ABSTRACT
Aim To investigate the protective effect of bezafibrate on renal injury and the changcs of 20- HETE in mice with diabetic nephropathy. Methods A diabctic model was established by long-term high-energy feeding combined with streptozotocin. The structural and functional changes of kidney were evaluated by renal pathological examination and blood urea nitrogen (BUN), serum creatinine ( Scr) , urinary albumin levels. The expressions of PPAIls and CYP4A protein were detected by Western blot. The level of 20-HETE was measured by ELISA kit. Results After four weeks with high-energy feeding, streptozotocin (40 mg • kg-1 • d"1 i. p) administration had been performed for five days. Then after seven days,fasting blood glucose (FBG) of mice exceeded 11.1 nimol • L"1, which suggested the establishment of the diabetic model. After four weeks of diabetic onset, the levels of BUN,Scr,urinary albumin increased significantly (P er and thickened basement membrane were observed in diabetic mice. Meanwhile,the expressions of PPARs and CYP4A protein in the kidney and the content of 20-HETE in serum decreased in model group (P <0. 01). With bezafibrate supplementation (75 mg • kg"' • d"1) for four weeks, both the structure and function of kidney were improved in diabetic mice,with the up-regulation of PPARs and CYP4A protein expressions and the increase of 20-HETE level (P <0. 01 ). Conclusions Bezafibrate can ameliorate renal injury in diabetic mice, which may be related to activating CYP4A-20-HETE.
ABSTRACT
Aim: To investigate the ameliorative effects of naringenin on renal injury and its roles in 20-HETE in diabetic mice. Methods: A diabetic model was established by long-term high-energy feeding combined with streptozotocin. The structural and functional changes of kidney were evaluated by renal pathological examination and blood urea nitrogen(BUN), serum creatinine(SCr), urinary albumin levels. The expression of CYP4A protein was detected by Western blot. The level of 20-HETE was measured by ELISA kit. Results: After four weeks of high-energy feeding, streptozotocin (40 mg. kg-1. d-1, IP) was given for five days. Then after seven days, fasting blood glucose of mice exceeded 11.1 mmol L-1, which suggested the establishment of diabetic model. After four weeks of diabetic onset, the levels of BUN, SCr, urinary albumin increased significantly(P <0. 01); kidney index(renal weight/body weight) and glomerular volume were raised(P <0. 01); increased collagen fiber and thickened basement membrane were observed in diabetic mice. Meanwhile, the expression of CYP4A protein in the kidney and the content of 20-HETE in serum decreased in model group (P < 0. 01). With naringenin supplementation(25 or 75 mg · kg-1 · d-1) for four weeks, both the structure and function of kidney were improved in diabetic mice, with the up-regulation of CYP4A protein expression and the increase of 20- HETE level(P < 0. 05). Conclusion: Naringenin can ameliorate renal injury in diabetic mice, which may be related to activating CYP4A and increasing 20-HETE.
ABSTRACT
Objective To clone the murine Na+-K+-2Cl-cotransporter (Nkcc2) gene promoter and analyze 20-HETE regulation of the murine Nkcc2 gene transcriptional activity. Methods A fragment of the murine Nkcc2 gene promoter was analyzed using bioinformatics software AliBaba and TRANSFAC TESS. The murine Nkcc2 gene promoter fragment (-1 462 bp-+40 bp) was amplified by PCR using murine genomic DNA as a template and then cloned into a pGL3-Basic vector to generate a luciferase reporter construct. The recombinant reporter construct was transiently transfected into HEK293 T cells using Lipofectamine 2000 for 24 h. The transfected HEK293 T cells were treated with 20-HETE for 2 h followed by measurement of the luciferase activity using the Dual-Luciferase Reporter Assay system. Results A luciferase reporter construct containing the murine Nkcc2 gene promoter was successfully generated. The results showed that 20-HETE significantly reduced the transcriptional activity of the construct. Conclusion 20-HETE may reduce the expression of the murine Nkcc2 gene through transcriptional regulation.
ABSTRACT
Objective To study the effect of 20-HETE on apoptosis in cultured neonatal rat cardiomyo-cytes and investigate its mechanism. Methods Neonatal rat cardiomyocytes were cultured in vitro.CCK-8 method was used to detect the cell activity and TUNEL assay was performed to analyze the cell apoptosis. Flou-3/AM la-belled assay was applied to measure the concentration of intracellular calcium([Ca2+]i). Western blot was per-formed to measure the expressions of RyR2,SERCA2a,CaMKII and phospho-CaMKII. Results Treatment with 20-HETE reduced the activity of cardiomyocytes and induced cell apoptosis obviously,while KN-93,an inhibitor of CaMKII,blocked the effects of 20-HETE. Treatment with 20-HETE significantly increased cardiomyocytes [Ca2+]i,up-regulated the expression of RyR2,and down-regulated the expression of SERCA2a,which could be blocked by KN-93. 20-HETE also increased the expressions of CaMKII and phospho-CaMKII in cardiomyocytes, indicating 20-HETE played a role in activating the CaMKII signaling pathway. Conclusions 20-HETE leads to altered functions of cardiac sarcoplasmic reticulum calcium-transport protein RyR2 and SERCA2a via activating the CaMKII signaling pathway,which causes calcium overload and induces apoptosis in neonatal rat cardiomyocytes.