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OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.
Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Doublecortin-Like Kinases , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Promyelocytic, Acute , Mitochondria , Oxidative Stress , Protein Serine-Threonine Kinases , Stem CellsABSTRACT
ObjectiveLymphatic epithelial cells (LECs) are important links involved in lymphatic metastasis in the microenvironment of cholangiocarcinoma. This study aims to detect the modulation of inflammatory factors and chemokines secreted by LECs after stimulation of cholangiocarcinoma cells, and observe the effects of highly expressed factors on lymphangiogenesis.MethodsThe culture medium of cholangiocarcinoma (RBE, HCCC9810), LECs stimulated by cholangiocarcinoma cell culture medium (CCM), and normal LECs were prepared. Inflammatory factors and chemokines in the culture medium were detected using protein chip. The experiments are divided into the following groups, including a blank control group, CCM group, CCM coupled with Anti-ENA-78 group, Anti-ENA-78 group, ENA-78 group, ENA-78 coupled with SB2252002, and SB225002 group. The relationship between the content of factor and time was investigated using ELISA, while the relation between target factors and lymphangiogenesis obtained by cell proliferation and tubule formation assay.ResultsWe found ENA-78, IP-10, GCP-2, MCP-2, MCP-3, MIP-3a, HCC-1, and Lymphotactin expression increased in LECs supernatant after CCM stimulation. However, I-TAC, MIP-1d, IL-10, MIG, PDGF-BB, and CXCL16 factors showed down-regulation. The secretion of ENA-78 in CCM was relatively low. By ELISA, we found that the ENA-78 protein in RBE-LECs and HCCC9810-LECs gradually increased over time, and reached the plateau phase at the point of 48h. The lymphatic tube forming ability of LECs cultured in CCM was significantly increased compared with that of the control group, and this ability could be partially weakened by ENA-78 neutralizing antibodies. In the exogenous ENA-78 protein group, the lymphatic tube formation ability was as well significantly increased compared with that in the control group, and this ability could be effectively blocked by the IL-8B inhibitor.ConclusionThe increased secretion ENA-78 of lymphatic epithelial cells induced by cholangiocarcinoma may play a role in promoting lymphangiogenesis through the IL-8B receptor.
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ObjectiveLymphatic epithelial cells (LECs) are important links involved in lymphatic metastasis in the microenvironment of cholangiocarcinoma. This study aims to detect the modulation of inflammatory factors and chemokines secreted by LECs after stimulation of cholangiocarcinoma cells, and observe the effects of highly expressed factors on lymphangiogenesis.MethodsThe culture medium of cholangiocarcinoma (RBE, HCCC9810), LECs stimulated by cholangiocarcinoma cell culture medium (CCM), and normal LECs were prepared. Inflammatory factors and chemokines in the culture medium were detected using protein chip. The experiments are divided into the following groups, including a blank control group, CCM group, CCM coupled with Anti-ENA-78 group, Anti-ENA-78 group, ENA-78 group, ENA-78 coupled with SB2252002, and SB225002 group. The relationship between the content of factor and time was investigated using ELISA, while the relation between target factors and lymphangiogenesis obtained by cell proliferation and tubule formation assay.ResultsWe found ENA-78, IP-10, GCP-2, MCP-2, MCP-3, MIP-3a, HCC-1, and Lymphotactin expression increased in LECs supernatant after CCM stimulation. However, I-TAC, MIP-1d, IL-10, MIG, PDGF-BB, and CXCL16 factors showed down-regulation. The secretion of ENA-78 in CCM was relatively low. By ELISA, we found that the ENA-78 protein in RBE-LECs and HCCC9810-LECs gradually increased over time, and reached the plateau phase at the point of 48h. The lymphatic tube forming ability of LECs cultured in CCM was significantly increased compared with that of the control group, and this ability could be partially weakened by ENA-78 neutralizing antibodies. In the exogenous ENA-78 protein group, the lymphatic tube formation ability was as well significantly increased compared with that in the control group, and this ability could be effectively blocked by the IL-8B inhibitor.ConclusionThe increased secretion ENA-78 of lymphatic epithelial cells induced by cholangiocarcinoma may play a role in promoting lymphangiogenesis through the IL-8B receptor.
