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Understanding how stress hormones induce apoptosis in oviductal epithelial cells (OECs) and mural granulosa cells (MGCs) can reveal the mechanisms by which female stress impairs embryonic development and oocyte competence. A recent study showed that tissue plasminogen activator (tPA) ameliorates corticosterone-induced apoptosis in MGCs and OECs by acting on its receptors low-density lipoprotein receptor-related protein 1 (LRP1) and Annexin A2 (ANXA2), respectively. However, whether tPA is involved in corticotropin-releasing hormone (CRH)-induced apoptosis and whether it uses the same or different receptors to inhibit apoptosis induced by different hormones in the same cell type remains unknown. This study showed that CRH triggered apoptosis in both OECs and MGCs and significantly downregulated tPA expression. Moreover, tPA inhibits CRH-induced apoptosis by acting on ANXA2 in both OECs and MGCs. While ANXA2 inhibits apoptosis via phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling, LRP1 reduces apoptosis via mitogen-activated protein kinase (MAPK) signaling. Thus, tPA used the same receptor to inhibit CRH-induced apoptosis in both OECs and MGCs, however used different receptors to inhibit corticosterone-induced apoptosis in MGCs and OECs. These data helps understand the mechanism by which female stress impairs embryo/oocyte competence and proapoptotic factors trigger apoptosis in different cell types.

In Brief: Genes expressed in cumulus cells might be used as markers for competent oocytes/embryos. This study identified and validated a new group of cumulus expansion and/or apoptosis-regulating genes, which may be used for selection of quality oocytes/embryos., Abstract: Studies on the mechanisms behind cumulus expansion and cumulus cell (CC) apoptosis are essential for understanding the mechanisms for oocyte maturation. Genes expressed in CCs might be used as markers for competent oocytes and/or embryos. In this study, both in vitro (IVT) and in vivo (IVO) mouse oocyte models with significant difference in cumulus expansion and CC apoptosis were used to identify and validate new genes regulating cumulus expansion and CC apoptosis of mouse oocytes. We first performed mRNA sequencing and bioinformatic analysis using the IVT oocyte model to identify candidate genes. We then analyzed functions of the candidate genes by RNAi or gene overexpression to select the candidate cumulus expansion and CC apoptosis-regulating genes. Finally, we validated the cumulus expansion and CC apoptosis-regulating genes using the IVO oocyte model. The results showed that while Spp1, Sdc1, Ldlr, Ezr and Mmp2 promoted, Bmp2, Angpt2, Edn1, Itgb8, Cxcl10 and Agt inhibited cumulus expansion. Furthermore, Spp1, Sdc1 and Ldlr inhibited CC apoptosis. In conclusion, by using both IVT and IVO oocyte models, we have identified and validated a new group of cumulus expansion and/or apoptosis-regulating genes, which may be used for selection of quality oocytes/embryos and for elucidating the molecular mechanisms behind oocyte maturation.

Background and Aims: Previous reports have shown that preventing excessive intestinal epithelial cell (IEC) apoptosis is a crucial approach for protecting the intestinal barrier in patients with Crohn's disease (CD). Magnolin (MGL) has various biological activities, including antiapoptotic activities, but its role in CD has largely not been determined. This study investigated how MGL impacts CD-like colitis and the underlying mechanism involved., Methods: Mice were treated with TNBS to establish a disease model, and these mice were used to assess the therapeutic effects of MGL on CD-like colitis. TNF-α-treated colon organoids were used to evaluate the impact of MGL on intestinal barrier function and IEC apoptosis. Enrichment analysis was performed to examine the potential pathways through which MGL inhibits IEC apoptosis. Finally, rescue experiments showed the mechanism by which MGL suppresses IEC apoptosis., Results: The animal experiments demonstrated that MGL treatment alleviated the weight loss, colon shortening, elevated disease activity index (DAI) scores, increased colitis histological scores and upregulated inflammatory factor expression that were observed in model mice. MGL ameliorated intestinal barrier dysfunction and the loss of tight junction (TJ) proteins (ZO-1 and Claudin-1) by inhibiting IEC apoptosis in both TNBS-treated mice and TNF-α-treated colon organoids. MGL inhibited the PI3K/AKT signalling pathway, thus safeguarding the intestinal barrier and alleviating CD-like colitis in vivo and in vitro., Conclusions: MGL improves the intestinal barrier integrity and prevents CD-like colitis by inhibiting IEC apoptosis. The potential mechanism of its anti-apoptotic impact on IECs could be associated with the PI3K/AKT pathway, presenting novel approaches and avenues for the clinical management of CD., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Hyperuricemia frequently accompanies dyslipidemia, yet the precise mechanism remains elusive. Leveraging cellular metabolomics analyses, this research probes the potential mechanisms wherein hyperuricemia provokes endothelial cell abnormalities, inducing disordered bile metabolism and resultant lipid anomalies., Methods and Results: We aimed to identify the differential metabolite associated with lipid metabolism through adopting metabolomics approach, and thereafter adequately validating its protective function on HUVECs by using diverse assays to measure cellular viability, reactive oxygen species, migration potential, apoptosis and gene and protein levels of inflammatory factors. Taurochenodeoxycholic acid (TCDCA) (the differential metabolite of HUVECs) and the TCDCA-involved primary bile acid synthesis pathway were found to be negatively correlated with high UA levels based on the results of metabolomics analysis. It was noted that compared to the outcomes observed in UA-treated HUVECs, TCDCA could protect against UA-induced cellular damage and oxidative stress, increase proliferation as well as migration, and decreases apoptosis. In addition, it was observed that TCDCA might protect HUVECs by inhibiting UA-induced p38 mitogen-activated protein kinase/nuclear factor kappa-B p65 (p38MAPK/NF-κB p65) pathway gene and protein levels, as well as the levels of downstream inflammatory factors., Conclusion: The pathogenesis of hyperuricemia accompanying dyslipidemia may involve high uric acid levels eliciting inflammatory reactions and cellular damage in human umbilical vein endothelial cells (HUVECs), mediated through the p38MAPK/NF-κB signaling pathway, subsequently impinging on cellular bile acid synthesis and reducing bile acid production., Competing Interests: Declaration of competing interest No author has any conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sevoflurane (SEV), usually causing neuronal damage and cognitive dysfunction, is one of the most commonly used anesthetics in clinical practice. However, the function of Trim47 in SEV-induced neuronal impairment remains elusive. The aim of this study was to study the effect of knocking down Trim47 on the nerve injury induced by SEV. Nerve injury was induced in rats by 3% SEV, and H19-7 was used to establish a pathological model, and sh-Trim47 was transfected into H19-7 to study the function of Trim47. The effects of SEV on the expression of Trim47 in the hippocampus and cognitive function of rats were studied by neurological function score and Moris water maze (MWM). The mRNA and protein expression of TNF-α, IL-1β and IL-6 in the cells, along with the neuronal apoptosis in the hippocampus of rats in each group were studied by TUNEL or WB. Flow cytometry was used to study the effect of knockdown of Trim47 on cell apoptosis. CCK-8 was used to detect cell viability of H19-7 cells. Finally, the potential signaling pathway affected by knockdown of Trim47 after abrogation of SEV induction was investigated by WB. The results showed that, knockdown of Trim47 ameliorated SEV-induced neurological damage and cognitive deficits, inflammation and neuronal cell apoptosis in rats, and promoted hippocampal neuronal activity. Knockdown of Trim47 can inhibit the NF-κB signaling pathway and improve neuronal cell damage and cognitive impairment induced by SEV in neonatal rats by regulating NF-κB signaling pathway, alleviating inflammatory response, and inhibiting neuronal apoptosis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

GPRASP2 is implicated in nervous system diseases, tumors and immune inflammation. In our previous study, G protein-coupled receptor associated sorting protein 2 (GPRASP2) was identified as a novel causal gene for X-linked recessive syndromic hearing loss (SHL). However, the role of GPRASP2 in auditory function has not been elucidated. The Gprasp2-knockout (KO) mouse HEI-OC1 auditory cells were constructed using CRISPR/Cas9-mediated gene editing. RNA-sequencing (RNA-seq) was used to investigate the differentially expressed genes (DEGs) and DEGs-enriched signaling pathways, which was verified by Western blot. Flow cytometry assay was used to examine cell apoptosis. The cytological pathology was evaluated by laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Mitochondrial damage was observed in Gprasp2-KO HEI-OC1 cells. RNA-seq analysis suggested that Gprasp2-KO was implicated in the apoptosis process, which could be mediated by Hedgehog (Hh) signaling pathway. The key molecules in Hh signaling pathway (Smo, Gli1, Gli2) were detected to be down-regulated in Gprasp2-KO HEI-OC1 cells. The differential expression of apoptosis molecules (Bcl2, Bax, Caspase-3/cleaved-Caspase-3) indicated that Gprasp2-KO induced apoptosis in HEI-OC1 cells. The treatment of smoothened agonist (Purmorphamine, PUR) activated the Hh-Gli signaling pathway and reduced apoptosis in Gprasp2-KO HEI-OC1 cells. This study revealed that Gprasp2-disruption inhibited Hh signaling pathway and led to cell apoptosis in HEI-OC1 cells, which might provide the potential molecular mechanism of GPRASP2 mutation associated with human SHL., Competing Interests: Declaration of competing interest The authors declare that no conflict of interests exists., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Many long non-coding RNAs (lncRNAs) can exert crucial roles in the pathogenesis of cataract, including lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1). We aimed to further elucidate the biological role and regulatory molecular mechanism of KCNQ1OT1 in cataract. The expression of KCNQ1OT1 and miR-223-3p and BCL2 like 2 (BCL2L2) was examined by qRT-PCR. Cataract cell model was constructed by treatment with hydrogen peroxide (H 2 O 2 ) in lens epithelial cells (SRA01/04). SRA01/04 cell viability and cell apoptosis were tested using CCK-8 assay and flow cytometry, respectively. Western blot (WB) was performed to measure the levels of apoptosis-related proteins and BCL2L2 protein. The oxidative stress factors were analyzed by corresponding kits. The interaction between miR-223-3p and KCNQ1OT1 or BCL2L2 was validated by dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. We found that KCNQ1OT1 was upregulated in cataract anterior lens capsule samples and H 2 O 2 -induced SRA01/04 cells. Knockdown of KCNQ1OT1 suppressed H 2 O 2 -induced SRA01/04 cell apoptosis and oxidative stress. KCNQ1OT1 acted as a sponge of miR-223-3p. Inhibition of miR-223-3p could abate the function of KCNQ1OT1 silence in H 2 O 2 -treated SRA01/04 cells. Additionally, BCL2L2 was a direct target of miR-223-3p, and miR-223-3p weakened H 2 O 2 -induced SRA01/04 cell apoptosis and oxidative stress by targeting BCL2L2. Collectively, the data suggest a role for the KCNQ1OT1/miR-223-3p/BCL2L2 axis in cataract formation but the data was generated using an epithelial cell line., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancers. Chemotherapy is the most important therapeutic option for TNBC, and chemotherapy-induced nausea and vomiting (CINV) is inevitable. Metoclopramide is a good and cost-effective therapeutic option for chemotherapy-induced nausea and vomiting. However, it is not commonly used in breast cancer because it can increase serum prolactin levels by blocking dopamine D2 receptor. This study aimed at elucidating the effect of metoclopramide on triple-negative breast cancer, MDA-MB-231 cells were treated with various concentrations of metoclopramide, the cell proliferation was detected by MTT method, the apoptosis rate was detected by Annexin V/PI double staining method, the expression change of death-related protein was detected by Western Blot. We found that metoclopramide inhibits cell proliferation and induces cell apoptosis of MDA-MB-231 in a concentration-dependent manner, and the Bcl family was involved in this process.

Liver damage is a common liver disorder, which could induce liver cancer. Oral antioxidant is one of the effective treatments to prevent and alleviate liver damage. In this study, three flavonoids namely myricetin, isoquercitrin, and isorhamnetin were isolated and identified from Laba garlic. The isolated compounds were investigated on the protective effects against H 2 O 2 -induced oxidative damages in hepatic L02 cells and apoptosis inducing mechanism in hepatic cancer cells HepG2 by using MTT assay, flow cytometry and western blotting analysis. Myricetin, isoquercitrin, and isorhamnetin showed proliferation inhibition on HepG2 cells with IC 50 value of 44.32 ± 0.213 µM, 49.68 ± 0.192 µM, and 54.32 ± 0.176 µM, respectively. While they showed low toxicity on normal cell lines L02. They could significantly alleviate the oxidative damage towards L02 cells (P < 0.05), via inhibiting the morphological changes in mitochondria and upholding the integrity of mitochondrial structure and function. The fluorescence intensity of L02 cells pre-treated with myricetin, isoquercitrin, and isorhamnetin (100 µM) was 89.23 ± 1.26%, 89.35 ± 1.43% and 88.97 ± 0.79%, respectively. Moreover, the flavonoids could induce apoptosis in HepG2 cells via Bcl-2/Caspase pathways, where it could up-regulate the expression of Bax and down-regulate the expression of Bcl-2, Bcl-xL, pro-Caspase-3, and pro-Caspase-9 proteins in a dose dependent manner. Overall, the results suggested that the flavonoids from Laba garlic might be a promising candidate for the treatment of various liver disorders. PRACTICAL APPLICATION: Flavonoids from Laba garlic showed selective toxicity towards HepG2 cells in comparison to L02 cells via regulating Bcl-2/caspase pathway. Additionally, the isolated flavonoids expressively barred the oxidative damage induced by H 2 O 2 in L02 cells. These results suggested that the flavonoids from laba garlic could be a promising agent towards the development of functional foods., (© 2021 Institute of Food Technologists®.)

