<Emphasis Type="Italic">Nigella sativa</Emphasis> Oil and Chromium Picolinate Ameliorate Fructose-Induced Hyperinsulinemia by Enhancing Insulin Signaling and Suppressing Insulin-Degrading Enzyme in Male Rats

Lead (Pb) pollution has become one of the most serious global ecological problems. In animals, Pb ingestion induces apoptosis in many tissues. However, the mechanisms by which Pb induces apoptosis in chicken splenic lymphocytes in vitro via the PI3K/Akt pathway and the antagonistic effect of selenium (Se) on Pb remain unclear. Therefore, we established the in vitro Se-Pb interaction model in chicken splenic lymphocytes and examined the frequency of apoptotic cells using acridine orange/ethidium bromide (AO/EB) staining and the TdT-mediated dUTP nick end labeling assay and detected the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The expression of PI3K/Akt pathway-related genes was also examined by qRT-PCR and western blotting. MDA and ROS levels were markedly increased, whereas the activities of GPx, SOD, and CAT were significantly decreased; the levels of the PI3K, Akt, and Bcl-2 messenger RNAs (mRNAs) and proteins were decreased; and the levels of the p53, Bax, cytochrome c (Cyt-c), caspase 3, and caspase 9 mRNAs and proteins were increased in the Pb group. In addition, the frequency of apoptotic cells was also significantly increased by the Pb treatment. However, Se supplementation during Pb exposure observably attenuated Pb-induced apoptosis; increased the levels of the PI3K, Akt, and Bcl-2 mRNAs and proteins; and decrease the levels of the p53, Bax, Cyt-c, caspase 3, and caspase 9 mRNAs and proteins in the chicken spleen. In conclusion, Pb exposure causes oxidative stress, inhibits the PI3K/Akt pathway, and subsequently induces apoptosis in chicken splenic lymphocytes in vitro, and these effects are partially attenuated by Se supplementation. To the best of our knowledge, this study is the first to reveal the antagonistic effect of Se on Pb-induced apoptosis of chicken splenic lymphocytes in vitro via the activation of the PI3K/Akt pathway.     

Copper-induced germline apoptosis in Caenorhabditis elegans: The independent roles of DNA damage response signaling and the dependent roles of MAPK cascades

Excess copper is toxic to life. Copper has been shown to induce apoptosis in various cell lines and tissues. However, due to the lack of appropriate gene knockout animal models, data concerning the underlying pathways of copper-induced apoptosis are insufficient, especially with regards to in vivo systems. The nematode Caenorhabditis elegans is a good model to study basic biological processes, including stress responses and apoptosis. In the present study, we investigated copper-induced germline apoptosis in the C. elegans strains carrying mutated alleles of homologs to known mammalian genes that are involved in apoptosis regulation. We show here that exposing C. elegans to copper causes dose- and time-dependent germline apoptosis. The knockout of checkpoint genes hus-1, clk-2, the Bcl-2 homolog ced-9, and the BH3-only domain egl-1 did not prevent cells of the germline from copper-induced apoptosis. The loss-of-function of the tumor suppressor gene, p53/cep-1, caused a significant increase in germline apoptosis with exposure to copper, and the depletion of p53 antagonist ABL1 significantly enhanced apoptosis. The knockout of the caspase gene ced-3 and the Apaf-1 homolog ced-4 abrogated both copper-induced and physiological germline apoptosis. Germline apoptosis stopped increase in the strains lin-45(ku51), mek-2(n1989), mpk-1(ku1) under copper stresses, respectively. Copper-induced apoptosis was blocked in the loss-of-function alleles of both JNK and p38 MAPK cascades excepting pmk-3, one of the three p38 MAPK components. Together, the results of this study suggest that caspase and Apaf-1 are required for copper-induced germline apoptosis while DNA damage response genes are not essential, and that the Raf-MEK-ERK, ASK1/2-MKK7-JNK, ASK1/2-MKK3/6-p38 signaling pathways are indispensable in mediating this apoptotic response.     

Expression profiles of seven glutathione S-transferase (GST) genes in cadmium-exposed river pufferfish (Takifugu obscurus)

Glutathione S-transferase (GST; EC 2.5.1.18) plays a critical role in detoxification pathways. In this study, we report cloning and expression of seven genes of the GST family of the pufferfish Takifugu obscurus together with mRNA tissue distribution pattern and time-course of expression in response to exposure to cadmium. At basal levels of tissue expression, GST-Mu is highly expressed in liver compared with other tissues. When fish were exposed to cadmium (5 mg/L for 96 h), expression of GST-MAPEG, GST-Mu, GST-Omega, and GST-Zeta was greatly increased, whereas GST-Alpha and GST-Kappa genes showed no significant response. These findings suggest that gene expression of a number of GST isoforms in T. obscurus is modulated in response to exposure to cadmium. We propose GST-Mu, GST-Theta, and GST-Zeta as candidate biomarkers for heavy metal exposure in this fish.     

