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Pseudolaric Acid B (PAB), a natural product with remarkable anti-tumor activity, is a starting point for new anticancer therapeutics. We designed and synthesized 27 PAB derivatives and evaluated their anti-proliferative activities against four cancer cell lines: MCF-7, HCT-116, HepG2, and A549. Compared with unmodified PAB, the PAB derivatives showed stronger anti-proliferative activity. The ability of compound D3 (IC 50  = 0.21 μM) to inhibit HCT-116 cells was approximately 5.3 times that of PAB (IC 50  = 1.11 μM) and the antiproliferative action was unrelated to cytotoxicity (SI=20.38), indicating its superior safety profile (PAB; SI=0.95). Compound D3 effectively suppressed the EdU-positive rate and reduced colony formation, arrested HCT-116 cells in the S and G2/M phases and induced apoptosis. In vivo experiments further demonstrated low toxicity of compound D3 while suppressing tumor growth in mice. In summary, given its strong anti-proliferative effect and relative safety, further development of compound D3 is warranted., 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 Inc. All rights reserved.)

The limitations of self-assembled polymeric nanoparticles for cancer therapy, including instability in the bloodstream, non-specific targeting of cancer cells, and unregulated intracellular drug delivery, were effectively addressed by the development of core-shell SNX@PLL-FPBA/mHA NPs. The core was SNX@PLL-FPBA NPs prepared from polylysine conjugated 3-fluoro-4-carboxyphenylboronic acid (PLL-FPBA) self-assembly and SNX encapsulation, while the shell was methacrylate-modified hyaluronic acid (mHA) adhering to the core by electrostatic interactions and subsequently stabilized by photo-crosslinking, without the use of any organic solvent. SNX@PLL-FPBA/mHA NPs exhibited good stability in varying ionic strengths (0-0.30 M NaCl), pH levels (6.8 and 7.4), and plasma environments mimicking the blood, ensuring their efficacy in systemic circulation. The drug delivery from the nanoparticles was highly sensitive to ATP/Hyals stimuli (82 % within 48 h), closely mimicking the intracellular environment of breast cancer cells. The nanoparticles demonstrated good hemocompatibility and non-toxicity towards human skin fibroblasts. Efficient internalization of SNX@PLL-FPBA/mHA NPs by MCF-7 and MDA-MB-231 breast cancer cells was observed by CLSM and flow cytometry. The intracellular ATP/Hyals stimuli triggered the rapid drug delivery and induced cellular apoptosis. Thus, SNX@PLL-FPBA/mHA NPs were a promising drug nanocarrier for breast cancer therapy, offering improved stability, targeted delivery, and controlled drug release to enhance treatment outcomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mcl-1 is a main antiapoptotic protein in acute myeloid leukemia (AML) and is used as a target to develop inhibitors. Currently, potent Mcl-1 inhibitors primarily interact with the P2-P4 pockets of Mcl-1, but pharmacological modulation by targeting the P1 pocket is less explored. We designed a series of 1 H -indole-2-carboxylic acid compounds as novel Mcl-1 inhibitors occupying the P1-P3 pockets and evaluated their Mcl-1 inhibition and apoptosis induction in AML cells. Two-dimensional 15 N-HSQC spectroscopy indicated that 47 ( K i = 24 nM) bound to the BH3 binding groove, occupied the P1 pocket in Mcl-1, and formed interactions with Lys234 and Val249. 47 exhibited good microsomal stability and pharmacokinetic profiles, with low potential risk of cardiotoxicity. 47 inhibited tumor growth in HL-60 and THP-1 xenograft models with growth inhibition rate of 63.7% and 57.4%, respectively. Collectively, 47 represents a novel Mcl-1 inhibitor targeting the P1-P3 pockets with excellent antileukemia effects.

Platelet apoptosis is irreversible under current storage conditions in blood banks. Studies have shown that programmed cell death ligand 1 (PD-L1) in tumour cells is required for neoplastic progression, tumour recurrence and metastasis by regulating apoptosis. However, whether PD-L1 is involved in storage-induced apoptosis in platelets remains poorly understood. In this study, we explored whether PD-L1 on platelets participated in the regulation of storage-induced apoptosis under blood bank conditions, as well as the underlying mechanism. Several apoptotic events in platelets from humans and PD-L1-knockout mice during storage under blood bank conditions were measured. The mechanism by which storage-induced apoptosis was regulated by platelet-intrinsic PD-L1 signalling was further investigated. Our results showed that PD-L1 in platelets progressively decreased. There was a strong negative correlation between platelet PD-L1 expression and the phosphatidylserine (PS) externalization rate and cleaved caspase-3 level and a positive correlation with anti-apoptosis protein Bcl-xl. Ex vivo, PD-L1-/- platelets stored at 22 °C showed rapid apoptosis via an intrinsic mitochondria-dependent pathway over time. Likewise, inhibiting PD-L1 signalling with BMS-1166 accelerated apoptosis by intrinsic mitochondria-dependent pathway. Coimmunoprecipitation analysis revealed that PD-L1 could bind AKT in platelets, and the binding capacity of both showed a progressive decrease with time. Finally, the decrease in PD-L1 expression levels during storage could be attributed to a complex process of progressive secretion. Therefore, platelet PD-L1 inhibits storage-induced apoptosis by sustaining activation of the AKT signalling pathway, which is expected to become a target for alleviating platelet storage lesions (PSLs) under current blood bank conditions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Osteoarthritis (OA) is a common musculoskeletal disorder characterized by chondrocyte apoptosis and extracellular matrix degradation. This study aimed to investigate the role of CCL4/CCR5 in regulating chondrocyte apoptosis and reactive oxygen species (ROS) levels in OA progression., Methods: Bioinformatics analysis was employed to identify CCL4 as the target gene, following which primary chondrocytes were treated with varying concentrations of CCL4. Apoptosis rate of chondrocytes and ROS levels were assessed using flow cytometry. The mechanism by which CCL4 regulated the extracellular matrix was investigated through Western blot and Immunofluorescence analyses. Additionally, maraviroc, a CCR5 inhibitor, was administered to chondrocytes in order to explore the potential signaling pathway of CCL4/CCR5., Results: Our study found that CCL4 was predominantly up-regulated among the top 10 hub genes identified in RNA-sequencing analysis. Validation through quantitative polymerase chain reaction (qPCR) confirmed elevated CCL4 expression in patients with Hip joint osteoarthritis, knee joint osteoarthritis, and facet joint osteoarthritis. The upregulation of CCL4 was associated with an increase in chondrocyte apoptosis and ROS levels. Mechanistically, CCL4, upon binding to its receptor CCR5, triggered the downstream phosphorylation of P65 in the nuclear factor-κB (NF-κB) signaling pathway. In vitro experiments demonstrated that treatment with maraviroc mitigated chondrocyte apoptosis, reduced intracellular ROS levels, and attenuated extracellular matrix degradation., Conclusion: The study highlights the critical role of CCL4/CCR5 in modulating chondrocyte apoptosis and ROS levels in OA progression. Targeting this pathway may offer promising therapeutic interventions for mitigating the pathogenic mechanisms associated with OA., 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 Ltd.. All rights reserved.)

Background: Renal cell carcinoma (RCC) is a common malignancy. Local anesthetics were displayed powerful effects against various cancers. This study aims to probe the functions and molecular mechanism of ropivacaine in RCC., Methods: Different concentrations of ropivacaine were performed to administrate RCC cells including 786-O and Caki-1 cells. Cell viability and cell apoptosis were examined using CCK-8 and flow cytometry, respectively. Cell migration and invasion were determined by transwell assay. RMRP and CCDC65 expression was firstly predicted using TCGA dataset and further validated in RCC cells using qRT-PCR and western blot. The interactions among RMRP, EZH2 and CCDC65 were verified by RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays., Results: Ropivacaine effectively suppressed RCC cell viability, migration and invasion and enhanced cell apoptosis rate. Aberrantly elevated RMRP expression in RCC tissues was predicted by TCGA database. Interestingly, overexpressed RMRP observed in RCC cells could be also blocked upon the administration of ropivacaine. Likewise, RMRP knockdown further strengthened ropivacaine-mediated tumor suppressive effects on RCC cells. In terms of mechanism, RMRP directly interacted with EZH2, thereby modulating the histone methylation of CCDC65 to silence its expression. Moreover, ropivacaine inhibited tumor growth in mice bearing RCC tumor through regulating RMRP/EZH2/CCDC65 axis., Conclusion: In sum up, our work revealed that ropivacaine suppressed capacities of RCC cell viability, migration and invasion through modulating the RMRP/EZH2/CCDC65 axis, which laid the experimental foundation of ropivacaine for clinical application in the future., (© 2023. The Author(s), under exclusive licence to Tehran University of Medical Sciences.)

