-2 family proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Peripheral blood-derived mesenchymal stem cells (PB-MSCs) show promise as a source of cells for autologous transplantation because they can be harvested through minimally invasive procedures. To ensure adequate numbers of cells for transplantation and tissue regeneration, PB-MSCs must first be cultured and expanded in vitro, but whether long-term passage modifies their properties has been poorly understood. In this study we triggered production of PB-MSCs in rabbits using granulocyte colony-stimulating factor (G-CSF) and AMD3100, and then isolated and expanded the cells in culture until they reached a state of senescence, usually after about 20 passages. Cultures of low-, middle-, and high-passage numbers were compared in terms of morphology, proliferative capacity, phenotype, differentiation potential, apoptosis, metabolic indicators, and senescence. As passage number increased, MSCs retained their elongated spindle shape, but became larger and flatter, slowed in growth gradually, and increased proportion of cells showed G1 arrest. The proportions of apoptotic cells, production of reactive oxygen species (ROS), and ADP/ATP ratio increased with passage number. Expression of senescence-associated β-galactosidase increased, while telomerase activity decreased. On the other hand, cultures did not show significant changes in phenotype or lose their ability to differentiate into three lineages as passage number increased. These results suggest that PB-MSCs maintain their stem cell properties during prolonged culturing, but they undergo senescence that may be due to apoptosis and production of ROS. These findings may help to standardize in vitro production of PB-MSCs for tissue engineering.

In the present study, the in vitro cytotoxicity of daidzein was evaluated in human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. BEL-7402 cells were sensitive to daidzein treatment, with an IC50 value of 59.7±8.1 µM. Daidzein showed no cytotoxic activity toward A549, HeLa, HepG-2 and MG-63 cells. Daidzein increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Morphological and comet assays showed that daidzein effectively induced apoptosis in BEL-7402 cells. Additionally, daidzein caused cell cycle arrest at the G2/M phase in the BEL-7402 cell line. Daidzein downregulated the expression of Bcl-2, Bcl-x and Baid proteins and upregulated the levels of Bim protein in the BEL-7402 cells. The results demonstrated that daidzein induced BEL-7402 cell apoptosis through an ROS-mediated mitochondrial dysfunction pathway.

Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up-regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)-induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG-induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short-hairpin RNA significantly decreased HG-induced cell apoptosis by reducing caspase-3 activation and ratio of Bcl2-associated X protein to B-cell lymphoma/leukemia-2 (bax/bcl-2). Furthermore, HG activated E26 transformation-specific sequence-1 (Ets-1), and HMGB1 inhibition attenuated HG-induced activation of Ets-1 via extracellular signal-regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets-1 significantly decreased HG-induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin-treated diabetic mice. Inhibition of HMGB1 by short-hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets-1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia-induced cardiomyocyte apoptosis by down-regulating ERK-dependent activation of Ets-1., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Ras association (RalGDS/AF-6) domain family member RASSF5 has been implicated in a variety of key biological processes, including cell proliferation, cell cycle regulation and apoptosis. It is believed to play an important role in tumorigenesis as a tumor suppressor in a number of malignancies. Yet, little is known concerning the function and underlying mechanisms of RASSF5 in human osteosarcoma (OS). The expression of RASSF5 was examined by immunohistochemical assay using a tissue microarray in 45 cases of OS tissues. A gain-of-function approach was used to observe the effects of lentiviral vector-mediated overexpression of RASSF5 (Lv-RASSF5) on cell growth, invasion and apoptosis, respectively, as indicated by MTT, Transwell and flow cytometry assays, and the expression levels of mammalian sterile 20-like (MST1) kinase, large tumor suppressor 1 (LATS1), proliferating cell nuclear antigen (PCNA), matrix metallopeptidase-9 (MMP-9) and p53 were detected by real-time PCR and western blot assays in OS cells (MG-63 and U-2 OS). The results indicated that the expression of RASSF5 protein was significantly downregulated in OS tissues compared to that in adjacent non-cancerous tissues (ANCT) (40.0 vs. 73.3%, P=0.002), and had a negative correlation with distant metastasis of the tumor (P=0.01). Overexpression of RASSF5 markedly suppressed cell proliferation and invasion, and induced cell apoptosis in the OS cell lines with increased expression of MST1, LATS1 and p53 and decreased expression of PCNA and MMP-9. Taken together, our findings demonstrate that RASSF5 expression is negatively correlated with distant metastasis of OS, and RASSF5 may function as a tumor suppressor in OS cells through activation of the MST1/LATS1 pathway.

