The Protective Effect Of Puerarin On Cadmium-induced Kidney Damage In Rats

Heavy metal cadmium is common in the environment of high toxic pollutants,acute or chronic cadmium exposure can cause a damage in variety of organs and tissues.Kidney is the important target organs and accumulation of cadmium toxicity damage parts,cadmium exposure caused oxidative stress and metabolic disorders can lead to kidney damage and cell apoptosis.Puerarin is a plant isoflavone extracted from Pueraria lobata,because of its low toxicity,metabolism faster and has antioxidant,regulate blood sugar level,and so on,it has been clinically used in the treatment of cardiovascular disease and diabetes complications.AMPK is the reaction center of the body energy metabolism,blood anoxic or poisoning caused by the body’s metabolic disorders can activate AMPK/AKT signaling pathway,to regulate the metabolic balance and protect the body from further damage.In this study,by using the method of combining the in vivo and in vitro,in rats and rats with the original generation of renal tubular epithelial cells for the mode,investigated the protective effect of puerarin when cadmium cause kidney damage.1.Cadmium exposure to renal injury in rats and the protective effect of puerarinThe study was carried out on male SD rats.Forty rats were allocated randomly to four groups of ten animals each:Control group(rats with no treatment);Cd treated group(CdAc2,2.5mg/kg·bw);Cd+PU treated group(PU,200mg/kg bw;CdAc2,2.5mg/kg·bw);PU treated group(PU,200mg/kg·bw).The experimental period was five weeks:The experimental period of PU treated(intragastric administration)was four weeks,The experimental period of Cd treated(intraperitoneal injection)was one week.Collection of renal cortex,whole blood,serum and urine for subsequent a series of tests.Using atomic absorption spectrometry measured cadmium content in rat renal cortex,serum,and urine;Blood routine analysis apparatus measured rats blood glucose level changes;Visible light spectrophotometer measured rat blood serum malondialdehyde(MDA),superoxide dismutase(T-SOD)and total antioxidant capacity(T-AOC);Paraffin section observation renal pathological changes;Blood biochemical instrument measured serum urea nitrogen(BUN),serum creatinine(CREA)content and urine beta-N-acetyl amino glycosidase enzymes(NAG)and urine beta 2 microglobulin(β2-MG);QRT-PCR to detect the expression of renal cortex of autophagy related genes and AMPK signaling pathway related gene;Western blot to detect expression of apoptosis related proteins in renal cortex of rats,autophagy related proteins and AMPK signaling pathway related protein.The results show that:(1)The volume of cadmium in the infected group rats was significantly higher than that of control group(P<0.01),while protecting group volume of cadmium was significantly lower than in mice infected group(P<0.01);(2)Infected rats serum MDA was significantly higher than that of control group(P<0.01),T-SOD and T-AOC were lower than that of control group(P<0.01),while protecting group rats serum MDA was significantly lower than infected group(P<0.01),T-SOD and T-AOC were significantly higher than that of the infected group(P<0.01 or P<0.05);(3)Infected group rats blood glucose significantly higher than that of control group(P<0.01),protect rats blood glucose significantly below infected group(P<0.05);(4)Infected group rat renal tubule degeneration,swelling,luminal stenosis,renal interstitial a large number of red blood cells infiltration,glomerular congestion,lesions were induced in rats of protect group.(5)Infected group rats serum BUN and CREA were significantly higher than that of control group(P<0.01),and protection group serum BUN and CREA were significantly lower than infected group(P<0.01);(6)Those infected group in rat urine NAG and β2-MG were significantly higher than control group(P<0.01),protect group NAG was significantly lower than infected group(P<0.05),but there was no significant alteration of the β2-MG;(7)infected group rat renal cortex Cleaved caspase 3,p-AKT/AKT,p-mTOR/mTOR quantity of protein expression is significantly higher than control group(P<0.01 or P<0.05),amount of Beclinl expression was significantly lower than control group(P<0.01),LC3Ⅱ/LC3I,ATG5,p62,p-AMPK/AMPK expression quantity had no obvious change;Protection group rat renal cortical LC3II/LC3I,ATG5,Beclinl,p-AMPK/AMPK expression were significantly higher than infected group(P<0.01 or P<0.05),p62,Cleaved caspase 3,p-AKT/AKT,p-mTOR/mTOR expression quantity significantly below infected group(P<0.01 or P<0.05);Group was infected rat renal cortical LC3,P62,BECN1,AKT,mTOR gene expression level was significantly higher than that of control group(P<0.01 or P<0.05),ATG5,AMPK expression level has no obvious change,protect group rat renal cortex LC3,ATG5,BECN1,AMPK expression level was significantly higher than that of the infected group(P<0.01 or P<0.05),P62,AKT,mTOR significantly below the level of gene expression infected group(P<0.01 or P<0.05).According to the all