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Background Exposure to diisononyl phthalate (DINP), an endocrine disruptor associated with metabolic diseases and widely used in plastic products, has been linked to the development of several adverse health outcomes in the liver, including non-alcoholic fatty liver disease (NAFLD). Objective To investigate the effects and the possible molecular mechanisms of DINP exposure on lipid metabolism in human hepatocellular carcinoma cells (HepG2 cells). Methods First, HepG2 cells were treated with DINP at three time spots (24, 48, and 72 h) and eleven doses (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 mmol·L−1). Cell viability were detected using cell counting kit 8 (CCK8). Intracellular lipid deposition was determined by oil red O staining and lipid content detection, and triglyceride (TG) and cholesterol (TC) were further detected. Finally, the mRNA expression levels were detected by fluorescence quantitative PCR, including fatty acid synthesis related genes [acetyl-CoA carboxylase alpha (Accα), fatty acid synthase (Fasn), malonyl-CoA decarboxylase (Mlycd), and sterol regulatory element binding protein 1 (Srebp1)] and β-oxidation related genes [peroxisome proliferator activated receptor alpha (Pparα), AMP-activated protein kinase (Ampk), carnitine palmitoyltransferase 1A (Cpt-1a), transcription factor A, mitochondrial (Tfam), nuclear respiratory factor 1 (Nrf1), and peroxisome proliferator-activated receptor gamma and coactivator 1 alpha (Pgc1-α)]. Results Compared with the control group (0 mmol·L−1), the no observed adverse effect levels (NOAEL) of HepG2 cell viability were 0.3, 0.1, and 0.1 mmol·L−1 after 24, 48, and 72 h exposure to DINP, respectively, and the corresponding lowest observed adverse effect levels (LOAEL) were 1, 0.3, and 0.3 mmol·L−1, respectively (P<0.05). After exposure to 30 mmol·L−1 and 100 mmol·L−1 DINP for 24 h, the intracellular lipid content, lipid deposition, TG, and TC levels were increased significantly compared with the control group (P<0.01). Compared with the control group, the mRNA expression levels of genes related to fatty acid synthesis, such as Mlycd, Srebp1, Fasn, and Accα, were down-regulated after the 100 mmol·L−1 DINP exposure for 24 h, while the mRNA expression level of Mlycd was up-regulated in the 30 mmol·L−1 group. The β-oxidation related genes such as Ampk, Pparα, and Tfam were up-regulated significantly after the 100 mmol·L−1 DINP exposure, while Cpt-1a mRNA expression level was down-regulated (P<0.05). Conclusion Exposure to DINP at 30 mmol·L−1 and 100 mmol·L−1 can interfere with fatty acid synthesis and β-oxidation in lipid metabolism of HepG2 cells, resulting in lipid deposition.
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KRAS‒PDEδ interaction is revealed as a promising target for suppressing the function of mutant KRAS. The bottleneck in clinical development of PDEδ inhibitors is the poor antitumor activity of known chemotypes. Here, we identified novel spiro-cyclic PDEδ inhibitors with potent antitumor activity both in vitro and in vivo. In particular, compound 36l (K D = 127 ± 16 nmol/L) effectively bound to PDEδ and interfered with KRAS-PDEδ interaction. It influenced the distribution of KRAS in Mia PaCa-2 cells, downregulated the phosphorylation of t-ERK and t-AKT and promoted apoptosis of the cells. The novel inhibitor 36l exhibited significant in vivo antitumor potency in pancreatic cancer patient-derived xenograft (PDX) models. It represents a promising lead compound for investigating the druggability of KRAS‒PDEδ interaction.