The high-mortality rate of cardiovascular diseases (CVDs) is associated with the myocardial ischemia and reperfusion (I/R). Recent investigations have revealed that microRNAs (miRNAs) exert vital functions in the apoptosis of cardiomyocyte cell. Nevertheless, the potential role of miR-30a-5p in the regulation of cardiomyocyte cell apoptosis needs to be illuminated. In the current study, we observed that hypoxia/reoxygenation (H/R) remarkably raised the level of miR-30a-5p but reduced the expression of E2F transcription factor 3 (E2F3) in H9c2 cardiomyocytes. In vivo, miR-30a-5p was found to be significantly upregulated in the hearts of rats following I/R. Downregulation of miR-30a-5p using anti-miR-30a-5p decreased H9c2 cardiomyocytes apoptosis caused by H/R and promoted the proliferation of H9c2 inhibited by H/R. Moreover, E2F3 was a possible target gene of miR-30a-5p and upregulation of miR-30a-5p reduced the expression level of E2F3 in H9c2 cardiomyocytes. We further identified that E2F3 silencing reversed the effect of anti-miR-30a-5p on the proliferation and apoptosis in H/R treated H9c2 cells. These studies suggested that downregulation of miR-30a-5p attenuated the impact of H/R on H9c2 cardiomyocytes through targeting E2F3., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Preeclampsia (PE) is a hypertensive disorder that occurs during pregnancy. Low-dose aspirin is used to reduce the occurrence of early-onset PE; however, the mechanisms are not clear. The aim of this study was to reveal the underlying mechanism of aspirin in reducing sFlt-1-mediated apoptosis of trophoblast cells in PE. Serum sFlt-1 and sEng profiles and placental oxidative stress levels were significantly decreased in PE patients treated with aspirin compared with untreated patients without it, whereas serum PLGF and placental SOD profiles were increased in PE patients with aspirin. Aspirin attenuated the role of sFlt-1 in oxidative stress and endothelial dysfunction and reduced apoptosis of trophoblasts by inactivating the NF-κB signalling pathway in HTR-8/SVneo trophoblast cells. Blood pressure, urine protein, swelling of the villous vessels and mitochondrial parameters were noted to be much better after aspirin administrated to sFlt-1 treated pregnant mice. In conclusion, aspirin reverses the endothelial dysfunction and oxidative stress caused by sFlt-1 and thus reduces apoptosis of preeclamptic trophoblasts by inactivating NF-κB signalling pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Long non-coding RNAs (lncRNAs) recently have been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Here we identified LOC285194 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of LOC285194 was dramatically down-regulated in a aortic atherosclerotic plaques of well-defined model of apolipoprotein-E knockout (ApoE -/- ) mice. Moreover, we found that targeting LOC285194 results in neointimal hyperplasia in vivo in carotid artery injury model. We also showed that targeting LOC285194 promotes cell proliferation and inhibits apoptosis in vascular smooth muscle cells (VSMCs) in vitro, and vice versa . In addition, targeting LOC285194 promotes cell invasion and migration in vitro. Our studies identify LOC285194 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

Background and Purpose: Endothelium exerts an important role in releasing vasoactive substances, maintaining the blood flow, regulating the growth of vessels, moderating the process of coagulation, and the balance of fibrinolytic system, the dysfunction of which is reported to result in arterial stiffness. The present study aimed to investigate the effects of loxoprofen sodium against HUVECs injury induced by angiotensin II., Methods: The injury model on HUVECs was established through incubation with angiotensin II. The expression levels of AT2R, NOX-4, Bax, Bcl-2, and caspase-3 were evaluated using qRT-PCR and Western Blot. DCFH-DA assay was used to detect the production of ROS and ELISA assay was used to evaluate the level of reduced glutathione. Mitochondrial membrane potential (MMP) was measured using dihydrorhodamine 123 assay. MTT and LDH assays were utilized to determine the proliferation ability of HUVECs. The apoptosis rate of HUVECs was evaluated using flow cytometry., Results: Loxoprofen sodium suppressed endothelial AT2R elevation by angiotensin II. Loxoprofen ameliorated Angiotensin II-induced production of ROS, reduced GSH, and NOX-2 and NOX-4 expression. Furthermore, Loxoprofen mitigated Angiotensin II, reduced mitochondrial membrane potential and improved cell viability, and suppressed LDH release by angiotensin II. Importantly, loxoprofen showed a beneficial role in protecting endothelial apoptosis by mitigating apoptotic machinery including the balanced expression of Bax, Bcl-2, and caspase-3 cleavage., Conclusion: Loxoprofen sodium might alleviate the high ROS levels and apoptosis induced by angiotensin II in HUVECs., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Ji et al.)

Local action of nicotine on oral mucosa contributes to the pathogenesis of precancerous and cancerous lesions. Nicotine participation in the mechanism of apoptosis in normal mucosa has not been established. Peroxiredoxin 1 (Prx1) is a cellular antioxidant that participates in regulating apoptosis. We investigated expression of Prx1 and proteins in apoptosis-related downstream signaling by mitogen-activated protein kinases (MAPKs) in nicotine-treated tongue tissues of wild-type and Prx1 knockout (Prx1 ± ) mice; we also investigated these processes in mouse embryonic fibroblast (MEF) cells in vitro. Nicotine increased the expression of Prx1 mRNA in tongue tissues in vivo. The rate of apoptosis was similar among the nicotine-treated mice, nicotine-treated + Prx1 ± mice and untreated controls. The expression of p-JNK was greater in Prx1 ± mice compared to control mice. In MEF cells, nicotine increased the expression of Prx1 and inhibited apoptosis and expression of p-p38 and p-JNK. Prx1 knockdown animals exhibited increased apoptotic rate and expression of p-p38 and p-JNK in MEFs. Nicotine-regulated apoptosis might occur via a Prx1-dependent pathway.

Continuous neuronal survival is vital for mammals because mammalian brains have limited regeneration capability. After neurogenesis, suppression of apoptosis is needed to ensure a neuron's long-term survival. Here we describe a robust genetic program that intrinsically attenuates apoptosis competence in neurons. Developmental downregulation of the splicing regulator PTBP1 in immature neurons allows neural-specific splicing of the evolutionarily conserved Bak1 microexon 5. Exon 5 inclusion triggers nonsense-mediated mRNA decay (NMD) and unproductive translation of Bak1 transcripts (N-Bak mRNA), leading to suppression of pro-apoptotic BAK1 proteins and allowing neurons to reduce apoptosis. Germline heterozygous ablation of exon 5 increases BAK1 proteins exclusively in the brain, inflates neuronal apoptosis, and leads to early postnatal mortality. Therefore, neural-specific exon 5 splicing and depletion of BAK1 proteins uniquely repress neuronal apoptosis. Although apoptosis is important for development, attenuation of apoptosis competence through neural-specific splicing of the Bak1 microexon is essential for neuronal and animal survival., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is a common chronic degenerative joint disease, and chondrocyte apoptosis is one of most important pathological changes of OA pathogenesis. Growing studies have shown that Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an important epigenetic regulatory factor that regulates cell proliferation and apoptosis of various tumors, but its role in OA remains ill-defined. In the present study, we found that UHRF1 expression was increased in human OA cartilage tissues, compared with normal cartilage tissues. Interleukin-1β (IL-1β), a major inflammatory cytokine that promotes cartilage degradation in OA, was used to stimulate primary human chondrocytes in vitro. The expression of UHRF1 was also enhanced in IL-1β-induced chondrocytes. Moreover, down-regulation of UHRF1 induced an increase on cell proliferation and autophagy, and a decrease on apoptosis of chondrocytes after IL-1β treatment. Further data indicated that silencing UHRF1 attenuated the up-regulation of IL-1β on phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway in chondrocytes. Then, an activator of PI3K weakened the effect of UHRF1 silencing on cell proliferation, autophagy, apoptosis of IL-1β-induced chondrocytes, and the cell autophagy special inhibitor 3-methyladenine (3-MA) also showed a same impact on UHRF1, hence suggesting that knockdown of UHRF1 enhances cell autophagy to protect chondrocytes from apoptosis in OA through PI3K/AKT/mTOR signaling pathway. In conclusion, our study suggests that UHRF1 may be a potential regulator of chondrocyte apoptosis in the pathogenesis of OA., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanisms by which female stress and particularly glucocorticoids impair oocyte competence are largely unclear. Although one study demonstrated that glucocorticoids triggered apoptosis in ovarian cells and oocytes by activating the FasL/Fas system, other studies suggested that they might induce apoptosis through activating other signaling pathways as well. In this study, both in vivo and in vitro experiments were conducted to test the hypothesis that glucocorticoids might trigger apoptosis in oocytes and ovarian cells through activating the TNF-α system. The results showed that cortisol injection of female mice (1.) impaired oocyte developmental potential and mitochondrial membrane potential with increased oxidative stress; (2.) induced apoptosis in mural granulosa cells (MGCs) with increased oxidative stress in the ovary; and (3.) activated the TNF-α system in both ovaries and oocytes. Culture with corticosterone induced apoptosis and activated the TNF-α system in MGCs. Knockdown or knockout of TNF-α significantly ameliorated the pro-apoptotic effects of glucocorticoids on oocytes and MGCs. However, culture with corticosterone downregulated TNF-α expression significantly in oviductal epithelial cells. Together, the results demonstrated that glucocorticoids impaired oocyte competence and triggered apoptosis in ovarian cells through activating the TNF-α system and that the effect of glucocorticoids on TNF-α expression might vary between cell types.