Background/objective: Bone marrow stromal cells (BMSCs) can support multiple myeloma (MM) disease progression and resistance to chemotherapy. The proliferation of MM cells may be suppressed by modifying the hematopoietic microenvironment (HME). We have previously isolated human umbilical cord blood-derived stromal cells (hUCBDSCs) and observed that hUCBDSCs suppressed proliferation and induced apoptosis in KM3 cells. To examine the mechanism by which hUCBDSCs drive the inhibition of MM, KM3 cells were co-cultured with hUCBDSCs., Methods: Interleukin (IL)-6 and soluble IL-6 receptor (sIL-6R) expression levels were measured by enzyme-linked immunosorbent assay. The expression levels of membrane IL-6 receptor (mIL-6R), intercellular cell adhesion molecule-1 (ICAM-1), B-cell lymphoma/leukemia-2 (Bcl-2), and Bcl-XL as well as the location of nuclear factor κB (NF-κB) were assessed by laser confocal microscopy. The expression profiles of mIL-6R and ICAM-1 were also more precisely examined by flow cytometry, and Bcl-2, Bcl-XL and inhibitor kappa B expression levels were analyzed by western blot. The mRNA expression levels of IL-6R, ICAM-1, Bcl-2, and Bcl-XL were assessed by real-time polymerase chain reaction. NF-κB DNA-binding activity was examined by electrophoretic mobility shift assay., Results: The protein expression levels of both sIL-6R and mIL-6R were reduced in culture conditions when KM3 cells were co-cultured with hUCBDSCs; moreover, the mRNA expression levels of IL-6R were also reduced. Nuclear translocation of the NF-κB p65 subunit was inhibited in KM3 cells by co-culture with hUCBDSCs. Moreover, hUCBDSCs inhibited NF-κB DNA-binding activity, thereby resulting in the downregulation of NF-κB-regulated proteins., Conclusion: hUCBDSCs can suppress proliferation and induce apoptosis in KM3 cells by both downregulating IL-6R expression and inhibiting NF-κB activity.

2,2',4,4'-tetrabromodiphenyl ether (PBDE-47)-elicited neurotoxicity is associated with neural apoptosis; however the underlying mechanisms remain unclear. To investigate whether the mitochondrial p53 pathway is involved in neuronal apoptosis induced by PBDE-47 and to correlate DNA hypomethylation with p53 activation, human neuroblastoma (SH-SY5Y) cells were treated with different concentrations of PBDE-47 (1, 5, 10 μmol/L) for 24h in vitro. The apoptosis and ultrastructural alterations in cells, levels of p53, Bcl-2, Bax, cytochrome c (Cyt c), caspase-3 and methylation status of p53 promoter were determined. Hoechst 33258 staining and transmission electron microscopy analysis showed that PBDE-47 induced SH-SY5Y cells apoptosis characterized by the typical apoptotic morphological changes. In addition, PBDE-47 activated the p53-dependent mitochondrial apoptotic pathway as evidenced by up-regulation of p53 and Bax, down-regulation of Bcl-2 and Bcl-2/Bax ration, enhancement of Cyt c release from mitochondria into the cytosol, activation of caspase-3 as well as ultrastructural abnormalities of mitochondria. However, no obvious decrease in p53 promoter methylation levels was observed in any of the treatment groups by bisulfite genomic sequencing. Collectively, these results suggest that the mitochondrial p53 pathway is involved in PBDE-47-induced SH-SY5Y cells apoptosis, nevertheless p53 promoter hypomethylation may not be implicated in this process., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We recently reported that Cx43 expression and gap junction intercellular communication (GJIC) between acute leukemic bone marrow stromal cells (BMSCs) were deficient, which could recovery after effective chemotherapy. However, the exact role of GJIC in the hematopoietic microenvironment in leukemic cell death and proliferation is not clear. We show here that following transfection with the Cx43 gene, GJIC function was increased between leukemic BMSCs. Furthermore, compared with leukemic cells alone, the proliferation and apoptosis of leukemic cells co-cultured with BMSCs were inhibited, the percentage of G0-phase cells was higher; and expression of p53 increased and bax decreased. However, after co-culturing leukemic cells with Cx43-modified BMSCs, the number of proliferative and spontaneously apoptotic Jurkat cells increased; the percentage of G0-phase cells decreased; the expression of p53 decreased; and bax increased. Compared with Jurkat cells co-cultured with BMSCs and Jurkat cells alone, the sensitivity of leukemic cells co-cultured with Cx43-modified BMSCs to chemotherapeutics increased. Our data suggests that GJIC between leukemia BMSCs is one of the impact factor to the proliferation, apoptosis and drug sensitivity of co-cultured leukemic cells. Up-regulating its function can inhibit the protective effects of leukemic BMSCS and enhance the efficacy of therapies in hematologic malignancies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Increasing evidence demonstrates that melatonin has an anti-apoptotic effect in somatic cells. However, whether melatonin can protect against germ cell apoptosis remains obscure. Cadmium (Cd) is a testicular toxicant and induces germ cell apoptosis. In this study, we investigated the effects of melatonin on Cd-evoked germ cell apoptosis in testes. Male ICR mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) every 8 hr, beginning at 8 hr before CdCl(2) (2.0 mg/kg, i.p.). As expected, acute Cd exposure resulted in germ cell apoptosis in testes, as determined by terminal dUTP nick-end labeling (TUNEL) staining. Melatonin significantly alleviated Cd-induced testicular germ cell apoptosis. An additional experiment showed that spliced form of XBP-1, the target of the IRE-1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an endoplasmic reticulum (ER) chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly increased testicular eIF2α and JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by CdCl(2). Interestingly, melatonin almost completely inhibited Cd-induced ER stress and the UPR in testes. In addition, melatonin obviously attenuated Cd-induced heme oxygenase (HO)-1 expression and protein nitration in testes. Taken together, these results suggest that melatonin alleviates Cd-induced cellular stress and germ cell apoptosis in testes. Melatonin may be useful as pharmacological agents to protect against Cd-induced testicular toxicity., (© 2011 John Wiley & Sons A/S.)

