Promotive role of eukaryotic translation initiation factor 4A isoform 3 in ovarian cancer cell growth and aerobic glycolysis through the pyruvate dehydrogenase kinase 4 signaling.

Ovarian cancer (OC) represents one of the most detrimental gynecological malignancies. RNA-binding protein eukaryotic translation initiation factor 4A isoform 3 (EIF4A3) is well-regarded as a definitive oncogene that contributes to the development of multiple malignant tumors. This study sought to elucidate the molecular mechanism of EIF4A3 in OC growth and aerobic glycolysis by regulation of pyruvate dehydrogenase kinase 4 (PDK4) mRNA stability. We determined the EIF4A3 and PDK4 expression levels in OC cell lines and normal ovarian epithelial cells, and subsequently evaluated the cell viability and colony formation by cell counting kit-8 and colony formation assays. The degree of cell aerobic glycolysis was evaluated by measurements of lactic acid production, glucose intake, adenosine triphosphate level, extracellular oxygen consumption, and protein levels of pyruvate kinase isozymes M2 and hexokinase-2. Afterwards, we verified the binding of EIF4A3 and PDK4 mRNA via RNA immunoprecipitation, and determined the mRNA stability after actinomycin D treatment. Finally, a series of rescue experiments was performed with pcDNA3.1-PDK4. EIF4A3 and PDK4 were upregulated in OC cells. Silencing EIF4A3 obstructed cell proliferation and aerobic glycolysis, while the same was annulled by EIF4A3 overexpression. Mechanically, EIF4A3 could bind to PDK4 mRNA to stabilize its mRNA and upregulate its protein levels. PDK4 overexpression inverted the inhibitory role of silencing EIF4A3 in proliferation and aerobic glycolysis. Overall, our findings highlighted that EIF4A3 induced OC progression by stabilizing PDK4 mRNA.

Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1.

BACKGROUND: Sinomenine (SIN) is an anti-inflammatory drug that has been used for decades in China to treat arthritis. In a previous study, SIN acted on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit inflammatory responses in macrophages, which indicates a new anti-inflammatory mechanism of SIN. However, the level of α7nAChR was increased in the inflammatory responses and was downregulated by SIN in vitro, so the underlying mechanisms of SIN acting on α7nAChR remain unclear. PURPOSE: To analyze the role of α7nAChR in inflammation and the effect and mechanism of SIN regulation of α7nAChR. METHODS: The effects of SIN on α7nAChR in endotoxemic mice and LPS-stimulated macrophages were observed. Nicotine (Nic) was used as a positive control, and berberine (Ber) was used as a negative control targeting α7nAChR. The antagonists of α7nAChR, α-bungarotoxin (BTX) and mecamylamine (Me), were used to block α7nAChR. In RAW264.7 macrophage cells in vitro, α7nAChR short hairpin RNA (shRNA) was used to knock down α7nAChR. Macrophage polarization was analyzed by the detection of TNF-α, IL-6, iNOS, IL-10, Arg-1, and Fizz1. U0126 was used to block ERK phosphorylation. The cytokines α7nAChR, ERK1/2, p-ERK1/2 and Egr-1 were detected. RESULTS: SIN decreased the levels of TNF-α, IL-6 and the expression of α7nAChR increased by LPS in endotoxemic mice. The above effects of SIN were attenuated by BTX. In the α7nAChR shRNA transfected RAW264.7 cells, compared with the control, α7nAChR was knocked down, and M1 phenotype markers (including TNF-α, IL-6, and iNOS) were significantly downregulated, whereas M2 phenotype markers (including IL-10, Arg-1, and Fizz1) were significantly upregulated when stimulated by LPS. SIN inhibited the expression of p-ERK1/2 and the transcription factor Egr-1 induced by LPS in RAW264.7 cells, and the above effects of SIN were attenuated by BTX. The expression of α7nAChR was suppressed by U0126, which lessened the expression of p-ERK1/2 and Egr-1. CONCLUSIONS: SIN acts on α7nAChR to inhibit inflammatory responses and downregulates high expression of α7nAChR in vivo and in vitro. The increase of α7nAChR expression is correlated with inflammatory responses and participates in macrophage M1 polarization. SIN downregulates α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1, which contributes to inhibiting macrophage M1 polarization and inflammatory responses.