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Objective A variety of miRNAs have been found to be involved in the occurrence and development of colorectal cancer. This paper aim to investigate the clinical and biological relevance of miR-217 and the pathway by which miR-217 may be involved in progression in colorectal cancer. Methods According to the diameter of tumor, the tumor was divided into tumors>5 cm(n=22) and tumors≤5 cm(n=28); 18 cases of colorectal cancer I-II specimens and 32 of III-IV patients; according to median expression of miR-217, the specimens were divided into high expression group and low expression group. The expression of miR-217 in 50 colon cancer patients’ carcinoma and adjacent tissues was determined by qRT-PCR. Cells were grouped: overexpression control Group (transfected control mimic) and miR-217 overexpression group(transfected miR-217 mimic), low expression control group((transfected control inhibitor) and miR-217 low expression group(transfected miR-217 inhibitor), miR-217&ERK1/2 low expression group(transfected miR-217 inhibitor+U0126). MiR-217 and HCT116 cells were over expressed in SW480 cells,and miR-217 was knocked down. A series of biological function assays were performed to assess cell viability (cck-8 assay), clony formation ability (clony formation assay), proliferation (edu assay), Changes in ERK1/2 expression were measured at protein level, and the relationship between miR-217 and ERK1/2 in colorectal cancer cells was explored by relevant rescue experiments. Results Compared with colorectal adjacent noncancerous tissues, the expression of miR-217 was significantly decreased in carcinoma tissues(-1.360±0.645 vs 2.244±0.168, P<0.01); the expression of miR-217 in tumors with a diameter >5cm was significantly lower than that of tumors with a diameter ≤5cm(-1.718±0.272 vs -0.587±0.288, P<0.01); the expression of miR-217 in stage I-II colorectal cancer tissues was significantly higher than that stage III-IV(-0.413±0.330 vs -01.463±0.230, P<0.05); the expression of miR-217 in the miR-217 overexpression group was significantly higher than miR-217 overexpression control group(15.120±0.522 vs 1.004±0.003, P<0.01), and the number of clone formation was significantly less than that of the overexpression control group(199.30±15.62 vs 439.70±18.91, P<0.01) . In HCT116 cells, the expression of miR-217 in the miR-217low expression group was significantly lower than miR-217 low expression control group(0.2070±0.021 vs 1.006±0.003, P<0.01), and the number of clone formation was significantly higher than that control group(237.30±12.14 vs 117.00±7.00, P<0.01) . When miR-217 overexpressed in SW480, the protein expression of ERK1/2 decreased; when miR-217 was inhibited in HCT116, the protein expression of ERK1/2 increased. The number of colons in ERK1/2 low expression group was significantly lower than that miR-217&ERK1/2 low expression group(221.70±12.73 vs 108.00±5.51) , the difference was statistically significant(P<0.01). Conclusion Low expression of miR-217 is observed in both colorectal cancer tissues and cells, and miR-217 can affect tumor cell proliferation in the progression of colorectal cancer partly by inhibiting the expression of ERK1/2.
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OBJECTIVE: To prepare cholesterol-PEG-modified amphotericin B liposomes and amphotericin B liposomes, and evaluate their encapsulation efficiency and pharmacokinetics. METHODS: It was employed to prepare both kinds of liposomes with the film-evaporation, the dynamic light scattering techniques and Ultrafree-CL were used to determine the mean diameter and the encapsulation efficiency, respectively. Pharmacokinetics was evaluated in Wista rats. RESULTS: Mean diameter of the liposomes was (115 ± 20) nm, and the encapsulation efficiency was over 99.9%. Moreover, the pharmacokinetic study in rats indicated that cholesterol- PEG-modified liposomes could significantly increase the ρmax and AUC. CONCLUSION: Amphotericin B liposomes prepared by film-evaporation method have high encapsulation efficiency, and cholesterol-PEG modification can prolong the circulation time of amphotericin B in vivo.
