Abstract
BackgroundDiabetic nephropathy (DN) is a serious complication of type 2 diabetes mellitus (T2DM). Hyperlipidemia plays a key role in occurrence and development of DN. The main saturated fatty acids of palmitic acid (PA) is closely related to glomeruli and tubules injury in T2DM. Astragaloside IV (AS-IV) has comprehensive pharmacological effects, such as anti-inflammation, anti-oxidation and anti-apoptosis. However, whether AS-IV can attenuate PA-induced renal tubular epithelial cells damages and its underlying mechanisms remain unclear.MethodsIn vitro, the HK-2 cells were stimulated with PA (200 μM) for 6, 9, 12, and 24 h respectively, then treated with AS-IV (10, 20 or 40 μM) and Apo (100 μM) for 24 h. In vivo, we investigated the effects of AS-IV in high-fat diets (HFD) and low-dose streptozotocin (STZ)-induced type 2 DN rats. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay kit. The lipid deposition was detected by ORO Staining assay kit. Annexin V-FITC/PI staining was used to detect the cell apoptosis. Immunofluorescence staining and Immunohistochemistry were used to measure the expressions of NLRP3 and NOX4. The ELISA kits were conducted to assess the levels of IL-1β, IL-18, GSH and SOD. Western blotting was used to assess the expressions of NLRP3, caspase-1, ASC, 1L-1β, NOX4, p22phox and p47phox proteins.ResultsOur results indicated that PA exposure not only significantly decreased cell viability, GSH and SOD expressions, and increased lipids deposition and apoptosis, but also increased the level of ROS and the expressions of IL-1β and IL-18. Moreover, PA exposure significantly increased the expressions of NADPH oxidase 4 (NOX4), NLRP3, ASC and caspase-1. However, all these abnormalities were significantly ameliorated by AS-IV and apocynin (Apo) treatment. In addition, our results indicated that AS-IV (80 mg/kg) could attenuate the renal tubular pathological injury and lipids deposition, and decrease the expressions of NLRP3, ASC, caspase-1, NOX4, p22phox and p47phox in renal tubules. ConclusionsThe study suggested that AS-IV could improve PA-induced HK-2 cells injury and renal tubular damage in T2DM rats, and the mechanism may be related to inhibition of NOX4-mediated NLRP3 inflammasome activation.
Immunoprecipitation of Acetyl-lysine And Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells
