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It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti-apoptosis effects of Sal B in a cultured HEI-OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK-8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase-3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC-1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl-2. The expression pattern of p-PI3K and p-Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin- and cisplatin-induced apoptotic features, enhanced cell viability and accompanied with decreased caspase-3 activity in the HEI-OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl-2 expression and down-regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin- and cisplatin-injured HEI-OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin- and cisplatin-induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug-induced HC apoptosis, offering a potential use in the treatment of hearing loss., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

To investigate the underlying molecular mechanism for connexin 26 (Cx26) knockout-induced apoptosis, we performed TUNEL assays to detect apoptosis in the cochlear sensory epithelium in Cx26 knockout mice. We also compared the miRNA expression profiles of Cx26 knockout and wild-type mice using microarray technology and bioinformatic analyses. Real-time PCR, luciferase reporter gene assays, and scala media microinjections were performed to identify the effect of a specific miRNA and its targets. The results showed that apoptosis increased in the cochlear sensory epithelium of Cx26 knockout mice. The abnormal expression of mir-27a and sgk1 in Cx26 knockout mice was verified with real-time PCR. Luciferase reporter gene assays showed that overexpression of mir-27a significantly decreased sgk1 reporter gene activity; an inhibitor of mir-27a blocked the effect. Mir-27a lentivirus also inhibited sgk1 expression in cultured cochlear tissue. Mir-27a shRNA treatment inhibited Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice and increased the expression of sgk1 mRNA. Thus, mir-27a was identified as an apoptotic molecule that participates in Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice by downregulating sgk1 expression.

CD4(+) T cell polarization plays a critical role in a number of immune disorders; the pathogenesis is unclear. Chromobox homolog 7 (Cbx7) is involved in the gene transcription of several cell types. This study aims to investigate the mechanism by which Cbx7 modulates the CD4(+) T cell polarization. Expression of Cbx7 was assessed by quantitative RT-PCR and Western blotting. Apoptosis of CD4(+) T cell was analyzed by flow cytometry. The FasL promoter methylation was evaluated by the methylation specific PCR. The results showed that CD4(+) CD25(-) T cells express Cbx7 that was increased significantly after activation by exposing to anti-CD3/CD28 Ab, but suppressed by exposing to specific antigens. More apoptotic cells were detected in CD4(+) T cells with the Cbx7 gene knockdown. Exposure to insulin-like growth factor-1 up regulated the expression of Cbx7 in CD4(+) T cells. After antigen-specific TCR activation, Cbx7-deficient CD4(+) T cells expressed more FasL and showed the FasL gene promoter hyper demethylation than wild CD4(+) T cells. In addition, CD4(+) T cells with overexpression of Cbx7 showed lower levels of FasL gene promoter demethylation. We conclude that CD4(+) T cells express Cbx7; the latter prevents FasL expression and the activation-induced CD4(+) T cell apoptosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cerebral ischemia represents a severe brain injury that could lead to significant neuronal damage and death. In this study, we performed a middle cerebral artery occlusion (MCAO) in adult rats and observed that a subunit of nuclear factor-Y (NF-Y) transcriptional factor, NF-YC, was accumulated in rat hippocampal CA1 neurons. Immunochemistrical and immunofluorescent analysis revealed that NF-YC was primarily expressed in the nucleus of neurons. Meanwhile, we found that the changes of bim, one of the target genes of NF-Y, were consistent with the expression of NF-YC and Bim was mainly located in the NF-YC positive cells. Moreover, there was a concomitant upregulation of active caspase-3 and TUNEL positive cells. Taken together, these results suggested that the upregulation of NF-YC might play an important role in the pathophysiology via proapoptotic protein Bim after MCAO and further research is needed to have a better understanding of its function and mechanism.

Comprehensive Summary: Actin filaments play important physiological functions, which have become potential targets of antitumor drugs. Using chemicals to intervene their polymerization‐depolymerization dynamics would generate new strategies for designing antitumor drugs. In this report, an artificial water channel appending acetazolamide moiety, a ligand that can selectively bind to carbonic anhydrase IX, has been prepared. We demonstrated that this conjugate can target colorectal cancer cells overexpressing carbonic anhydrase IX and trigger the depolymerization of actin filaments of the cancer cells by selectively mediating water transmembrane transport. Moreover, the conjugate‐promoted actin depolymerization led to tumor cell apoptosis and its high antitumor activity in vitro and in vivo against colorectal cancer. The method described herein represents a new and general strategy for designing antitumor drugs by using artificial channel‐mediated selective water transport to promote actin depolymerization. [ABSTRACT FROM AUTHOR]

