Effects Of Caffeic Acid Para-nitro Phenethly Ester On Diabetic Nephropathy And Its Metabolites Detecting In Renal Tissue

Diabetic nephropathy（DN）is the most common chronic complication of diabetes mellitus,which is the main cause of end-stage renal disease.Its characterized by glomerular hypertrophy,thickening basement membranes of glomerular,and extracellular matrix deposition in mesangial in the early stage.In the later stage,the glomerular renal tubule fibrosis and renal failure resulted from mass extracellular matrix deposition.The present study showed that its pathogenesis is related with inflammation,oxidative stress,renal fibrosis,renin-angiotensin system and the end-stage of glycosylation products and so on.For the treatment of diabetic nephropathy,there is no effective method.So it is urgent to find effective and lower toxicity drug for the DN,which is positive significance.CAPE is one of the main active compounds of propolis and has anti-inflammatory,anti-oxidation and anti-tumor activity.In our previous studies,CAPE-pNO₂was designed and synthesized from CAPE,which possessed the better effect on the phagocytosis of macrophage in mice,anti-platelet aggregation,protected of myocardial ischemia-reperfusion injury and anti-cancer.This study was aimed to explore the effect of CAPE-pNO₂ on the diabetic nephropathy via the animal and cell,and mainly divided into three parts:the effect of CAPE-pNO₂ on the STZ induced diabetic mice,the protective of CAPE-pNO₂ on the 30mM D-glucose induced glomerular mesangial cell and the metabolites of kidney tissue analyzed by LC-MS/MS.In the animal experiment,the diabetic mice were induced by peritoneal injection of STZ for continuous 5 days（50 mg/kg）,and the fasting blood glucose value above 11.1mM was regarded as diabetic mice.Which divided into four groups:1)the diabetes group;2)CAPE group（20μmol/kg/day）;3)low-dose CAPE-pNO₂（20μmol/kg/day）;4)high-dose CAPE-pNO₂（40μmol/kg/day）.The another 10 normal mice were injected the same volume of saline and regarded as the control group.During the eight weeks administration,the fasting blood glucose was determined once a week and body weight was measured every three days.At the end of injection,the 24h urine was collected and then executed all the mice and collected serum and kidney tissues.The serum and kidney tissue related indicators were determined by kits;The HE,PAS and Masson staining aimed to examine the morphological changes of renal tissue;The expressions of inflammatory,oxidative stress and fibrotic proteins were investigated by western blot.The results showed that CAPE and CAPE-p NO₂ could control fasting blood glucose,lower kidney index ratio,increase body weight in diabetic mice and decrease the levels of BUN,sCr,24-h Alb,TC and TG.The HE,PAS and Masson staining results showed that in the diabetic mice,the basement membrane thickening obviously,the glomerular hypertrophy,renal tubule shrinking and renal tissue arranged messy with kidney fibrosis.Treatment with CAPE and CAPE-pNO₂ could improve the structure of glomerulus and renal tubule,and alleviate the deposition of collagen in the glomerular and renal tubular obviously.CAPE and CAPE-pNO₂ could decrease MDA content and increase SOD activity and reduce the oxidative stress related protein of NOX4expression significantly.CAPE and CAPE-pNO₂ also could reduce TNF-α,IL-β1,IL-6,nuclear p65,p-IκBα,p-Akt and p-PI3K expression and increase cytoplasm p65expression to slow the inflammatory in kidney;decrease the TGF-β1,Collagen IV,Fibronectin,α-SMA and p-Smad2/3 expression to prevent renal fibrosis.Similarly,the treatment of CAPE and CAPE-pNO₂ could increase the NO content and iNOS expression.In the cell experiment,the glomerular mesangial cell was induced by 30mM D-glucose for 24h and divided into 5 groups:1)the control group;2)high glucose group;3)CAPE treatment group;4)CAPE-pNO₂ treatment group;5)PDTC inhibitor group.The effects of CAPE and CAPE-pNO₂ on cell proliferation were investigated by MTT method;DCFH-DA were investigated the levels of ROS and the changes of cell morphology;Flow cytometry detected the proportion of cells in each cycle and Western Blot was used to detect the expression of cycle and extracellular matrix related proteins.The results showed that cultured in 30 mM D-glucose for 24h could promote the cell proliferation significantly,inhibit its proliferation.CAPE and CAPE-pNO₂ could increase the SOD activity,reduce the lipid peroxide MDA level and the intracellular ROS content with reducing the oxidative stress related protein NOX4 expression.The30 mM D-glucose could reduce the G0/G1 phase cell number,increase S phase cell number and unchange the G2/M phase number.Treatment with the CAPE and CAPE-pNO₂ could increase the proportion of cell number in G0/G1 phase and arrest the cell cycle at G0/G1 phase.The western blot results showed the CAPE and CAPE-pNO₂treatment could down-regulate the Cyclin E,Cdk2,Fibronectin,Collagen IV,p-IκBα,TGF-β1 and nuclear p65 protein expression and up-regulate p21^Cip1,p27^Kip1,cytoplasm p65 and iNOS expression.In the analysis of the metabolites in kidney tissue,the kidney tissues were cut into small pieces and add extracting agent（chloroform:methanol:water=1:2:1）,and then homogenated and centrifuge to collected the supernatant as the sample to be tested.The samples were analyzed by LC-MS/MS.The results of LC-MS/MS found that the metabolites of CAPE and CAPE-pNO₂ in diabetic mice were different,and the metabolites of the CAPE in renal tissues were C₈H₁₁O₄P and C₁₄H₁₆N₂O₆;and CAPE-pNO₂ possessed three metabolites:C₁₀H₁₄N₂O₆S,C₈H₁₁O₄P and C₁₄H₁₆N₂O₆.In the above experiments,the CAPE and CAPE-pNO₂ had a better effect on diabetic nephropathy,and the CAPE-pNO₂ showed better results in general（p<0.05）.The possible mechanism of CAPE-pNO₂ on DN was related with the anti-oxidative stress,anti-inflammatory,anti-fibrotic effect of CAPE-pNO₂,and inhibiting the proliferation of glomerular mesangial cells.The metabolites analysis,CAPE and CAPE-pNO₂ possessed the different types of metabolite,this maybe the reason for CAPE-pNO₂ possesse the better effective than CAPE on DN.