OBJECTIVE: To explore the effects of Eriodictyol to the growth, apoptosis and oxidative stress of Burkitt lymphoma (BL) cells and phosphorylation of protein kinase B (AKT) in children. METHODS: The effects of Eriodictyol (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320 μmol/L) to viability of BL cell line DG-75 cells were detected by CCK-8. The effects of Eriodictyol (0, 10, 20, 40 μmol/L) to the proliferation activity of DG-75, apoptosis rate, levels of apoptosis-related proteins, oxidative stress indexes ［superoxide dismutase (SOD), malondialdehyde (MDA)］, mitochondrial membrane potential (MMP) and phosphorylation level of phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycinm (mTOR) were detected by clony formation assay and Wester blot. RESULTS: When the treatment concentration of Eriodictyol was 20 μmol/L, the proliferation activity of the cells was decreased (P<0.05). The concentrations at 10, 20, 40 μmol/L were selected for subsequent experiments. Compared with 0 μmol/L Eriodictyol, the proliferation activity of DG-75, SOD activity, MMP, phosphorylation levels of PI3K, AKT and mTOR in 20 and 40 μmol/L Eriodictyol treatment groups were significantly decreased (P<0.05), while cells apoptosis rate, Cleaved-Caspase-3/Caspase-3, Bax/Bcl-2 and MDA level were significantly increased (P<0.05). CONCLUSION Eriodictyol may promote the mitochondrial apoptosis pathway by inhibiting the abnormal activation of PI3K/AKT/mTOR to reduce the proliferation activity of DG-75, and inhibit oxidative stress response to increase the apoptosis rate and play anti-tumor roles.
Apoptosis ; Flavanones ; Phosphatidylinositol 3-Kinases/metabolism* ; Signal Transduction

This study aimed to examine the effect of the 24 N-terminal amino acids (N24) of p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), on the endotoxin lipopolysaccharide (LPS)-stimulated release of the cytokines (CKs) by HaCaT cells. The fusion protein, trans-acting activator of transcription (TAT)-N24 (an experimental peptide, EP) containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E·coli. HaCaT cells (a human keratinocyte cell line) was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 μg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) in HaCaT cells. The expression of the NF-κB inhibitor alpha (IκB-α) protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The results showed that EP treatment increased TNF-α secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-α, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-α, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-κB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-κB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-κB p65 protein expression was inhibited after the addition of EP. Western blotting showed that IκB-α expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. IκB-α expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater IκB-α expression was found in cells treated with LPS and EP combined than those treated with LPS alone. It was concluded that EP can effectively inhibit the LPS-stimulated expression of TNF-α, IL-6, and IL-8, which involves the inhibition of the hydrolysis of IκB-α and thereby blockage of the nuclear translocation of NF-κB p65.
Amino Acids ; metabolism ; Cell Line ; Cytokines ; metabolism ; Endotoxins ; metabolism ; Humans ; Inflammation ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism

OBJECTIVE: To investigate the expression of the cell division- associated gene NUF2 in breast cancer and its clinical significance. METHODS: The expression of NUF2 in breast cancer tissues was analyzed using Oncomine database. The relationship between the expression of NUF2 and the prognosis of breast cancer was analyzed using the Kaplan-Meier Plotter database. Gene set enrichment analysis (GSEA) and GEO database were used to investigate the effect of NUF2 on gene enrichment. The String database was utilized to analyze the proteins associated with NUF2. The TIMER database was analyzed to assess the correlations of NUF2 with BUB1, MAD2L1 and MYC. The expressions of NUF2 mRNA in 8 pairs of breast cancer tissues and adjacent tissues were verified by q-PCR. RESULTS: Compared with that in normal breast tissue, NUF2 was significantly overexpressed in breast cancer ( < 0.001). The overall survival time (HR = 1.52, = 0.015) and the recurrence-free survival time (HR = 1.85, = 3.2e-14) of the patients with high NUF2 expression were significantly shorter than those of patients with low NUF2 expression. In patients with high NUF2 expression, the enriched genes were involved mainly in cell cycle, P53, G2/M, DNA repair, MYC, and PI3K-AKT-MTOR signaling pathways, which were associated with tumor proliferation, invasion, metastasis and stemness. Combination of the results of String database, gene enrichment and TIMER database analyses suggested that NUF2 interacted directly with BUB1, MAD2L1, and MYC, which could promote the progression of breast cancer. The results of q-PCR showed that NUF2 expression was up-regulated in 6 cancer tissues and down-regulated in 2 cancer tissues. CONCLUSIONS NUF2 gene is overexpressed in breast cancer, and its expression level is important in predicting the prognosis of breast cancer.
Breast Neoplasms ; metabolism ; Cell Cycle Proteins ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Phosphatidylinositol 3-Kinases ; Prognosis