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

Nickel (Ni) is the most important environmental pollution in the world. Ni has been confirmed to have multi-organ toxicology and carcinogenicity. Recently, Ni also can impair the male reproductive system, however, its precious mechanism still has not been clarified. The current work found that nickel chloride (NiCl 2 ) induced histopathological lesions in testis. And, the Johnsen's score, seminiferous tubule diameter, and spermatogenic epithelium thickness were decreased in NiCl 2 -treated mice. The number of spermatogonium, primary spermatocyte, and round spermatid also were significantly reduced after Ni treatment. Next the potential molecular mechanism was measured. NiCl 2 treatment elevated ROS production in the testis. Additionally, NiCl 2 was found to induce apoptosis with features including up-regulation of Bax, cleaved-caspase-3, cleaved-caspase-8, caspase-9, and caspase-12, while down-regulation of Bcl-2 expression. In the meantime, the marker protein of DNA damage γ-H2AX was significantly increased in NiCl 2 -primed mice testis. To clarify effects of reactive oxygen species (ROS) in apoptosis and DNA damage induced by NiCl 2 , NiCl 2 was used to co-treat antioxidant NAC (N-Acetyl-L-cysteine). NAC weakened ROS production induced by NiCl 2 , and played an inhibition role in apoptosis and DNA damage. Moreover, co-treatment using NiCl 2 and NAC group also eliminated spermatogenesis disorders. In summary, research results reveal the relations of spermatogenesis disorder induced by NiCl 2 with apoptosis and DNA damage mediated by ROS and apoptosis in the testis., 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. Published by Elsevier Inc.)

Background: Cancer stem cells (CSCs) play vital roles in lung adenocarcinoma (LUAD) recurrence, metastasis, and drug resistance. Cuproptosis has provided a novel insight into the treatment of lung CSCs. However, there is a lack of knowledge regarding the cuproptosis-related genes combined with the stemness signature and their roles in the prognosis and immune landscape of LUAD., Methods: Cuproptosis-related stemness genes (CRSGs) were identified by integrating single-cell and bulk RNA-sequencing data in LUAD patients. Subsequently, cuproptosis-related stemness subtypes were classified using consensus clustering analysis, and a prognostic signature was constructed by univariate and least absolute shrinkage operator (LASSO) Cox regression. The association between signature with immune infiltration, immunotherapy, and stemness features was also investigated. Finally, the expression of CRSGs and the functional roles of target gene were validated in vitro ., Results: We identified six CRSGs that were mainly expressed in epithelial and myeloid cells. Three distinct cuproptosis-related stemness subtypes were identified and associated with the immune infiltration and immunotherapy response. Furthermore, a prognostic signature was constructed to predict the overall survival (OS) of LUAD patients based on eight differently expressed genes (DEGs) with cuproptosis-related stemness signature (KLF4, SCGB3A1, COL1A1, SPP1, C4BPA, TSPAN7, CAV2, and CTHRC1) and confirmed in validation cohorts. We also developed an accurate nomogram to improve clinical applicability. Patients in the high-risk group showed worse OS with lower levels of immune cell infiltration and higher stemness features. Ultimately, further cellular experiments were performed to verify the expression of CRSGs and prognostic DEGs and demonstrate that SPP1 could affect the proliferation, migration, and stemness of LUAD cells., Conclusion: This study developed a novel cuproptosis-related stemness signature that can be used to predict the prognosis and immune landscape of LUAD patients, and provided potential therapeutic targets for lung CSCs in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yang, Liu, Yang, Ji, Qin, Deng and Wang.)

Direct activation of the pro-apoptotic protein BAX represents a potential therapeutic strategy to trigger apoptosis in cancer. Herein, structural optimization of the reported BAX trigger site activator BTSA1 turned out into a series of pyrazolone derivatives, where compound 6d exhibited significantly enhanced antiproliferative effects and apoptosis induction ability compared to BTSA1. Mechanism of action studies revealed that compound 6d could initiate the BAX activation cascade, promoting BAX insertion into mitochondrial membranes and activating MOMP, ultimately leading to the release of cytochrome c and apoptosis. Furthermore, 6d showed significantly improved in vitro stability and CYPs profile compared to BTSA1. This work may lay a foundation to develop potent BAX trigger site activators for the treatment of BAX-expressing malignancies., 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 © 2022. Published by Elsevier Masson SAS.)

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant that is widely distributed in the natural environment. Cohort study showed that PFOA-producing workers displayed a significant increase for mortality of liver cancer and liver cirrhosis. However, the underlying mechanism of PFOA-induced hepatotoxicity is far from clear. In this research, cell viability, apoptosis rate, reactive oxygen species, mitochondrial membrane potential (ΔΨm), calcium ion levels, and protein expressions of human liver L02 cells in response to PFOA were determined. Results indicated that a 24 h-treatment with 64 and 256 μM PFOA could remarkably induce mitochondrial-mediated apoptosis via initiating the vicious cycle between endoplasmic reticulum stress and oxidative stress, thereby increasing the level of calcium ion and decreasing the level of ΔΨm, simultaneously elevating the protein expressions of Cyclophilin D (CYPD), Bcl-2 homologous antagonist/killer (Bak), Bcl-2-associated X protein (Bax), Bcl-2-like protein 11 (Bim), cytochrome C (Cyt-C), 78 kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), and thioredoxin-interacting protein (TXNIP), while inhibiting the protein expression of tumor necrosis factor receptor-associated protein 1 (TRAP1), Lon protease 1 (Lonp1), Pro-caspase-9, B-cell lymphoma-2 (Bcl-2), and Sigma 1-type opioid receptor (Sig-1R) (p < 0.05). To sum up, PFOA-induced hepatocellular endoplasmic reticulum stress and mitochondrial-mediated apoptosis in vitro was regulated by endoplasmic reticulum (ER)-mitochondria communication via mitochondria-associated ER membranes (MAMs)., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aim: We evaluated the effect of H3K9ac modification on the transcriptional level of Pik3ca and the apoptosis induction effects of Pik3ca in the PI3K/AKT pathway in rat H9C2 cells., Main Methods: H9C2 cells were cultured with 1uM doxorubicin for 8 h. The acetylation level of histone H3K9 in the transcriptional initiation area of Pik3ca was determined by CHIP-seq. The enrichment of mRNA fragment of Pik3ca transcription initiation region by H3K9ac antibody was detected by CHIP-qPCR, and the expression of Pik3ca was detected by real-time Polymerase Chain Reaction(rt-PCR) and western blot. The transcription efficiency of Pik3ca siRNA was detected by immunofluorescence and western blot. Cell Counting Kit8(CCK8) was used to detect the cell activity and flow cytometry was used to detect the apoptosis rate. Western blot was applied to assess the protein expression level of PI3K, P-AKT, AKT, Bcl2, BAX and cleaved-caspase3 in H9C2 cells., Key Findings: In doxorubicin-inducedH9C2 cells, the acetylation levels of histone H3K9 in the Pik3ca transcriptional initiation region significantly increased and promoted the transcription of Pik3ca. After knockdown of Pik3ca, the PI3K/AKT signaling pathway was inhibited, and the protein expression of Bcl2 was increased, the protein expression of BAX and cleaved-caspase3 were decreased. PI3K/AKT pathway activator partially reversed the effect of silencing Pik3ca., Significance: In summary, the elevated H3K9ac level in the Pik3ca transcriptional initiation region promoted the transcription of Pik3ca, and Pik3ca promoted doxorubicin induced apoptosis in H9C2 cells by activating the PI3K/AKT pathway, providing a new theoretical basis for the study of doxorubicin cardiomyopathy., (Copyright © 2018. Published by Elsevier Inc.)