Antioxidant Activity of Novel Casein-Derived Peptides with Microbial Proteases as Characterized via Keap1-Nrf2 Pathway in HepG2 Cells.

Casein-derived antioxidant peptides by using microbial proteases have gained increasing attention. Combination of two microbial proteases, Protin SD-NY10 and Protease A "Amano" 2SD, was employed to hydrolyze casein to obtain potential antioxidant peptides that were identified by LCMS/ MS, chemically synthesized and characterized in a oxidatively damaged HepG2 cell model. Four peptides, YQLD, FSDIPNPIGSEN, FSDIPNPIGSE, YFYP were found to possess high 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability. Evaluation with HepG2 cells showed that the 4 peptides at low concentrations (< 1.0 mg/ml) protected the cells against oxidative damage. The 4 peptides exhibited different levels of antioxidant activity by stimulating mRNA and protein expression of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as nuclear factor erythroid-2-related factor 2 (Nrf2), but decreasing the mRNA expression of Kelch-like ECH-associated protein 1 (Keap1). Furthermore, these peptides decreased production of reactive oxygen species (ROS) and malondialdehyde (MDA), but increased glutathione (GSH) production in HepG2 cells. Therefore, the 4 casein-derived peptides obtained by using microbial proteases exhibited different antioxidant activity by activating the Keap1-Nrf2 signaling pathway, and they could serve as potential antioxidant agents in functional foods or pharmaceutic preparation.

A network pharmacology-based study on the anti-hepatoma effect of Radix Salviae Miltiorrhizae.

BACKGROUND: Radix Salviae Miltiorrhizae (RSM), a well-known traditional Chinese medicine, has been shown to inhibit tumorigenesis in various human cancers. However, the anticancer effects of RSM on human hepatocellular carcinoma (HCC) and the underlying mechanisms of action remain to be fully elucidated. METHODS: In this study, we aimed to elucidate the underlying molecular mechanisms of RSM in the treatment of HCC using a network pharmacology approach. In vivo and in vitro experiments were also performed to validate the therapeutic effects of RSM on HCC. RESULTS: In total, 62 active compounds from RSM and 72 HCC-related targets were identified through network pharmacological analysis. RSM was found to play a critical role in HCC via multiple targets and pathways, especially the EGFR and PI3K/AKT signaling pathways. In addition, RSM was found to suppress HCC cell proliferation, and impair cancer cell migration and invasion in vitro. Flow cytometry analysis revealed that RSM induced cell cycle G2/M arrest and apoptosis, and western blot analysis showed that RSM up-regulated the expression of BAX and down-regulated the expression of Bcl-2 in MHCC97-H and HepG2 cells. Furthermore, RSM administration down-regulated the expression of EGFR, PI3K, and p-AKT proteins, whereas the total AKT level was not altered. Finally, the results of our in vivo experiments confirmed the therapeutic effects of RSM on HCC in nude mice. CONCLUSIONS: We provide an integrative network pharmacology approach, in combination with in vitro and in vivo experiments, to illustrate the underlying therapeutic mechanisms of RSM action on HCC.

Alpha7 nAChR Expression Is Correlated with Arthritis Development and Inhibited by Sinomenine in Adjuvant-Induced Arthritic Rats.