Although the effects of Gonadotropin on ovarian physiology have been known for many decades, its action on glucose uptake in the rat ovary remained poorly understood. Evidence also suggests that glucose uptake is mediated by a number of glucose transporter proteins (Glut). Therefore, we examined the rat ovary for the presence of Glut1-4 and blood glucose level after eCG (equine chorionic gonadotropin) and anti-eCG antiserum treatment. All of the glucose transports were present in the ovarian oocyte, granulosa cells and theca cells in different stage follicles. The expression of Glut in ovary was up-regulated by eCG, however, anti-eCG antiserum reversed eCG action. Western blot analysis also demonstrated the content of Glut1 was higher in eCG treatment group compared with anti-eCG antiserum and control group. The same tendency was shown in other glut isoforms. Moreover, there were no significant difference between the anti-eCG antiserum and control group. In additional, the level of serum glucose in eCG treatment group was significantly higher than others, which is similar with glut expression pattern. High glucose level in blood is correlated with increased expression of glucose transporter proteins in rat ovary. Meanwhile, anti-eCG antiserum increased granulosa cell apoptosis in antral follicle compared with those in eCG group. Our observations provide potential explanation for the effects of Glut on follicular development in rat ovary and a role for eCG in the regulation of ovarian glucose uptake.

Cadmium (Cd) is associated with male infertility and poor semen quality in humans. Increasing evidence demonstrates that Cd induces testicular germ cell apoptosis in rodent animals. However, the molecular mechanisms of Cd-induced testicular germ cell apoptosis remain poorly understood. In the present study, we investigated the role of endoplasmic reticulum (ER) stress on Cd-evoked germ cell apoptosis in testes. We show that spliced form of XBP-1, the target of the IRE1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an ER chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly caused eIF2α and JNK phosphorylation in testes, indicating that the unfolded protein response pathway in testes was activated by Cd. Interestingly, phenylbutyric acid (PBA), an ER chemical chaperone, attenuated Cd-induced ER stress and protected against germ cell apoptosis in testes. In addition, PBA significantly attenuated Cd-evoked release of cytochrome c from mitochondria to cytoplasm in testes. Taken together, these results suggest that crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis.

Fenvalerate has a potentially adverse effect on male reproduction and spermatogenesis, whereas the precise mechanism remains obscure. The present study investigated the effects of fenvalerate on germ cell apoptosis in testes. Adult male mice were administered with fenvalerate (15 or 60 mg/kg) by gavage for 28 days. Germ cell apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). The number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with fenvalerate in a dose-dependent manner. TUNEL+ germ cells were observed mainly in stages VII-VIII and also stages IX-XII seminiferous tubules in testes. Additional experiments showed that fenvalerate increased the level of active caspase-8 and caspase-3 in testes. In addition, fenvalerate upregulated the expression of Fas and FasL in testes. No significant difference on the expression of Bcl-2 and Bax in testes was observed between fenvalerate-treated mice and controls. Fenvalerate did not affect the leakage of cytochrome c from mitochondria into cytoplasm. In addition, fenvalerate did not cause the activation of caspase-9 in testes. Taken together, these results suggest that fenvalerate induces germ cell apoptosis in testes through the Fas/FasL signaling pathway.