Myocardial apoptosis contributes to acute ethanol-induced cardiac injury. Improving immoderate apoptosis has become the potential therapeutic strategy for acute ethanol-induced heart damage. Previous studies reported that Tanshinone IIA (Tan IIA), a key ingredient extracted from Salvia miltiorrhiza Bunge, performed an anti-apoptotic role against acute ethanol-related cell damage. In this study, we investigated whether Tan IIA protected the acute ethanol-induced cardiac damage in vivo and in vitro. C57BL/6 mice were treated with acute ethanol and then treated with Tan IIA. The results showed that Tan IIA significantly improved heart function and blocked myocardial apoptosis. Acute ethanol exposure induced H9C2 cells apoptosis. Treatment with Tan IIA abrogated acute ethanol-induced H9C2 cells apoptosis. Mechanistically, Tan IIA inhibited apoptosis by downregulating the programmed cell death protein 4 (PDCD4) expression and activating the phosphoinositide 3-kinase (PI3K)/Akt pathway. Furthermore, PDCD4 overexpression abrogated Tan IIA-mediated anti-apoptotic role and activation on the PI3K/Akt pathway. Interestingly, the PI3K inhibitor (LY294002) application significantly attenuated the main protective effects of Tan IIA. In conclusion, Tan IIA improves acute ethanol-induced myocardial apoptosis mainly through regulating the PDCD4 expression and activating the PI3K/Akt signaling pathway. We provide evidence that Tan IIA is a new treatment approach for acute ethanol-induced heart damage., (© 2021 John Wiley & Sons Ltd.)

Mesenchymal stem cell (MSC)-mediated immunomodulation has been harnessed for the treatment of human diseases, but its underlying mechanism has not been fully understood. Dead cells, including apoptotic cells have immunomodulatory properties. It has been repeatedly reported that the proportion of nonviable MSCs in a MSC therapeutic preparation varied from 5~50% in the ongoing clinical trials. It is conceivable that the nonviable cells in a MSC therapeutic preparation may play a role in the therapeutic effects of MSCs. We found that the MSC therapeutic preparation in the present study had about 5% dead MSCs (DMSCs), characterized by apoptotic cells. Namely, 1 × 10 6 MSCs in the preparation contained about 5 × 10 4 DMSCs. We found that the treatment with even 5 × 10 4 DMSCs alone had the equal therapeutic effects as with 1 × 10 6 MSCs. This protective effect of the dead MSCs alone was confirmed in four mouse models, including concanavalin A (ConA)- and carbon tetrachloride (CCl 4 )-induced acute liver injury, LPS-induced lung injury and spinal cord injury. We also found that the infused MSCs died by apoptosis in vivo. Furthermore, the therapeutic effect was attributed to the elevated level of phosphatidylserine (PS) upon the injection of MSCs or DMSCs. The direct administration of PS liposomes (PSLs) mimic apoptotic cell fragments also exerted the protective effects as MSCs and DMSCs. The Mer tyrosine kinase (MerTK) deficiency or the knockout of chemokine receptor C-C motif chemokine receptor 2 (CCR2) reversed these protective effects of MSCs or DMSCs. These results revealed that DMSCs alone in the therapeutic stem cell preparation or the apoptotic cells induced in vivo may exert the same immunomodulatory property as the "living MSCs preparation" through releasing PS, which was further recognized by MerTK and participated in modulating immune cells., (© 2021. The Author(s).)

Background: Laryngeal squamous cell carcinoma (LSCC) brings a heavy blow to the patient's voice. Transfer RNA (tRNA) is a common RNA, the roles of tRNAs in LSCC are largely unknown., Methods: The tRNA expression profile in LSCC tissues and adjacent normal tissues was measured by a tRNA qRT-PCR array. The expression level of tRNA Ini CAT in LSCC tissues and plasmas was detected by qRT-PCR. The receiver operating characteristic (ROC) curve was established. tRNA Ini CAT was upregulated by a lentivirus vector in the LSCC cell line. Moreover, tRNA Ini CAT was upregulated in LSCC xenograft nude mouse model and the xenografts were used for pathological analysis and transmission electron microscope (TEM) observation., Results: The top 10 upregulated tRNAs were tRNA Lys CTT -1, tRNA Leu TAA , tRNA Phe GAA , tRNA Leu CAG , tRNA Tyr ATA , tRNA Met CAT , tRNA Tyr GTA -1, tRNA Thr CGT , tRNA Tyr GTA -2, tRNA Ala AGC ; and the top 10 downregulated tRNAs were tRNA Ini CAT inhibited LSCC cell proliferation and promoted apoptosis. The in vivo results showed that tRNA Glu inhibited the growth of the xenografts and promoted apoptosis.TTC , tRNA Val CAC -3, mt-tRNA Trp TCA , mt-tRNA Tyr GTA , mt-tRNA Lys TTT , mt-tRNA Thr TGT , mt-tRNA Asp GTC , mt-tRNA Asn GTT , mt-tRNA Pro TGG . tRNA Ini CAT was downregulated in LSCC tissues and plasma. The area under the ROC curve (AUC) in LSCC tissues and the plasma of patients with LSCC was 0.717 and 0.808, respectively. tRNA Ini CAT inhibited LSCC cell proliferation and promoted apoptosis. The in vivo results showed that tRNA Ini CAT inhibited the growth of the xenografts and promoted apoptosis., Conclusions: This is the first study to provide tRNA expression profiles for LSCC tissues. tRNA Ini CAT inhibits cell proliferation and promotes apoptosis in vitro and in vivo.Ini CAT inhibits cell proliferation and promotes apoptosis in vitro and in vivo., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Ethanol-induced myocardial injury involves multiple pathophysiological processes including apoptosis. Empagliflozin (EMPA), is a novel hypoglycaemic drug which possesses multiple pharmacologically relevant protective effects, including anti-apoptotic, anti-inflammatory and antioxidant effects. However, whether EMPA treatment has a protective effect on ethanol-induced myocardial injury has not been assessed, to the best of our knowledge. Therefore, the aim of this study was to determine the effect of EMPA treatment on ethanol-induced myocardial injury and the underlying mechanism. An ethanol-induced myocardial injury model was established by culturing H9c2 cells treated with 200 mmol/L ethanol for 24 hours, and additional groups of ethanol treated cells were also treated with EMPA with or without SIRT1 inhibitors prior to ethanol treatment. Cell viability and apoptosis were assessed using a CCK-8 assay and flow cytometry, respectively. The expression of apoptosis-related proteins was assessed using western blotting. The results showed that EMPA pretreatment resulted in increased cell viability and a decrease in LDH activity. Moreover, EMPA pretreatment significantly reduced apoptosis of cardiomyocytes, and reduced the expression of cleaved caspase 3. Furthermore, EMPA increased the expression of SIRT1, increased the phosphorylation levels of Akt, and reduced the expression of PTEN. EMPA also reduced ethanol-induced mitochondrial apoptosis, increasing the Bcl-2/Bax ratio and the mitochondrial membrane potential. However, the cardioprotective effects of EMPA were abrogated when cells were pretreated with a SIRT1 inhibitor. In conclusion, EMPA can alleviate ethanol-induced myocardial injury by inhibiting mitochondrial apoptosis via the SIRT1/PTEN/Akt pathway. Therefore, EMPA may be a novel target for treatment of ethanol-induced myocardial injury., (© 2021 John Wiley & Sons Australia, Ltd.)

Apoptosis, also named programmed cell death, is a highly conserved physiological mechanism. Apoptosis plays crucial roles in many life processes, such as tissue development, organ formation, homeostasis maintenance, resistance against external aggression, and immune responses. Apoptosis is regulated by many genes, among which Apoptosis Inhibitor-5 (API5) is an effective inhibitor, though the structure of API5 is completely different from the other known Inhibitors of Apoptosis Proteins (IAPs). Due to its high expression in many types of tumors, API5 has received extensive attention, and may be an effective target for cancer treatment. In order to comprehensively and systematically understand the biological roles of API5, we summarized the evolution and structure of API5 and its roles in anti-apoptosis in this review., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Bladder cancer is one of the most commonly diagnosed and fatal malignancies of the urinary tract. Noncoding RNAs have been reported to be new biomarkers and effective treatment targets for bladder cancer. In the present study, we identified a novel bladder cancer-related circRNA-miRNA-mRNA network, the circ&#95;0004463/miR-380-3p/FOXO1 axis. circ&#95;0004463 is significantly downregulated, whereas miR-380-3p is upregulated in bladder carcinoma tissue samples and cells. circ&#95;0004463 acts as a tumor suppressor by inhibiting bladder cancer cell proliferation. Genes that negatively correlated with miR-380-3p and genes that miR-380-3p might target are enriched in mitochondrial respiration chain-related pathways. miR-380-3p promotes the proliferation of bladder cancer cells and mitochondrial respiration by acting as an oncogenic miRNA. circ&#95;0004463 competes with FOXO1 for miR-380-3p binding to counteract miR-380-3p-mediated repression of FOXO1. Circ&#95;0004463 overexpression inhibits cancer cell proliferation and mitochondrial respiration in bladder cancer cell lines, while miR-380-3p overexpression dramatically reverses the roles of circ&#95;0004463 overexpression. In conclusion, the circ&#95;0004463/miR-380-3p/FOXO1 axis could regulate mitochondrial respiration and bladder cancer cell apoptosis via FOXO1 signaling.

