The Inhibitory Effect of siRNAs on The High Glucose-Induced Overexpression of TGF-beta1 in Mesangial Cells

Diabetic nephropathy is characterized by an expansion of the glomerular mesangium, caused by mesangial cell proliferation and an excessive accumulation of extracellar matrix (ECM) proteins, which eventually leading to glomerulosclerosis. TGF-beta1 was found to play an important role in the accumulation of ECM in the kidney. In this study, TGF-beta1 RNA interference was used as an effective therapeutic strategy. The inhibitory effect of TGF-beta1 small interfering RNAs (siRNAs) on the high glucose-induced overexpression of TGF-beta1 in rat mesangial ceys (RMCs). A high levels of glucose induces TGF-beta1 mRNA and protein, and TGF-beta1 siRNAs reduce the ability of high glucose to stimulate their expression. We also examined the inhibitory effect of TGF-beta1 siRNAs on the expression of plasminogen activator inhibitor (PAI)-1 and Collagen Type I which are down-regulators of TGF-beta1. The expression of TGF-beta1, PAI-1 and Collagen Type I was increased in RMCs that were stimulated by 30 mM glucose. TGF-beta1 siRNAs reduces high glucose-induced TGF-beta1, PAI-1, and Collagen Type I mRNA and protein expression in a dose-dependent manner. In conclusion, the present study demonstrates that TGF-beta1 siRNAs effectively inhibits TGF-beta1 mRNA and protein expression in RMCs. These suggest that TGF-beta1 siRNAs through RNAi may be a useful tool for developing new therapeutic applications for the treatment of diabetic nephropathy.

The Effect of the Transcriptional Regulation of Sp1 for TGF-beta1 and CTGF Expression in Scar Formation

This study is to examine the relationship between TGF-b1 expression and CTGF expression, and to evaluate the effect of Sp1 blockade on the expression of TGF-b1, CTGF and extracellular genes, clones of fibroblasts stably transfected with Sp1 decoy ODN. R-Sp1 decoy ODN was highly resistant to degradation by nucleases or serum, compared to the linear or phosphorothioated-Sp1 decoy ODN. Skin wounds were created on the back of 36 anesthetized rats. They were divided into four groups-the rats with normal skin, with wounded skin without decoy, with wounded skin injected with R-Sp1 decoy, and with wounded skin injected with mismatched R-Sp1 decoy, respectively. Skins were collected at 3rd, 5th, 7th, 14th day after wounding. Cellular RNA was extracted by RT-PCR analysis. TGF-beta1 and CTGF were deeply related with skin fibrosis during scar formation and it appeared that TGF-beta1 may cause the induction of CTGF expression. R-Sp1 decoy ODN inhibited TGF-beta1 and CTGF expression both in cultured fibroblasts and in the skin of rats. These results indicate that targeting Sp1 with R-type decoy efficiently blocks extracellular matrix gene expression, and suggest an important new therapeutic approach to control the scarring in normal wound healing and fibrotic disorders.

Effects of Intracoronary Calcium Chloride and Epinephrine on Coronary Blood Flow, Oxidative Metabolism, and Mechanical Function in Canine Normal and Stunned Myocardium / 대한마취과학회지

BACKGROUND: The effects of calcium chloride (CaCl2) on regional mechanical function, coronary blood flow (CBF) and myocardial oxygen consumption (MVO2) were examined in normal and stunned myocardium in an open-chest canine model. The effects were compared with those of epinephrine. METHODS: Thirty-one dogs were acutely instrumented under enflurane anesthesia to measure aortic and left ventricular pressure, pulmonary and left anterior descending (LAD) coronary flow, and subendocardial segment length the in LAD region. CaCl2 (0.1, 0.25, 0.5, 0.75 mg/ml of LAD flow, n = 16) or epinephrine (4, 10, 20, 30 ng/ml of LAD flow, n = 15) was directly infused into the LAD before (normal) and after 15 min of its occlusion and reperfusion (stunned). Simultaneous measurements of arterial and coronary venous contents of oxygen and lactate were made to calculate oxygen (EO2) and lactate (Elac) extraction ratio during CaCl2 or epinephrine infusion. RESULTS: Both CaCl2 and epinephrine infusions in normal myocardium resulted in dose-dependent increases in mechanical functions and MVO2. These changes were accompanied by parallel increases in CBF, resulting in no changes of EO2 with CaCl2, while CBF increased more than MVO2 with epinephrine, resulting in a decrease in EO2. After ischemia and reperfusion, mechanical functions and Elac were significantly depressed, but similar mechanical responses to both agents with resultant unaltered EO2 were observed. Elac was decreased further during epinephrine, while it remained unaltered during CaCl2 infusion in stunned myocardium. CONCLUSIONS: CaCl2, similar to epinephrine, exerts positive inotropic and lusitropic effects in normal and stunned myocardium in dogs. In addition, CaCl2 has no direct effect on coronary vascular tone in either normal or stunned myocardium, while epinephrine causes direct vasodilation in normal but not in stunned myocardium.