RESUMO
There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.
RESUMO
The freshwater clam is a widely-consumed shellfish and is used as a remedy for chronic hepatitis in Asia. However, its contribution to acute liver injury (ALI) remains unclear. The aim of this study is to assess the protective effects of freshwater clam extract (CE) in ALI induced by hemorrhage in rats. Rats were randomly divided into 5 groups, (1) blood loss (BL) 40%, (2) CE 150 mg/kg plus BL 40%, (3) CE 75 mg/kg plus BL 40%, (4) CE 150 mg/kg, and (5) CE 75 mg/kg groups. CE was given by femoral vein catheter in Groups 2 to 5. Initial hemorrhage was induced by withdrawing blood (loss 40% of total blood volume) from a femoral arterial catheter after CE administration in Groups 2 and 3. The levels of blood tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured at several time points during the experimental period. Rats were sacrificed after 48 hours, and the liver was harvested for hematoxylin and eosin (HE) stain to show liver tissue injury. The results indicated that hemorrhage significantly decreased mean arterial pressure (MAP), increased blood AST, ALT and LDH levels and induced liver injury. Pre-treatment with the CE increased MAP and IL-10 levels and decreased AST, ALT, LDH and TNF-alpha levels after hemorrhage. The HE stains showed diminished organ injury in the CE groups. In conclusion, freshwater clam extract is a potential immunomodulating agent and ameliorates acute liver injury.