Previously, Nucleolar protein 66 (NO66) was reported to be closely associated with alcohol exposure-induced injury. However, the role of NO66 in alcohol-induced cytotoxicity remains unclear. In this study, we explored the potential effect and mechanism of NO66 on ethanol-induced apoptosis in human AC16 cardiomyocytes. The AC16 cell lines with NO66 and phosphatase and tensin homolog (PTEN) overexpression were constructed. Cell counting kit-8 (CCK-8), lactate dehydrogenase (LDH) assay, Annexin V-FITC/PI staining, and flow cytometry were used to evaluate the cell viability, membrane damage, and apoptosis, respectively. Quantitative real-time PCR (qRT-PCR) and western blot analysis were applied to measure mRNA and protein expression. The results showed that acute ethanol exposure markedly augmented cytotoxicity and reduced NO66 level in AC16 cardiomyocytes. Overexpression of NO66 partially reversed ethanol-induced apoptosis. NO66 upregulation reversed the decrease in phosphorylation of protein kinase B (Akt) and B-cell lymphoma-2/Bcl-2-associated x (Bcl-2/Bax) ratio and the increase in PTEN, p53, and caspase-3 activity induced by ethanol treatment. Meanwhile, the application of PI3K inhibitor (LY294002) and PTEN overexpression attenuated the inhibition efficiency of NO66 on cell apoptosis. In addition, PTEN overexpression weakened the effect of NO66 on PI3K/Akt activation, without affecting the level of NO66. Our data suggested that NO66 overexpression might play an anti-apoptotic role in ethanol-induced cell injury via reducing PTEN and upregulating the PI3K/Akt pathway., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Our previous study demonstrated mechanical stretch (MS) could induce the apoptosis of retinal pigment epithelial (RPE) cells, but the related mechanisms remained unclear. This study was to characterize the protein expression profile in RPE cell line ARPE-19 exposed to MS, cytochalasin D (CD; an inhibitor of actin polymerization) or CD + MS at 2-time points (6, 24 hr; n = 3, at each time point) by using proteomics technique. Our data highlighted that compared with control, ECE1 was continuously downregulated in ARPE-19 cells treated by MS or CD + MS from 6 to 24 hr. Function and protein-protein interaction network analyses showed ATAD2 was downregulated in all three treatment groups compared with control, but successive upregulation of RPS13 and RPL7 and downregulation of AHSG were specifically induced by MS. ATAD2 was enriched in cell cycle; AHSG was associated with membrane organization; RPS13 and RPL7 participated in ribosome biogenesis. Furthermore, transcription factor CREB1 that was upregulated in MS group at 24 hr after treatment, may negatively regulate ATAD2. The expressions of all crucial proteins in ARPE-19 cells were confirmed by western blot analysis. Overexpression of ATAD2 and AHSG were also shown to reverse the apoptosis of ARPE-19 cells induced by MS or CD + MS, with significantly decreased apoptotic rates and caspase-3 activities. Accordingly, our findings suggest downregulation of ATAD2 and AHSG may be potential contributors to the apoptosis of RPE cells induced by MS. Overexpression of them may represent underlying preventive and therapeutic strategies for MS-induced retinal disorders., (© 2020 Wiley Periodicals LLC.)

In this study, we investigated a gene-edited (Zp2MT/MT) rat model of infertility caused by the failure to express the zona pellucida glycoprotein 2 (ZP2) due to the significant reduction of mRNA amount. We examined the defects in the zona pellucida (ZP) caused by ZP2 nullification and the influence of these defects on aspects of oocyte development, including apoptosis and fertilization ability. To investigate the cause of the influence to the oocytes' development, we evaluated the morphology of follicular transzonal projections (TZPs), known as 'bridges', which mediate the bidirectional signaling between the oocyte and surrounding granulosa cells and the level of reactive oxygen species (ROS) in ovulated eggs. Our results showed that two types of ZP defects were generated in the Zp2MT/MT rat,that is, ZP intact but thinned and ZP cracked (or even absent). The fertilization rate of the ovulated eggs reduced in both types, while increased oocyte apoptosis was observed only in the latter type. Moreover, the increased oocyte apoptosis rate correlated closely with the reduction in follicular TZPs and increased ROS levels in ovulated egg. In conclusion, nullification of rat ZP2 destroyed the integrity of the ZP, impaired the bidirectional signaling between the oocyte and surrounding granulosa cells. Therefore, the resulting infertility likely occurs via elevation of oxidative stress and oocytes apoptosis.

Transcription factors are known to mediate the conversion of somatic cells to induced pluripotent stem cells (iPSCs). Transcription factor TFAP2C plays important roles in the regulation of embryonic development and carcinogenesis; however, the roles of Tfap2c in regulating somatic cell reprogramming are not well understood. Here we demonstrate Tfap2c is induced during the generation of iPSCs from mouse fibroblasts and acts as a facilitator for iPSCs formation. Mechanistically, the c-Myc-dependent apoptosis, which is a roadblock to reprogramming, can be significantly mitigated by Tfap2c overexpression. Meanwhile, Tfap2c can greatly promote mesenchymal-to-epithelial transition (MET) at initiation stage of OSKM-induced reprogramming. Further analysis of gene expression and targets of Tfap2c during reprogramming by RNA-sequencing (RNA-seq) and ChIP-qPCR indicates that TFAP2C can promote epithelial gene expression by binding to their promoters directly. Finally, knockdown of E-cadherin (Cdh1), an important downstream target of TFAP2C and a critical regulator of MET antagonizes Tfap2c-mediated reprogramming. Taken together, we conclude that Tfap2c serves as a strong activator for somatic cell reprogramming through promoting the MET and inhibiting c-Myc-dependent apoptosis.

Background and Objectives: The injury burden after head trauma is exacerbated by secondary sequelae, which leads to further neuronal loss. B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein and a key modulator of the programmed cell death (PCD) pathways. The current study evaluates the clinical evidence on Bcl-2 and neurological recovery in patients after traumatic brain injury (TBI). Materials and Methods: All studies in English were queried from the National Library of Medicine PubMed database using the following search terms: (B-cell lymphoma 2/Bcl-2/Bcl2) AND (brain injury/head injury/head trauma/traumatic brain injury) AND (human/patient/subject). There were 10 investigations conducted on Bcl-2 and apoptosis in TBI patients, of which 5 analyzed the pericontutional brain tissue obtained from surgical decompression, 4 studied Bcl-2 expression as a biomarker in the cerebrospinal fluid (CSF), and 1 was a prospective randomized trial. Results: Immunohistochemistry (IHC) in 94 adults with severe TBI showed upregulation of Bcl-2 in the pericontusional tissue. Bcl-2 was detected in 36-75% of TBI patients, while it was generally absent in the non-TBI controls, with Bcl-2 expression increased 2.9- to 17-fold in TBI patients. Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling (TUNEL) positivity for cell death was detected in 33-73% of TBI patients. CSF analysis in 113 TBI subjects (90 adults, 23 pediatric patients) showed upregulation of Bcl-2 that peaked on post-injury day 3 and subsequently declined after day 5. Increased Bcl-2 in the peritraumatic tissue, rising CSF Bcl-2 levels, and the variant allele of rs17759659 are associated with improved mortality and better outcomes on the Glasgow Outcome Score (GOS). Conclusions: Bcl-2 is upregulated in the pericontusional brain and CSF in the acute period after TBI. Bcl-2 has a neuroprotective role as a pro-survival protein in experimental models, and increased expression in patients can contribute to improvement in clinical outcomes. Its utility as a biomarker and therapeutic target to block neuronal apoptosis after TBI warrants further evaluation.

