Toxicity studies of condensed fuzheng extract in mice and rats.

Nutritional supplements have been used to improve immune function. Condensed fuzheng extract (CFE) is a well-known traditional Chinese medicine (TCM) formula that is predominantly made from sheep placenta, Astragalus mongholicus Bunge, and Polygonatum kingianum Collett & Hemsl. However, the toxicological profile of CFE has not been determined. In this study, we investigated the acute (14 days) and sub-chronic (90 days) oral toxicities of CFE in mice and rats and the phytochemical composition of CFE. Materials and methods: For the assessment of acute toxicity, 80 ICR mice of both sexes were randomly divided into four groups. Three groups were treated with 4500, 2250 and 1125 mg/kg/d bw CFE daily (n = 10/group per sex) for 14 days; a separate group was used as control. To test the sub-chronic toxicity, male and female Sprague Dawley rats were orally administered 8150, 4075 or 2037 mg/kg bw of CFE for 90 days; a control group was included. Hematological, biochemical, and histopathological markers were tested at the end of the experiment. The chemical composition of CFE was determined by UPLC-HRMS method. Results: In both acute and sub-chronic toxicity studies, no mortalities, indications of abnormality, or treatment-related adverse effects were observed. The LD50 of CFE was higher than 4500 mg/kg. There were no significant changes in the hematological and biochemical data in the treatment group compared with the control group (p > 0.05). Histopathological analyses of the heart, liver, spleen, lungs, kidneys, thymus, testes (male rats) and ovaries (female rats) revealed no anatomical changes of each organ. Phytochemical analysis of CFE revealed the presence of flavonoids (highest abundance), phenols and alkaloids. In conclusion, our results showed that CFE is a safe and non-toxic formula. We also reported phytochemicals in CFE that may possess important pharmacological effects.

PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1.

Protein arginine methyltransferase 5 (PRMT5) is a major type II enzyme responsible for symmetric dimethylation of arginine (SDMA), and plays predominantly roles in human cancers, including in ovarian cancer. However, the exactly roles and underlying mechanisms of PRMT5 contributing to the progression of ovarian cancer mediated by reprogramming cell metabolism remain largely elusive. Here, we report that PRMT5 is highly expressed and correlates with poor survival in ovarian cancer. Knockdown or pharmaceutical inhibition of PRMT5 is sufficient to decrease glycolysis flux, attenuate tumor growth, and enhance the antitumor effect of Taxol. Mechanistically, we find that PRMT5 symmetrically dimethylates alpha-enolase (ENO1) at arginine 9 to promotes active ENO1 dimer formation, which increases glycolysis flux and accelerates tumor growth. Moreover, PRMT5 signals high glucose to increase the methylation modification of ENO1. Together, our data reveal a novel role of PRMT5 in promoting ovarian cancer growth by controlling glycolysis flux mediated by methylating ENO1, and highlights that PRMT5 may represent a promising therapeutic target for treating ovarian cancer.

PRMT6 promotes tumorigenicity and cisplatin response of lung cancer through triggering 6PGD/ENO1 mediated cell metabolism.

Metabolic reprogramming is a hallmark of cancer, including lung cancer. However, the exact underlying mechanism and therapeutic potential are largely unknown. Here we report that protein arginine methyltransferase 6 (PRMT6) is highly expressed in lung cancer and is required for cell metabolism, tumorigenicity, and cisplatin response of lung cancer. PRMT6 regulated the oxidative pentose phosphate pathway (PPP) flux and glycolysis pathway in human lung cancer by increasing the activity of 6-phospho-gluconate dehydrogenase (6PGD) and α-enolase (ENO1). Furthermore, PRMT6 methylated R324 of 6PGD to enhancing its activity; while methylation at R9 and R372 of ENO1 promotes formation of active ENO1 dimers and 2-phosphoglycerate (2-PG) binding to ENO1, respectively. Lastly, targeting PRMT6 blocked the oxidative PPP flux, glycolysis pathway, and tumor growth, as well as enhanced the anti-tumor effects of cisplatin in lung cancer. Together, this study demonstrates that PRMT6 acts as a post-translational modification (PTM) regulator of glucose metabolism, which leads to the pathogenesis of lung cancer. It was proven that the PRMT6-6PGD/ENO1 regulatory axis is an important determinant of carcinogenesis and may become a promising cancer therapeutic strategy.

