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AIM To investigate the effects of diosgenin on autophagy of human osteosarcoma cells.METHODS Human osteosarcoma MG63 and U2OS cells with or without exposure to diosgenin had their proliferation detected by MTT assay,their ultrastructure observed by transmission electron microscopy,their expression of autophagy protein Beclin1 observed by immunofluorescence staining,and their expressions of autophagy molecular markers LC3,Beclin1 and PI3K/Akt/mTOR signaling pathway related proteins detected by Western blot.The MG63 and U2OS cells cotreated with diosgenin and PI3K pathway inhibitor LY294002 had the expression of Beclin1 mRNA detected by RT-qPCR.The MG63 and U2OS cells cotreated with autophagy inhibitor 3-methyladenine(3-MA)had their inhibition rate of proliferation detected by MTT assay,their expression of cleaved-caspase3 protein detected by Western blot,and their expression of caspase3 mRNA detected by RT-qPCR.RESULTS Upon osteosarcoma MG63 and U2OS cells,diosgenin inhibited their proliferation,promoted the generation of autophagosomes,increased the protein expression of LC3 Ⅱ and Beclin1(P<0.05,P<0.01),reduced the protein expression of LC3 I(P<0.01),and inhibited the protein phosphorylation level of PI3K/Akt/mTOR pathway(P<0.05,P<0.01),whose effects were offset by the intervention with autophagy inhibitors in terms of the reduced proliferation inhibition and down-regulated expressions of caspase3 mRNA and cleaved-caspase3 protein(P<0.01).CONCLUSION Diosgenin can inhibit the proliferation of osteosarcoma cells and induce their autophagy leading to their death and autophagy apoptosis,which may be related to the activation of PI3K/Akt/mTOR signaling pathway and up-regulation of the expression of LC3 Ⅱ and Beclin1 proteins.
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Objective::To explore the pharmacological mechanism of Xiao Xianxiongtang in treating type 2 diabetes mellitus (T2DM) by network pharmacology. Method::The main active ingredients, corresponding targets and target genes of Xiao Xianxiongtang were searched on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) website. Relevant target genes of T2DM were obtained through Gene Cards. The targets of drug active ingredients were mapped to the targets of T2DM, and the intersection targets were obtained as the predictive targets of Xiao Xianxiongtang on T2DM. Cytoscape 3.7.1 software was used to construct the drug active ingredient-intersection target network model and select the key active ingredients. Interactive protein-protein interaction network (PPI) was constructed by STRING website, and key target genes were selected. Gene function analysis (GO) and enrichment analysis based on the Kyoto encyclopedia of genes and genomes (KEGG) pathway were performed on the intersecting targets using DAVID6.8 online tool. Result::Xiao Xianxiongtang had 30 active ingredients, 156 relevant targets, 14 key active ingredients and 18 key target genes on T2DM. GO analysis showed that the biological functions of Xiao Xianxiongtang in the treatment of potential genes of T2DM mainly involved transcriptional regulation, oxidative stress, protein binding and inflammatory reaction. KEGG pathway enrichment showed that the main pathways of Xiao Xianxiongtang in the treatment of T2DM were hypoxia inducible factor-1 (HIF-1) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor signaling pathway and thyroid hormone signaling pathway, phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway, hepatitis B, hepatitis C, tyrosine kinase receptor2(ErbB) signaling pathway, calcium signaling pathway and nuclear factor-kappa B (NF-κB) signaling pathway. Conclusion: Xiao Xianxiongtang is a multi-component, multi-target and multi-pathway process in the treatment of T2DM. It plays an important role in the treatment of T2DM by regulating transcription, oxidative stress, protein binding and inflammatory reaction. Conclusion::The mechanism of Xiao Xianxiongtang in treating T2DM may alleviate insulin resistance, increase insulin sensitivity and reduce blood sugar by inhibiting the secretion of inflammatory factors, participating in anti-inflammatory response, reducing oxidative stress, increasing intracellular calcium concentration, blocking glucagon signaling pathway and activating PI3K/Akt pathway.