Aims/introduction: The purpose of the present study was to observe the relationship between serum α-klotho (KL) protein level and diabetic retinopathy (DR), and to further examine the effects of KL protein on apoptosis induced by palmitic acid (PA) in human retinal endothelial cells., Materials and Methods: A total of 17 healthy people and 60 type 2 diabetes patients were included. According to the results from fundus fluorescein angiography, the diabetes patients were divided into three subgroups: without DR, non-proliferative DR and proliferative DR. Serum KL level was measured by enzyme-linked immunosorbent assay. In vitro, human retinal endothelial cells were exposed to PA with or without KL protein. Apoptosis rates were analyzed by flow cytometry analysis. Apoptotic-related protein expressions were detected by western blotting analysis., Results: Serum KL level was lower in diabetes patients than that in healthy participants (P = 0.007), and was gradually decreased among the without DR, non-proliferative DR and proliferative DR subgroups (P = 0.045). A logistic regression analysis showed that after adjusting for the other confounding factors, serum KL level was independently and negatively related with DR (P = 0.049). Furthermore, the increased apoptosis rates induced by PA were inhibited with the addition of KL protein. Consistently, KL protein reversed the expression levels of the increased pro-apoptotic protein Bax and the decreased anti-apoptotic protein Bcl-2 induced by PA. However, the anti-apoptotic effect of KL protein was attenuated by LY294002 through the phosphatidylinositol 3 kinase-serine∕threonine kinase pathway., Conclusions: The data suggested that KL protein was probably a potential protective factor against retinopathy in type 2 diabetes patients., (© 2019 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Object: Cisplatin is a type of broad-spectrum anti-carcinogen that has been widely used in anti-gastric cancer therapy. Drug resistance prevails in gastric cancer therapy. α-Hederin has been reported to exert anti-tumour ability by inducing apoptosis in many cancers in vitro and in vivo. A combination chemotherapy regimen can improve the outcome of patients., Methods: In the present study, we used a CCK-8 assay, Hoechst 33258 staining, Annexin V-PE/7-AAD, intracellular reactive oxygen species (ROS) measurement, mitochondrial membrane potential (MMP) assay kit and western blotting to detect apoptosis and mitochondrial function in gastric cancer (GC) cells. A xenograft tumour model in nude mice was used to evaluate the anti-tumour effects of cisplatin and α-Hederin in vivo., Results: Combination treatment of cisplatin and α-Hederin increased the apoptotic effects in cisplatin-induced GC cell lines. In the xenograft mouse model, the combination of cisplatin and α-Hederin remarkably increased the tumour inhibition effect compared to either drug alone. Interestingly, combination of cisplatin and α-Hederin increased the expression of apoptosis-related proteins. Using in vitro experiments, we verified that the combination of cisplatin and α-Hederin increased the accumulation of ROS in GC cell lines and also reduced the MMP, thus inhibiting proliferation and promoting apoptosis in GC cells., Conclusion: α-Hederin enhances cisplatin-induced anti-tumour effects in GC both in vitro and in vivo by promoting the accumulation of ROS and decreasing MMP. Our data strongly suggested that α-Hederin is a promising candidate for intervention in gastric cancer., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The effect of miR-182 on the expressions of CRR9 in laryngeal squamous cell carcinoma (LSCC) cells, and the impact on invasion and metastasis of LSCC were investigated in the present paper., Methods: The expressions of miR-182 in LSCC tissue and cell line were detected by RT-qPCR. MTT assay and Annexin V staining were used to detect the effects of miR-182 on tumor cells proliferation. Target gene prediction and screening, and luciferase reporter assay were designed to verify downstream target genes of miR-182. The mRNA and protein expressions of CRR9 were detected by qRT-PCR and Western blot. Finally, the expressions of CRR9 were measured by transfecting cells with miR-182 in mice., Results: Compared with normal tissue and cell, the expressions of miR-182 in tumor tissues and cells were much lower. Over-expressions of miR-182 can increase apoptosis rate. Luciferase reporter assay revealed that CRR9 was a downstream gene of miR-182. Reintroduction of CRR9 abolished miR-182-induced LSCC cell growth inhibition. In animal models, over-expressions of miR-182 can reduce tumor weight and promote apoptosis., Conclusion: miR-182 can inhibit the proliferation of LSCC cells by directly inhibiting the expressions of CRR9, thereby suppressing the occurrences and developments of LSCC., (© 2019 The Author(s).)

A series of novel oridonin derivatives bearing various substituents on the 14-OH position were designed and synthesised. Their antitumour activity was evaluated in vitro against three human cancer cell lines (HCT116, BEL7402, and MCF7). Most tested derivatives showed improved anti-proliferative activity compared to the lead compound oridonin and the positive control drug 5-fluorouracil (5-Fu). Among them, compound C7 (IC 50  = 0.16 μM) exhibited the most potent anti-proliferative activity against HCT116 cells; it was about 43- and 155-fold more efficacious than that of oridonin (IC 50  = 6.84 μM) and 5-Fu (IC 50  = 24.80 μM) in HCT116 cancer cells. Interestingly, the IC 50 value of compound C7 in L02 normal cells was 23.6-fold higher than that in HCT116 cells; it exhibited better selective anti-proliferative activity and specificity than oridonin and 5-Fu. Furthermore, compound C7 possibly induced cell cycle arrest and apoptosis by regulating the p53-MDM2 signalling pathway. Notably, C7 displayed more significant suppression of tumour growth than oridonin in colon tumour xenograft models where the tumour growth inhibition rate was 85.82%. Therefore, compound C7 could be a potential lead compound for the development of a novel antitumour agent., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: TGF-β1 contributes to chronic heart failure. It is known that lncRNA GASL1 can inactivate TGF-β1 in cancer biology., Methods: All the participants were enrolled in the First People's Hospital of Zhaoqing during the period June 2012 to June 2013. ELISA, RT-qPCR, vectors, transient transfections and western blot were carried out during the research., Results: We found that plasma levels of TGF-β1 were significantly higher, while levels of GASL1 in plasma were significantly lower in chronic heart failure (CHF) patients compared to the control group. TGF-β1 and GASL1 were inversely correlated in CHF patients. Low pretreatment plasma levels of GASL1 were closely associated with poor survival of CHF patients. GASL1 expression was not significantly affected by TGF-β1 overexpression in cardiomyocytes, while cardiomyocytes with GASL1 overexpression showed downregulated TGF-β1. Overexpression of GASL1 led to a decreased, while TGF-β1 overexpression led to an increased apoptotic rate of cardiomyocytes under H 2 O 2 treatment. In addition, TGF-β1 overexpression attenuated the effect of GASL1 overexpression., Conclusion: In conclusion, GASL1 was downregulated in CHF. GASL1 overexpression may improve CHF by inhibiting cardiomyocyte apoptosis through the inactivation of TGF-β1., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

Fluoride overexposure is an environmental health hazard and can cause enamel and skeletal fluorosis. Previously we demonstrated that fluoride increased acetylated-p53 and its downstream target p21 in ameloblast-derived LS8 cells. However, p21 function in fluoride toxicity is not well characterized. This study seeks to gain a better understanding of how p53 down-stream mediators, p21 and MDM2, respond to fluoride toxicity. LS8 cells were treated with NaF with/without MG-132 (proteasome inhibitor) or Nutlin-3a (MDM2 antagonist). NaF treatment for 2-6 h increased phospho-p21, which can inhibit apoptosis. However, phospho-p21 and p21 were decreased by NaF at 24 h, even though p21 mRNA was significantly increased at this time point. MG-132 reversed the fluoride-mediated p21 decrease, indicating that fluoride facilitates p21 proteasomal degradation. MG-132 suppressed fluoride-induced caspase-3 cleavage, suggesting that the proteasome plays a pro-apoptotic role in fluoride toxicity. NaF increased phospho-MDM2 in vitro and in mouse ameloblasts in vivo. Nutlin-3a suppressed NaF-mediated MDM2-p21 binding to reverse p21 degradation which increased phospho-p21. This suppressed apoptosis after 24 h NaF treatment. These results suggest that MDM2-mediated p21 proteasomal degradation with subsequent phospho-p21 attenuation contributes to fluoride-induced apoptosis. Inhibition of MDM2-mediated p21 degradation may be a potential therapeutic target to mitigate fluoride toxicity.

Pyroptosis is a novel manner of cell death that can be mediated by chemotherapy drugs. The awareness of pyroptosis is significantly increasing in the fields of anti-tumor research and chemotherapy drugs. Invoking the occurrence of pyroptosis is an attractive prospect for the treatment of lung cancer. Here, the compound L61H10 was obtained as a thiopyran derivative to compare its activity with curcumin. It was indicated that L61H10 exhibited good anti-tumor activity both in vitro and in vivo via the switch of apoptosis-to-pyroptosis, which was associated with the NF-κB signaling pathway. In addition, L61H10 had no obvious side effects both in vitro and in vivo. In brief, L61H10 is shown to be a potential anti-lung cancer agent and research on its anti-tumor mechanism provides new information for chemotherapy drug research.