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Objective:To investigate the genetic characteristics of pancreatic cancer-associated diabetes mellitus (PCDM) and screen out the possible molecular markers for PCDM.Methods:The clinical data of pancreatic cancer (PC) cohort from The Cancer Genome Atlas (TCGA) were selected and collected, and the patients were divided into PCDM( n=11) and PC groups( n=109) according to whether the patients were diagnosed as diabetes within 2 years of PC diagnosis. Then, the mRNA microarray data of genome expression were extracted from TCGA PC cohort, and the differentially expressed genes (DEGs) were screened out by the " limma" package of R software based on (|log2 fold change|>2 and P<0.05). The functions of DEGs were revealed with gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, a protein-protein interaction (PPI) network was constructed with the STRING database, and the hub genes were identified by the molecular complex detection (MCODE) module of Cytoscape software. Results:The analysis showed that among 20 531 genes, 47 genes were significantly upregulated, and 60 genes were significantly downregulated in the PCDM group. GO analyses revealed that 107 DEGs were mainly involved in the positive regulation of secretory function in terms of biological function (gene number=9, P<0.01); in the regulation of receptor function of molecular function (gene number=10, P<0.01); and in the intracavitary components of cytoplasmic microtubules of cellular components (gene number=8, P<0.01). The results of KEGG pathway enrichments revealed that DEGs mainly affected PCDM via cytokine interactions (gene number=8, P<0.01). Finally, five hub genes, including GNG8, CNR2, GALR2, CXCL13, and NPY2R, were identified for PCDM in PPI network analysis. Conclusions:The feature genes of PCDM are mainly different from PC in terms of secretion function, receptor function, cytoplasmic microtubule composition, and cytokine interaction. Five genes including GNG8, CNR2, GALR2, CXCL13, and NPY2R may become potential molecular markers for PCDM.
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Objective:To explore the relationship between CD244 and the phenotype and function of CD56bright NK cells of patients with active pulmonary tuberculosis.Methods: PBMCs were isolated from peripheral blood by density gradient centrifugation.The expression of CD244,CD94,NKG2D on the CD56bright NK cells from the active pulmonary tuberculosis patients and healthy controls was detected by flow cytometry.And then analyzed the relationship of the expression of CD244 with Tim3,CD27,CD62L,CCR7,IFN-γ and CD107a in CD56bright NK cells by flow cytometry.Results: The expression of CD244 on the CD56bright NK cells showed no significant difference between the patients with active pulmonary tuberculosis and healthy controls without MTB antigen.The expression of CD244 was significantly increased on CD56bright NK cells of patients with tuberculosis stimulated with MTB antigen.The expression of CD94 and NKG2D on CD56bright NK cells showed no difference between patients and healthy controls.The proportion of Tim3+ cells in CD244+CD56bright NK cells was significantly higher than CD244-CD56bright NK cells.While the expression of CD62L and IFN-γ decreased significantly in CD244+CD56bright NK cells.The expression of CD107a on CD56bright NK cells was not significantly different between CD244+ cells and CD244-cells.Conclusion: The expression of CD244 on CD56bright NK cells in patients with active pulmonary tuberculosis increased significantly,maybe inhibit IFN-γ co-work with Tim3.CD244 has nothing to do with degranulation of CD56bright NK cells.
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Objective:To investigate the impact and mechanisms of TAM to the imbalance of Treg /Th17 in the Eoc microenvi-ronment.Mte hods:Build the in vitro M2 macrophage model ,which was like TAMs .Use flow cytometry to detect the difference of the Treg/Th17 before and after the co-culture of M2 macrophage and CD 4+T cells.Use Western bolt to detect the change of T cell transcription factor and ELISA to detect the IL-10 levels in the supernatant after co-culture.Use crystal violet methods to detect the influence to the ovarian tumor cell proliferation between the different co-culture supernatants and the Transwell to detect the influence to the ovarian tumor cell migration.Thus to analysis the how TAMs influence the imbalance of Treg/Th17 in Eoc microenvironments.R esults:①After coc-ultured with M2 macrophage ,the ratio of Treg/Th17 was( 0.76 ±0.33 ) significant increased compared with control (0.41±0.25) ,M0( 0.40±0.32) and M1(0.31±0.16) (P<0.05).②After co-cultured,the supernatant of M2 group has a significant ability to promote the proliferation of Skov-3 cells.After co-cultured for 1 day, the Skov-3 cell number of M2 group was 14 942.43 ±434.19 , which was significantly higher than the control group ( 12 445.57 ±179.34 ) and CD3/28 group (12 470.32±434.18)(P<0.001).After co-cultured for 2 days,the Skov-3 cell number of M2 group was 30 129.09±520.53 ,which was significantly higher than the control group (25 622.81±897.07) and CD3/28 group(25 721.62±1 808.60) (P<0.05).③After co -cultured with M2 macrophage , the Treg-specific transcription factor Foxp 3 increased ( P=0.047 ) compared with control and M 1 group .④After co-cultured with M2 macrophage for 3 days,the concentration of IL1-0 in the supernatant was(264.04±75.9)pg/ml, which was significantly higher than CD 3/28 group ( 60.89 ±46.54 ) pg/ml,M 0 group ( 44.81 ±32.93 ) pg/ml, M1 group ( 42.71 ± 26.09)pg/ml(P=0.001).Conclusion: M2 macrophage induces the increase of the radio of Treg /Th17 as well as the increase of Treg-specific transcription factor Foxp 3 and the decrease of Th17 -specific transcription factor ROR-γt.Meanwhile , the co-culture supernatant of M2 macrophage and CD4+T cell have the ability to promote the proliferation and migration of ovarian cancer cell ,the mechanism which ,may related to the IL -10 in the supernatant .