Effects of astragalus polysaccharide on apoptosis of myocardial microvascular endothelial cells in rats undergoing hypoxia/reoxygenation by mediation of the PI3K/Akt/eNOS signaling pathway

The study explores the effect of astragalus polysaccharide (APS) mediating P13K/Akt/eNOS signaling pathway on apoptosis of myocardial microvascular endothelial cells (MMECs) in hypoxia/reoxygenation (H/R). MMECs were classified into blank, H/R, H/R + 25 mg/L APS, H/R + 50 mg/L APS, H/R + 100 mg/L APS, H/R + LY, and HR + 100 mg/L APS + LY groups. Cell viability was detected using MTT assay and apoptotic cell morphological changes by Hoechst staining. NO content, cell cycle and apoptosis, PI3K/Akt/eNOS signaling pathway proteins were detected using nitrate reductase assay, flow cytometry and Western blotting. An increased cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins, and a decreased apoptosis rate was observed in the H/R + 50 mg/L APS and H/R + 100 mg/L APS groups compared with the H/R and H/R + 25 mg/L APS groups. Compared with the H/R + 50 mg/L APS group, the apoptosis rate decreased, whereas the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins increased in the H/R + 100 mg/L APS group. The H/R + LY and HR + 100 mg/L APS + LY groups followed opposite trends. In comparison to the HR + 100 mg/L APS group, the apoptosis rate in the H/R + LY and HR + 100 mg/L APS + LY groups increased, and the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins decreased. Collectively, APS improves the damage caused by H/P by mediating PI3K/Akt/eNOS signaling pathway.     

Cyclotrisiloxan and β-Sitosterol rich Cassia alata (L.) flower inhibit HT-115 human colon cancer cell growth via mitochondrial dependent apoptotic stimulation

Cancer traits dependent chemo and radiotherapy display acute toxicity and long-term side effects. Since last two decades, researchers investigated a new anticancer agents derived from plants. Cassia alata (L.) is a medicinal herb distributed in the tropical and humid regions. In this study, C. alata flower methanol extract (CME) have been prepared using cold percolation method and the phytochemical components were identified using GC–MS analysis. CME have been used to study the antiproliferative and apoptosis properties against human colon cancer HT-115 colon cancer cells, its molecular mechanism have been explored. 0.2 mg/mL dose of CME, inhibited 50% of HT-115 colon cancer cell growth after 48hr was confirmed the significant antiproliferation effect. In normal cells such as Vero cells and hMSCs, 0.2 mg/mL dose of CME shown only 4% and 5% growth inhibition confirmed the HT-115 cell specific cytotoxic effect. This effect might be due to the availability of phytoactive biomolecules in CME such as, cyclotrisiloxan, beta-sitosterol and alpha-tocopherol have been confirmed by GC–MS. Most interestingly, PI and AO/ErBr staining of CME treated HT-115 cells shown early (25%), pro (17%) and late (8%) apoptotic and 3% necrotic cells after 48 hr. Treatment with CME extract showed potential effect on the inhibition of protumorigenic inflammatory and oxidative stress genes. Protumorigenic COX-2/PGE-2 and TNF-α/NF-κB immune axis were normalized after CME treatment. Amounts of both apoptosis related mRNA p53, Bax, caspase 3 and p21 genes were upregulated, whereas it resulted in significant reduction in the anti-apoptotic marker mdm2 and Bcl-2 genes. In conclusion, bioactive compounds present in CME potentially inhibit HT-115 colon cancer cell proliferation via an inhibition of protumorigenic immune axis and stimulation of mitochondria dependent apoptotic pathway without necrotic effect.     

