The Mechanism Of Endogenous N-3 Polyunsaturated Fatty Acids Ameliorating Diabetic Nephropathy

Objective: Diabetic nephropathy,one of the complications of diabetes mellitus,is the major cause of death in patients with diabetes.Evidence indicates that n-3 PUFAs and their metabolites play a key role in anti-inflammatory and anti-oxidative stress.However,the mechanism of n-3 PUFAs against diabetic nephropathy has remained obscure so far.In the present study,the fat-1 transgenic mice which are rich in endogenous n-3 PUFAs were used to establish diabetes model and further investigate the mechanism of n-3 PUFAs against diabetic nephropathy.Our resluts will provide fundamental basis for using n-3 PUFAs diet in prevention and treatment of diabetic nephropathy.Method: Heterozygous fat-1 mice were mated with wild type(WT)C57BL/6 mice and fat-1 gene was identified using DNA from tail by PCR.Fat-1 mice and WT mice were divided into two groups randomly and kept on diet containing 10% safflower seed oil.After 7 weeks,4 groups mice respectively received a single intraperitoneal injection of streptozotocin(STZ,60 mg/kg·d)over 5 days.Random blood glucose,impaired glucose tolerance test(OGTT),metabolites parameters and weight were measured.After 12 weeks,the blood glucose level reached to about 30 mM and microalbuminurea appeared.Mice were sacrificed for tissues and plasma collection.Plasma was used to check the biochemical indicators and the IL-1? levels of adipose tissues were detected by ELISA.The slice of Kidney and pancreas were made to pathology changes and the expression of IL-1? and its related signaling pathway proteins in kidney tissue were measured using western blot.Results: 1.fat-1 gene was identified by genotype using PCR.2.Diabetes nephropathy model:(1)The blood glucose level after STZ injection reached to 16.7 mM for 72 h and 25 mM 12 weeks later.(2)Urine,water quantity,and 24 h urine protein levels increased in STZ-treatment WT and fat-1 mice(P<0.05).3.The beneficial effects of endogenously increased n-3 PUFAs in kidneytissues.WT+STZ mice exhibited increased glomerular,tubular basement membrane,mesangial matrix and interstitial fibrosis expansion,compared to fat-1+STZ mice.4.The underlying mechanism of endogenously increased n-3 PUFAs against diabetic nephropathy.(1)BUN,Scr levels of plasma,the IL-1? levels of adipose tissues,and urine protein in WT+STZ mice were much higher than those in fat-1+STZ mice(P<0.05).(2)Fatty acid composition: n-3 PUFAs and their lipid mediators in fat-1 mice and fat-1+STZ mice were higher than in WT mice and WT+STZ mice,and the ratio of n-6/n-3 PUFAs in plasma were relatively higher in WT mice and WT+STZ mice than fat-1 mice and fat-1+STZ mice.(3)The molecular mechanism of n-3 PUFAs againt diabetic nephropathy: there was no significance difference in the expression levels of cleaved caspase-1,pro-caspase-1,cleaved IL-1?,IL-1?,NF-?B,p-NF-?B,NLRP3 between WT mice and fat-1 mice.But their expression levels significantly increased in WT+STZ mice and fat-1+STZ mice.It was found that the protein levels of SOD1,cleaved caspase-1 and cleaved IL-1? obviously were upregulated in WT+STZ mice than in fat-1+STZ mice.Conclusions: In the present study,on one hand endogenously synthesized n-3 PUFAs in fat-1 mice,upregulated the SOD1 expression,which inhibited the activation of inflammasome NLRP3.Inactivated NLRP3 decreased the levels of cleaved IL-1?.On the other hand,endogenously increased n-3 PUFAs upregulated adhension molecular E-cadherin and downregulated mesenchymal marker N-cadherin,which impaired nephropathy fibrosis.In conclusion,our study have shown that n-3 PUFAs alleviate diabetic nephropathy through inhibiting inflammasome NLRP3 activation and epithelial-mesenchymal transition of renal tubular endothelia cell,which provides theoretical basis for using n-3 PUFAs diet in prevention and treatment of diabetic nephropathy.