Effect Of Ellagic Acid And Its Metabolism On Diabetic Nephropathy And Its Mechanism

Objective:Diabetic nephropathy(DN)is the major chronic complications of diabetes mellitus,many studies have shown that the mechanism of diabetic nephropathy is related to inflammation.The effect of ellagic acid and its metabolites on diabetic nephropathy was elucidated.The effects of ellagic acid and its metabolites on diabetic nephropathy were studied.Methods:1)ICR mice were divided into six groups:normal group(SHAM),model group(MODEL),irbesartan group(IRB),ellagic acid low dose group(LEA),ellagic acid medium dose group(MEA),ellagic acid high dose group(HEA).Except the normal group,the remaining mice were injected with 180 mg/kg streptomycin(STZ)intraperitoneally to establish diabetic nephropathy model.Four weeks after DN model was successfully established,the LEA group was treated with 50mg/kg ellagic acid,MEA group was treated with 100mg/kg ellagic acid,HEA group was treated with 150mg/kg ellagic acid,IRB group was treated with 20mg/kg irbesartan,SHAM group and model groups were given the same dose of saline.Administration lasts for 4 weeks.Twenty-four-hour urine and serum were collected,kidney tissue was made into renal tissue homogenate.The levels of serum albumin,serum TNF-?were detected using ELISA.Urea nitrogen and creatinine levels in urine and serum,T-SOD and MDA in renal tissue and serum were measured by corresponding kit.The expression of TLR4\IRAK4\TRAF6\IKK?\NF-?b P65\HMGB1 in renal tissue was detected by western blot.HE and PAS staining was performed in renal tissue.2)Rat kidney NRK-52E cells was cultured,and was stimulated with different concentrations of LPS,observe TLR4 secretion of cells using immunofluorescence assay.LPS-stimulated cells were incubated with EA and UroA,and observe whether the secretion of TLR4 was reduced using immunofluorescence assay.cells were also incubated with 100 mg/ml EA,200 mg/ml UroA,100 mg/ml UroA and 5umol/L TAK-242(inhibitor of TLR4),and then were stimulated with LPS.the level of TLR4,IRAK1 and NF-?b p65 were estimate by western blot.Results:1)The daily state of mice in LEA,MEA,HEA and IRB group was significantly improved,and the body weight was higher than that of model group,but there was no significant difference,blood glucose is lower in treated mouse,but the difference was not significant too.Irbesartan and each dose of EA did not improve the kidney index.(P<0.01).The levels of microalbumin in urine was tested using ELISA,m-ALB level in IBR group,LEA group,MEA group and HEA group(P<0.01)were significantly lower than that in model group.The results of pathological staining showed that IRB,LEA,MEA and HEA could improve the renal pathology of DN mice in different degrees.TNF-a was measured by ELISA,and the level of TNF-a in DN mice decreased in a dose-dependent manner(p<0.01),and the effect was better than that of IRB.The creatinine and blood BUN were significantly increased(P<0.01),while urinary creatinine and urine BUN were significantly decreased(p<0.01).Serum creatinine was significantly decreased(p<0.01),and the improvement of BUN in blood and urine was not obvious at all doses of EA and medium dose and high dose of EA.There was no significant difference in MDA and T-SOD between the model group and the normal group.In the serum,the activity of T-SOD in the model group was significantly decreased(P<0.01)and MDA was significantly increased(P<0.01).The decrease of T-SOD activity and the increase of MDA in EA group serum was improved(p<0.05,p<0.01).The expressions of TLR4,IRAK4,TRAF6,IKK?,NF-Kbp65 and HMGB1 in renal tissue were detected by western blot,the level of these proteins in renal tissue of LEA,MEA and HEA group were significantly increased compared to that in the model group.The reduction of these protein in renal tissue was dose-independent,and in irbesartan group,the reduction was in the most obvious manner.2)Immunofluorescence staining showed that TLR4 was mainly expressed on the cell membrane.The expression of TLR4 in 0.1 ?g/ml LPS stimulated cells was significantly higher than that in control group(P<0.05),and the expression of TLR4 was significantly increased by 100?g/ml,50?g/ml and 25?g/ml UroA.Two hundred mg/ml UroA and 100mg/mlEA showed significant reduction of TLR4,IRAK4 and NF-Kb p65(p<0.01),but the effect was weaker than the LPS inhibitor TAK-242.Conclusion:Ellagic acid can reduce the production of inflammatory factors such as TNF-a,by anti-oxidation,and reduce the inflammation by down-regulating the TLR4/NF-?B pathway.Urolithin A makes one of the major metabolites of ellagic acid.The second part of this paper shows that Urolithin A can significantly reduce the expression of TLR4 induced by LPS,so it is speculated that ellagic acid is closely related to Urolithin A.