Bladder cancer is the ninth most common type of cancer, and its surgery is always followed by chemotherapy to prevent recurrence. Berberine is non-toxic to normal cells but has anti-cancer effects in many cancer cell lines. This study was aimed to determine whether berberine inhibits the cell proliferation and induces cell cycle arrest and apoptosis in BIU-87 and T24 bladder cancer cell line. The superficial bladder cancer cell line BIU-87 and invasive T24 bladder cancer cells were treated with different concentrations of berberine. MTT assay was used to determine the effects of berberine on the viability of these cells. The cell cycle arrest was detected through propidium iodide (PI) staining. The induction of apoptosis was determined through Annexin V-conjugated Alexa Fluor 488 (Alexa488) staining. Berberine inhibited the viability of BIU-87 and T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G0/G1 in a dose-dependent manner and induced apoptosis. We observed that H-Ras and c-fos mRNA and protein expressionswere dose-dependently and time-dependently decreased by berberine treatment. Also, we investigated the cleaved caspase-3 and caspase-9 protein expressions increased in a dose-dependent manner. Berberine inhibits the cell proliferation and induces cell cycle arrest and apoptosis in BIU-87, bladder cancer cell line and T24, invasive bladder cancer cell line. Berberine can inhibit the oncogentic H-Ras and c-fos in T24 cells, and can induce the activation of the caspase-3 and caspase-9 apoptosis. Therefore, berberine has the potential to be a novel chemotherapy drug to treat the bladder cancer by suppressing tumor growth., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Thyroid hormone (TH) is important for normal reproductive function. Our previous studies indicate that FSH increases preantral follicle growth in vitro, a response markedly enhanced by triiodothyronine (T3). However, the nature of this hormonal interaction is poorly understood. The objective of this study was to determine if and how T3 modulate FSH-induced expression and actions of granulosa cell intracellular survival and death intermediates. We investigated the possible involvement of Src and PI3K/Akt pathway in the regulation of granulosa cell survival. We demonstrated that, while ineffective alone (0.1-100 nM), T3 markedly enhanced FSH (100 ng/ml)-induced granulosa cell phospho-Src and phospho-Akt contents and Xiap expression in vitro. The effects of T3 were concentration-dependent, with maximal responses at 1.0 nM. FSH alone decreased Fas Ligand (FasL) content irrespective of the presence of T3. Co-treatment of cell with T3 and FSH decreased Fas content, although neither hormone alone elicited a significant response. Taken together, the present study demonstrates that T3 potentiates the cell survival action of FSH through Src- and PI3K-mediated Xiap up-regulation and decreased Fas and FasL expression.

Bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. An earlier study showed that BPA-induced germ cell apoptosis through the Fas/FasL apoptotic pathway. In the present study, we aimed to investigate whether the mitochondrial pathway is also involved in the process of BPA-mediated germ cell apoptosis in testes. Male mice were administered with BPA (160 or 480 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND49. Germ cell apoptosis in testes was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). As expected, the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII-VIII seminiferous tubules in testes. An increase in the level of Fas and FasL was observed in testes of mice exposed to BPA during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. Interestingly, pubertal BPA exposure also caused the translocation of cytochrome c from mitochondria into cytosol. In addition, pubertal BPA exposure upregulated the level of Bax and active caspase-9 in testes. Taken together, these results suggest that pubertal BPA exposure induces germ cell apoptosis in testes through not only the Fas/FasL signaling pathway but also the mitochondrial apoptotic pathway., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: To study the effect of multiglycosides of Tripterygium wilfordii (GTW) on sperm apoptosis in male rats and its possible mechanisms., Methods: Sixteen male SD rats were equally assigned to two groups to receive GTW and carboxymethylcellulose (CMC) intragastrically, both at 20 mg/(kg x d) for 6 weeks. Then the epididymal sperm was collected for the measurement of the apoptosis rate, sperm membrane lipid fluidity and the contents of NO, MDA and SOD by flow cytometry and spectrophotometric determination., Results: After 6 weeks of medication, the GTW group showed a significant increase in sperm apoptosis and contents of NO and MDA (P < 0.01) and a remarkable decrease in sperm membrane lipid fluidity (P < 0.05) and SOD content (P < 0.01) as compared with the CMC control group., Conclusion: GTW can damage sperm membrane lipid peroxidation and sperm membrane structure, increase sperm apoptosis, and reduce sperm membrane lipid fluidity.