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[Objective] To discuss the clinical effect of Jianpi Qingwei Decoction on chronic erosive gastritis(CEG). [Method] Choose the said patients 68 cas-es, randomly divide them into treatment group(n=38) and control one(n=30).The treatment group take the self-made Jianpi Qingwei Decoction for treat-ment;the control one, lansoprazole and col oid pectinBi. Both take 4w as a course;after 1 course, compare their cure effect and side effects;fol ow up the re-currence. [Result] In the treatment group, 22 cases were cured, 13 better, 3 uncured, the total effective rate 92.10%;for the control one, they were respective-ly 13,11,6 and 80.00%;by comparison, their difference had statistical meaning;there ’s no recurrence fol owing up for half year. [Conclusion] Jianpi Qingwei Decoction has marked cure effect and little side effects on CEG, without easy recurrence, worth promotion.
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Objective To investigate the feature of Th1 cytokines induced by Mtb-specific antigen in patients with refractory pulmonary tuberculosis.Methods 28 patients with refractory pulmonary tuberculosis,67 patients with first-treated pulmonary tuberculosis,and 25 healthy controls with positive T.spot (LTBI group) were enrolled.IFN-γ,IL-2 and TNF-α in supernatants from PBMCs stimulated with ESAT-6 and CFP-10 were analyzed with Bender Flowcytomix on flow cytometry.Results The levels of the three cytokines were utmost high in patients with first-treated pulmonary tuberculosis.The lowest level of IFN-γ and IL-2 were induced in patients with refractory pulmonary tuberculosis,and were significantly lower than the LTBI group(Mann-Whitney U ＝ 105.5,162.5,P ＜ 0.01).Conclusions The immunotherapy with IFN-γ and IL-2 may play a role in treatment for refractory pulmonary tuberculosis but not for most of first-treated pulmonary tuberculosis.
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Objective To study the feature of MIG and IFN-γ obtained from PBMCs stimulated with Mtb specific antigens and the potential value in the differential diagnosis of active pulmonary tuberculosis from bacterial pneumonia and primary lung cancer. Methods 90 patients with active pulmonary tuberculosis and 31 patients with bacterial pneumonia and primary lung cancer were enrolled. MIG and IFN-γin supernatants from PBMCs stimulated with Mycobacterium tuberculosis-specific antigens were analyzed with Bender Flowcytomix on flow cytometry. The diagnostic values were established based on receiver operating characteristic curve analysis. Results PBMCs stimulated with Mtb-specific antigens produced significantly higher levels of MIG compared with IFN-γ The level of MIG in active pulmonary TB patients was significantly higher than in controls(3023.0 pg/ml vs 112.5 pg/ml, P ＜0.0001). The MIG and IFN-γtests were positive in 96. 8 and 86. 7% of the TB patients, the specificity was up to 94. 4 and 87. 1%. With combination of MIG and IFN-γtests, the positive rate increased among TB patients to 97. 8% without a significant decrease in specificity. Conclusions The responses of the MIG and IFN-γagainst to Mtb-specific antigens could be used to discriminate newly-treated active pulmonary tuberculosis fiom bacterial pneumonia and primary lung cancer. Combination of MIG and IFN-γ might be a simple and quick approach to diagnosis newly-treated active pulmonary tuberculosis.
