Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over-activation and consequently attenuate DCM. Streptozotocin-induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure-volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15-F2t-Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme-linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT-PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end-systolic volume (LVVs) as compared to non-diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15-F2t-Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase-1 (HO-1) and Keap1. ALP reverted all the above-mentioned diabetes-induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia-induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over-activation may represent major mechanisms whereby ALP attenuates DCM.

Background

Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).

Results

We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.

Conclusion

The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.