Dendrobine enhances bovine oocyte maturation and subsequent embryonic development and quality.

Strategies for improving the quality of oocytes have important theoretical and practical significance for increasing the efficiency of livestock breeding. In this respect, the accumulation of reactive oxygen species (ROS) is a major factor affecting the development of oocytes and embryos. This study investigated the effects of Dendrobium nobile extract (DNE) on the in vitro maturation of bovine oocytes and embryonic development after IVF. DNE is an extract from Dendrobium rhizomes that contains alkaloids with anti-inflammatory, anti-cancer and anti-ageing functions. Various concentrations of DNE (0, 5, 10, 20 and 50 µmol/L) were added during oocyte maturation in vitro, and we found that 10 µmol/L of DNE remarkably increased the oocyte maturation rate, the subsequent blastocyst formation rate and embryo quality. Further, we found that DNE treatment decreased the frequency of spindle/chromosome defects and ROS and increased the oocyte glutathione and mitochondrial membrane potential in oocytes. Moreover, DNE upregulated the expression of oxidative stress-related genes (Sirt1, Sirt2, Sirt3 and Sod1) in oocytes and apoptosis-related genes (Caspase-3, Caspase-4, Bax, Bcl-xl and Survivin) in blastocysts. These results suggest that DNE supplementation can promote oocyte maturation and subsequent embryonic development by regulating redox reactions and inhibiting embryonic apoptosis.

SOCS1 Regulates the Immunomodulatory Roles of MSCs on B Cells.

BACKGROUND AND OBJECTIVES: The effective use of MSCs for the treatment of some B cell-mediated immune diseases is quite limited. The main reason is that the immunomodulatory effects of mesenchymal stem cells (MSCs) on B cells are unclear, and their underlying mechanisms have not been fully explored. METHODS AND RESULTS: By co-culturing B cells with MSCs without (MSC/CTLsh) or with suppressor of cytokine signaling 1 (SOCS1) knockdown (MSC/SOCS1sh), we found that MSCs inhibited B cell proliferation, activation and terminal differentiation. Remarkably, the highest inhibition of B cell proliferation was observed in MSC/SOCS1sh co-culture. Besides, MSC/SOCS1sh reversed the inhibitory effect of MSCs in the last stage of B cell differentiation. However, MSC/SOCS1sh had no effect on inhibiting B cell activation by MSCs. We also showed that IgA+ B cell production was significantly higher in MSC/SOCS1sh than in MSC/CTLsh, although no difference was observed when both MSCs co-cultures were compared to isolated B cells. In addition, MSCs increased PGE2 production after TNF-α/IFN-γ stimulation, with the highest increase observed in MSC/SOCS1sh co-culture. CONCLUSIONS: Our results highlighted the role of SOCS1 as an important new mediator in the regulation of B cell function by MSCs. Therefore, these data may help to develop new treatments for B cell-mediated immune diseases.

Pterostilbene alleviates hydrogen peroxide-induced oxidative stress via nuclear factor erythroid 2 like 2 pathway in mouse preimplantation embryos.

Pterostilbene (PTS) in blueberries is a phytoalexin with antioxidant properties. PTS exerts strong cytoprotective effects on various cells via Nuclear Factor Erythroid 2 like 2 (NFE2L2) pathway. We evaluated the antioxidant PTS treatment in mouse preimplantation embryos. In vitro culture media were supplemented with different concentrations of PTS. Treatment of zygotes with 0.25 µM PTS improved the development of day 4 blastocysts (P < 0.05). Moreover, H2O2 treatment significantly increased the reactive oxygen species level and reduced the glutathione level in mouse blastocyst, whereas PTS treatment counteracted these effects. The fluorescence intensity of apoptotic positive cell was higher in the H2O2 group than in the PTS group. Furthermore, PTS-treated embryos significantly increased the protein expression of NFE2L2 in the nucleus and decreased Kelch-like ECH-associated protein1 (KEAP1). PTS treatment significantly increased the expression of downstream target genes involved in the NFE2L2 pathway, such as catalase (CAT), heme oxygenase1 (HMOX1), glutathione peroxidase (GPX), and superoxide dismutase (SOD); these genes confer cellular protection. In addition, PTS treatment significantly increased the expression of anti-apoptotic B-cell lymphoma 2 (BCL2), with a concomitant reduction in the apoptotic Bcl-2-associated X protein (BAX) and Caspase-3 genes in the embryo. PTS treatment also increased the protein expression of BCL2 and reduced the protein expression of BAX in the mouse embryo. In conclusion, PTS activated NFE2L2 signaling pathway in the development of mouse embryos by altering downstream expression of genes involved in the antioxidant mechanisms and apoptosis.

