ABSTRACT
ObjectiveLipopolysaccharide （LPS）-induced zebrafish inflammation model was used to evaluate the anti-inflammatory activity of different extracts from Lianggesan （LGS） and its component Glycyrrhiza Radix et Rhizoma. MethodDifferent polar fractions of LGS and Glycyrrhiza Radix et Rhizoma were obtained by the principle of similar miscibility. For toxicity observation， the zebrafish （3 day-post-fertilization） was exposed to different concentrations of extracts for 24， 48 and 72 h. The yolk sac of the zebrafish was microinjected with 0.5 g·L-1 LPS to establish the inflammation model， and then the embryos were soaked with different concentrations of extracts to observe their survival status at 72 h and the aggregation of neutrophils in yolk sac at 12 h after treatment. Hematoxylin-eosin staining was used to analyze the yolk sac of the zebrafish microinjected with LPS. Quantitative Real-time polymerase chain reaction （Real-time PCR） was performed to further investigate the anti-inflammatory effects and mechanisms of LGS and Glycyrrhiza Radix et Rhizoma. ResultThe toxicity of LGS and Glycyrrhiza Radix et Rhizoma was decreased with the increase of polarity， and the descending order was petroleum ether>ethyl acetate>n-butanol>water. Compared with model group， the extracts from different fractions of LGS and Glycyrrhiza Radix et Rhizoma prolonged the survival time of the zebrafish， and inhibited the recruitment and aggregation of neutrophils and decreased the infiltration of inflammatory cells in the yolk sac， among which the water fraction of LGS and the ethyl acetate fraction of Glycyrrhiza Radix et Rhizoma had the most significant effect （P<0.01）. In addition， compared with model group， the water fraction of LGS and the ethyl acetate fraction of Glycyrrhiza Radix et Rhizoma down-regulated the mRNA expression of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α）， and suppressed the expression of toll like receptor 4 （TLR4） and nuclear factor kappa-B （NF-κB） in LPS-stimulated zebrafish （P<0.01）. ConclusionThe extracts from different fractions of LGS and Glycyrrhiza Radix et Rhizoma exerted protective effects in LPS-induced zebrafish by inhibiting the TLR4 and NF-κB signaling pathways. Moreover， in zebrafish model， the method of administration by soaking was applicable to the high-throughput screening of anti-inflammatory Chinese medicine， which was suitable for the evaluation of anti-LPS activity of Chinese medicine and the different extracts.
ABSTRACT
Exosome, a membranous vesicle with biological activity, not only transmits active substances between cells but also transfers information between cells to participate in cell communication. Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire migratory and invasive properties to become mesenchymal stem cells. EMT is essential for the development of a spectrum of diseases. Studies have shown that exosomes have dual effects on EMT to, on the one hand, promote EMT and tumor cell invasion and metastasis and accelerate angiogenesis and tumor growth; on the other hand, exosomes can suppress tumor cell invasion, inhibit fibrosis of the heart, liver and kidney, and improve myocardial infarction by inhibiting EMT. Exosomes modulate EMT-related signaling pathways by carrying EMT-related proteins or miRNA to exert their bi-directional regulatory effects.
Subject(s)
Humans , Epithelial-Mesenchymal Transition , Exosomes , MicroRNAs , Neoplasms , Signal TransductionABSTRACT
A microfluidic chip with micropillar arrays for three-dimensional (3D) cell culture was designed and validated.The chip consisted of a polydimethylsiloxane (PDMS) channel plate and a glass cover plate.One cell culture chamber composed of two rows of micropillar arrays and two lateral channels for transporting the culture medium were integrated on the PDMS channel plate.The spacing between micropillars directly affects the chip performance, which is critical for the design of the chip.In this work, the spacing between micropillars was optimized by numerical simulation and experimental validation.With the optimized microfluidic chip, the mixture of cells and extracellular matrix mimics could be steadily injected into the cell culture chamber, the nutrients in the culture medium from the lateral channels could quickly diffuse into the chamber, and the cell metabolites could also timely diffuse out of the chamber.To test the stability of the microenvironment in the microfluidic chip, neural stem cells were three-dimensionally cultured.
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Objective To investigate the changes in Th17 cells and CD4+CD25+regulatory T lym-phocytes ( Treg) as well as transcription factors and cytokines relating to them in children with Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) and to analyze their role and clinical significance. Methods Thirty-two children with newly diagnosed EBV-associated HLH in the Hematology/Oncology Department of Zhengzhou Children′s Hospital from January 2012 to December 2016 were enrolled in this study. Thirty healthy children taking physical examination in the same hospital in the corresponding period were recruited as controls. Percentages of Th17 and Treg cells in peripheral blood T lymphocytes were detected by flow cytometry. Expression of RORγt and Foxp3 at mRNA level in peripheral blood mononuclear cells was detected by real-time PCR. Levels of IL-6, IL-17, IL-10 and TGF-β1 in serum samples were measured by ELISA. Results Compared with the control group, the EBV-associated HLH group showed in-creased percentage of Th17 cells [(1. 09±0. 43)％ vs (0. 39±0. 19)％, P<0. 05] and enhanced expres-sion of RORγt at mRNA level [(1. 41±0. 37) vs (0. 67±0. 13), P<0. 05], but decreased percentage of Treg cells [(3. 66±1. 13)％ vs (6. 80±1. 15)％, P<0. 05] and inhibited expression of Foxp3 at mRNA level [(15. 97±5. 11) vs (30. 23±4. 95), P<0. 05]. All of the above mentioned changes were reversed af-ter treatment (P<0. 05). Serum levels of IL-6 and IL-17 of EBV-associated HLH group were higher than those of control group, while serum levels of IL-10 and TGF-β1 were lower (P<0. 05). Conclusion Im-balanced Th17/Treg cells might play an important role in the pathogenesis of EBV-associated HLH. Cyto-kines relating to the maintenance of Th17/Treg cell balance could be used as indicators of disease develop-ment.