Angiogenesis is an important pathway for revascularization of ischemic tissues after acute myocardial infarction (AMI). It is unclear what role CXCR7 plays in angiogenesis in the ischemic area after AMI, although some researchers have shown that the activation of CXCR7 protectsthe heart under those conditions. Here, we hypothesize that the activation of CXCR7 promotes angiogenesis, reduces cell apoptosis and alleviates cardiac deficiency after AMI. C57BL/6 J mice were subjected to AMI and treated with TC14012 (10 mg/kg) for 24 days. HUVECs were cultured in a hypoxic (2% O 2 ) environment to generate a model of hypoxia. CXCR7 was knocked down in HUVECs by sh-CXCR7 transfection, and CXCR7 was activated by TC14012 (30 μM) treatment. The results showed that CXCR7 was downregulated in infarcted heart tissue and hypoxic HUVECs. The global activation of CXCR7 may alleviate the decrease in cardiac function indexes - (ejection fraction and fraction shortening), and reduce infarct size after AMI.. Moreover, CXCR7 activation has been shown to enhance the level of angiogenesis in ischemic heart tissue. In vitro, hypoxia-induced angiogenic functional loss and apoptosis are aggravated by CXCR7 knockdown in HUVECs. Both angiogenic impairment and cell apoptosis are rescued by CXCR7 activation. In conclusion, the present study indicates that activation of CXCR7 plays an important protective role for ischemic cells in hypoxic endothelial cells and AMI model mice by promoting angiogenesis and reducing apoptosis, which suggests that CXCR7 may be a potential therapeutic target to rescue the ischemic myocardium.., Competing Interests: Declaration of Competing Interest The authors did not report any conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Studies have observed that restraint stress (RS) and the associated elevation in corticotrophin-releasing hormone (CRH) impair oocyte competence by triggering apoptosis of ovarian cells but the underlying mechanisms are largely unclear. Although one study demonstrated that RS and CRH elevation triggered apoptosis in ovarian cells and oocytes via activating Fas/FasL signalling, other studies suggested that RS might damage cells by activating other pathways as well as Fas signalling. The objective of this study was to test whether RS and CRH elevation impairs oocytes by activating tumour necrosis factor α (TNF-α) signalling. Our invivo experiments showed that RS applied during oocyte prematuration significantly increased expression of TNF-α and its receptor (TNFR1) while inducing apoptosis in both oocytes and mural granulosa cells (MGCs). Invitro treatment of MGCs with CRH significantly increased their apoptotic percentages and levels of TNF-α and TNFR1 expression. Invitro knockdown by interfering RNA, invivo knockout of the TNF-α gene or injection of TNF-α antagonist etanercept significantly relieved the adverse effects of RS and CRH on apoptosis of MGCs and/or the developmental potential and apoptosis of oocytes. The results suggest that RS and CRH elevation in females impair oocyte competence through activating TNF-α signalling and that a TNF-α antagonist might be adopted to ameliorate the adverse effects of psychological stress on oocytes.

Dioctyl phthalate (DOP) (200, 500, and 1000 mg kg -1 bw, i.g.), Pb (Ac) 2 (50 mg L -1 , p.o.), and NaAsO 2 (10 mg L -1 , p.o.) were administered individually and as mixtures to weanling male mice for 8 weeks. It was observed that Pb, As, and DOP exposure could significantly inhibit the growth and development of mice. Compared with the Pb, As, and Pb + As groups, the activities of iNOS and TNOS were significantly increased, the levels of AChE and SOD were significantly decreased, and the level of MDA was significantly increased in the Pb + DOP-H, As + DOP-H, and Pb + As + DOP-H groups. The factorial analysis shows that the iNOS, TNOS, and AChE present synergistic effects on Pb, As, and DOP. A significant increase of escape latency and a significant decrease of original platform quadrant stops were observed between Pb + As + DOP-H and Pb + As groups. The factorial analysis shows that there was a synergistic effect on Pb, As, and DOP. Compared with that of the control group, the expression levels of caspase-3 and Bax expression in Pb + As, DOP-H, Pb + DOP-H, As + DOP-H, and Pb + As + DOP-H groups were significantly increased in the hippocampus. The expression levels of Bcl-2 expression decreased significantly and the Bax/Bcl-2 ratio increased significantly. Pathological alterations on the hippocampus were found in exposed groups. This result shows that combined exposure of Pb, As, and DOP could induce neurotoxicity, of which possible mechanism is hippocampal neuronal apoptosis. Graphical abstract This study shows that there were three components with eigenvalues greater than 1, which together explained 89.40% of total variance. The first component (PC1) showed high loadings on B-SOD, L-SOD, B-MDA, L-MDA, K-MDA, iNOS, tNOS, and AChE and accounted for 46.55% of the total variance after Varimax rotation. PC2 accounted for 23.81% of the total variance with high loadings on B-As, L-As, K-As, and K-SOD, whereas PC3 showed high loadings on B-Pb, L-Pb, and K-Pb and accounted for 19.04% of the total variance.