Pterostilbene exerts a protective effect via regulating tunicamycin-induced endoplasmic reticulum stress in mouse preimplantation embryos.

Pterostilbene (PTS) mainly enriched in small fruits such as berries and grapes exerts an antioxidant effect. However, the protective effects of PTS against endoplasmic reticulum stress (ERS) have not yet been elucidated in mouse preimplantation embryo. ERS plays an important role in regulating the pathological and physiological processes, including embryonic development. We explored the protective effect of PTS on the tunicamycin (TM)-induced ERS in mouse preimplantation embryos. In vitro, culture medium was supplemented with different concentrations of TM and PTS. Our result indicated that treatment of zygotes with 0.5 µg/ml TM significantly decreased the development of day 4 blastocysts (P < 0.05), whereas 0.25 µM PTS supplementation improved the development rate of blastocysts. Moreover, TM treatment significantly increased (P < 0.05) the apoptotic index and reduced the total cell number of the blastocyst, whereas PTS treatment counteracted these effects. Additionally, TM potently increased expression levels of ERS-related proteins, such as GRP78, ATF6, PERK, p-Perk, IRE1, ATF4, and CHOP (P < 0.05). However, PTS and PTS + TM treatment decreased expression levels of ERS-related proteins (P < 0.05). Furthermore, expression level of the anti-apoptotic protein and gene BCL2 significantly decreased (P < 0.05) in TM-treated embryo but increased by PTS treatment (P < 0.05), whereas expression levels of the pro-apoptotic protein and gene BAX increased (P < 0.05) with TM but significantly decreased (P < 0.05) with co-treatment with PTS. In summary, PTS treatment significantly increased the development potential of mouse embryo by reduction of ERS.

Quinalizarin Induces Apoptosis through Reactive Oxygen Species (ROS)-Mediated Mitogen-Activated Protein Kinase (MAPK) and Signal Transducer and Activator of Transcription 3 (STAT3) Signaling Pathways in Colorectal Cancer Cells.

BACKGROUND Quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) exhibits potentially useful anticancer effects by inducing apoptosis in several types of cancer, but its underlying mechanism of action remains unknown. The present study examined the effects of quinalizarin on the induction of cell cycle arrest, apoptosis, the generation of reactive oxygen species (ROS), other underlying mechanisms, and its role in modifying colorectal cancer cell lines. MATERIAL AND METHODS The MTT assay was used to evaluate the viability of SW480 and HCT-116 cells that had been treated with quinalizarin and 5-fluorouracil (5-FU). Cell cycle arrest and apoptosis were analyzed by flow cytometry. Western blotting was used to investigate the mitochondrial pathway; Akt, MAPK, and STAT3 signaling pathways were also investigated. The relationship between ROS generation and apoptosis was analyzed by flow cytometry and western blotting. RESULTS The results indicated that quinalizarin significantly inhibits the viability of SW480 and HCT-116 cells in a dose-dependent manner. Quinalizarin induced SW480 cell cycle arrest at G2/M by regulating cyclin B1 and CDK1/2. The apoptosis-related protein expression levels of p-p53, Bad, cleaved caspase-3, cleaved PARP and p-JNK were increased in quinalizarin-treated cells, while protein expression levels Bcl-2, p-Akt, p-ERK, and p-STAT3 were decreased. Quinalizarin induced apoptosis in colorectal cancer cells by regulating MAPK and STAT3 signaling pathways via ROS generation. CONCLUSIONS Quinalizarin induces apoptosis via ROS-mediated MAPK/STAT3 signaling pathways.

Novel 1,4-naphthoquinone derivatives induce apoptosis via ROS-mediated p38/MAPK, Akt and STAT3 signaling in human hepatoma Hep3B cells.

