Effects Of Endogenous Antioxidant Levels On Diabetic Nephropathy In Lias^H/HLepr^db/db Mice

BackgroundOxidative stress and inflammatory processes are thought to be involved in the development of diabetic nephropathy（DN）but antioxidant therapy has produced conflicting results during the treatment of diabetic nephropathy.It is postulated that the antioxidant does not reach the effective concentration in the mitochondria.Lipoic acid（LA）is produced by lipoic acid synthase in mitochondria and is a strong antioxidant with the potential to protect mitochondria.It can remove many free radicals in the body and recycle other antioxidants.It plays an important role in the regulation of antioxidant system in vivo.Therefore,the present study has quantitatively evaluated the effect of endogenous antioxidant levels on diabetic nephropathy using diabetic mouse model with overexpression of lipoic acid synthase（Lias）gene.ObjectiveThe aim of this study was to establish a mutant Lias^H/HLepr^db/db mouse model to investigate whether the mice with enhanced endogenous antioxidant capacity could improve diabetic nephropathy and explore the possible antioxidant mechanism.MethodsThirty SPF Lias^+/+Lepr^db/+,Lias^+/+Lepr^db/dband Lias^H/HLepr^db/db male mice were selected,and glucose tolerance experiments and metabolic experiments were performed at28 weeks.The body weight and fasting blood glucose（FBG）levels of three groups of mice were measured after fasting for 8 hours after 28 weeks.Blood of mice was obtained from femoral artery before euthanasia.The serum is separated after centrifugation,and the lower layer of red blood cells is used to detect the concentration of glycated hemoglobin（GHb,Hb A1c）.The right kidney was weighed.Half of the left kidney was fixed with 4%paraformaldehyde for pathological observation andanother half was cut into three pieces of kidney cortex with a size of about 2³ mm to fix in 2.5%glutaraldehydefor transmission electron microscopy.Serum oxidative stress index and renal injury index were detected by biochemical kit;serum inflammatory cytokine levels were detected by enzyme-linked immunosorbent assay（ELISA）.the expression of nuclear factor E2 related factor 2（Nrf2）andnuclear factor-κB（NF-κB）were detected using Western blotting.The mitochondria of renal tissue cells were extracted,and the expression level of lipoic acid synthase（LIAS）protein in mitochondria was detected.Results1.Compared with Lias^+/+Lepr^db/dbmice,Lias ^H/HLepr^db/db mice had higher body weight,the 24-hour food and water consumption,24-hour urine volume,FPG,Hb A1c levels decreased,and the difference was statistically significant（P<0.01）.The results of glucose tolerance showed that blood sugar decreased at each time point,and there was significant difference between 90 minutes and 120 minutes after injection of glucose（P<0.05）.2.Histopathological observation showed that Lias^+/+Lepr^db/+mice had intact renal cell structure.the extent of MME was significantly larger in Lias^+/+Lepr^db/db mice than Lias^+/+Lepr^db/+mice but the expansion was attenuated in Lias^H/HLepr^db/dbmice.Semi-quantitative analysis revealed significantly higher MME scores in Lias^+/+Lepr^db/dbmice compared with Lias^H/HLepr^db/dbmice（P<0.05）.Podocyte foot process effacement and mitochondrial damage,assessed using transmission electron microscopy,were server in Lias^+/+Lepr^db/db mice,compared to Lias^+/+Lepr^db/+mice but significantly improved in Lias^H/HLepr^db/dbmice.3.The concentrations of T-AOC,GSH in serum and T-AOC in renal cortex mitochondria of Lias^H/HLepr^db/dbmice were higher than those of Lias^+/+Lepr^db/dbmice（P<0.05）,and the levels of GSH in kidney tissue increased,while the concentrations of SOD decreased slightly（P>0.05）.4.The levels of IL-1β,CRP and MCP-1 in Lias ^H/HLepr^db/db mice were significantly lower than those of Lias^+/+Lepr^db/dbmice（P<0.01）,and the levels of TNF-αwere lower than those in Lias^+/+Lepr^db/db mice（P>0.05）.5.Compared with Lias^+/+Lepr^db/+mice,the serum CRE,BUN and urinary MAU concentrations in Lias^+/+Lepr^db/db mice were significantly elevated（P<0.05）;compared with Lias^+/+Lepr^db/db mice,the serum CRE and urinary MAU concentrations in Lias^H/HLepr^db/db mice decreased significantly（P<0.05）;Mean serum BUN in Lias ^H/HLepr^db/dbmice increased（P>0.05）.6.Compared with Lias^+/+Lepr^db/+mice,the expression of LIAS and Nrf2 protein in renal mitochondria of Lias^+/+Lepr^db/db mice decreased,while the expression of NF-kappa B protein in renal tissue increased significantly（P<0.05）.The expression of LIAS and Nrf2protein in renal mitochondria of Lias^H/HLepr^db/dbmice recovered significantly than those of Lias^+/+Lepr^db/dbmice.,while the expression of NF-kappa B protein was partially inhibited（P<0.05）.ConclusionsLias^H/HLepr^db/db mice with high endogenous lipoic acid expression mainly improve diabetic nephropathy by reducing blood sugar,improving oxidative stress in the body and kidney mitochondria,and reducing inflammatory response in vivo.