Using laser-induced photoactivation of intravenously administered rose Bengal in rats, we generated an ischemic infarction of the intrascleral portion of the optic nerve (ON) comparable to that which occurs in humans to investigate optic nerve axon degenerative events following optic nerve infarct and the potential for axon re-growth. Animals were euthanized at different times post infarct. Axon degeneration was evaluated with SMI312 immunolabeling, and GAP-43 immunostaining was used to identify axon regeneration. Terminal dUTP nick end labeling (TUNEL) was used to evaluate retinal ganglion cell (RGC) death. There was significant axon structural disruptinot ion at the anterior intrascleral portion of the ON by 3d post-infarct, extending to the posterior ON by 7d post-stroke. Destruction of normal axon structure and massive loss of axon fibers occurred by 2 weeks. GAP-43 immunoreactivity occurred in the anterior ON by 7d post-infarct, lasting 3-4 weeks, without extension past the primary ischemic lesion. TUNEL-positive cells in the RGC layer appeared by 7d post-insult. These results indicate that following induction of ischemic optic neuropathy, significant axon damage occurs by 3d post-infarct, with later neuronal death. Post-stroke adult rat retinal ganglion cells attempt to regenerate their axons, but this effort is restricted to the unmyelinated region of the anterior ON. These responses are important in understanding pathologic process that underlies human non-arteritic anterior ischemic optic neuropathy (NAION) and may guide both the appropriate treatment of NAION and the window of opportunity for such treatment., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Our previous reports indicated that cyclic AMP response element-binding (CREB) protein was involved in the regulation of Bcl-2 expression in apoptotic HeLa cells induced by trichosanthin (TCS). Here we presented that blockade the binding site of CREB to Bcl-2 by a CRE decoy oligonucleotide abrogated the TCS-decreased Bcl-2 expression. Furthermore, overexpression of phosphorylated CREB (p-CREB) in cells transfected with p-CREB/GFP fusion construct resulted in an increase of Bcl-2 protein content, however, this increase was attenuated by TCS treatment. Therefore, this data supports the hypothesis that CREB is a possible upstream regulator of Bcl-2 in apoptotic HeLa cells induced by TCS. The study provides new insights into understanding the mechanism of TCS in the treatment of cervical cancer.

Objective: To explore influence of oxidative stress reaction on apoptosis rate and expression of apoptosis-related genes bax and bcl-2 of enterocytes in severely burned rats with delayed resuscitation on the plateau., Methods: One hundred and twenty rats subjected to 30% TBSA full-thickness scald on the back were devided into plateau experimental group (PE, altitude 3840 m) and Lanzhou experimental group (LE, altitude 1517 m). Then LE and PE groups were subdivided into Lanzhou immediate fluid resuscitation group (LIFR, with immediate intraperitoneal injection of isotonic saline after scald, 40 mL/kg), Lanzhou delayed fluid resuscitation group [LDFR, with intraperitoneal injection of isotonic saline at 6 post scald hour (PSH), 40 mL/kg], and plateau immediate fluid resuscitation group (PIFR, with immediate intraperitoneal injection of isotonic saline after scald, 40 mL/kg), plateau delayed fluid resuscitation group (PDFR, with intraperitoneal injection of isotonic saline at 6 PSH, 40 mL/kg). Another 12 rats were divided into Lanzhou sham scald group (LS) and plateau sham scald group (PS), with 6 rats in each group. Rats in LS and PS groups were sham scalded in a water bath for 15 s without fluid infusion. Rats were sacrificed at 6, 12, 24, 48, 72 PSH for collection of small intestine samples to determine the contents of malonaldehyde (MDA) and total hydrosulfide (TSH). The apoptosis of enterocytes was determined by TUNEL, and the expression of bax and bcl-2 in epithelial cells were observed by immunohistochemical method. Intestinal sample of LS and PS groups were collected to determine the contents of MDA and TSH., Results: After being scalded, content of MDA in intestinal tissue of rats in LDFR group and PDFR group was respectively greater than that in LIFR group and PIFR group (P < 0.05 or P < 0.01). Intestinal tissue content of MDA of rats in LDFR group (9.8 +/- 4.0 nmol/mg) and PDFR group (10.2 +/- 1.3 nmol/mg) was respectively greater than that in LIFR group (9.5 +/- 2.7 nmol/mg) and PIFR group (9.6 +/- 1.1 nmol/mg) (P < 0.05) at 24 PSH. After being scalded, intestinal content of TSH of rats in PE group and PDFR group was respectively smaller than that in LE group and LIFR group (P < 0.05). A multitude of brown positive apoptotic cells were observed in PDFR at 6 and 12 PSH. Absorbance values in LDFR group, PIFR group, and PDFR group were higher than that in LIFR group at each time point (P < 0.05 or P < 0.01). A multitude of bax positive cells were observed in intestinal mucous membrane in PDFR at 6 and 12 PSH. Expression of bal-2 was negative in PE group, and LIFR and LDFR groups at 6, 12 PSH, and it was weakly positive in LIFR and LDFR groups at 24, 48, 72 PSH., Conclusions: Enhancement of bax expression and weakening of bcl-2 expression in enterocytes are induced by the severe oxidative stress reaction in intestinal mucous membrane after burn with delayed resuscitation on the plateau, which may be one of the important reasons in inducing an increase in apoptosis of enterocytes.