1,4-Naphthoquinone and its derivatives have shown some efficacy as therapeutic compounds for cancer and inflammation, though their clinical application is limited by their side-effects. To reduce the toxicity of these compounds and optimize their effects, we synthesized two 1,4-naphthoquinone derivatives-2-butylsulfinyl- 1,4-naphthoquinone (BSNQ) and 2-octylsulfinyl-1,4-naphthoquinone (OSNQ)-and investigated their effects and underlying mechanisms in hepatocellular carcinoma cells. BSNQ and OSNQ decreased cell viability and significantly induced apoptosis, accompanied by the accumulation of reactive oxygen species (ROS). However, pretreatment with N-acetyl-l-cysteine, a specific ROS scavenger, blocked apoptosis. Western blot results indicated that BSNQ and OSNQ up-regulated the phosphorylation of p38 and JNK, and down-regulated the phosphorylation of ERK, Akt and STAT3, and that these effects were blocked by N-acetyl-l-cysteine. Furthermore, BSNQ and OSNQ suppressed tumor growth and modulated MAPK and STAT3 signaling in mouse xenografts without detectable effects on body weight or hematological parameters. These results indicate that BSNQ and OSNQ induce apoptosis in human hepatoma Hep3B cells via ROS-mediated p38/MAPK, Akt and STAT3 signaling pathways, suggesting that these 1,4-naphthoquinone derivatives may provide promising new anticancer agents to treat HCC.

Quinalizarin exerts an anti-tumour effect on lung cancer A549 cells by modulating the Akt, MAPK, STAT3 and p53 signalling pathways.

Quinalizarin may be a potential chemical agent for cancer therapy, as it exerts anti­tumour effects against a variety of different types of cancer. However, the underlying regulatory mechanism and signalling pathways of quinalizarin in lung cancer cells remains unknown. The present study sought to investigate the effects of quinalizarin on proliferation, apoptosis and reactive oxygen species (ROS) generation in lung cancer. MTT assays were used to evaluate the effects of quinalizarin on the viability of lung cancer A549, NCI­H460 and NCI­H23 cells. Flow cytometry was employed to evaluate the effects of quinalizarin on the cell cycle, apoptosis and ROS generation in A549 cells. Western blotting was performed to detect cell cycle and apoptosis­associated protein expression levels in A549 cells. Quinalizarin inhibited A549, NCI­H460 and NCI­H23 cell proliferation and induced A549 cell cycle arrest at the G0/G1 phase. Quinalizarin induced apoptosis by upregulating the expression of B­cell lymphoma 2 (Bcl­2)­associated agonist of cell death, cleaved­caspase­3 and cleaved­poly (adenosine diphosphate­ribose) polymerase, and downregulating the expression of Bcl­2. Furthermore, quinalizarin activated mitogen­activated protein kinase (MAPK) and p53, and inhibited the protein kinase B and signal transducer and activator of transcription­3 (STAT3) signalling pathways. In addition, quinalizarin increased ROS generation. The ROS scavenger N­acetyl­L­cysteine restored quinalizarin­induced cell apoptosis, and inactivated the MAPK and STAT3 signalling pathways. The results of the present study demonstrated that quinalizarin induces G0/G1 phase cell cycle arrest and apoptosis via ROS mediated­MAPK and STAT3 signalling pathways.

Reactive oxygen species-mediated unfolded protein response pathways in preimplantation embryos.

Excessive production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress-mediated responses are critical to embryonic development in the challenging in vitro environment. ROS production increases during early embryonic development with the increase in protein requirements for cell survival and growth. The ER is a multifunctional cellular organelle responsible for protein folding, modification, and cellular homeostasis. ER stress is activated by a variety of factors including ROS. Such stress leads to activation of the adaptive unfolded protein response (UPR), which restores homeostasis. However, chronic stress can exceed the toleration level of the ER, resulting in cellular apoptosis. In this review, we briefly describe the generation and impact of ROS in preimplantation embryo development, the ROS-mediated activation mechanism of the UPR via the ER, and the subsequent activation of signaling pathways following ER stress in preimplantation embryos.

Effect of potential role of p53 on embryo development arrest induced by H2O2 in mouse.

During mammalian embryo development in vitro, mechanism of embryonic development arrest caused by oxidative stress has not been clear so far. The tumor suppressor protein p53 controls cell cycle and programmed cell death by regulating relevant signal pathway. Recent researches revealed that the concentration and distribution of p53 are closely related with reactive oxygen species (ROS). The main objective of this experiment was to explore the role of p53 on embryonic development arrest caused by oxidative stress. Results showed that embryo arrest at two-four-cell stage was significantly increased in the presence of 50 µM H2O2 (39.01 ± 2.74 vs. 77.20 ± 5.34%, p < 0.05). Supplementation of N-acetyl-L-cysteine (NAC) obviously reduced the ratio of development arrest (39.01 ± 2.74 vs. 71.18 ± 5.34%, p < 0.05), which was accompanied by an increase in ROS level, and H2O2 treatment sharply increased messenger RNA (mRNA) expression and protein levels of p53 and p53-ser15. Further increased transcription of GADD45a and p21, a downstream of p53, has an especially significant effect on the mRNA expression of GADD45a. However, expressions of cdc2 were reduced by H2O2. In addition, using Pifithrin-α (PFT-α), the suppresser of p53, the result showed that GADD45a and p21 were significantly downregulated, but the cell cycle gene cdc2 was significantly upregulated, while the protein level of p53 and p53-ser15 was significantly decreased. Taken together, these results demonstrate that ROS could activate p53 and regulate p53 target genes to influence early embryo development in in vitro culture.