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are aerobic microorganisms that can remove nitrogen under high-salt conditions, but their performance in practical applications are not satisfactory. As a compatible solute, trehalose helps microorganisms to cope with high salt stress by participating in the regulation of cellular osmotic pressure, and plays an important role in promoting the nitrogen removal efficiency of microbial populations in the high-salt environment. We investigated the mechanism of exogenous-trehalose-enhanced metabolism of HN-AD community under high-salt stress by starting up a membrane aerobic biofilm reactor (MABR) to enrich HN-AD bacteria, and designed a C₁₅₀ experimental group with 150 μmol/L trehalose addition and a C₀ control group without trehalose. The reactor performance and the community structure showed that NH₄⁺-N, total nitrogen (TN) and chemical oxygen demand (COD) removal efficiency were increased by 29.7%, 28.0% and 29.1%, respectively. The total relative abundance of salt-tolerant HN-AD bacteria (with Acinetobacter and Pseudofulvimonas as the dominant genus) in the C₁₅₀ group reached 66.8%, an 18.2% increase compared with that of the C₀ group. This demonstrated that trehalose addition promoted the enrichment of salt-tolerant HN-AD bacteria in the high-salt environment to enhance the nitrogen removal performance of the system. In-depth metabolomics analysis showed that the exogenous trehalose was utilized by microorganisms to improve proline synthesis to increase resistance to high-salt stress. By regulating the activity of cell proliferation signaling pathways (cGMP-PKG, PI3K-Akt), phospholipid metabolism pathway and aminoacyl-tRNA synthesis pathway, the abundances of phosphoethanolamine, which was one of the glycerophospholipid metabolites, and purine and pyrimidine were up-regulated to stimulate bacterial aggregation and cell proliferation to promote the growth of HN-AD bacteria in the high-salt environment. Meanwhile, the addition of trehalose accelerated the tricarboxylic acid (TCA) cycle, which might provide more electron donors and energy to the carbon and nitrogen metabolisms of HN-AD bacteria and promote the nitrogen removal performance of the system. These results may facilitate using HN-AD bacteria in the treatment of high-salt and high-nitrogen wastewater.
Nitrification ; Denitrification ; Trehalose ; Phosphatidylinositol 3-Kinases/metabolism* ; Heterotrophic Processes ; Salt Stress ; Nitrogen/metabolism* ; Aerobiosis ; Bioreactors/microbiology*

OBJECTIVE: To investigate the expressions and time-dependent changes of phosphatidylinositol-3-kinase (PI3K), phospho-PI3K (p-PI3K), protein kinase B (PKB/Akt) and phospho-Akt (p-Akt) during wound healing process of mice skin. METHODS: The changes of PI3K, p-PI3K, Akt and p-Akt expression in skin wound were detected by immunohistochemistry, Western blotting and real-time PCR. RESULTS: Immunohistochemistry showed the expression of PI3K and p-Akt were observed in mononuclear and fibroblast after skin wound, and reached peak in reconstruction. The positive bands of PI3K, p-PI3K, Akt and p-Akt were observed in all time points of the wound healing process by Western blotting. The expression peak of p-PI3K and p-Akt showed in inflammation and proliferation; the expression peak of PI3K and Akt in reconstruction. Real-time PCR showed the expression peak of PI3K mRNA in inflammation and reconstruction and the peak of Akt mRNA in reconstruction. CONCLUSION During the wound healing process, the expressions of PI3K, Akt, p-PI3K and p-Akt show different changes with significant correlation to wound time. The expression of PI3K/Akt may be a valuable marker for wound time estimation.
Animals ; Blotting, Western ; Class I Phosphatidylinositol 3-Kinases ; Fibroblasts/metabolism* ; Mice ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Skin/injuries* ; Wound Healing