Ischemic manifestations of atherosclerosis are mainly due to thrombus formation upon superficially eroded (denudation of luminal endothelium) plaques or deeply ruptured (fibrous cap rupture) plaques. Eroded plaques, atherosclerotic plaques without rupture, are found common in young patients of sudden death with coronary thrombosis. Our study aimed to investigate the role of endothelial cell (EC) apoptosis in eroded plaque with thrombosis using an atherosclerotic rabbit model. Atherosclerotic plaques were established by post-balloon-injury high-cholesterol feeding in 33 rabbits. After three-month feeding, a 2-cm segment of plaque-rich abdominal aorta for each animal was clamped in vivo and filled with staurosporine, which induces endothelial apoptosis, or saline for 20 minutes. Three days later, serum lipids and high sensitive C-reactive protein (hs-CRP), a valuable inflammatory parameter, were quantified, and abdominal aorta angiography was conducted. In addition, immunohistochemistry staining was performed for all processed aortae. In the staurosporine-treated aorta, endothelium integrity of plaques was disrupted partially or in large areas, but fibrous cap rupture was not observed, the findings of which were similar to eroded plaques detected in human subjects. As compared to saline controls, staurosporine-treated rabbits showed higher apoptosis scores and thrombotic scores, and more angiographic overt thrombosis and histological thrombosis (P < 0.01, respectively), despite the similar serum levels of lipids and hs-CRP. We further confirmed that apoptosis score was linearly associated with thrombotic score. These results suggest that endothelial apoptosis may be an independent risk factor for thrombosis. In conclusion, the increase in endothelial apoptosis is involved in the formation of thrombotic eroded plaques.

Background and Objective: The role of apoptosis in lung destruction in emphysema/COPD is increasingly being recognized. The relationship between anti-oxidants and alveolar septal cell apoptosis in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of the anti-oxidant, N-acetylcysteine (NAC), on the development of emphysema and alveolar septal cell apoptosis in smoking-induced COPD in rats., Methods: Sprague-Dawley rats (n = 48) were randomly assigned to normal, COPD, sham and NAC groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) in BAL fluid by ELISA, VEGF and VEGF receptor-2 (VEGFR2) protein expression by western blotting, and the apoptotic index (AI) of alveolar septal cells by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. Histopathological evaluations (mean linear intercept (MLI), destructive index (DI)) and lung function measurements were performed., Results: FEV(0.3)/FVC and PEF were lower in the COPD group than in the normal group. MLI and DI were lower in the NAC-treated group than in the COPD or sham-treated groups. As confirmed by western blotting, the levels of VEGF in BAL fluid were higher in the NAC-treated group than in the COPD group. VEGFR2 protein expression was higher in the NAC-treated group than in the COPD group. The AI was significantly lower in the NAC-treated group than in the COPD group. There was an inverse correlation between levels of VEGF in BAL fluid and the AI of alveolar septal cells., Conclusions: NAC attenuates lung damage, pulmonary emphysema and alveolar septal cell apoptosis by partly reversing the decrease in VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