Gastric cancer (GC), one of the most common malignant tumors and the second most common leading cause of cancer-related death worldwide, is a biologically heterogeneous disease accompanied by various genetic and epigenetic alterations. However, the molecular mechanisms underlying this disease are complex and not completely understood. Increasing studies have shown that aberrant microRNA (miRNA) expression is associated with GC tumorigenesis and growth. MiR-1297 has been confirmed to be a cancer suppressor in diverse tumors in humans. However, to date, the function and mechanism of miR-1297 in GC have not been determined. Here, we found that the expression of miR-1297 was significantly reduced in GC tissues or GC cell lines compared with paracarcinoma normal tissue or normal cell lines. Exogenic overexpression of miR-1297 in GC cell lines can inhibit cell proliferation and colony formation and induce apoptosis, and inhibition of miR-1297 in GC cell lines can promote cell proliferation and colony formation, and reduce apoptosis in vitro. We further confirmed that miR-1297 acted as a tumor suppressor through targeting cell division control protein 6 (CDC6) in GC. Moreover, the inverse relationship between miR-1297 and CDC6 was verified in GC cell lines. Our results indicated that miR-1297 is a potent tumor suppressor in GC, and its antiproliferative and gene-regulatory effects are, in part, mediated through its downstream target gene, CDC6. These findings implied that miR-1297 might be used as a novel therapeutic target of GC.

The pathogenesis of abdominal aortic aneurysm remains unclear. The aim of the present study was to establish whether isoleucyl-tRNA synthetase (Iars) regulates the differentiation and apoptosis of vascular smooth muscle cells (VSMCs) during the development of abdominal aortic aneurysm (AAA). In addition, the contribution of various signaling pathways towards this process was ascertained. The study demonstrated that the expression of Iars, p-p38, osteopontin (OPN) and Bcl-2-associated X protein (Bax) clearly increased, while levels of p-PI3K and smooth muscle 22 alpha (SM22α) decreased significantly in AAA tissues. Inhibition of Iars significantly reduced the incidence of angiotensin II (AngII)-induced AAA in mice, coincident with decreased activity of the p38 MAPK pathway and increased PI3K pathway activity. AngII-induced phenotypic switching and apoptosis of VSMCs decreased following the inhibition of Iars in vitro. Upregulation of the IARS gene induced phenotypic switching and apoptosis in VSMCs in addition to increased p38 MAPK pathway activation and reduced PI3K pathway activation. Following pretreatment with an activator of the PI3K pathway, expression of Iars and the phenotypic markers of VSMCs were not affected, while apoptosis of VSMCs decreased. Similarly, inhibition of the p38 MAPK pathway in VSMCs did not affect the expression of Iars or the degree of cell apoptosis, but reduced phenotypic switching was observed. Conclusively, upregulation of Iars regulates the phenotypic switching and apoptosis of VSMCs. Targeting Iars may be a promising strategy to prevent abdominal aortic aneurysm., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In our previous study, chondrocyte apoptosis in femoral head necrosis (FHN)-affected broilers was found to be associated with the endoplasmic reticulum stress (ERS) signaling pathway. In the present study, we further explored the role of ERS-induced chondrocyte apoptosis in FHN-affected broilers and the parallel test was carried out with articular chondrocytes cultivated in vitro. The broilers and chondrocytes were treated with methylprednisolone (MP). The main pathological changes in FHN-affected broilers included the proximal femoral head separated from its articular cartilage and growth plate lesions. MP-treated chondrocytes demonstrated morphology changes, cell viability reduction, secretory capacity dysfunction, and apoptosis. The mRNA expressions of pro-apoptotic genes controlled by ERS signaling pathway were up-regulated both in vivo and in vitro experiments. It showed that MP induced FHN in broilers, activated apoptosis-related genes on ERS signaling pathway, and affected the survival and apoptosis of chondrocytes, and bone growth., (© 2018 Poultry Science Association Inc.)

Our previous study showed that the tea extract, epigallocatechin-3-gallate (EGCG), protects against microcystin-LR (MC-LR) -mediated apoptosis of human umbilical vein endothelial cells (HUVECs); however, the mechanism underlying MC-LR-induced HUVEC apoptosis remains incompletely understood. In this study, we investigated whether the nuclear factor erythroid-like 2 (NRF2)/heme oxygenase-1 (HO-1) pathway, which regulates antioxidant transcriptional regulation of oxidative stress and apoptosis, is involved in this process. Mitochondrial membrane potential (MMP) and caspase-3/-9 activities were evaluated in HUVECs by JC-1 staining and colorimetric activity assay, and a DCFH-DA fluorescent probe assay was used to quantitate reactive oxygen species (ROS) generation. The effects of MC-LR, EGCG, NF2, and HO-1 on HUVEC apoptosis were explored by western blotting and small interfering RNA (siRNA) analyses. MC-LR treatment downregulated HUVEC mitochondrial membrane potential, and decreased levels of cytochrome c release and activated caspase-3/-9, ROS generation, consequently inducing HUVEC apoptosis. EGCG treatment attenuated MC-LR-mediated HUVEC oxidative stress and mitochondria-related apoptosis. EGCG induced NRF2/HO-1 expression and activation in MC-LR treated HUVECs, while downregulation of NRF2/HO-1 by specific siRNAs revealed that NRF2/HO-1 signaling was involved in EGCG attenuation of MC-LR-induced HUVEC apoptosis. Our findings indicate that EGCG treatment protects against MC-LR-mediated HUVEC apoptosis via activation of NRF2/HO-1 signaling., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The objective of this study was to explore the role of HOTAIR in the development of cervical cancer, as well as its downstream signaling pathway. We conducted computational analysis, luciferase assay to explore downstream of HOTAIR and miR-331-3p. Real-time PCR and Western blot were carried out to detect the relationship among E7, HOTAIR, miR-331-3p, NRP2, and P53. Finally, MTT assay and flow cytometry analysis were performed to validate the effect of E7 and miR-331-3p on cell apoptosis and proliferation. NRP2 was identified as a virtual target gene of miR-331-3p with a binding site of miR-331-3p, and HOTAIR was directly sponged to miR-331-3p, miR-331-3p reduced luciferase activity of wild-type of NRP2 3'UTR and HOTAIR, but not those of mutant NRP2 3'UTR and HOTAIR. MiR-331-3p down-regulated NRP2 and E7 expression levels, and further promoted cell apoptosis, while inhibited cell proliferation. Cell transfected with HPV16 E7 displayed lower levels of HOTAIR, NRP2 and P53, a higher level of miR-331-3p, over-expression of E7 further repressed cell apoptosis, while improved cell proliferation compared with control. Normal HPV (+) group exhibited a higher miR-331-3p, and lower mRNA levels of HOTAIR and NRP2 than HPV (-) group. According to the result of IHC (immunohistochemistry), we found that NRP2 protein was highly expressed in HPV (-) group compared to that in HPV (+) group. E7-HOTAIR-miR-331-3p-NRP2-E7 formed a regulatory loop, and could be involved in the pathogenesis of cervical cancer., (© 2017 Wiley Periodicals, Inc.)

HG30, a tetrahydroanthraquinone compound isolated from the roots of Prismatomeris connate, was previously shown to inhibit the proliferation of A549 cells. The aim of this study was to evaluate the antitumor activity of HG30 in two non-small cell lung cancer cell lines, A549 and H1299, and to explore potential underlying mechanisms. In cell viability and colony formation assays, HG30 treatment suppressed the proliferation and number of colonies formed by A549 and H1299 cells. Western blot analysis further demonstrated that induction of apoptosis by HG30 in A549 and H1299 cells involves both caspase-dependent apoptosis pathways, including mitochondria- and death receptor-mediated pathways, and an apoptosis-inducing factor (AIF) -associated caspase-independent apoptosis pathway. Specifically, HG30 treatment affected Bcl-2 family proteins and inhibitor of apoptosis protein (IAP) family proteins by down-regulating of Mcl-1, survivin and XIAP and up-regulation of Bid, and Bim., (Copyright © 2018. Published by Elsevier Masson SAS.)

Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12-ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.

Background: Chemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells' fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu's cytotoxicity in vitro and in vivo., Methods: Cell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice., Results: Curcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells., Conclusion: Pre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.

The present study aimed to analyze novel mechanisms underlying Nrf2-mediated anti-apoptosis in periodontal ligament stem cells (PDLSCs) in the periodontitis oxidative microenvironment. We created an oxidative stress model with H₂O₂-treated PDLSCs. We used real-time PCR, Western blotting, TUNEL staining, fluorogenic assay and transfer genetics to confirm the degree of oxidative stress and apoptosis as well as the function of nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrated that with upregulated levels of reactive oxygen species (ROS) and malondialdehyde (MDA), the effect of oxidative stress was obvious under H₂O₂ treatment. Oxidative molecules were altered after the H₂O₂ exposure, whereby the signaling of Nrf2 was activated with an increase in its downstream effectors, heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1) and γ-glutamyl cysteine synthetase (γ-GCS). Additionally, the apoptosis levels gradually increased with oxidative stress by the upregulation of caspase-9, caspase-3, Bax and c-Fos levels in addition to the downregulation of Bcl-2. However, there was no alterations in levels of caspase-8. The enhanced antioxidant effect could not mitigate the occurrence of apoptosis. Furthermore, Nrf2 overexpression effectively improved the anti-oxidative levels and increased cell proliferation. At the same time, overexpression effectively restrained TUNEL staining and decreased the molecular levels of caspase-9, caspase-3, Bax and c-Fos, but not that of caspase-8. In contrast, silencing the expression of Nrf2 levels had the opposite effect. Collectively, Nrf2 alleviates PDLSCs via its effects on regulating oxidative stress and anti-intrinsic apoptosis by the activation of oxidative enzymes., Competing Interests: The authors declare no conflict of interest.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis, accounting for approximately 12-24% of breast cancer cases. Accumulating evidence has indicated that there is no effective targeted therapy available for TNBC. Dipalmitoylphosphatidic acid (DPPA) is a bioactive phospholipid. However, the function of DPPA in the growth of TNBC has not yet been studied. In this study, we employed TNBC cells and a subcutaneous tumor model to elucidate the possible effect of DPPA on tumor growth in TNBC. We showed that DPPA significantly inhibited tumor growth in the mouse subcutaneous tumor model and suppressed cell proliferation and angiogenesis in TNBC tumor tissues. This inhibition was mediated partly by suppressing the expression of cyclin B1 (CCNB1), which directly promoted the accumulation of cells in the G2 phase and arrested cell cycle progression in human TNBC. In addition, the inhibition of tumor growth by DPPA may also be mediated by the suppression of tumor angiogenesis in TNBC. This work provides initial evidence that DPPA might be vital as an anti-tumor drug to treat TNBC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Aims: Asbestos is harmful to human health. However, the pathogenicity of chrysotile is a controversial matter. This study aimed to investigate the apoptosis of a human bronchial epithelial cell line (BEAS-2B) exposed to chrysotile that may function in part through the Fas death receptor pathway., Methods: Cultured human BEAS-2B cells were treated with chrysotile and cell viability was evaluated by CCK-8 assay. The induction of cell apoptosis was evaluated by FACS analysis. mRNA expression levels of Fas, caspase-3, and caspase-8 were evaluated quantitatively by real-time PCR. The expression of Fas, caspase-3, and caspase-8 proteins were evaluated by Western blot. Meanwhile, cells were preincubated with various concentrations of anti-Fas antibody (CH11) and antagonistic anti-Fas antibody (ZB4)., Results: Chrysotile inhibits proliferation, induces apoptosis, and upregulates the expression of Fas, caspase-3, and caspase-8. The role of Fas as a regulator of chrysotile-induced apoptosis in BEAS-2B cells was tested by the prominent increase in and partial blockade of the apoptotic rate with CH11 and ZB4. When CH11 was pretreated, a synergistic effect was apparent on chrysotile-induced apoptosis and the mRNA and protein expression levels of Fas and cleaved caspase-3., Conclusion: Chrysotile causes the apoptosis of BEAS-2B cells via the Fas death receptor pathway. The Fas-mediated apoptosis pathway plays an important role in chrysotile-induced apoptosis of BEAS-2B cells in vitro., (© 2017 S. Karger AG, Basel.)

Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (P<0.01) than those from BALB/c mice by flow cytometry. A total of 15 apoptosis-associated genes including the major components of an intrinsic cell-death pathway were identified from S. japonicum in this study, suggested that a similar apoptosis pathway might occur in S. japonicum. Real-time PCR analyses revealed that the expression levels of most of the tested apoptosis-associated genes, except CASP7, were significantly higher or at the similar level in adult worms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing apoptosis and host interactions in schistosome infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na⁺/H⁺ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells., Materials and Methods: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis., Results: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions., Conclusion: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.

Hypoxia-induced inhibition of apoptosis in pulmonary artery endothelial cells (PAECs) has an important role in pulmonary arterial remodeling leading to aggravated hypoxic pulmonary arterial hypertension. However, the mechanisms involved in the hypoxia-induced inhibition of PAEC apoptosis have not been elucidated. e-selectin and biliverdin reductase (BVR) have been reported to contribute to the cascade of apoptosis in several cell lines but not in PAECs. In the present study, we show that the expression of e-selectin and BVR was both up-regulated by hypoxia in PAECs. Moreover, hypoxia attenuated the decreased cell survival and apoptotic protein expression, and increased DNA fragmentation induced by serum deprivation in the PAECs, which was mediated by the e-selectin/BVR pathway. In addition, by examining the mitochondrial membrane potential and mitochondrial membrane proteins (Bcl-2 and BAX), we show that the mitochondrial-dependent apoptosis pathway was necessary for the e-selectin/BVR pathway inducing the anti-apoptotic effect of hypoxia in PAECs. Taken all together, our data show that the e-selectin/BVR pathway participates in the inhibitory process of hypoxia in PAEC apoptosis which is mediated by the mitochondrial-dependent apoptosis pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Neuronal cell apoptosis is an important pathological change in Alzheimer's disease (AD). Hydrogen sulfide (H(2)S) is known to be a novel gaseous signaling molecule and a cytoprotectant in many diseases including AD. However, the molecular mechanism of the antiapoptosis activity of H(2)S in AD is not yet fully understood. The aim of the present study is to evaluate the inhibitory effects of H(2)S on Abeta (Aβ)-induced apoptosis and the molecular mechanisms underlying primary neuron cells. Our results showed that sodium hydrosulfide (NaHS), a donor of H(2)S, significantly ameliorated Aβ-induced cell apoptosis. NaHS also reversed the Aβ-induced translocation of the phosphatase and tensin homologs deleted on chromosome 10 (PTEN) from the cytosol to the mitochondria. Furthermore, H(2)S increased the level of p-AKT/AKT significantly. Interestingly, the antiapoptosis effects of H(2)S were blocked down by specific PI3K/AKT inhibitor wortmannin. In conclusion, these data indicate that H(2)S inhibits Aβ-induced neuronal apoptosis by attenuating mitochondrial translocation of PTEN and that activation of PI3K/AKT signaling pathway plays a critical role in H(2)S-mediated neuronal protection. Our findings provide a novel route into the molecular mechanisms of neuronal apoptosis in AD., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