Fluorine or fluoride can have toxic effects on bone tissue and soft tissue at high concentrations. These negative effects include but not limited to cytotoxicity, immunotoxicity, blood toxicity, and oxidative damage. Apoptosis plays an important role in fluoride-induced toxicity of kidney, liver, spleen, thymus, bursa of Fabricius, cecal tonsil, and cultured cells. Here, apoptosis activated by high level of fluoride has been systematically reviewed, focusing on three pathways: mitochondrion-mediated, endoplasmic reticulum (ER) stress-mediated, and death receptor-mediated pathways. However, very limited reports are focused on the death receptor-mediated apoptosis pathways in the fluoride-induced apoptosis. Therefore, understanding and discovery of more pathways and molecular mechanisms of fluoride-induced apoptosis may contribute to designing measures for preventing fluoride toxicity.

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.)

In sepsis, the liver plays a crucial role in regulating immune responses and is also a target organ for immune-related injury. Despite the critical function of CD8 + T cells against opportunistic viral infections, the CD8 immune response in the liver during sepsis remains elusive. Here we found that Tim-3 is highly up-regulated in liver CD8 + T cells in a mouse cecal ligation and puncture model and in peripheral blood CD8 + T cells of human patients with sepsis. The expression of Tim-3 in liver CD8 + T cells displayed a bi-phasic pattern and deletion of Tim-3 led to reduction of CD8 + T cell apoptosis. Administration of α-lactose, a molecule with a similar structure to galactin-9, reduced Tim-3 expression and liver injury in sepsis. Our results demonstrate that targeting Tim-3 to boost CD8 + T cell immune response may offer an improved outcome in patients with sepsis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

IgA nephropathy (IgAN) is characterized by predominant IgA deposition in the glomerular mesangium. It has been considered that the deposited IgA is synthesized by B cells, although recent reports have suggested the implication of other cell types. Therefore, the present study investigated whether glomerular mesangial cells could produce IgA by themselves. Semi‑quantitative reverse transcription-polymerase chain reaction, and immunostaining analysis revealed that the IgA protein and gene transcripts were expressed in primary human renal mesangial cells (HRMCs). Furthermore, the IgA heavy chain (α1 and α2) and the light chain (κ and λ) were localized in the cytoplasm or were located on the cell membranes of human mesangial cells (HMCs). Mass spectrometry results indicated that Ig α1 and Ig α2 were secreted in the culture media of HMCs. The transcripts of Ig α, Ig κ and Ig λ constant regions were detected. The predominant rearrangement pattern of the variable region of Ig κ, was Vκ3‑20*01/Jκ1*01 in HMCs and Vκ1‑12*01/Jκ4*01 in HRMCs. In addition, knockdown of Ig α1 expression by small interfering RNA (siRNA) inhibited cell adhesion and promoted apoptosis. Our findings demonstrate that HMCs can express IgA, and that this expression is associated with cell functions, which may contribute to the deposition of IgA in patients with IgAN.

Background Substantial evidence from clinical reports has established that most cerebral palsy (CP) patients benefit from a comprehensive rehabilitation exercise training programme. Such advances are enhanced when scalp electroacupuncture (EA), applied at a location corresponding to the projection of the motor area, is combined with rehabilitation exercise training. However, little information exists regarding the mechanistic basis for these effects. Objective To examine whether EA stimulation within the scalp projection location of the motor area can inhibit apoptosis of hippocampal neurons by regulating the PI3k/Akt signalling pathway in a rat model of CP. Methods Fifty male Sprague-Dawley rats underwent surgical modelling of CP. Five were used to confirm successful establishment of the model and the remaining 45 rats were randomly divided into one of three groups that remained untreated (CP group, n=15) or received EA treatment alone (CP+EA group, n=15) or EA in combination with a PI3K/Akt inhibitor (CP+EA+LY294002 group, n=15)). An otherwise healthy negative control group of rats undergoing sham surgery was also included (Control group, n=15). In the CP+EA and CP+EA+LY294002 groups, EA was applied to the scalp surface at alocation corresponding to the projection of the motor area. Basso, Beattie and Bresnahan (BBB) locomotor scores, hippocampal protein expression of Akt and p-Akt (by Western blot analysis) and neuronal apoptosis in hippocampal tissue (by histopathology) were assessed at 7, 14 and 21 days post-CP induction. Results CP rats receiving scalp EA treatment demonstrated improved behavioural scores, less hippocampal neuronal apoptosis and higher expression levels of Akt and p-Akt (p<0.05) at all time points studied compared with untreated CP rats. There were no significant differences observed between CP+EA+LY294002 and untreated CP model groups. Conclusions The effects of scalp EA on the PI3K/ Akt signalling pathway may represent one of the mechanisms involved in the inhibition of hippocampal neuronal apoptosis and improvement of deficits associated with CP in a rat model., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Hyperglycemia is a key factor in the development of diabetic complications, including the processes of atherosclerosis. Receptor‑interacting protein 3 (RIP3), a mediator of necroptosis, is implicated in atherosclerosis development. Additionally, hydrogen sulfide (H2S) protects the vascular endothelium against hyperglycemia‑induced injury and attenuates atherosclerosis. On the basis of these findings, the present study aimed to confirm the hypothesis that necroptosis mediates high glucose (HG)‑induced injury in human umbilical vein endothelial cells (HUVECs), and that the inhibition of necroptosis contributes to the protective effect of exogenous H2S against this injury. The results revealed that exposure of HUVECs to 40 mM HG markedly enhanced the expression level of RIP3, along with multiple injuries, including a decrease in cell viability, an increase in the number of apoptotic cells, an increase in the expression level of cleaved caspase‑3, generation of reactive oxygen species (ROS), as well as dissipation of the mitochondrial membrane potential (MMP). Treatment of the cells with sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to HG significantly attenuated the increased RIP3 expression and the aforementioned injuries by HG. Notably, treatment of cells with necrostatin‑1 (Nec‑1), an inhibitor of necroptosis, prior to exposure to HG ameliorated the HG‑induced injuries, leading to a decrease in ROS generation and a loss of MMP. However, pre‑treatment of the cells with Nec‑1 enhanced the HG‑induced increase in the expression levels of cleaved caspases‑3 and ‑9. By contrast, pre‑treatment with Z‑VAD‑FMK, a pan ‑caspase inhibitor, promoted the increased expression of RIP3 by HG. Taken together, the findings of the present study have demonstrated, to the best of our knowledge for the first time, that exogenous H2S protects HUVECs against HG‑induced injury through inhibiting necroptosis. The present study has also provided novel evidence that there is a negative interaction between necroptosis and apoptosis in the HG‑treated HUVECs.

Neuronal apoptosis after intracerebral hemorrhage (ICH) plays an essential role in neurological deterioration. Preclinical studies have shown that EGb761, an extract of Ginkgo biloba, is neuroprotective in some other neurological diseases with apoptosis. This study was conducted to investigate the potential neuroprotective effect of EGb761 on neuronal apoptosis in experimental ICH. A model of ICH was induced in C57BL/6 mice by injecting collagenase. EGb761 was administered for 21 days and neurologic behaviors were assessed at 1, 3, 7, 14, and 21 days after ICH. RNAi-mediated knockdown of p90 ribosomal S6 kinase 1 (RSK1) was used to further investigate the role of RSK1 in EGb761-induced neuroprotective effects. Neuronal death was determined by TUNEL staining. The image datasets of neurovascular networks were acquired via micro-optical sectioning tomography (MOST). The glycogen synthase kinase-3β (GSK3β) activity was assayed using commercial kit. Primary cultured cortical neurons were exposed to ferrous iron and treated with EGb761. Apoptotic neurons were counted by flow cytometry. RSK1, GSK3β, phosphorylated-GSK3β (pGSK3β), Bcl2, Bax, cleaved-caspase3 (CC3), and VEGF were measured by Western blot. The pGSK3β was also detected by immunofluorescence staining. We found that mice in EGb761 group performed better on rotarod test. Reduced TUNEL-positive neurons and richer microvascular networks were observed in mice treated with EGb761. EGb761 attenuates neuronal apoptosis induced by ferrous iron counted by flow cytometry in vitro. Decreased GSK3β activity was observed in EGb761-treated mice compared with mice with ICH. EGb761 increased the expression of pGSK3β (Ser9), RSK1 and the Bcl2/Bax ratio, and VEGF and decreased CC3 expression. In conclusion, EGb761 reduces neuronal apoptosis and promotes angiogenesis in experimental intracerebral hemorrhage via RSK1/GSK3β pathway.