Previous studies have revealed that elevated expression of high mobility group A2 (HMGA2) is closely associated with the occurrence of pituitary adenomas (PAs). The expression of microRNA (miR)-16 is deregulated in PA tissues. Bioinformatics analysis has demonstrated that there is a complementary region between seed region of miR-16 and 3′-untranslated region (3′-UTR) of HMGA2 gene. In the present study, it was investigated whether miR-16 may regulate the expression of HMGA2 and whether it is involved in the pathogenesis of PAs. A total of 52 patients with PAs were recruited. Normal brain tissues obtained from 12 patients with traumatic brain injury were used as controls. The association between miR-16 and HMGA2 was validated using dual-luciferase reporter gene assay. HP75 cells were cultured in vitro and divided into the following groups: miR control, miR-16 mimic, small interfering RNA (si)-negative control, si-HMGA2 and miR-16 mimic+si-HMGA2 groups. The expression of miR-16 and HMGA2 in HP75 cells was determined using qRT-PCR and western blot. Cell proliferation was detected using the Cell Counting Kit-8 assay and apoptosis was detected using the TdT-UTP nick end labeling assay. Compared with normal pituitary tissues, the expression of miR-16 in PA tissues was significantly decreased, while the mRNA and protein levels of HMGA2 were significantly increased. miR-16 targeted the 3′-UTR of HMGA2 gene and regulated the expression of HMGA2. Transfection with siRNAs targeting HMGA2 and/or miR-16 mimics inhibited the expression of HMGA2 and the proliferative ability of HP75 cells, whereas it increased apoptosis of HP75 cells. The downregulation of miR-16 and upregulation of HMGA2 were involved in the pathogenesis of PAs. Thus, it is hypothesized that miR-16 inhibited the proliferation and promoted apoptosis of HP75 cells by inhibiting HMGA2 expression. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is in the forefront of malignancies for its high incidence and mortality. 5-Fluorouracil (5-FU) is one of the most widely used effective drugs for the treatment of CRC. However, there is an urgent need in reducing its systemic side effects and chemoresistance, in order to make 5-FU-based chemotherapy more effective in the treatment of CRC. In this study, engineered CRC cells were established to overexpress miR-323a-3p, which was a tumor suppressor that targeted both EGFR and TYMS. Then miR-323a-3p-loaded exosomes (miR-Exo) were obtained with suitable methods of collection and purification. We found that miR-Exo significantly inhibited CRC cell proliferation and induced apoptosis by the way of targeting EGFR directly in the cells, which eventually led to desirable tumor regression in the cell derived xenograft (CDX) and patient derived xenograft (PDX) tumor mice models. Moreover, we discovered that miR-323a-3p released from miR-Exo directly inhibited the upregulation of thymidylate synthase (TYMS) induced by 5-FU-resistence in CRC cells, resulting in the revival of tumor cytotoxicity from 5-FU. MiR-Exo could effectively induce the CRC cell apoptosis by targeting EGFR and TYMS, and enhance the therapeutic effects of 5-FU on CRC. Our work demonstrates the potency of miR-Exo for advanced CRC biotherapy. [ABSTRACT FROM AUTHOR]

Pikeperch (Sander lucioperca) is a sub-cold water fish species with high aquaculture potential. Its culture is seriously affected by increasing summer temperatures in recent years. Aim to investigate the effects of heat stress on apoptosis, oxidative stress, and the immune response in pikeperch. the fish were heat stressed at 30 °C, 32 °C and 34 °C for 2h respectively, followed by a 48h recovery period. The results showed that as temperature increased, the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2) in the liver increased significantly. Meanwhile, acute heat stress results in progressive deleterious alterations in liver tissue, especially vascular rupture, blood infiltration, and severe vacuolation at 34 °C. TUNEL staining revealed that the apoptosis level increased significantly with the rising temperature. Acute heat stress significantly induced the mRNA expression of apoptosis-related genes, including tumor suppressor (p53), B-cell lymphoma-2 (bcl-2), bcl-2-associated X (bax), apoptotic protease activating factor-1 (apaf-1), cysteinyl aspartate specific proteinase (caspase-3 and caspase-9), and the expression of p53 was also positively correlated with bax expression and the bax/bcl-2 ratio. Additionally, caspase-3 and caspase-9 activity increased significantly at 34 °C compared with the control group (23 °C). Innate immune genes, including tumor necrosis factor (tnf-α), interleukins (il-7 , il-8 , il-10 and il-1β), complement 3 (c3) were activated under acute heat stress, and H 2 O 2 content was positively correlated with the expressions of tnf-α and il-1β. After the temperature reached again 23 °C, most measured indexes in heat-stressed groups didn't return to stress-free levels, and liver tissue also didn't return to its normal state in the histopathology. It was found that p53 -mediated mitochondrial apoptosis pathway was triggered in pikeperch under acute heat stress, and there may be a vicious cycle between oxidative stress and inflammation. In summary, the present study is helpful to elucidate how acute heat stress mediates liver injury of pikeperch through mitochondrial pathway, inflammation and oxidative stress. • Acute heat stress induces mitochondrial pathway apoptosis regulated by P53 in the liver of pikeperch. • Acute heat stress leads to a vicious circle between H 2 O 2 and pro-inflammatory cytokines in the liver of pikeperch. • The interaction among oxidative stress, apoptosis and inflammation under acute heat stress leads to liver injury. [ABSTRACT FROM AUTHOR]