Cryptotanshinone induces ROS-mediated apoptosis in human gastric cancer cells.

Cryptotanshinone (CT), isolated from the plant Salvia miltiorrhiza Bunge, has been reported to have potential anticancer effects on human prostate and breast cancer cells. However, the mechanisms of action of CT on gastric cancer (GC) cells are not well understood. Here we investigated the antitumor effects of CT on GC cells and its possible molecular mechanism. We found CT suppressed viability of twelve GC cell lines in a dose-dependent manner. CT induced cell cycle arrest at the G2/M phase and mitochondrial apoptosis accompanying the accumulation of reactive oxygen species (ROS). Pretreatment with ROS inhibitor N-acetyl-L-cysteine (NAC) blocked CT-induced apoptosis. CT increased p-JNK and p-p38, and decreased p-ERK and p-STAT3 protein expression, these effects were prevented by NAC. Furthermore, a xenograft assay showed that CT significantly inhibited MKN-45 cell-induced tumor growth in vivo by increasing expression of pro-apoptotic proteins (p-JNK, p-38 and cleaved-caspase-3) and reducing expression of anti-apoptotic proteins (p-ERK and p-STAT3) without adverse effects on nude mice weight. In conclusion, CT induced apoptosis and cell cycle arrest in GC cells via ROS-mediated MAPK and AKT signaling pathways, and this CT may be a useful compound for the developing anticancer agents for GC.

Delta-Like-1 Changes the Immunomodulatory Property of OP9 Cells.

As stromal cells and recently confirmed mesenchymal stem cells, OP9 cells support hematopoiesis stem cell (HSC) differentiation into the B lymphocyte lineage, yet Delta-like-1 (DL1) overexpressing OP9 (OP9DL1) cells promote the development of early T lymphocytes from HSC. However, the immunomodulatory capacity of OP9 or OP9DL1 on mature B and T cell proliferation has not been elucidated. Here, we show that OP9 and OP9DL1 have similar proliferation capacities and immunophenotypes except DL1 expression. Compared with OP9, OP9DL1 displayed more osteogenesis and less adipogenesis when cultured in the respective induction media. Both OP9 and OP9DL1 inhibited mature B and T cell proliferation. Furthermore, OP9 showed stronger inhibition on B cell proliferation and OP9DL1 exhibited stronger inhibition on T cell proliferation. With stimulation, both OP9 and OP9DL1 showed increased nitrate oxide (NO) production. The NO levels of OP9 were higher than that of OP9DL1 when stimulated with TNFα/IFNγ or LPS/IL4. Taken together, our study reveals a previously unrecognized role of OP9 and OP9DL1 in mature B and T cell proliferation. DL1 overexpression alone changed the properties of OP9 cells in addition to their role in early B cell development.

SOCS1 regulates the immune modulatory properties of mesenchymal stem cells by inhibiting nitric oxide production.

Mesenchymal stem cells (MSCs) have been shown to be highly immunosuppressive and have been employed to treat various immune disorders. However, the mechanisms underlying the immunosuppressive capacity of MSCs are not fully understood. We found the suppressor of cytokine signaling 1 (SOCS1) was induced in MSCs treated with inflammatory cytokines. Knockdown of SOCS1 did not bring much difference on the proliferation and differentiation properties of MSCs. However, MSCs with SOCS1 knockdown exhibited enhanced immunosuppressive capacity, showing as inhibiting T cell proliferation at extremely low ratio (MSC to T) in vitro, significantly promoting tumor growth and inhibiting delayed-type hypersensitivity response in vivo. We further demonstrated that SOCS1 inhibited the immunosuppressive capacity of MSCs by reducing inducible nitric oxide synthase (iNOS) expression. Additionally, we found the significantly lower SOCS1 expression and higher nitric oxide (NO) production in MSCs isolated from synovial fluid of rheumatoid arthritis patients. Collectively, our data revealed a novel role of SOCS1 in regulating the immune modulatory activities of MSCs.