Sinomenine (SIN) is the active ingredient of the Chinese herb Sinomenium acutum that has been used to treat rheumatoid arthritis (RA) for about 30 years in China. Marked expression of the alpha7 nicotinic acetylcholine receptor (α7nAChR) in the joint synovium of RA patients suggested a relationship between α7nAChR and RA. This study investigated the relationship between α7nAChR and RA development and the effects of SIN on α7nAChR expression in vivo and in vitro. Sprague-Dawley rats were injected with complete Freund's adjuvant to induce arthritis and then treated with SIN or methotrexate (MTX) from day 0 to day 30. Four clinical parameters-paw volume, arthritic index (AI), serum TNF-α concentration, and erythrocyte sedimentation rate (ESR)-were measured. Splenic lymphocytes were isolated for Bacille Calmette Guerin (BCG) stimulation. α7nAChR expression in tissues and cells was examined by RT-PCR, western blot, immunofluorescence, flow cytometry, and immunohistochemistry. Cell proliferation was evaluated by the CCK-8 assay. The relationship between α7nAChR expression and the four clinical parameters was analyzed by single-factor correlation analysis. Our results showed that the paw volume, AI, TNF-α concentration, and ESR in adjuvant-induced arthritic (AIA) rats were reduced by SIN or MTX treatment. SIN decreased α7nAChR expression in tissues and cells compared to the model group, while MTX had no significant effect on α7nAChR expression. Moreover, there was a positive relationship between α7nAChR expression and paw swelling, AI, and TNF-α concentration. Splenic lymphocyte activation was accompanied by increased α7nAChR expression, while SIN treatment inhibited cell activation and downregulated α7nAChR expression. α7nAChR expression showed a positive correlation with the progression of RA in AIA rats that may involve lymphocyte activation. Different from MTX, the inhibition of SIN on α7nAChR expression might contribute to its antiarthritic effect, suggesting that SIN could be an important supplement to the treatment strategy for RA.

Sinomenine regulates CD14/TLR4, JAK2/STAT3 pathway and calcium signal via α7nAChR to inhibit inflammation in LPS-stimulated macrophages.

Objective: To investigate the cellular mechanism that sinomenine (SIN) inhibits inflammation in macrophages induced by LPS through α7 nicotinic acetylcholine receptor (α7nAChR). Materials and methods: RAW264.7 cells were stimulated with LPS and treated by SIN or nicotine (Nic). A selective antagonist of α7nAChR, α-bungarotoxin (BTX) was used to block α7nAChR. AG490 was used to inhibit JAK2 activation. ELISA was performed to detect the levels of TNF-α and MCP-1. Western blotting was used to analyze the expression of MIF, MMP-9, CD14, TLR4, STAT3 and p-STAT3. Intracellular-free calcium level was measured by Fluorescent probe fluo-3/AM Results: SIN inhibited the production of TNF-α, MCP-1, MIF, and MMP-9, decreased the expression of CD14 and TLR4, and inhibited the release of intracellular-free calcium from intracellular stores in RAW 264.7 cells stimulated by LPS. JAK-specific inhibitor AG490 attenuated the inhibitory effect of SIN on TNF-α. SIN increased the phosphorylation of STAT3. And the above effects of SIN were attenuated by antagonist of α7nAChR. Conclusions: SIN can decrease the expression of CD14/TLR4 and intracellular free calcium level, activate JAK2/STAT3 pathway to inhibit inflammatory response through α7nAChR in macrophages.

Sinomenine inhibits fibroblast-like synoviocyte proliferation by regulating α7nAChR expression via ERK/Egr-1 pathway.