The T helper 1 (Th1) polarization plays a critical role in the pathogenesis of a number of inflammatory disorders in the body; the remedies in the correction of polarized Th1 cells are limited. This study aims to investigate the role of T cell immunoglobulin mucin domain molecule 4 (TIM4) in the induction of Th1 cell apoptosis. In this study, polarized Th1 cells were generated from naive Th1 cells from the mouse spleen. Recombinant TIM4 was added to the culture to stimulate the polarized Th1 cells. The apoptosis of Th1 cells was assessed by flow cytometry. The expression of FasL was analyzed by chromatin immunoprecipitation, real time RT-PCR, and Western blotting. The results showed that the polarized Th1 cells expressed high levels of TIM3. After exposure of the polarized Th1 cells to TIM4 in the culture, a complex of TIM3 and TIM4 was detected on the surface of Th1 cells, which induced the Th1 cell apoptosis. The engagement of TIM3 by TIM4 increased p300 phosphorylation in Th1 cells, which further increased the levels of Fas ligand in the cells and induced Th1 cell apoptosis. In conclusion, TIM4 binds TIM3 on the surface of polarized Th1 cells to induce Th1 cell apoptosis, which may contribute to the development of Th2-dominant immune disorders.

Insulin is a protein secreted by pancreatic β-cells, which plays an important role in the regulation of ovarian function. However, the specific molecular mechanism of its function remains largely unknown. This study aimed to assess the effect of insulin on mouse folliculogenesis using an in vitro ovary-culture model. The results demonstrated that insulin promoted the proliferation of ovarian granulosa cells in vitro, and thereby accelerated the progress of folliculogenesis (the percentage of oocytes in cysts declined from 42.6% to 29.3%); however, the percentage of apoptotic oocytes increased after insulin treatment. Further investigation indicated that apoptosis occurred mainly in germ-cell cysts. After 3 days of insulin treatment, oestrogen in the culture medium of mouse ovaries significantly increased (P<0.01), while the lower dose of oestrogen promoted primordial-follicle assembly in vitro. In conclusion, insulin promoted folliculogenesis by facilitating germ-cell apoptosis within the cysts and upregulating oestrogen levels.

Objective: The aim of this study was to determine the changes and underlying mechanisms of erectile organ structure and function in castrated rats. In addition, the regulatory effects of an androgen on autophagy and apoptosis in corpus cavernosum smooth muscle cells (CCSMCs), especially the regulatory effect of androgen on the BECN 1-Bcl-2 interaction, were investigated., Methods: Male Sprague-Dawley rats were divided into three groups (30/group): control group, castration group, and castration with testosterone supplementation group. The erectile function was examined both in vivo and in vitro, by electric stimulation of the cavernous nerve and corpus cavernosum strip bath test, respectively. Transmission electron microscopy, TUNEL assay, Masson's trichrome staining, immunohistochemistry, and western blotting were performed to determine the levels of autophagy and apoptosis, and the structural changes in corpus cavernosum., Results: Compared with control group, the castration group showed (1) lower erectile function: lower intracavernosal pressure/mean arterial pressure ratio, lower systolic and diastolic capability of corporal strips, and reduced expressions of eNOS and nNOS; (2) greater fibrosis: decreased smooth muscle/collagen ratio, lower expression of α-SMA, and higher expression of TGF-β1; (3) inhibited autophagy: decreased autophagosomes, lower expressions of BECN1 and LC3-II; and (4) enhanced apoptosis: higher apoptotic index and decreased Bcl-2/Bax ratio. Testosterone supplementation partially improved the effects of castration., Conclusions: Castration attenuates erectile function and induces corporeal fibrosis by inhibiting autophagy and promoting apoptosis of CCSMCs in rats. Therefore, our study highlights the important role of androgens in maintaining the integrity of the structure and function of corpus cavernosum in rats through counter-regulation of autophagy and apoptosis, mainly by regulating BECN 1-Bcl-2 interaction.

Death signaling provided by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS) are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC), a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI)+ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1), and siRNA-mediated knockdown of RIP3 decreased the annexin V-/PI+ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.

Arsenic trioxide has been successfully used for the treatment of patients with acute promyelocytic leukemia (APL) worldwide. Recently, it has also been further developed to treat solid tumors in clinical trials. However, the therapeutic effects on malignant tumors appeared to be unsatisfactory, as these cells exhibited resistance towards arsenic. In this study, we explored new therapeutic strategies for treatment of human breast cancer MCF-7 cells based on arsenic metabolites. The MCF-7 cells were exposed to three arsenic species, namely, inorganic arsenite (iAs(III)) and its intermediate metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) either alone or in combination with cryptotanshinone (CPT) to establish their anticancer effects against MCF-7 cells. Surprisingly, MCF-7 cells were shown to be resistant to both iAs(III) and CPT when used alone; however, they were shown to be relatively sensitive to treatment when exposed to MMA(III) and DMA(III) alone. Conversely, the combination of MMA(III) with CPT showed significantly enhanced anticancer effects on MCF-7 cells at low doses, but no appreciable effect was observed upon exposure to the other two arsenic species with CPT. In addition, remarkable redistribution of pro-apoptosis related proteins Bax and Bak was observed in the mitochondria, together with activation of poly(ADP-ribose) polymerase (PARP) and caspase-9 after exposure to the combination of MMA(III) with CPT. Furthermore, we clearly found that induction of apoptosis in MCF-7 cells was predominantly triggered by endoplasmic reticulum (ER) stress after exposure to the combination of MMA(III) with CPT.

Multidrug resistance (MDR) has become a significant challenge in chemotherapeutic treatment of cancer. Quercetin, a naturally occurring flavonoid, has been found to possess anti-proliferative, anti-inflammatory and immunoregulatory bioactivities. The present study was performed to examine the effect of quercetin on human leukemic MDR K562/adriamycin (ADR) cells. Treatment of K562/ADR cells with a combination of quercetin and ADR resulted in potentiation of cytotoxicity, which was measured using a cell counting kit-8 assay. Flow cytometric analysis revealed that quercetin dose-dependently promoted cell apoptosis and treatment with a combination of quercetin and ADR caused synergistic enhancement of the apoptotic effect. In addition, treatment of K562/ADR cells with quercetin alone or in combination with ADR resulted in loss of mitochondrial membrane potential, activation of caspase-8, -9 and -3, reduced expression of the anti-apoptotic proteins B-cell lymphoma (Bcl)-2 and Bcl-extra large and enhanced expression of the pro-apoptotic proteins Bcl-2-interacting mediator of cell death, Bcl-2-associated death promoter and Bcl-2-associated X protein in the cells. Furthermore, the combined treatment of quercetin and ADR synergistically increased the expression of phosphorylated (p-)c-Jun N-terminal kinase and p-p38 mitogen-activated protein kinase and decreased the expression of p-extracellular signal-regulated kinase in the K562/ADR cells. In addition, the expression of P-glycoprotein was significantly decreased following treatment with quercetin alone or in combination with ADR. These findings demonstrated that quercetin is important in MDR and may be developed into a new reversal agent for cancer chemotherapy.