OBJECTIVE: To investigate the effect and potential mechanism of dihydromyricetin (Dmy) on H9C2 cell proliferation, apoptosis, and autophagy. METHODS: H9C2 cells were randomly divided into 7 groups, namely control, model, EV (empty pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector), IV (circHIPK3 interference), Dmy (50 µ mol/L), Dmy+IV, and Dmy+EV groups. Cell proliferation and apoptosis were detected by cell counting kit-8 assay and flow cytometry, respectivley. Western blot was used to evaluate the levels of light chain 3 II/I (LC3II/I), phospho-phosphoinositide 3-kinase (p-PI3K), protein kinase B (p-AKT), and phospho-mammalian target of rapamycin (p-mTOR). The level of circHIPK3 was determined using reverse transcriptase polymerase chain reaction. Electron microscopy was used to observe autophagosomes in H9C2 cells. RESULTS: Compared to H9C2 cells, the expression of circHIPK in H9C2 hypoxia model cells increased significantly (P<0.05). Compared to the control group, the cell apoptosis and autophagosomes increased, cell proliferation rate decreased significantly, and the expression of LC3 II/I significantly increased (all P<0.05). Compared to the model group, the rate of apoptosis and autophagosomes in IV, Dmy, and Dmy+IV group decreased, the cell proliferation rate increased, and the expression of LC3 II/I decreased significantly (all P<0.05). Compared to the control group, the expressions of p-PI3K, p-AKT, and p-mTOR in the model group significantly reduced (P<0.05), whereas after treatment with Dmy and sh-circHIPK3, the above situation was reversed (P<0.05). CONCLUSION Dmy plays a protective role in H9C2 cells by inhibiting circHIPK expression and cell apoptosis and autophagy, and the mechanism may be related to PI3K/AKT/mTOR pathway.
Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Phosphatidylinositol 3-Kinases/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Autophagy

OBJECTIVE: To investigate the effect of glycolytic enzyme pyruvate kinase type 2 (PKM2) on the proliferation and apoptosis of human leukemia HL-60 cells. METHODS: si-PKM2 plasmid was transfected into HL-60 cells (set as si-PKM2 group), and blank vector transfected cells were set as control group (si-Ctl group). The expression levels of PKM2 mRNA and protein in si-Ctl group and si-PKM2 group were detected by RT-qPCR and Western blot. CCK-8 cell detection kit was used to detect the proliferation ability of the cells in the two groups. Flow cytometry was used to detect the changes of cell cycle and apoptosis. Western blot and RT-qPCR were used to detect the changes of p-Akt and p-mTOR protein levels in PI3K/Akt/mTOR signaling pathway and the changes of glycolysis-related mRNA levels of the cells in the two groups. The changes in glucose consumption and lactic acid production of the cells were assayed. Over expressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002 or galactose, the changes in cell proliferation ability, cell cycle and apoptosis, as well as changes in glucose consumption and lactic acid production were detected. RESULTS: Interfered by si-PKM2, mRNA and protein levels of PKM2 in si-PKM2 group significantly decreased, and proliferation ability of the cells was also reduced (P<0.05). After PKM2 knockdown, the cells were significantly blocked at G CONCLUSION PKM2 knockdown can inhibit the proliferation and induce apoptosis of HL-60 cells, and its molecular mechanism may be related to the PKM2-mediated PI3K/Akt/mTOR-glycolysis, which suggesting that PKM2 may serve as a molecular target for the prevention and treatment of leukemia.
Apoptosis ; Cell Proliferation ; Glycolysis ; Humans ; Phosphatidylinositol 3-Kinases/metabolism* ; Pyruvate Kinase