CCl4诱导的大鼠急性肝衰竭与恢复过程中基因表达变化及作用分析

急性肝［功能］衰竭(acute liver failure, ALF)是一种危害较大的肝疾病,因其诱发和影响因素众多,导致其发生和发展机制尚不完全清楚。本文构建了四氯化碳(CCl4)诱导的大鼠ALF模型,通过检测血清ALT和AST活性、肝系数及形态结构进行建模评估,用大鼠基因组 230 2.0芯片和生物信息学方法检测和分析了相关基因表达变化,用qRT-PCR和Western印迹检测了其机制相关基因在mRNA和蛋白质水平的表达变化。结果表明,本研究成功建立了可靠的大鼠ALF模型。检测发现,6 681个基因发生了有意义的表达变化。其中,4 819个基因与ALF相关。在急性肝［功能］衰竭过程中,细胞存活、增殖和分化等生理活动及IL-1、IL-6和IL-8等信号通路的信号传导活性增强,而细胞凋亡以及p53、ATM和AMPK等信号通路减弱。基于本文结果推测,在ALF的损伤和进展阶段,炎症因子IL-1R1、TNFR1和TNFR2等通过IL-1α→IL-1R1→→MAPK8→FOS/JUN途径和/或TNF-α→TNFR1/B→→ NF-κB→→ Caspases途径促进细胞凋亡、炎症反应和免疫应答;抑癌基因TP53在进展和恢复阶段,通过p53途径调节细胞凋亡;TNF-α和IL-10在恢复阶段,通过NF-κB、JAK-STAT和MAPK等信号通路激活增殖相关基因表达,促进肝细胞增殖。本文推测,在CCl4诱导的急性肝［功能］衰竭中,IL-1R1、TNFR1、TNFR2、CASPASE3、TP53、PCNA和NF-κB等基因发挥重要作用。本文为了解急性肝［功能］衰竭的发生和发展机制提供了有用的信息。     

Cyclotrisiloxan and β-Sitosterol rich Cassia alata (L.) flower inhibit HT-115 human colon cancer cell growth via mitochondrial dependent apoptotic stimulation

Cancer traits dependent chemo and radiotherapy display acute toxicity and long-term side effects. Since last two decades, researchers investigated a new anticancer agents derived from plants. Cassia alata (L.) is a medicinal herb distributed in the tropical and humid regions. In this study, C. alata flower methanol extract (CME) have been prepared using cold percolation method and the phytochemical components were identified using GC–MS analysis. CME have been used to study the antiproliferative and apoptosis properties against human colon cancer HT-115 colon cancer cells, its molecular mechanism have been explored. 0.2 mg/mL dose of CME, inhibited 50% of HT-115 colon cancer cell growth after 48hr was confirmed the significant antiproliferation effect. In normal cells such as Vero cells and hMSCs, 0.2 mg/mL dose of CME shown only 4% and 5% growth inhibition confirmed the HT-115 cell specific cytotoxic effect. This effect might be due to the availability of phytoactive biomolecules in CME such as, cyclotrisiloxan, beta-sitosterol and alpha-tocopherol have been confirmed by GC–MS. Most interestingly, PI and AO/ErBr staining of CME treated HT-115 cells shown early (25%), pro (17%) and late (8%) apoptotic and 3% necrotic cells after 48 hr. Treatment with CME extract showed potential effect on the inhibition of protumorigenic inflammatory and oxidative stress genes. Protumorigenic COX-2/PGE-2 and TNF-α/NF-κB immune axis were normalized after CME treatment. Amounts of both apoptosis related mRNA p53, Bax, caspase 3 and p21 genes were upregulated, whereas it resulted in significant reduction in the anti-apoptotic marker mdm2 and Bcl-2 genes. In conclusion, bioactive compounds present in CME potentially inhibit HT-115 colon cancer cell proliferation via an inhibition of protumorigenic immune axis and stimulation of mitochondria dependent apoptotic pathway without necrotic effect.     

Polygonatum cyrtonema lectin induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras–Raf and PI3K–Akt signaling pathways

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which PCL induces cancer cell death are still elusive. In the current study, we found that PCL could induce apoptosis and autophagy in murine fibrosarcoma L929 cells. Subsequently, we demonstrated that inhibition of Ras could promote L929 cell death, suggesting that Ras–Raf signaling pathway plays the key negative regulator in PCL-induced apoptosis. And, we showed that Ras-Raf signaling pathway was also involved in PCL-induced autophagy as the negative regulator. In addition, we found that class I phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway could play the negative regulator in PCL-induced apoptosis and autophagy. Taken together, these results demonstrate that PCL induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K–Akt signaling pathways.     

Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: a role for NF-κB-STAT3-directed gene expression

Mitochondrial DNA depleted (ρ⁰) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ⁺ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ⁰ cells compared to ρ⁺ HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ⁺ HSF, but this response was substantially decreased in ρ⁰ HSF. Suppression of the IKK–NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2–STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ⁺ HSF. Inhibitory antibodies against IL6, the main activator of JAK2–STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ⁰ HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6–JAK2–STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.