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Seminario biologia molecular universidad pontificia bolivariana
- 1. SARA VELEZ GONZALEZ III SEMESTER UPB
- 2. Warburg Effect Is a metabolic phenomenon observed in cancer cells where they predominantly rely on glycolysis for energy production, even in the presence of oxygen.
- 3. Angiotensin Is a peptide hormone that plays a key role in regulating blood pressure and fluid balance in the body. It is part of the renin- angiotensin system. The most biologically active form is angiotensin II.
- 4. Objective This study investigates whether Ang-(1-7) mitigates inflammation in LPS-induced macrophages and septic mice by regulating the Warburg effect in immune metabolism.
- 5. Methods WESTERN BLOT Levels of TNF-a an IL-6 in cell culture supernatant and mouse serum was measured using an Elisa kit (Enzyme-linked immunosorbent assay). The kit was brought to room temperature, standard and samples wells were prepared. The standards, samples and reagents were added according to the instructions. Absorbance at 450 nm was then measured, and sample concentrations were calculated following the manual’s instructions. In this study, RAW264.7 cells and kidney tissues were lysed to release proteins, which were then separated, transferred onto membranes, and blocked. Primary antibodies were applied overnight at 4°C, followed by secondary antibodies at room temperature. Chemiluminescent signals were used for detection. Target proteins included β-actin, TNF- α, IL-6, SLC25A1, HK2, PFKFB3, PKM2, NDUFB8, UQCRFS1, ACLY, and HIF-1α. ELISA
- 6. Methods QRT-PCR Tissues were processed with xylene and ethanol, followed by antigen retrieval and blocking with 5% BSA. They were then incubated overnight at 4°C with primary antibodies and with secondary antibodies at room temperature. DAPI staining was applied for nuclear visualization, and slides were sealed. Analysis was done using a fluorescence microscope, capturing microscopic images. The total RNA was extracted from cells or tissues using the SteadyPure Quick RNA extraction kit. The concentration and purity of the RNA samples were assessed. Next, the RNA was reverse- transcribed into cDNA using RT Master Mix for qPCR II. qPCR reactions were then carried out using SYBR Green qPCR Master Mix and the CFX96 RT- PCR system. IMMUNOFLUORE- SCENCE STAINIG
- 7. Results
- 8. Results
- 10. Conclusions The study underscores the significance of molecular biology by elucidating how Angiotensin-(1-7) influences the Warburg effect, a key metabolic pathway, in LPS-induced macrophages and septic mice By targeting molecular pathways involved in cellular metabolism, such as the Warburg effect, Angiotensin- (1-7) presents a promising approach to mitigating inflammation in septic conditions, emphasizing the intersection between molecular biology and clinical interventions.