Nanosized copper particles induced mesangial cell toxicity via the autophagy pathway.

Nanosized copper particles (nano Cu) have been incorporated into products in multiple industries, although studies have demonstrated that these particles are nephrotoxic. We investigated the cytotoxicity of nanosized copper particles on rat mesangial cells and measured rates of apoptosis, the expression of caspase-3, and generation of reactive oxygen species. We also measured autophagy through the acridine orange (AO) staining and expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62 to screen the underlying mechanism of toxicity. Nanosized copper particles inhibited mesangial cell viability, up-regulated the activity of caspase-3, and increased the rates of apoptosis and the generation of reactive oxygen species in a concentration-dependent manner. Exposure to nano Cu increased the formation of acidic vesicular organelles and the expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62, and treatment with an autophagy inhibitor reduced nephrotoxicity. This indicated that the autophagy pathway is involved in the toxicity induced by nanosized copper particles to mesangial cells. This finding can contribute to the development of safety guidelines for the evaluation of nanomaterials in the future.

Neuroprotection of Triptolide against Amyloid-Beta1-42-induced toxicity via the Akt/mTOR/p70S6K-mediated Autophagy Pathway.

Triptolide is a natural active compound that has significant neuroprotective properties and shows promising effects in the treatment of Alzheimer's disease (AD). Recent studies have shown that autophagy occurs in AD. In this study, we determined whether autophagy regulated by triptolide ameliorates neuronal death caused by amyloid-Beta1-42 (Aß1-42). We examined the effects of triptolide on cell viability, autophagy, apoptosis, and the protein kinase B/mammalian target of the rapamysin/70 kDa ribosomal protein S6 kinase (Akt/mTOR/p70S6K) signaling pathway in PC12 cells. The results indicated that triptolide treatment exhibited a cytoprotective effect against cell injury induced by Aß1-42. Triptolide also reduced apoptosis and enhanced cell survival by decreasing autophagosome accumulation and inducing autophagic degradation. Furthermore, our results also showed that activating the Akt/mTOR/p70S6K mechanism was one reason for the protection of triptolide. Triptolide treatment protected against Aß1-42-induced cytotoxicity by decreasing autophagosome accumulation, and inducing autophagic degradation in PC12 cells. These findings also suggest that the reduction of autophagosome accumulation observed in triptolide-treated cells was Akt/mTOR/p70S6K pathway dependent. Overall, triptolide exhibits a neuron protective effect and this study provides new insight into AD prevention and treatment.

Nanosized copper particles induced mesangial cell toxicity via the autophagy pathway

Nanosized copper particles (nano Cu) have been incorporated into products in multiple industries, although studies have demonstrated that these particles are nephrotoxic. We investigated the cytotoxicity of nanosized copper particles on rat mesangial cells and measured rates of apoptosis, the expression of caspase-3, and generation of reactive oxygen species. We also measured autophagy through the acridine orange (AO) staining and expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62 to screen the underlying mechanism of toxicity. Nanosized copper particles inhibited mesangial cell viability, up-regulated the activity of caspase-3, and increased the rates of apoptosis and the generation of reactive oxygen species in a concentration-dependent manner. Exposure to nano Cu increased the formation of acidic vesicular organelles and the expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62, and treatment with an autophagy inhibitor reduced nephrotoxicity. This indicated that the autophagy pathway is involved in the toxicity induced by nanosized copper particles to mesangial cells. This finding can contribute to the development of safety guidelines for the evaluation of nanomaterials in the future.

PIKE-A promotes glioblastoma growth by driving PPP flux through increasing G6PD expression mediated by phosphorylation of STAT3.

Reprogramming of energy metabolism is a hallmarkofcancer, and the pentose phosphate pathway (PPP) is a major glucose metabolic pathway important for meeting the cellular demands of biosynthesis and anti-oxidant defense. Our previous study showed that phosphoinositide 3-kinase enhancer-activating Akt (PIKE-A) plays an important role in glioblastoma cell survival and growth under cellular energy stress condition. However, the crucial functions of PIKE-A in cancer energy metabolism are poorly understood.In the present study, we show that PIKE-A promotes DNA biosynthesis, NADPH production and inhibits reactive oxygen species (ROS) production, leading to increasing proliferation and growth of glioblastoma cell and suppressing cellular senescence. Mechanistically, PIKE-A binds to STAT3 and stimulates its phosphorylation mediated by tyrosine kinase Fyn, which enhances transcription of the rate-limitting enzyme glucose-6-phosphate dehydrogenase (G6PD) in the PPP. Finally, targeting PIKE-A-G6PD axis sensitizes glioblastoma to temozolomide (TMZ)treatment. This study reveals that STAT3 is a novel binding partner of PIKE-A which recruits Fyn to phosphorylate STAT3, contributing to the expression of G6PD, leading to promoting tumor growth and suppressing cellular senescence. Thus, the PIKE-A/STAT3/G6PD axis strongly links the PPP to carcinogenesis and may become a promising cancer therapeutic target.