As a common polycyclic aromatic hydrocarbon compound, benzo[a]pyrene (BaP) is readily produced in processing of oil and fatty foods. It is not only a strong carcinogen but also a substance with strong immunotoxicity and reproduction toxicity. Autophagy and pyroptosis are two types of programmed cell death. Whether or not BaP damages body tissues via autophagy or pyroptosis remains unknown. The present study investigated the effects of BaP on autophagy and pyroptosis in HL-7702 cells. The results showed that BaP induced cell death in HL-7702 cells enhanced the intracellular levels of ROS and arrested the cell cycle at the S phase. Additionally, BaP resulted in cell death through autophagy and pyroptosis. Compared with the BaP group, the autophagy inhibitor 3-MA significantly (p < 0.01) inhibited the release of LDH by 70.53% ± 0.46 and NO by 50.36% ± 0.80, the increase of electrical conductivity by 12.08% ± 0.55, and the expressions of pyroptotic marker proteins (Caspase-1, Cox-2, IL-1β, IL-18). The pyroptosis inhibitor Ac-YVAD-CM also notably (p < 0.01) blocked BaP-induced autophagic cell death characterized by the increase of autophagic vacuoles and overexpression of Beclin-1 and LC3-II. In conclusion, BaP led to injury by inducing autophagy and pyroptosis simultaneously, the two of which coexisted and promoted each other in HL-7702 cells.

Deoxynivalenol (DON) and zearalenone (ZEA) are the two most common mycotoxins in animal feed. In this study, we examined oxidative injury and apoptosis of porcine splenic lymphocytes induced by DON or ZEA and their combination in vitro. Based on IC 50 values, porcine splenic lymphocytes were treated with 0.06, 0.3, 1.5, and 7.5μg/mL DON, 0.08, 0.4, 2, and 10μg/mL ZEA, or both DON and ZEA at 0.06 and 0.08μg/mL, 0.3 and 0.4μg/mL, and 1.5 and 2μg/mL, respectively. After 48h of DON and/or ZEA exposure, the cells were analyzed for antioxidant functions, apoptosis, and mRNA and protein expression of apoptosis-related genes p53, Bcl-2, Bax, caspase-3, and caspase-8 to determine their apoptosis and oxidative damage effects and mechanisms. The results showed that, compared with the control group, SOD, CAT, GPx, GSH, and Bcl-2 mRNA and protein expression levels were significantly reduced in exposed groups (P<0.05 or P<0.01). Furthermore, MDA contents, apoptosis rates, and p53, Bax, caspase-3 and caspase-8 protein and mRNA expression levels were increased significantly (P<0.01). The effects of DON and ZEA were dose dependent and synergistic in combination. These data suggest that DON and ZEA induce oxidative damage and apoptosis of porcine splenic lymphocytes., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The current study was conducted to investigate the effect of sodium fluoride (NaF) on the oxidative stress and apoptosis as well as their relationship in the mouse liver by using methods of flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, biochemistry and experimental pathology. 240 four-week-old ICR mice were randomly divided into 4 groups and exposed to different concentration of NaF (0 mg/kg, 12 mg/kg, 24 mg/kg and 48 mg/kg) for a period of 42 days. The results showed that NaF caused oxidative stress and apoptosis. NaF-caused oxidative stress was accompanied by increasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreasing mRNA expression levels and activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-PX) and glutathione-s-transferase (GST). NaF induced apoptosis via tumor necrosis factor recpter-1 (TNF-R1) signaling pathway, which was characterized by significantly increasing mRNA and protein expression levels of TNF-R1, Fas associated death domain (FADD), TNFR-associated death domain (TRADD), cysteine aspartate specific protease-8 (caspase-8) and cysteine aspartate specific protease-3 (caspase-3) in dose- and time-dependent manner. Oxidative stress is involved in the process of apoptotic occurrence, and can be triggered by promoting ROS production and reducing antioxidant function. NaF-caused oxidative stress and apoptosis finally impaired hepatic function, which was strongly supported by the histopathological lesions and increased serum alanine amino transferase (ALT), aspartic acid transferase (AST), alkaline phosphatase (AKP) activities and TBIL contents.

Targeting the autophagic pathway is currently regarded as an attractive strategy for cancer drug discovery. Our previous work showed that IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells, yet the effect of IMB-6G on autophagy and pancreatic cancer cell death remains unknown. Here, we show that IMB-6G inhibits the growth of MiaPaCa-2 and HupT-3 pancreatic cancer cells and induces caspase-mediated apoptosis, which is correlated with an accumulation of autophagic vacuoles. IMB-6G promotes autophagosome accumulation from the early stage of treatment but blocks autophagic flux in the degradation stage, mainly through attenuation of lysosomal cathepsin activity in pancreatic cancer cells. Moreover, IMB-6G triggers lysosomal membrane permeabilization (LMP), followed by cathepsin B/CTSB and cathepsin D/CTSD release from lysosomes into the cytoplasm. Inhibition of autophagosome formation with siRNA against autophagy protein 5 (Atg5) attenuates IMB-6G-induced LMP and apoptosis. Furthermore, cathepsin inhibitors relieve IMB-6G-induced apoptosis as well. Altogether, our findings demonstrate that IMB-6G is a novel autophagy inhibitor, which induces autophagy-dependent apoptosis through autophagosomal-cathepsin axis in pancreatic cancer cells and indicate the potential value of IMB-6G as a novel antitumor drug candidate., Competing Interests: The authors declare no competing financial interests.

At present, there are no reports on the relationship between fluoride-induced apoptosis and endoplasmic reticulum (ER) stress (ER stress) in the spleen of human and animals in vivo and in vitro . Therefore, the aim of this study was to define sodium fluoride (NaF)-induced apoptosis mediated by ER stress in the spleen of mice in vivo and in vitro . Apoptosis and expression levels of the ER stress-related proteins were detected by flow cytometry and western blot, respectively. The results showed that NaF treatment increased lymphocytes apoptosis, which was consistent with NaF-caused ER Stress. NaF-caused ER stress was characterized by up-regulating protein expression levels of glucose-regulated protein 78 (BiP) and glucose-regulated protein 94 (GRP94), and by activating unfolded protein response (UPR). The signaling pathway of ER stress-associated apoptosis was activated by up-regulating protein expression levels of cleaved cysteine aspartate specific protease-12 (cleaved caspase-12), growth arrest and DNA damage-inducible gene 153 (Gadd153/CHOP) and phosphorylation of JUN N-terminal kinase (p-JNK). Additionally, our in vitro study found that apoptotic rate was decreased with remarkable down-regulation of the cleaved caspase-12, CHOP, p-JNK after ER stress was inhibited by 4-Phenylbutyric acid (4-PBA) treatment. In conclusion, NaF-induced apoptosis may mediated by ER stress in the spleen., Competing Interests: The authors declare no conflicts of interest.

In an effort to better understand the biological efficacy of the tridentate aroyl hydrazone Cu(II) complexes, the Cu(II) complex of di-2-pyridyl ketone isonicotinoyl hydrazone ligand (HL), {[Cu(L)(H 2 O)]·H 2 O·NO 3 } n (C1) was synthesized and characterized. Single crystal X-ray study reveals that complex C1 forms 1D zigzag chains in solid state. In water, the hydrolysis of the 1D zigzag chains was observed, and finally formation of monomeric species. In vitro studies revealed that complex C1 showed significantly more anticancer activity than the ligand alone. Investigation of the anticancer mechanisms of C1, confirmed that the Cu(II) complex exhibit a strong capacity to promote productions of reactive oxygen species (ROS), leading to caspase-dependent apoptotic cell death.

Though fluorine has been shown to induce apoptosis in immune organs in vivo, there has no report on fluoride-induced apoptosis in the cultured lymphocytes. Therefore, this study was conducted with objective of investigating apoptosis induced by sodium fluoride (NaF) and the mechanism behind that in the cultured splenic lymphocytes by flow cytometry, western blot and Hoechst 33258 staining. The splenic lymphocytes were isolated from 3 weeks old male ICR mice and exposed to NaF (0, 100, 200, and 400 μmol/L) in vitro for 24 and 48 h. When compared to control group, flow cytometry assay and Hoechst 33258 staining showed that NaF induced lymphocytes apoptosis, which was promoted by decrease of mitochondria transmembrane potential, up-regulation of Bax, Bak, Fas, FasL, caspase 9, caspase 8, caspase 7, caspase 6 and caspase 3 protein expression (P < 0.05 or P <0.01), and down-regulation of Bcl-2 and Bcl-xL protein expression (P <0.05 or P <0.01). The above-mentioned data suggested that NaF-induced apoptosis in splenic lymphocytes could be mediated by mitochondrial and death receptor pathways.