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<p><b>BACKGROUND</b>The calcium clearance and reactive oxygen species (ROS) generations in the coronary artery smooth muscle cells in chronic heart failure (HF) have not been fully investigated. Therefore, we attempted to understand the gene expressions underlying the mishandling of calcium clearance and the accumulations of ROS.</p><p><b>METHODS</b>We initially established an animal model of chronic HF by making the left anterior descending coronary artery ligation (CAL) in rats, and then isolated the coronary artery vascular smooth muscle cells from the ischemic and the nonischemic parts of the coronary artery vessels in 12 weeks after CAL operation. The intracellular calcium concentration and ROS level were measured using flow cytometry, and the gene expressions of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), encoding sarcoplasmic reticulum Ca2+-ATPase 2a, encoding sodium-calcium exchanger (NCX), and p47phox encoding a subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were examined using real-time quantitative reverse transcription polymerase chain reaction and Western blotting, respectively.</p><p><b>RESULTS</b>We found that the calcium accumulation and ROS generation in the coronary artery smooth muscle cells isolated from either the ischemic or the nonischemic part of the CAL coronary artery vessel were significantly increased irrespective of blood supply (all P < 0.01). Moreover, these were accompanied by the increased expressions of NCX and p47phox, the decreased expression of SERCA2a, and the increased amount of phosphorylated forms of p47phox in NADPH oxidase (all P < 0.05).</p><p><b>CONCLUSIONS</b>Our results demonstrated that the disordered calcium clearance and the increased ROS generation occurred in the coronary artery smooth muscle cells in rats with chronic HF produced by ligation of the left anterior descending coronary artery (CAL), and which was found to be disassociated from blood supply, and the increased generation of ROS in the cells was found to make concomitancy to the increased activity of NADPH oxidase in cytoplasm.</p>
Subject(s)
Animals , Male , Rats , Calcium , Metabolism , Cells, Cultured , Coronary Vessels , Metabolism , Disease Models, Animal , Flow Cytometry , Heart Failure , Metabolism , Myocytes, Smooth Muscle , Metabolism , Random Allocation , Rats, Sprague-Dawley , Reactive Oxygen Species , MetabolismABSTRACT
Complement activation-related pseudo-allergic reactions (CARPA) may represent 77% of all immune-mediated immediate hypersensitivity reactions. Because of the universality of the CARPA response and correlation between it and drug properties, complement activity tests are recommended as one of the tests for immunotoxicity and bioequivalence of drugs. However, in-vivo tests of complement activation are complicated, and the immunological differences between different individuals and between human and animal, making it very necessary to establish a standard and sample evaluation model for testing the effects of drugs on complement activity. In this study, the standard reaction serum was prepared by pooling sera collected from 40 healthy blood donors; a standard positive control was prepared by incubation with a heat-agglutinated IgG and zymosan A; SC5b-9, C5a, C4d and Bb were chosen as the test targets and evaluation criteria of the results was defined, all of these constituted the in-vitro model. By using this in-vitro model, the immunological toxicity of the different prescription of antifungal drug amphotericin B, and voriconazole for injection, and the bioequivalence of amphotericin B liposome formulations were studied.
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Objective:To research the solubilization of sulfobutyl ether-β-cyclodextrin ( SEB-β-CD) on voriconazole and the sta-bility of voriconazole with different pH values. Methods:Phase-solubility diagram was applied to study the solubilization of SEB-β-CD on voriconazole. Meanwhile, the impurity of voriconazole was determined to study the stability. Results:SBE-β-CD could increase the solubility of voriconazole significantly and the apparent stability constant was similar within the pH range of 5. 0-8. 0. The stability of voriconazole was decreased with the increase of pH. Conclusion:SEB-β-CD is an ideal solubilizer for voriconazole, while the stability is influenced by pH.
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<p><b>OBJECTIVE</b>To select an effective way to enhance vigor of Prunella vulgaris seeds.</p><p><b>METHOD</b>Three population seeds were treated at the 20 degrees C and dark enviroment.</p><p><b>RESULT</b>Priming with 20% - 30% PEG and 200 - 400 mg x L(-1) GA3 could enhance seeds germination and vigor. Germination percentage of three population seeds treated with 0. 6% - 3.0% NaCl reduced, but they started to germinate in advance. Treated with 0.6% - 2.4% KNO3-KH2PO4, germination rate and vigor of seeds in Zijinshan and Pan' an both increased and the one in Bozhou decreased.</p><p><b>CONCLUSION</b>Vigor of P. vulgaris seed treated with PEG and GA3 under proper concentration increases, while treated with KNO3-KH2PO, and NaCl low vigor seeds germination rate reduces.</p>
Subject(s)
Darkness , Germination , Radiation Effects , Gibberellins , Pharmacology , Nitrates , Pharmacology , Phosphates , Pharmacology , Polyethylene Glycols , Pharmacology , Potassium Compounds , Pharmacology , Prunella , Physiology , Radiation Effects , Seeds , Radiation Effects , Sodium Chloride , Pharmacology , TemperatureABSTRACT
OBJECTIVE@#To observe the length heteroplasmy and point heteroplasmy in human mtDNA control region.@*METHODS@#The peripheral blood, buccal cell, and single hair shaft from 50 individuals and 16 family members, related in their maternallineage were analyzed by direct sequencing, and clones from 20 individuals whose mtDNA sequences have a T-C transition at 16189 nt were sequenced.@*RESULTS@#No point heteroplasmy were observed in peripheral blood, buccal cell, single hair shaft from the same individual, neither in maternally related individuals. Length heteroplasmy was observed in those individuals with a homopolymeric tract and the different clones from the same individual has different proportions of length variants, but the hair shafts from the same individual were very similar to the measurements made from blood DNA. No length heteroplasmy was observed between different tissues from the same individual.@*CONCLUSION@#mtDNA sequences have a characteristic of high consistency and genetic stability, mtDNA sequencing is a suitable tool for forensic applications such as individual identification.