Quercetin Inhibits Breast Cancer Stem Cells via Downregulation of Aldehyde Dehydrogenase 1A1 (ALDH1A1), Chemokine Receptor Type 4 (CXCR4), Mucin 1 (MUC1), and Epithelial Cell Adhesion Molecule (EpCAM).

BACKGROUND Quercetin, nature's most common flavonoid, possesses anticarcinogenic properties against various forms of cancer. The aim of this study was to investigate the effect of quercetin on breast cancer stem cells in the MDA-MB-231 cell line, and to elucidate the possible mechanisms for those effects. MATERIAL AND METHODS We evaluated breast cancer stem cell proliferation, clone generation, and mammosphere formation to determine the effect of quercetin treatment on breast cancer stem cells. RESULTS In our study, quercetin suppressed breast cancer stem cell proliferation, self-renewal, and invasiveness. It also lowered the expression levels of proteins related to tumorigenesis and cancer progression, such as aldehyde dehydrogenase 1A1, C-X-C chemokine receptor type 4, mucin 1, and epithelial cell adhesion molecules. CONCLUSIONS These results indicate that quercetin targets and destroys breast cancer stem cells, making it a potential novel drug in the fight against cancer.

Aortic wall proteomic analysis in spontaneously hypertensive rats with a blood pressure decrease induced by 6-week load-free swimming.

Decreased arterial compliance is one of the earliest detectable manifestations of adverse structural and functional changes within the vessel wall in hypertension. The proteomic approach is a powerful technique to analyze a complex mixture of proteins in various settings. Physical activity level was negatively associated with blood pressure. Sixteen 4-week-old male spontaneously hypertensive rats (SHR) and 16 Wistar-Kyoto (WKY) rats were randomly divided into four groups: i) SHR exercise group, ii) SHR rest group, iii) WKY exercise group and iv) WKY rest group. In the SHR and WKY exercise groups, rats were treated with a 6-week load-free swimming protocol (1 h/day, 5 days/week). The blood pressure of the rats was tested by the CODATM2 single non-invasive blood pressure measurement appliance. After the 6-week swimming protocol, the total aorta excluding abdominal aorta was extracted. The proteins were separated by two-dimensional gel electrophoresis and identified via LC-mass spectrometry (MS)/MS. After 6-week load-free swimming, blood pressure decreased in the SHRs. Compared with sedentary SHRs, 11 spots on the 2D-gel showed a significant difference in exercised SHRs. Nine of these were chosen for further identification. There were 5 upregulated proteins (long-chain specific acyl-CoA dehydrogenase, heat shock protein ß-1, isocitrate dehydrogenase subunit α, actin, α cardiac muscle 1 preprotein and calmodulin isoform 2) and 4 downregulated proteins (adipocyte-type fatty acid-binding protein, tubulin ß-2C chain, 78 kDa glucose-regulated protein precursor and mimecan). Proteomics is an effective method to identify the target proteins of exercise intervention for hypertension.

[Effects of sodium tanshinone B on the protein expression of NMDAR1 in rat hippocampal subfields following focal ischemia/reperfusion injury].