Background: Recent studies have revealed that lung cell apoptosis plays an important role in pathogenesis of cigarette-induced chronic obstructive pulmonary disease (COPD). Tumor necrosis factor alpha (TNF-alpha) is one of the most important cytokines which are involved in COPD. This study aimed at investigating the influence of its inhibitor, recombinant human necrosis factor-alpha receptor II:IgG Fc fusion protein (rhTNFR:Fc) on alveolar septal cell apoptosis in passive smoking rats., Methods: Forty-eight rats were randomly divided into a normal control group, a passive smoking group, an rhTNFR:Fc intervention group and a sham intervention group. The passive smoking rats were treated by exposure to cigarette smoking daily for 80 days. After smoking for one month the rhTNFR:Fc intervention group was treated with rhTNFR:Fc by subcutaneous injection, the sham intervention group injected subcutaneously with a neutral preparation (normal saline 0.1 ml, manicol 0.8 ml, cane sugar 0.2 mg, Tris 0.024 mg as a control. Lung function was determined and the levels of TNF-alpha in serum and broncho-alveolar lavage fluid (BALF) were measured with enzyme-linked immunosorbnent assay (ELISA). Lung tissue sections stained by hematoxylin and eosin (HE) were observed for study of morphological alternations. Mean linear intercept (MLI) and mean alveolar numbers (MAN) were measured and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was carried out to determine the percentage of positive cells and distribution of apoptotic cells., Results: Increased MLI and decreased MAN were found in the passive smoking group compared with both the normal control group and the rhTNFR:Fc intervention group (P < 0.05). Forced expiratory volume in 0.3 second (FEV 0.3)/forced vital capacity (FVC) and peak expiratory flow (PEF) were lower in the passive smoking group than that in the normal control group (P < 0.05). Compared with the sham intervention group, FEV 0.3/FVC and PEF increased in the rhTNFR:Fc intervention group (P < 0.05). The levels of TNF-alpha in serum were higher in the passive smoking group than that in the normal control group (P < 0.05) and rhTNFR:Fc intervention group (P < 0.05). Significant differences were found between the levels of TNF-alpha in the serum of the rhTNFR:Fc intervention group and sham intervention group (P < 0.05). The levels of TNF-alpha in BALF were higher in the passive smoking group than that in the normal control group (P < 0.05), but no significant differences of TNF-alpha levels in BALF were found between the passive smoking group and rhTNFR:Fc intervention group. The number of TUNEL positive cells in alveolar septa was significantly increased in the passive smoking group as compared with the normal control group and the rhTNFR:Fc intervention group (P < 0.05)., Conclusion: This study provides preliminary evidence that rhTNFR:Fc may interfere with TNF-alpha and reduce alveolar septal apoptosis in smoking rats.

Aim: To investigate the effects of N-acetylcysteine on D-galactosamine (GalN)/ lipopolysaccharide (LPS)-induced apoptotic liver injury in mice., Methods: When given together with a low dose of LPS, GalN highly sensitizes animals to produce apoptotic liver injury with severe hepatic congestion, resulting in rapid death. In the GalN/LPS model, TNF-alpha is the major mediator leading to apoptotic liver injury. Reactive oxygen species (ROS) are involved in GalN-induced sensitization to TNF-alpha-evoked hepatocyte apoptosis. N-acetylcysteine (NAC) is an antioxidant and a glutathione (GSH) precursor. In this study, we investigated the effects of NAC on LPS-induced apoptotic liver injury in GalN-sensitized mice., Results: Pretreatment with NAC significantly reduced GalN/LPS-induced elevation of serum alanine aminotransferase levels. In parallel, GalN/LPS-induced hepatic necrosis and congestion were obviously improved by NAC. Furthermore, NAC pretreatment significantly alleviated GalN/LPS-induced hepatic apoptosis, measured by the inhibition of hepatic caspase-3 activity and attenuation of DNA laddering. NAC pretreatment had no effect on LPS-evoked nitric oxide production in GalN-sensitized mice. Increases in serum TNF-alpha concentration, which were observed in GalN/LPS-treated mice, were not significantly reduced by NAC. Although NAC pretreatment significantly alleviated LPS-induced hepatic GSH depletion, DL-buthionine-(SR)-sulfoximine, an inhibitor of GSH synthesis, did not influence the protective effect of NAC on GalN/LPS-induced apoptotic liver injury., Conclusion: NAC attenuates GalN/LPS-induced apoptotic liver injury via its strong ROS scavenging and anti-apoptotic effects.