The leaves of sea buckthorn(Hippophae rhamnoides), considered as common food raw materials, have records of medicinal use and diverse pharmacological activities, showing a potential medicinal value. However, the active substances in the sea buckthorn leaves and their mechanisms of action remain unclear. In addition, due to the extensive source and large variety variations, the quality evaluation criteria of sea buckthorn leaves remain to be developed. To solve the problems, this study predicted the main active components, core targets, key pathways, and potential pharmacological effects of sea buckthorn leaves by network pharmacology and molecular docking. Furthermore, ultra-performance liquid chromatography with diode-array detection(UPLC-DAD) was employed to determine the content of active components and establish the chemical fingerprint, on the basis of which the quality markers of sea buckthorn leaves were predicted and then verified by the enzyme activity inhibition method. The results indicated that sea buckthorn leaves had potential therapeutic effects on a variety of digestive tract diseases, metabolic diseases, tumors, and autoimmune diseases, which were consistent with the ancient records and the results of modern pharmacological studies. The core targets of sea buckthorn leaves included PTPN11, AKT1, PIK3R1, ESR1, and SRC, which were mainly involved in the PI3K-AKT, MAPK, and HIF-1 signaling pathways. In conclusion, the active components of sea buckthorn leaves are associated with the rich flavonoids and tannins, among which quercitrin, narcissoside, and ellagic acid can be used as the quality markers of sea buckthorn leaves. The findings provide a reference for the quality control and further development and utilization of sea buckthorn leaves as medicinal materials.
Hippophae/chemistry* ; Network Pharmacology ; Molecular Docking Simulation ; Phosphatidylinositol 3-Kinases/metabolism* ; Flavonoids/analysis* ; Fruit/chemistry*

The synthesis and decomposition of glycogen adjust the blood glucose dynamically to maintain the energy supply required by the cells. As the only hormone that lowers blood sugar in the body, insulin can promote glycogen synthesis by activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and increasing glucose transporter translocation, and inhibit gluconeogenesis to lower blood glucose. In the endometrium, glycogen metabolism is active, but gluconeogenesis does not occur. The glycogen metabolism in the endometrium is controlled not only by the classical glucose regulating hormones, but also by the ovarian hormones. The functional activities related to implantation of the endometrium during the implantation window require glucose as energy source. A large amount of glucose is used to synthesize glycogen in the endometrium before implantation, which could meet the increased energy demand for embryo implantation. In diabetes, glycogen metabolism in the endometrium is impaired, which frequently leads to implantation failure and early abortion. This article reviews the glycogen metabolism in the endometrium and discusses its role in embryo implantation, which provide new ideas for embryo implantation research and infertility treatment.
Blood Glucose/metabolism* ; Embryo Implantation ; Endometrium ; Female ; Glucose/metabolism* ; Glycogen/metabolism* ; Humans ; Insulin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Pregnancy

Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway (PAM pathway) plays an important role in the development of breast cancer and are closely associated with the resistance to endocrine therapy in advanced breast cancer. Therefore, anti-cancer treatment targeting key molecules in this signaling pathway has become research hot-spot in recent years. Randomized clinical trials have demonstrated that PI3K/AKT/mTOR inhibitors bring significant clinical benefit to patients with advanced breast cancer, especially to those with hormone receptor (HR)-positive, human epidermal growth factor receptor (HER) 2-negative advanced breast cancer. Alpelisib, a PI3K inhibitor, and everolimus, an mTOR inhibitor, have been approved by Food and Drug Administration. Based on their high efficacy and relatively good safety profile, expanded indication of everolimus in breast cancer have been approved by National Medical Products Administration. Alpelisib is expected to be approved in China in the near future. The members of the consensus expert panel reached this consensus to comprehensively define the role of PI3K/AKT/mTOR signaling pathway in breast cancer, efficacy and clinical applications of PI3K/AKT/mTOR inhibitors, management of adverse reactions, and PIK3CA mutation detection, in order to promote the understanding of PI3K/AKT/mTOR inhibitors for Chinese oncologists, improve clinical decision-making, and prolong the survival of target patient population.
Breast Neoplasms/metabolism* ; Consensus ; Everolimus/therapeutic use* ; Female ; Humans ; MTOR Inhibitors ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphoinositide-3 Kinase Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; Sirolimus/therapeutic use* ; TOR Serine-Threonine Kinases/metabolism*