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Objective To study the feature of MCP-1 in plasma of active pulmonary tuberculosis patients and the correlation of MCP-1 obtained from PBMCs stimulated with specific Mtb peptides with IFNγ.Methods 20 patients with active pulmonary tuberculosis and 16 healthy controls with positive PPD were enrolled.The concentration of MCP-1 and IFN-γwas analyzed with Bender Flowcytomix on flow cytometry.Results No statistical difference of MCP-1 concentration in plasma was found between TB patients and controls [(263.8 ± 25.31)pg/ml and(212.1 ± 18.04)pg/ml,P ＞ 0.05].But TB patients with continuous respiratory symptoms showed higher MCP-1 in plasma.The concentration of MCP-1 and IFN-γwas significantly elevated in PBMCs culture supernatants.It was significantly higher in TB patients than in controls [(21460 ±3376)pg/ml vs(10910 ±2141)pg/ml,P ＜0.01].There was no correlation between the concentration of MCP-1 and IFN-γ.Conclusions The concentration of MCP-1 in plasma may be related to the progress of the pulmonary tuberculosis.MCP-1 stimulated by Mtb-specific peptides may be one of the biomarkers for TB diagnosis.
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Objective To study the expression of CD27 and CD28 in antigen-specific CD4~+T cells in patients with pulmonary tuberculosis and healthy people, and understand the role of differentiated stages of CD4~+T cells in the pathogenesis of tuberculosis. Methods The expression of CD27 and CD28 was analyzed by CD4, CD154, CD27 and CD28 staining and flow cytometry. The distributions of CD27 and CD28 in antigen-specific CD4~+T cells were compared between patients with pulmonary tuberculosis and healthy controls. Results In patients of pulmonary tuberculosis, the frequencies of CD27 + CD28 + (early differentiated stage), CD27~- CD28~+ and CD27~+ CD28~- (intermediate differentiated stage), CD27~- CD28~-(fully differentiated stage) T cell subsets in antigen specific CD4~+T cells were (49. 55 ±6. 15)%, (26. 85 ±3. 87)% ,(7. 2 ± 1.37)% and ( 16. 35 ±3.97)%, respectively. In healthy controls, the frequencies of the four subsets in antigen-specific CD4~+T cells were ( 51.81 ± 4. 94 ) %, ( 29. 83 ± 5.33 ) %, ( 12. 65 ±4. 48)% and (5.7±2)%, respectively. The early differentiated CD4~+T cell was the major subset both in patients and healthy people, however, which had significant difference compared with the fully differentiated subset ( t = 2. 26, P < 0. 05 ). Conclusion The population frequency of the fully differentiated CD4~+T cells in patients with pulmonary tuberculosis was significantly higher than that in healthy people. This suggested that the differentiation degree of the antigen-specific CD4~+T cell might be related with pulmonary tuberculosis.
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Objective To study population frequencies of CD4+,CD154+ T cell subset in patients with pulmonary tuberculosis and controls with positive PPD reaction. Methods Flow cytometry was used to detect the CD4+,CD154+ T cell subset, the population frequen-cies in patients with pulmonary tuberculosis and controls were compared. Results The expression level of CD154 was higher when PE-la-beled CD154 antibody was added during stimulation period, compared with CD154 labeling after stimulation(1.51±0. 36/0. 40±0. 13, P <0.05). The CD154+ cells were not detectable in fresh isolated CD4+ T cells, but significantly increased after stimulation with specific anti-gens. The population of CD4+, CD154+ T cell subset was significantly reduced in patients with active pulmonary tuberculosis, compared with healthy controls with PPD positive reaction(0. 72±0. 32/1.65±0. 76, P <0. 01). Conclusions The population of CD4± ,CD154± T cell subset was significantly reduced in patients with active pulmonary tuberculosis, which indicated that it may play an important role in the de-velopment of tuberculosis.