Fibroblast like synoviocyte (FLS) is a crucial in the pathogenesis of rheumatoid arthritis (RA), and involved in inflammation and joint destruction. Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has anti-inflammatory and analgesic effect and been used for RA treatment in China. Alpha 7 nicotinic acetylcholine receptors (α7nAChR), as the key receptor in cholinergic anti-inflammatory pathway (CAP) to inhibit inflammation, has been detected in RA patients synovium, but its role is still unclear. Here we investigated the association between the aggressive proliferation of FLS and α7nAChR expression and the effect of sinomenine. FLS was isolated from synovial tissues of adjuvant-induced-arthritis (AIA) rat. Tumor necrosis factor(TNF)-α was used to induce the aggressive proliferation of FLS. MTT assay was applied to evaluate the proliferation of FLS. The messenger RNA (mRNA) and protein levels of α7nAChR and early growth response gene-1 (Egr-1) were measured. The results showed that TNF-α induced FLS proliferation in vitro (P < .01) and increased the phosphorylation of ERK1/2 and the expression of Egr-1 and α7nAChR (P < .05 or P < .01). U0126, the inhibitor of ERK1/2 inhibited α7nAChR expression and FLS proliferation significantly (P < .05 or P < .01). Specific short interference RNA(siRNA) of α7nAChR decreased α7nAChR expression and inhibited FLS proliferation as well. SIN inhibited the proliferation of FLS and decreased the phosphorylation of ERK1/2, and the expression of Egr-1 and α7nAChR induced by TNF-α (P < .05). In conclusion, the expression of α7nAChR involved in the aggressive proliferation of FLS induced by TNF-α and was regulated by ERK/Egr-1 signal pathway. SIN inhibited FLS proliferation and α7nAChR expression through inhibiting ERK/Egr-1 signal pathway, this may contribute to the anti-inflammatory and anti-arthritic effect of SIN.

[Differentiation of K562 Cells Induced by Pulsatilla Saponin A into Erythroid Lineage].

OBJECTIVE: To explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells. METHODS: Pulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells. RESULTS: K562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated. CONCLUSION: Pulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.

[Study on the Measurement of Infrared Spectrum for Discontinuous Sample].

The samples to be measured with infrared instrument are usually required to be homogeneous. However, discontinuous sample is quite common in the real infrared measurements, such as, high viscosity liquids containing air bubbles. In this paper, the spectral distortion of discontinuous sample was analyzed with simple theoretical simulation and experimental examples quantitatively. The spectral properties of the discontinuous sample were studied and discussed systematically. Both theoretical computation and experimental results demonstrated that the infrared spectrum of a discontinuous sample was highly similar to that of the uniform sample if suitable experimental conditions were adopted. Our results showed that if infrared samples can be prepared to be thin enough, the infrared spectrum of the discontinuous sample can fulfill the requirement of analysis work nearly perfectly.

[How to Remove Water Absorption Bands Completely from IR Spectra of Aqueous Solution].

Infrared spectral studies of aqueous solution have been severely limited by the strong water absorption. Usually, it is very difficult to experimentally achieve perfect compensation between the amount of water that contributes to the sample and background spectra. In the present paper, we introduced a new method to make the infrared bands of water disappear during the measurement of an infrared spectrum for aqueous solution. Both background spectrum of the empty ATR cell and that of the water on ATR cell are used in sequence. Qualified infrared spectra of 10% K2CO3 and 10% BSA aqueous solutions were successfully obtained by ATR accessory (single-reflection) without water interference. The experimental results demonstrated that the new method is fast, effective and powerful. The limitation of the new method is also discussed.

The effect of electroacupuncture on T cell responses in rats with experimental autoimmune encephalitis.

Successive electroacupuncture (EA) stimulation on Zusanli ST36 acupoints of rats with experimental autoimmune encephalitis (EAE), which is an inflammatory disease mediated by autoreactive T cells, relieved disease severity, inhibited specific T cell proliferation and rebuilt the CD4+ T cell subset balance. In addition, EA-treated rats had significantly higher ACTH concentrations in vivo compared to untreated EAE rats. These results indicated that EA stimulation could relieve the severity of EAE by restoring balance to the Th1/Th2/Th17/Treg Th cell subset responses by stimulating the hypothalamus to increase ACTH secretion.

A role for the parafascicular thalamic nucleus in the development of morphine dependence and withdrawal.