results we can draw the following conclusions:(1)Cadmium exposure caused rat body oxidative damage and metabolic disorders;(2)Cadmium exposure caused renal tubular and glomerular injury in rats,and lead to cell apoptosis;(3)Puerarin can relieve oxidative damage caused by cadmium,regulating metabolism balance;(4)Puerarin by AMPK/AKT signaling pathways regulating autophagy,antagonism cell apoptosis caused by cadmium.2.Cadmium exposure to renal tubular epithelial cell injury in rats and the protective effect of puerarinMechanical mesh combined with enzyme digestion method is adopted to establish the rat primary renal tubular epithelial cells(rPT)model,in the logarithmic phase,rPT cells be divided into four groups:Control group(rats with no treatment);Cd treated group(10μM CdAc2);Cd+PU treated group(50μM PU,10μM CdAc2);PU treated group(50μM PU),or:Control group(rats with no treatment);Cd treated group(10μM CdAc2);Cd+AICAR treated group(500μM AICAR,10μM CdAc2);PU treated group(500μM AICAR),or:Control group(rats with no treatment);Cd+Dor group(50μM Dor,10μM CdAc2);Cd+PU+Dor group(50μM Dor,50pM PU,10μM CdAc2);Dor group(50μM Dor).Then a series of tests were carried out.CCK 8 method is used to detect each group rPT cell vitality;The firefly luciferase luminescence method is adopted to detect the contents of ATP in the cell;DCFH-DA probe method is used to detect each group ROS levels in the cell;By using transmission electron microscopy(SEM)observated ultrastructure of cell in each group;With Annexin V-FITC/PI double dye and flow cytometry were used to detect each cell apoptosis rate;By DAPI probe dyeing,using confocal laser technology to detect the morphology of nucleus apoptosis;By the MDC probe dyeing,using laser confocal technique to observe the number of autophagic vacuoles in cells;By RFP-LC3 plasmid transfection,using laser confocal fluorescent observation accumulation point;Through the GFP-RFP-LC3 double standard plasmid transfection,using laser confocal observation cell autophagy flow;Using Western blot test cell apoptosis related proteins,autophagy related proteins and AMPK signaling pathway related protein expression.The results show that:(1)Infected group cell vitality and ATP level was significantly lower than control group(P<0.01 or P<0.05),the level of ROS is significantly higher than the control group(P<0.01),protect the cell vitality and ATP level was significantly higher than in infected group(P<0.01 or P<0.05),ROS were significantly lower than infected group(P<0.01);(2)Infected cells damaged organelles,mitochondria swelling and appeared different degree of damage and cavitation,autolysosome appear in the cells of protect group for degrade damaged organelles;(3)Infected cell apoptosis rate is significantly higher than the control group(P<0.01),protect the apoptosis rate was significantly lower than infected group(P<0.01);(4)Infected cell autophagy body quantity was significantly less than control group(P<0.01 or P<0.05)and autophagy flow blocked,protect cell autophagy body significantly more than the number infected group(P<0,05)and autophagy flow smoothly;(5)In infected cells,LC3Ⅱ/LC3I,ATG5,Cleaved caspase 3,p-AKT/AKT,p-mTOR/mTOR quantity of protein expression is significantly higher than control group(P<0.01 or P<0.05),amount of Beclinl expression was significantly lower than control group(P<0.01),p-AMPK/AMPK,p62 expression had no obvious change,protect cells LC3II/LC3I,ATG5,Beclinl,p-AMPK/AMPK expression amount were significantly higher than that of the infected group(P<0.01 or P<0.05),p62,Cleaved caspase 3,p-AKT/AKT,p-mTOR/mTOR expression quantity significantly below infected group(P<0.01 or P<0.05);(6)By AMPK excited agent AICAR,Cd+AICAR group of intracellular LC3Ⅱ/LC3I,ATG5,Beclinl expression were significantly higher than that of Cd group(P<0.01 or P<0.05),p62,Cleaved caspase 3 expression quantity were significantly lower than control group(P<0.01 or P<0.05),Cd group cell LC3Ⅱ/LC3I,ATG5,p62,Cleaved caspase 3 expression quantity were significantly higher than that of control group(P<0.01 or P<0.05),Beclinl expression significantly lower than that of control group(P<0.01);(7)By AMPK inhibitors treated,group of Cd+PU+Dor intracellular p-AKT/AKT expression was significantly higher than Cd+PU group(P<0.01),p-AMPK/AMPK expression was significantly lower than that of Cd+PU+Dor group(P<0.05),p-mTOR/mTOR expression quantity has no obvious change;Cd+PU group cell p-AMPK/AMPK,p-AKT/AKT,p-mTOR/mTOR expression is significantly higher than control group(P<0.01 or P<0.05).According to the above results can draw the following conclusions:(1)Cadmium can cause rPT cell vitality decline,morphology and ultrastructure damage and oxidative damage and metabolic abnormalities;(2)Cadmium can cause rPT cell autophagy flow blocked,leading to cell apoptosis;(3)Puerarin can alleviate rPT cell damage caused by cadmium,maintain metabolic balance;(4)Puerarin can be adjusted autophagy through the activation of AMPK signaling pathway,antagonism rPT cells apoptosis caused by cadmium.