Purpose: To elucidate the role of activated microglia in the photoreceptor apoptosis of rd mice by identifying sequential events and factors associated with microglial activation, migration, and cytotoxicity during retinal degeneration., Methods: Photoreceptor apoptosis in rd mice at postnatal days (P)8, 10, 12, 14, 16, and 18 was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglia were identified by CD11b antibody. Expression of chemokine mRNA, including monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T-cell expressed and secreted (RANTES), interferon-gamma-inducible 10-kDa protein (IP-10), and fractalkine in the retina were examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Production of tumor necrosis factor (TNF)-alpha in the dystrophic retina was studied by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry analysis. Microglial expression of TNF-alpha was determined by double immunolabeling., Results: Whereas photoreceptor apoptosis in the rd mice started at P10 and reached a peak at P16, activation and migration of microglial cells were observed at P10 and peaked at P14. The expression of MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES transcripts were noted at P8 and reached a peak at P12. Production of TNF-alpha was noted in the outer nuclear layer (ONL) of the rd mice at P8 and reached a peak at P12. At the peak of microglial activity, TNF-alpha was predominantly expressed in the activated microglial cells in the ONL., Conclusions: Activation of microglia, as well as expression of their signaling molecules (chemokines) and microglia-derived toxic factor (TNF-alpha), coincides with or precedes the occurrence of photoreceptor apoptosis, suggesting activated microglia play a major role in retinal degeneration in rd mice. The chemokines MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES are involved in activation and recruitment of the microglia to the degenerating photoreceptor cell layer. TNF-alpha, produced by the activated microglia, may accentuate the photoreceptor cell death.

We have discovered a spontaneous mutation in the Sprague-Dawley rat with a novel eye phenotype that we have named Nuc1. The Nuc1 mutation behaves as a single semi-dominant locus with an intermediate phenotype in the heterozygotes. Heterozygotes exhibit nuclear cataracts. Homozygous Nuc1 rats are fully viable and have microphthalmia, retinal abnormalities and disruption of lens structure shortly before birth. The homozygous mutant shows no obvious pathology outside of the eye, indicating that the mutation is highly eye specific in its effects. An unusual feature of the mutation is that it prevents the normal programmed loss of nuclei from lens fiber cells, but does not affect the loss of other organelles. TUNEL, light, and electron microscopic studies show normal intact nuclei in lens fibers, in contrast to many other models with degenerate nuclei and unlike normal lenses where no such nuclei remain. The beaded filament protein, filensin, is down-regulated in fibers of Nuc1, while heat shock cognate 70 is up-regulated. Homozygous retinas are thicker than normal, and TUNEL labeling indicates roughly half the number of apoptotic cells compared to a wild-type retina. The transient layer of Chievitz persists in adult Nuc1 retina, indicative of delayed development. Hence, Nuc1 is a novel mutation that could be an eye-specific regulator of apoptosis.

Background: This study aimed to evaluate the relation between apoptosis of enterocytes and oxygen-free radical injury in scalded rats with delayed resuscitation as well as the role of antioxidants in the prevention of enterocyte apoptosis., Methods: For this study, 150 male Wistar rats were divided randomly into four groups representing early resuscitation (ER), delayed resuscitation (DR), N-acetylcysteine (NAC) treatment, and allopurinol (Allo) treatment. The animals were subjected to a 30% total body surface area, full-thickness scald. Fluid therapy was started 6 hours after the injury in the DR and treatment groups. Apoptosis of enterocytes was identified by DNA fragmentation (ap%), DNA agarose gel electrophoresis, and terminal deoxynucleotidyl transferace (TdT)-mediated dUPT-biotin nick end labeling (TUNEL). The contents of malondialdehyde (MDA), total sulfhydryl (TSH), and nonprotein sulfhydryl (NPSH) and the activity of xanthine oxidase in intestinal mucosa were determined after the burn in the four groups., Results: Apoptosis of enterocytes increased significantly in all the groups. The animals in the DR group showed an earlier and greater increase in ap% than the animals in the ER group. Similar results were seen for electrophoresis, TUNEL assay, and levels of MDA, xanthine oxidase (XO), TSH, and NPSH. Treatment with NAC was associated with a decrease in ap% and MDA, but not XO, as compared with the levels in the DR group, whereas treatment with Allo was associated with a decrease in MDA and XO, but not ap%. Delayed resuscitation was associated with significant decreases in TSH and NPSH, as compared with the levels in the ER group, whereas both the NAC and Allo groups had significantly higher levels of TSH and NPSH than the DR group., Conclusions: Significant apoptosis of enterocytes was induced by oxidative stress in the intestinal mucosa after a burn in rats. The findings show that NAC blunted intestinal apoptosis induced by oxygen-free radical, which was generated in the process of ischemia-reperfusion injury after a burn because of delayed resuscitation.