Relaxation Effect of Schisandra Chinensis Lignans on the Isolated Tracheal Smooth Muscle in Rats and Its Mechanism.

Schisandra chinensis (S. chinensis) is one of the core drugs used for relieving cough and asthma in traditional Chinese medicine. However, there are few basic studies on the treatment of respiratory diseases with S. chinensis in modern pharmacology, and the material basis and mechanism of its antiasthmatic effect are still unclear. Lignans are the main active components of S. chinensis. The aim of this study was to observe the relaxation effect of S. chinensis lignans (SCL) on the tracheal smooth muscle of rats by in vitro tracheal perfusion experiments, and to explore the mechanism by preincubation with L-type calcium channel blocker verapamil, four potassium channel blockers glibenclamide, tetraethylamine, 4-aminopyridine and barium chloride (BaCl2), ß-adrenoceptor blocker propranolol, nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME), and the cyclooxygenase inhibitor indomethacin, respectively. The results showed that SCL (0.25-1.75 mg/mL) reduced the contraction of isolated tracheal smooth muscle induced by acetylcholine, the preincubation with verapamil and glibenclamide could attenuate the relaxation effect, whereas propranolol, 4-aminopyridine, BaCl2, tetraethylamine, L-NAME, and indomethacin had no such effect. These results suggest that SCL has a significant relaxation effect on the isolated tracheal smooth muscle of rats, and the mechanism may be related to the inhibition of extracellular calcium influx and intracellular calcium release from the sarcoplasmic reticulum, as well as the activation of ATP-sensitive potassium channels. These findings may provide a pharmacological basis for the traditional use of S. chinensis to treat asthma.

[Effect of AML1-ETO Fusion Protein on Transcriptional Regulation of p14ARF].

OBJECTIVE: To investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14ARF. METHODS: P14ARF expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14ARF promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14ARF promoter in AML1-ETO positive leukemia cell line. And the p14ARF mRNA expression level was detected by qRT-PCR after treatment with 5-Aza. RESULTS: AML1-ETO-expressing cell subclone displayed low level of p14ARF mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14ARF mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14ARF gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14ARF promoter. 5-Aza could increase the expression of p14ARF. CONCLUSION: P14ARF is a possible target gene of AML1-ETO. The p14ARF silencing induced by hyper-methlylation may be an important factor for occurrence and development of the M2b subtype of acute myeloid leukemia.

Schisandra polysaccharide inhibits hepatic lipid accumulation by downregulating expression of SREBPs in NAFLD mice.

BACKGROUND: Hepatoprotective effects of Chinese herbal medicine Schisandra Chinensis (Schisandra) have been widely investigated. However, most studies were focused on its lignan extracts. We investigated the effects of Schisandra polysaccharide (SCP) in a mouse model of non-alcoholic fatty liver disease (NAFLD), and studied its effect on sterol regulatory element binding proteins (SREBPs) and the related genes. METHODS: The mouse model of NAFLD was established by feeding mice with a high-fat diet for 16 weeks. Effect of SCP-treatment (100 mg/kg, once daily for 12 weeks) on biochemical parameters and liver histopathology was assessed. Relative levels of sterol regulatory element-binding proteins (SREBPs) and their gene expressions were determined by quantitative real-time polymerase chain reaction and Western Blot. RESULTS: SCP significantly reduced the liver index by 12.0%. Serum levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol, alanine aminotransferase and aspartate aminotransferase were decreased by 31.3, 28.3, 42.8, 20.1 and 15.5%, respectively. Serum high-density lipoprotein cholesterol was increased by 26.9%. Further, SCP lowered hepatic TC and TG content by 27.0% and 28.3%, respectively, and alleviated fatty degeneration and necrosis of liver cells. A significant downregulation of mRNA and protein expressions of hepatic lipogenesis genes, SREBP-1c, fatty acid synthase and acetyl-CoA carboxylase, and the mRNA expression of liver X receptor α (LXRα) was observed in NAFLD mice treated with SCP. SCP also significantly reduced the hepatic expression of SREBP-2 and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). CONCLUSION: These findings demonstrate the hepatoprotective effects of SCP in a mouse model of NAFLD; the effects may be mediated via downregulation of LXRα/SREBP-1c/FAS/ACC and SREBP-2/HMGCR signaling pathways in the liver.

[Effect of AML1-ETO fusion protein on the expression of BCL-2].

This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.

Epigenetic silencing of Bcl-2, CEBPA and p14(ARF) by the AML1-ETO oncoprotein contributing to growth arrest and differentiation block in the U937 cell line.

The AML1-ETO fusion transcription factor generated by the t(8;21) translocation is considered to deregulate the expression of genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors, resulting in acute myelogenous leukemia by recruiting co-repressor complexes to DNA. To investigate the role of AML1-ETO in leukemogenesis, we transfected the cloned AML1-ETO cDNA and expressed the AML1-ETO protein in U937 myelomonocytic leukemia cells. By focusing on the anti-apoptotic gene Bcl-2, the key regulator gene of granulocytic differentiation CCAAT/enhancer-binding protein α (CEBPA) and the tumor suppressor gene p14(ARF), we found that both AML1-ETO-expressing cell lines and t(8;21) leukemia samples displayed low levels of these three genes. Chromatin immunoprecipitation assays demonstrated that Bcl-2, CEBPA and p14(ARF) were direct transcriptional targets of AML1-ETO. The universal binding of AML1-ETO to genomic DNA resulted in recruitment of methyl-CpG binding protein 2 (MeCP2), reduction of histone H3 or H4 acetylation and increased trimethylation of histone H3 lysine 9 as well as lysine 27 indicating that AML1-ETO induced heterochromatic silencing of Bcl-2, CEBPA and p14(ARF). These results suggested that the aberrant transcription factor AML1-ETO epigenetically silenced the function of the Bcl-2, CEBPA and p14(ARF) genes by inducing repressed chromatin configurations at their promoters through histone modifications.

[Construction of AML1-ETO eukaryotic expression vector and its effects on proliferation and differentiation of U937 cells].

OBJECTIVE: To construct a pcDNA3.1-AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells. METHODS: AML1-ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry. RESULTS: The recombinant pcDNA3.1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1-AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0.05). The expression of CD11b in transfected group \[(4.17 ± 0.31)%\] was lower than that in empty plasmid transfected group and non-transfected group \[(11.40 ± 0.17)% and (11.03 ± 0.15)%\] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CDl1b was unchanged in transfected cells treated with TPA (P > 0.05). CONCLUSION: The eukaryotic expression vector for AML1-ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1-ETO in leukemogenesis.