The combined subchronic effects of exposure to lead acetate and cadmium chloride on apoptosis protein expression were detected in the liver and kidney of rats to investigate the hazards of environmentally relevant, low-dose exposure to these compounds. The TUNEL assay showed that there were increased numbers of apoptotic cells. Immunohistochemical tests showed increased numbers of positive cells under Bax and caspase-3 protein detection and decreased Bcl-2 protein. Furthermore, mitochondrial injury and increased numbers of apoptotic cells with condensed nuclei were observed by TEM. These results suggested that low-dose exposure to Pb and Cd can cause significant hepatic and renal apoptosis and finally impair their function. Hepatic and renal apoptosis induced by low-dose exposure is associated with mitochondrial injury and changes in levels of apoptogenic proteins, such as Bcl-2, Bax, and caspase-3.

Rationale: Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple pathogeneses. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supranormal Ca2+ influx required for initiating of apoptosis are not understood., Objective: Here, we addressed the role of TRPM2 and its interaction with the short splice variant TRPM2 short variant (TRPM2-S) in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis., Methods and Results: We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma, and this interaction functioned to suppress TRPM2-dependent Ca2+ gating constitutively. Reactive oxygen species production in endothelial cells or directly applying reactive oxygen species induced protein kinase C-α activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The protein kinase C-α-activated phosphoswitch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase., Conclusions: Here, we describe a fundamental mechanism by which activation of the trp superfamily TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves reactive oxygen species-induced protein kinase C-α activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.

Background: Though we have reported the neuroprotective effect of exogenous ActA on oxygen-glucose deprivation (OGD) injury, the endogenous role of Smad6 remains not well understood. Smad6 is an important regulator of the ActA/smads signaling via a negative feedback circuit., Methods: In this study, nerve growth factor (NGF) and OGD were used to stimulate (rat adrenal pheochromocytoma) PC12 cells converting them into neurons to establish an ischemia in vitro model. Combined with the small interfering technology of Smad6 and FCM, Hoechst and Western blot were used to identify the apoptosis rate. The effect of silencing of Smad6 with siRNA was observed., Results: These results showed that the apoptosis rate was 21.54% by 16-h OGD. For the combined Smad6-siRNA, the apoptosis rate was 10.55%., Conclusions: The expression of procaspase-3 protein was increased by Smad6-siRNA.The expression of ActA and p300 was also increased. The apoptosis rate was decreased in the ischemic injury with Smad6-siRNA. At the same time, it provided a reference to study the mechanism of Smad6 and its signaling in response to the acute ischemic damage.

Kaempferol (Kae), a natural flavonoid, is widely distributed in fruits and vegetables. Previous studies have identified Kae as a possible cancer preventive and therapeutic agent. We found Kae to exhibit potent antiproliferation and anti-migration effects in human bladder cancer EJ cells. Kaempferol robustly induced apoptosis in EJ cells in a dose-dependent manner, as evidenced by increased cleavage of caspase-3. Furthermore, we found Kae-induced apoptosis in EJ cells to be associated with phosphatase and the tensin homolog deleted on the chromosome 10 (PTEN)/PI3K/Akt pathway. Kae significantly increased PTEN and decreased Akt phosphorylation. Kae-induced apoptosis was partially attenuated in PTEN-knockdown cells. Our findings indicate that Kae could be an alternative medicine for bladder cancer, based on a PTEN activation mechanism.

A picosecond pulsed electric field (psPEF) is a localized physical therapy for tumors that has been developed in recent years, and that may in the future be utilized as a targeted non‑invasive treatment. However, there are limited studies regarding the biological effects of psPEF on cells. Electric field amplitude and pulse number are the main parameters of psPEF that influence its biological effects. In this study, we exposed HeLa cells to a psPEF with a variety of electric field amplitudes, from 100 to 600 kV/cm, and various pulse numbers, from 1,000 to 3,000. An MTT assay was used to detect the growth inhibition, while flow cytometry was used to determine the occurrence of apoptosis and the cell cycle of the HeLa cells following treatment. The morphological changes during cell apoptosis were observed using transmission electron microscopy (TEM). The results demonstrated that the cell growth inhibition rate gradually increased, in correlation with the increasing electric field amplitude and pulse number, and achieved a plateau of maximum cell inhibition 12 h following the pulses. In addition, typical characteristics of HeLa cell apoptosis in the experimental groups were observed by TEM. The results demonstrated that the rate of apoptosis in the experimental groups was significantly elevated in comparison with the untreated group. In the treatment groups, the rate of apoptosis was greater in the higher amplitude groups than in the lower amplitude groups. The same results were obtained when the variable was the pulse number. Flow cytometric analysis indicated that the cell cycle of the HeLa cells was arrested at the G2/M phase following psPEF treatment. Overall, our results indicated that psPEF inhibited cell proliferation and induced cell apoptosis, and that these effects occurred in a dose-dependent manner. In addition, the results demonstrated that the growth of the HeLa cells was arrested at the G2/M phase following treatment. This study may provide a foundation for further in vivo experiments, and for the potential clinical application of psPEF in the treatment of cervical cancer.

Objective: To investigate the role of p38 mitogen-activated protein kinases (MAPKs) in the apoptosis of human bronchial epithelial cells (BEAS-2B) induced by refractory ceramic fibers (RCFs)., Methods: BEAS-2B cells were exposed to 10, 20, 40, 80, and 160 µg/cm(2) RCF1, RCF2, and RCF3 for 24 h, and the cell viability was measured by CCK-8 assay. BEAS-2B cells were exposed to 20, 40, and 100 µg/cm(2) RCF1, RCF2, and RCF3 for 24 h, and the cell apoptosis rate was measured by flow cytometry. BEAS-2B cells were exposed to 40 µg/cm(2) RCF1, RCF2, and RCF3, and the expression levels of phospho-p38 MAPK and caspase-3 were measured by Western blot. In each of the above treatments, the BEAS-2B cells were divided into positive control, p38 inhibitor SB203580 intervention, and normal groups., Results: As the concentration of RCFs rose, the RCF exposure groups showed decreased cell viability and increased cell apoptosis rate. After SB203580 intervention, the intervention groups (all concentrations of asbestos + SB, 20, 40, 80, and 160 µg/cm(2)RCF1+SB, and 40, 80, and 160 µg/cm(2) RCF2 and RCF3+SB) had significantly increased cell viabilities (P < 0.05), and the intervention groups (asbestos + SB and 20, 40, and 100 µg/cm(2) RCF1, RCF2, and RCF3 + SB) had significantly decreased cell apoptosis rates (P < 0.05). Compared with the normal group, the RCF (40 µg/cm(2)) exposure and positive control groups had significantly increased expression of phospho-p38 MAPK (P < 0.05), and the RCF (40 µg/cm(2)) exposure group had significantly increased expression of caspase-3 (P < 0.05). The intervention groups (asbestos + SB and 40 µg/cm(2) RCF1, RCF2, and RCF3 + SB) had significantly decreased expression of caspase-3 after SB203580 intervention., Conclusion: p38 MAPKs play an important role in RCF-induced apoptosis of BEAS-2B cells.

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.