UNLABELLED: OBJECTIVE To observe the changing laws of the protein expression of N-methyl D-aspartate receptor (NMDAR) in rat hippocampal subfields following focal ischemia/reperfusion injury, and to study the effects of sodium tanshinone B (STB) on it, thus exploring the possible mechanism of STB for treating cerebral ischemia. METHODS: The rat model of focal cerebral ischemia/reperfusion injury was established using middle cerebral artery occlusion (MCAO) by reversibly inserting a nylon thread. The Wistar rats were randomly divided into the sham-operation group, the I/R model group, and the low, middle, and high dose STB groups. The neural functional disturbance was scored referring to the 5-grade Zea Longa EL standard. The protein expression of NMDAR1 in the ischemic side was detected using immunohistochemical assay. RESULTS: There was statistical difference in the scores of the neural functional disturbance in the middle and high dose STB groups when compared with the model group (P < 0.01). Results of the immunohistochemical assay showed the expression of NMDAR1 in CA1 region was obviously higher in the I/R model group, the low and middle dose STB groups than in the sham-operation group (P < 0.01). The expression of NMDAR1 in CA1 region was obviously lower in the high dose STB group than in the I/R model group (P < 0.01), the low (P < 0.01) and middle dose STB groups (P < 0.05). The expression of NMDAR1 in CA3 region was obviously higher in the low dose STB group and the I/R model group than in the sham-operation group, the middle and high dose STB groups (P < 0.01). The expression of NMDAR1 in CA3 region was obviously higher in the high and middle dose STB groups than in the sham-operation group (P < 0.05). CONCLUSIONS: STB could promote the recovery of neural functions in cerebral ischemia/reperfusion injury rats. STB fought against cerebral ischemia/reperfusion injury by lowering excitable neurotransmitter glumatic acid and reducing the protein expression of NMDAR1.

Neutroprotective efficacy of sodium tanshinone B on hippocampus neuron in a rat model of focal cerebral ischemia.

OBJECTIVE: To investigate the protective effects of sodium tanshinone B (STB) on brain damage following focal ischemia-reperfusion (I/R) injury through interfering with N-methyl-D-aspartic acid receptor (NMDAR) and excitatory and inhibitory amino acids, and evaluate the potential mechanisms of the neuroprotective activity of STB. METHODS: Transient forebrain ischemia was induced by middle cerebral artery occlusion (MCAO). The rats were randomized into a sham operated group, a model group (I/R) and three STB different dose groups. Rats were pretreated with STB at the doses of 4, 8, 16 mg/kg (STB(1), STB(2), STB(3)) for 3 days before MCAO. The expression of NMDAR1 was detected by immunohistochemistry and Western blotting. The concentrations of glutamate and γ-aminobutyric acid (GABA) were analyzed using high performance liquid chromatography. RESULTS: STB treatment reduced neurological defect scores, cerebral infarction volume and brain water content. The levels of NMDAR1 were significantly higher in the l/R and STB(1) groups than that of the sham and the STB(3) groups (P<0.01). Optical density of NMDAR1 was significantly increased in cornu ammonis (CA)1 region of the l/R group (P<0.05). STB treatment reduced NMDAR1 optical density in the CA1 region (P<0.01). The levels of glutamate were significantly lower in the hippocampus in the STB(3) group than that of the l/R, STB(1) and STB(2) groups (P<0.01). CONCLUSION: Preconditioning with STB appears to be a simple and promising strategy to reduce or even prevent cerebral l/R injury and has potential for future clinical application.

[Effects of salvianolic acid B preconditioning endothelial progenitor cells on expressions of myocardial genes in bone mesenchymal stem cells at the early cell differentiation stage of rats].

OBJECTIVE: To investigate the effects of Salvianolic acid B preconditioned endothelial progenitor cells (EPCs) on the Nkx2.5 and GATA-4 gene expressions at the early stage of cell differentiation of bone mesenchymal stem cells (BMSc) transplanted into infarcted myocardium, in order to find out the best synergism for co-transplantation of the two kinds of cells. METHODS: BMSc and EPCs of rats were isolated and cultured, and rats were modeled into acute myocardial infarction (AMI) by left coronary artery ligation. Then the EPCs preconditioned with different concentrations of Salvianolic acid B and BMSc or DMEM medium were implanted into heart ischemia area. Expressions of Nkx2.5 and GATA-4 mRNA expressions in myocardium were detected by Real-time RT-PCR 4 weeks later. RESULTS: Compared with those in the non-implanted model rats' myocardium, the gene expression of Nkx2.5 and GATA-4 mRNA were significantly higher in all the transplantation receptive groups, comparisons between the implanted groups showed that the highest value of expressions (2. 654 +/- 0.606 of Nkx2.5 and 1.573 +/- 0.372 of GATA-4) displayed in the group contained more EPCs, for 8-fold to BMSc in volume. CONCLUSION: BMSc can differentiate into cardiac muscle like cells, and condition of their differentiation is related with the degree of the internal environment improved.