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Objective: To explore the mechanism of Dahuang Huanglian Xiexintang in the treatment of type 2 diabetes mellitus based on network pharmacology. Method: Major chemical constituents, corresponding targets and target genes of Dahuang Huanglian Xiexintang were obtained by Traditional Chinese Medicine Systems Pharmacology(TCMSP), and target genes of type 2 diabetes mellitus were obtained by GeneCards. The target genes of drug and disease were mapped to predict target genes of Dahuang Huanglian Xiexintang for type 2 diabetes mellitus. Cytoscape3.7.1 software was used to construct the compound-target network and protein-protein interaction network (PPI) of traditional Chinese medicine. Gene ontology (GO) analysis of potential genes and enrichment analysis of gene encyclopedia kyoto encyclopedia of genes and genomes (KEGG) pathway were carried out using DAVID 6.8 online tool. Result: There were 17 active ingredients, 94 related targets, 17 key active ingredients and 16 key targets in Dahuang Huanglian Xiexintang on type 2 diabetes mellitus. GO analysis showed that the biological functions of potential genes of Dahuang Huanglian Xiexintang in the treatment of type 2 diabetes were mainly related to oxidative stress, apoptosis, protein binding, inflammatory reaction, et al. KEGG pathway enrichment results showed that the pathways of potential genes of Dahuang Huanglian Xiexintang in the treatment of type 2 diabetes mainly involved hypoxia inducible factor(HIF), tumor necrosis factor(TNF), phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt), nuclear transcription factor-кB(NF-кB), and vascular endothelial growth factor(VEGF) signaling pathways. Conclusion: Dahuang Huanglian Xiexintang is a complex process of multi-component, multi-target and multi-pathway in the treatment of type 2 diabetes mellitus. It plays an important role in the treatment of type 2 diabetes mellitus by participating in oxidative stress, apoptosis, protein binding and inflammatory reaction.
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Objective: To investigate the expression of microRNA (miRNA, miR)-126 in esophageal carcinoma tissues and the effect of miR-126 on the proliferation and migration of esophageal cell line EC109. Methods: The expressions of miR-126 and sex determining region Y-box 2 (SOX2) mRNA in esophageal cancer tissues and their paracancerous tissues from 24 patients were detected by real-time fluorescence-based quantitative PCR. The expression of miR-126 in EC109 cells after transfection with miR-126 mimics or miR-negative control (NC) was detected by realtime fluorescence-based quantitative PCR. The abilities of cell proliferation and migration of EC109 cells transfected with miR-126 mimics were measured by MTT and Transwell assays, respectively. The dual luciferase reporter vectors containing 3'-untranslated region (3'-UTR) with miR-126 binding site of wild type or mutant SOX 2 gene were constructed by using Dual-Luciferase Reporter Assay System, then the relative activity of firefly luciferase was detected to confirm the binding site of miR-126 on SOX 2 gene. The expression levels of SOX2 mRNA and protein in EC109 cells transfected with miR-126 mimics were detected by real-time fluorescence-based quantitative PCR and Western blotting, respectively. The expression of SOX2 protein in esophageal cancer tissues and their paracancerous tissues was examined by immunohistochemistry. Results: The expression level of miR-126 in esophageal cancer tissues was lower than that in paracancerous tissues (P < 0.01), but the expression level of SOX2 mRNA was opposite (P < 0.05). The expression of miR-126 was negatively correlated with the expression of SOX2 (r =-0.837, P < 0.001). After transfection with miR-126 mimics, the expression level of miR-126 in EC109 cells was higher than that in miR-NC group (P < 0.01), but the abilities of cell proliferation and migration were opposite (P < 0.05, P < 0.01). miR-126 can directly target the SOX 2 3'-UTR through two predicted binding sites. The expression levels of SOX2 mRNA and protein in EC109 cells after transfection with miR-126 mimics were lower than those in miR-NC group (P < 0.01). The positive expression rate of SOX2 protein in esophageal cancer tissues was higher than that in paracancerous tissues (P < 0.01). Conclusion: The expression level of miR-126 is lower in esophageal carcinoma tissues, and miR-126 can inhibit the proliferation and migration of EC109 cells, in which SOX 2 may be one of the targeted genes.
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Course construction is the groundwork for vocational college to improve education quality. The first thing for the excellent course construction is to raise awareness. The fundamental starting point and destination are benefitial to students. It must start from the teachers themselves, and have entire optimization in the teaching content,teaching methods ,teaching materials, the means of teaching and so on.
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The method of PBL (problem based learning) teaching in China is widely recognized. However, the characteristics of Vocational Medical Education limit its wider application. The paper discusses the mode which is suitable for our country by analyzing the major issues,improving methods of teaching and doing experiments.
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Objective Protein N-SRCR derived from salivary agglutinin (SAG) inhibits HIV-1 infection.An N-SRCR monoclonal antibody was prepared for the study of the interaction between N-SRCR and HIV-1 envelop glycoprotein (gp120).Methods The purified recombinant N-SRCR expressed by 293 cells was used to immunize four weeks old BALB/c mice.After the final boost,the mouse spleen cells were isolated and fused with mouse myeloma cell line SP2/0-Ag-14,and the resulting hybridomas were screened for the production of N-SRCR-specific antibodies by ELISA assay.The monoclonal antibody against N-SRCR was purified by HiTrap Protein G kit,the purity determined by SDS-PAGE and the antibody titers by ELISA.The antibody specificity.was charqacterized by western blotting.Results A strain of hybridoma cell clones stably secreting N-SRCR antibody,named 1D6,was obtained.The high purity of the IgG was demonstrated by SDS-PAGE,and the ELISA titers of 1D6 was more than 100?25.Conclusion A monoclonal antibody against N-SRCR was successfully prepared,which laid the ground for further studies on the biological function of N-SRCR and the interactions between SRCR domains and HIV-1 Env gp120.