Intervention Of Two Kinds Of Carotenoids From Marine Sources On Diabetic Nephropathy

Aim:To evaluate the effect of astaxanhin and fucoxanthin on diabetic nephropathy and determind the underlying mechanisms.Methods:The research on astaxanthin including in vivo and in vitro studies.We established diabetic rat model for in vivo studies:male Sprague-Dawley rats were housed under standard conditions with food and water freely obtained.After being fed with a regular diet for 1 week,they were assigned to a diabetic model group(n = 34),which were fed with a high-fat,high-glucose diet for the following 4 weeks,a normal control group(n = 8),which were fed a normal diet,and a vehicle group(n = 8).After 4 weeks of feeding,diabetic model group rats were given a single intraperitoneal injection of STZ on 30 mg/kg,freshly prepared.The normal group and vehicle group rats were injected with an equal volume of citrate buffer.Diabetic rats were accepted by the fasting blood glucose measurement 11.1 mM after a 72 h injection.Diabetic rats were randomized into an administration group(n = 8)to receive AST(25 mg/kg daily i.g.),and the other diabetic rats and vehicle group rats received an equal volume of olive oil.Rats were sacrificed after a 12-week treatment.Kidney cortex samples were rapidly excised,frozen in liquid nitrogen quickly,and then stored at-80℃ or fixed in 10%neutral buffered formalin.The blood sample was collected from the abdominal vein,and serum was obtained by centrifugation at 3000 g for 15 min and stored at-80℃.Glomerular mesangial cells(GMCs)were cultured in DMEM(normal glucose,5.6 mM)supplemented with 10%fetal bovine serum at 37 ℃ under an atmosphere of 5%C02 for in vitro studies.At subconfluence,the GMCs were serum-strave for 24h and then divide into 4 groups:(a)control,where cells were kept in DMEM without fetal bovine serum(control group),(b)high glucose treatment group,in which cells were kept in DMEM contain 30 mM glucose without fetal bovine serum(model group),(c)astaxanthin treatment group treat with 5μm astaxanthin,(d)NAC treatment group treatment with 5μm NAC.Fibronectin(FN)level in GMCs was devected by western blot and immunofluorescence to evaluate the protective effect of AST in vitro.The renal protective effect of fucoxanthin was explored in high glucose-treated GMCs.The protein expression of FN and collagen IV(Col IV)were detected by western blot.Intracellular content of FN was also detected using immunofluorescence.The reactive oxygen species(ROS),F-actin,superoxide dismutase(SOD)and malondialdehyde(MDA)were measured by commercially available kits.Meanwhile,western blot assay was also applied to detect the protein expression of ICAM-1 and TGF-β1 and nuclear content of p65.Dual luciferase reporter gene assay is to detect the effect of fucoxanthin on transcriptional activity of NF-κB under high glucose.Results:1.Kidney weight and kidney hypertrophy index(KW/BW),fasting blood glucose,blood urea nitrogen,serum creatinine,and urine protein over 24 h were significantly increased in STZ-induced diabetic rats compared with those in control rats(P<0.05).After 12 weeks of treatment with AST(25 mg/kg daily),the diabetic rats exhibited a significant reduction in these parameters except blood glucose(P<0.05).The PAS-positive matrix in the glomeruli was increased in diabetic rats compared with control rats;however,treatment with AST reversed this change.HE staining showed that the kidney of diabetic rats exhibited structural damage,glomerular hypertrophy,mesangial hyperplasia,and basement membrane thickening.Treatment with AST significantly improved the pathological changes in the kidney of diabetic rats.Reduced FN and Col IV protein expression were found in the kidney of diabetic rats.In GMCs,FN in decreased significantly which was evaluated both western blot and immunofluorescence.Furthermore,AST promoted the nuclear translocation of Nrf2 and increased its downstream protein heme oxygenase-1 and superoxide dismutase 1 expression.AST also increased the activity of SOD and decreased malondialdehyde generation in the serum of diabetic rats.2.The expression of FN and Col IV in high glucose-treated GMCs were upregulated significantly while fucoxanthin reversed it,as well as the tendency of hypertrophy.Fucoxanthin significantly increased the activity of SOD and decreased the content of MDA and ROS level that were abnormal under the high glucose condition.Moreover,fucoxanthin inhibited the activation of NF-κB signaling induced by high glucose.Conclusion:1.Astaxanthin improved kidney performance and ameliorated glomerular fibrosis in vivo and in vitro.Astaxanthin also can improve the antioxidative ability of diabetic rats and reduce the damage caused by oxidative stress.Astaxanthin improve the antioxidative ability by activating Nrf2-ARE signaling pathway.2.Fucoxanthin could obviously decrease the accumulation of ECM components and alleviate hypertrophy in high glucose-treated GMCs.The protective effect of fucoxanthin is relevant to its role of decreasing the oxidative stress and inflammation in GMCs.Our study suggests that fucoxanthin could be used as a potential drug for diabetic nephropathy.