ABSTRACT
Objective To investigate the changes in Th17 cells and CD4+CD25+regulatory T lym-phocytes ( Treg) as well as transcription factors and cytokines relating to them in children with Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) and to analyze their role and clinical significance. Methods Thirty-two children with newly diagnosed EBV-associated HLH in the Hematology/Oncology Department of Zhengzhou Children′s Hospital from January 2012 to December 2016 were enrolled in this study. Thirty healthy children taking physical examination in the same hospital in the corresponding period were recruited as controls. Percentages of Th17 and Treg cells in peripheral blood T lymphocytes were detected by flow cytometry. Expression of RORγt and Foxp3 at mRNA level in peripheral blood mononuclear cells was detected by real-time PCR. Levels of IL-6, IL-17, IL-10 and TGF-β1 in serum samples were measured by ELISA. Results Compared with the control group, the EBV-associated HLH group showed in-creased percentage of Th17 cells [(1. 09±0. 43)％ vs (0. 39±0. 19)％, P<0. 05] and enhanced expres-sion of RORγt at mRNA level [(1. 41±0. 37) vs (0. 67±0. 13), P<0. 05], but decreased percentage of Treg cells [(3. 66±1. 13)％ vs (6. 80±1. 15)％, P<0. 05] and inhibited expression of Foxp3 at mRNA level [(15. 97±5. 11) vs (30. 23±4. 95), P<0. 05]. All of the above mentioned changes were reversed af-ter treatment (P<0. 05). Serum levels of IL-6 and IL-17 of EBV-associated HLH group were higher than those of control group, while serum levels of IL-10 and TGF-β1 were lower (P<0. 05). Conclusion Im-balanced Th17/Treg cells might play an important role in the pathogenesis of EBV-associated HLH. Cyto-kines relating to the maintenance of Th17/Treg cell balance could be used as indicators of disease develop-ment.
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A microfluidic chip with integrated microelectrode for real-time dopamine detection was designed and fabricated. The chip consisted of a polydimethylsiloxane ( PDMS) channel plate and a glass electrode plate. One central channel as the culture chamber of neural stem cells and two lateral channels for transport of the culture medium were integrated on the PDMS channel plate. Microelectrodes for real-time dopamine detection were integrated on the glass electrode plate. To solve the problem in demoulding the PDMS channel plate from the silicon mould, a novel demoulding method was developed. An Au-Au-Au three-electrode system was constructed, and it performed well in electrochemical detection. The performance of the microfluidic chip was primarily studied by detecting dopamine dissolved in the medium for the culture of neural stem cells. The limit of detection was 3. 92 μmol/L, the linear detection range was from 10 μmol/L to 500 μmol/L, and the detection reproducibility from different chips was less than 4%.
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This study is to investigate the anti-angiogenetic effect of arenobufagin in vitro and in vivo. The anti-proliferation effect of arenobufagin on CNE-2, Hep2, SH-SY5Y, LOVO, PC-3 and DU145 cells as well as human umbilical vein endothelial cells (HUVECs) was determined by MTT assay. Cell morphological changes of LOVO and HUVECs after arenobufagin treatment were observed by microscopy. Arenobufagin inhibited the proliferation of CNE-2, Hep2, SH-SY5Y, LOVO, PC-3, DU145 and HUVECs in a dose-dependent manner. Furthermore, it was obviously observed that the subcytotoxic concentration of arenobufagin in human carcinoma cells induced a marked decrease in the viability of HUVECs. Chick embryo chorioallantoic membrane (CAM) model was used to detect the anti-angiogenetic effect of arenobufagin in vivo. Arenobufagin significantly suppressed the angiogenesis of CAM. Cell cycle analysis demonstrated that G2/M phase was arrested and the sub-G1 peak appeared with the increase of arenobufagin concentration. PI/Annexin V double staining assay further demonstrated that arenobufagin could induce apoptosis in a dose- and time-dependent manner. Mitochondrial potential collapse detected by flow cytometric analysis was increased after arenobufagin treatment. It also observed that PARP was cleaved to p85 active form by Western blotting. Taken together, arenobufagin has significant anti-angiogenetic effect in vitro and in vivo, and the action mechanisms behind its anti-angiogenesis may be associated with cell cycle arrest and apoptosis of vein endothelial cells.