Glutamine provides cancer cells with the energy required to synthesize macromolecules. Methods which block glutamine metabolism in treatment of breast cancer inhibit oncogenic transformation and tumor growth. We investigated whether inhibiting glutamine metabolism produces effects that are synergistic with those produced by drugs which damage DNA in triple-negative breast cancer cells. HCC1937 and BT-549 breast cancer cells were co-treated with either cisplatin or etoposide in combination with BPTES (a specific inhibitor of glutaminase 1) or exposure to a glutamine-free medium, and the cell proliferation and cell apoptosis were measured by flow cytometry, immunoblotting studies, and CCK-8 assays. The results showed that both glutamine deprivation and BPTES pretreatments increased the toxic effects of cisplatin and etoposide on HCC1937 cells, as demonstrated by their reduced proliferation, increased expression of apoptosis-related proteins (cleaved-PARP, cleaved-caspase 9, and cleaved-caspase 3) and decreased Bcl-2/BAX ratio. However, in BT-549 cells, glutamine deprivation and BPTES treatment increased etoposide-induced apoptosis only when used with higher concentrations of etoposide, and the effect on cisplatin-induced apoptosis was minimal. These results suggest that the anti-cancer effects produced by a combined approach of inhibiting glutamine metabolism and administering common chemotherapeutic agents correlate with the tumor cell type and specific drugs being administered.

The accumulation of reactive oxygen species is detrimental to the health of the ovarian follicle. The protective, antioxidant properties of melatonin, an endogenous component of porcine follicular fluid, on apoptosis of granulosa cells were evaluated in this study. Porcine granulosa cells from medium-sized (3-5 mm), healthy follicles were cultured in serum-free conditions with melatonin (0, 0.01, 0.1, 1.0, 10, and 100 ng/mL) with or without its receptor antagonist, luzindole, followed by evaluation of apoptotic markers in the treated cells. Results revealed that endogenous, intrafollicular melatonin concentration decreased as follicular atresia progressed, whereas the percentage of apoptotic granulosa cells increased. Spontaneous apoptosis of granulosa cells, triggered by serum deprivation in vitro, was remarkably blocked by melatonin (1.0 ng/mL melatonin, 32.7 ± 0.5%, vs. control, 47.0 ± 1.0%; P < 0.05). Treatment with 1.0 ng/mL of melatonin also significantly elevated MT2, SOD1, and GPX4 while lowering FASL, CHOP, and GRP78 mRNA abundance compared to the untreated control. The anti-apoptotic effect and some changes of apoptotic-relevant genes in granulosa cells invoked by melatonin supplementation were markedly blocked by luzindole, suggesting that melatonin could prevent the apoptosis of porcine granulosa cells during follicular atresia via its membrane receptors and its free-radical-scavenging activity. These findings provide new insights into the regulatory mechanism of melatonin in follicular atresia-related functions. Mol. Reprod. Dev. 83: 692-700, 2016 © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Pancreatic carcinoma is a still unsolved health problem all over the world with poor prognosis and high mortality rate. YLT256, a novel synthesized chemical small inhibitor, displays potent antineoplastic activities via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT256 showed growth inhibition against a broad spectrum of human cancer cell lines and pancreatic cancer cell line BxPc-3 was the most sensitive with an IC50 of 0.42μM. We also found YLT256 could induce apoptosis of BxPc-3 cells in a dose-dependent manner. Western blot analysis revealed that the occurrence of its apoptosis was associated with activation of caspases-3 and -9, up-regulation of pro-apoptotic Bak, and down-regulation of anti-apoptotic Bcl-2. Moreover, YLT256-treated resulted in changes of mitochondrial membrane potential (Δψm), and generation of reactive oxygen species (ROS). Furthermore, our data also revealed that YLT256 suppressed the growth of established tumor-bearing xenograft models without obvious side effects. Immunohistochemical analyses and TUNEL assay revealed an increase in cleaved caspase-3-positive cells and TUNEL-positive cells, a decrease in Ki67-positive cells upon YLT256. Together, all the results of present study provided evidence demonstrating that YLT256 could be a promising potential drug candidate for pancreatic cancer therapy., (Copyright © 2016. Published by Elsevier Masson SAS.)

Bone marrow-derived mesenchymal stem cells (BMSCs) are being broadly investigated for treating numerous inflammatory diseases. However, the low survival rate of BMSCs during the transplantation process has limited their application. Autophagy can maintain cellular homeostasis and protect cells against environmental stresses. Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine that can induce both autophagy and apoptosis of BMSCs. However, the actual role of autophagy in TNF-α-induced apoptosis of BMSCs remains poorly understood. In the current study, BMSCs were treated with TNF-α/cycloheximide (CHX), and cell death was examined by the Cell Counting Kit-8, Hoechst 33342 staining, and flow cytometric analysis as well as by the level of caspase-3 and caspase-8. Meanwhile, autophagic flux was examined by analyzing the level of microtubule-associated protein light chain 3 B (LC3B)-II and SQSTEM1/p62 and by examining the amount of green fluorescent protein-LC3B by fluorescence microscopy. Then, the cell death and autophagic flux of BMSCs were examined after pretreatment and cotreatment with 3-methyladenine (3-MA, autophagy inhibitor) or rapamycin (Rap, autophagy activator) together with TNF-α/CHX. Moreover, BMSCs pretreated with lentiviruses encoding short hairpin RNA of beclin-1 (BECN1) were treated with TNF-α/CHX, and then cell death and autophagic flux were detected. We showed that BMSCs treated with TNF-α/CHX presented dramatically elevated autophagic flux and cell death. Furthermore, we showed that 3-MA and shBECN1 treatment accelerated TNF-α/CHX-induced apoptosis, but that Rap treatment ameliorated cell death. Our results demonstrate that autophagy protects BMSCs against TNF-α-induced apoptosis. Enhancing the autophagy of BMSCs may elevate cellular survival in an inflammatory microenvironment.

Diabetic osteoporosis represents a serious health condition with increasing incidence. Previous studies have shown that microRNA (miR)-335-5p is highly expressed in MC3T3-E1 osteoblasts and promotes their differentiation via downregulating the expression of dickkopf‑1 (DKK1). The present study investigated the effects of miR‑335‑5p on apoptosis of osteoblasts induced by high glucose (HG), as well as the underlying molecular mechanisms. MC3T3‑E1 osteoblasts were transfected with miR‑335‑5p mimics or control miR and cultured under HG conditions for seven days. Reverse‑transcription PCR and showed that, compared with the control group, the expression levels of miR‑335‑5p were significantly downregulated in the HG group. However, no significant differences were observed in the mRNA expression levels of DKK1 between these groups. Furthermore, flow cytometric analysis showed that the apoptotic rate was increased by >2‑fold in the HG group compared with that in the control group, while miR‑335‑5p overexpression significantly decreased the apoptotic rate in these model cells by ~40%. In addition, western blot analysis revealed that the protein expression levels of DKK1 and caspase‑3 were significantly elevated in the HG group, which was significantly inhibited by overexpression of miR‑335‑5p. These results may indicate that miR‑335‑5p overexpression inhibited HG‑induced apoptosis of MC3T3‑E1 osteoblasts through decreasing the protein expression levels of DKK1. The results of the present study suggested that miR‑335‑5p may represent a potential target for the treatment of diabetic osteoporosis.

Sophoridinic acid derivatives have received considerable attentions for their potencies in cancer therapy. IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells. In the present study, we explored the antitumor abilities of IMB-6G in human hepatocellular carcinoma (HCC) cells and investigated the underlying mechanisms. We found that IMB-6G inhibited cell growth and induced mitochondrial-dependent apoptosis in HepG2 and SMMC7721 cells. Analyses of the molecular mechanism of IMB-6G-induced apoptosis indicated IMB-6G induced endoplasmic reticulum (ER) stress activation. Incubation of HCC cells with IMB-6G induced increase in Bip and CHOP levels, which precede induction of apoptosis. Further study showed IMB-6G activated IRE1α and PERK pathways but did not stimulated ATF6 pathway in HCC cells. Moreover, silencing of IRE1α dramatically abrogated IMB-6G-induced pro-apoptotic ASK1-JNK signaling. Importantly, interruption of CHOP rendered HCC cells sensitive to IMB-6G-induced apoptosis via inactivation of Bim, PUMA and Bax. Thus, the IRE1α-ASK1 and PERK-CHOP pathways may be a novel molecular mechanism of IMB-6G-induced apoptosis. Collectively, our study demonstrates that IMB-6G induces ER stress-mediated apoptosis by activating IRE1α and PERK pathways. Our findings provide a rationale for the potential application of IMB-6G in HCC therapy., Competing Interests: The authors have declared no conflicts of interest to this work.