The parafascicular thalamic nucleus (nPf) is a critical relay in the ascending system that mediates motor control in the central nervous system (CNS). Yet, little is known about whether or not the nPf is involved in the development of morphine dependence and withdrawal. In the present study, kainic acid was used to chemically destroy the nPf in Wistar rats, and morphine dependence and withdrawal models were established. Morphine withdrawal symptoms score was evaluated in each group. An electrophysiological method was used to measure the changes in spontaneous discharge of nPf neurons. mu-Opioid receptor (MOR) mRNA level in nPf was detected using semi-quantitative RT-PCR. The ultrastructural alterations were examined by transmission electron microscopy. Results showed that the bilateral lesion of nPf had a marked influence on the development of morphine dependence and withdrawal. In order to address the mechanisms underlying, we found: (1) the average frequency and sum of nPf neurons that exhibited spontaneous discharge were increased in the morphine withdrawal group in comparison with the sham model group (P<0.05); (2) MOR mRNA level in the nPf of the morphine dependence group was decreased in comparison with that of the sham model group (1.45+/-0.38 vs. 5.37+/-0.94, P<0.01). In the morphine withdrawal group, which underwent 40 h withdrawal, the MOR mRNA level was higher than that in the morphine dependence group (2.97+/-0.73 vs. 1.45+/-0.38, P<0.05) but still lower than that in the sham model group (P<0.05); (3) the ultrastructural injuries of nPf neurons, which were in the nucleus, organelles and neuropil, were marked in the morphine dependent and withdrawal groups. Our study indicated that nPf played an important role in the development of morphine dependence and withdrawal. The results suggest that nPf may become a therapeutic target for treating morphine withdrawal syndrome.

Administration of bone marrow stromal cells ameliorates experimental autoimmune myasthenia gravis by altering the balance of Th1/Th2/Th17/Treg cell subsets through the secretion of TGF-beta.

Bone marrow stromal cells (BMSCs) are strong candidates for cell therapy against human autoimmune diseases. Intravenous administration of syngenic BMSCs to EAMG-model rats effectively ameliorated the disease, partially through a TGF-beta-dependent mechanism. The proliferative ability of T or B cells from EAMG rats was inhibited by BMSCs at proper cocultured ratios. And the imbalance of Th1, Th2, Th17 and Treg cell subsets accompanied with the development of EAMG was corrected by the administration of BMSCs. These results provide further insights into the pathogenesis of MG, EAMG, and other immune-mediated diseases, and support a potential role for BMSCs in their treatment.

RAGE expression is up-regulated in human cerebral ischemia and pMCAO rats.

To determine whether the receptor for advanced glycation endproducts (RAGE) contributes to cerebral ischemia, we evaluated RAGE expression in human cerebral ischemia and a model of permanent middle cerebral artery occlusion (pMCAO) in rats. Biopsy specimens were obtained from 12 patients with unilateral cerebral infarction. For the pMCAO model, the middle cerebral artery (MCA) of Sprague-Dawley (SD) rats was permanently occluded. Immunohistochemistry and Western blotting were used to measure RAGE expression in the ischemic hemisphere relative to the normal hemisphere. PC12 cells subjected to oxygen and glucose deprivation (OGD) were used to evaluate the role of RAGE in cell injury. As expected, cerebral ischemia patients expressed elevated levels of RAGE in the ischemic hemisphere. In 1 and 2 days pMCAO rats, levels of RAGE were higher in the ischemic hemisphere relative to the non-ischemic hemisphere, and expression was primarily located in the penumbra of the ischemic hemisphere. In PC12 cells, levels of RAGE increased after 7h of OGD culture. Notably, blockade of RAGE with a selective RAGE antibody in vitro reduced the cytotoxicity caused by OGD. The present data suggest that RAGE is up-regulated in human cerebral ischemia and pMCAO rats, suggesting a role for RAGE in brain ischemia.