Anti-fibrosis Activity And Mechanism Research Of Rapeseed-derived Antioxidant Peptide RAP In Diabetic Nephropathy

Background and purpose: Diabetic nephropathy(DN)is one of the most common and serious microvascular complications of diabetes.Kidney fibrosis is the main pathological change in diabetic nephropathy(DN),which is the major cause of end stage renal disease(ESRD).The pathological changes of DN mainly include the loss of the normal nephron,hyperplasia of a large number of fibroblasts and muscle fibroblasts,and excessive accumulation of extracellular matrix(ECM)including collagen and fibronectin(FN),thus the glomeruli Kimmelsstiel-Wilson(KW)nodules are formed.In addition,glomerulus and renal tubular basement membrane thickening and renal tubular interstitial fibrosis,eventually lead to glomerular sclerosis.The strategy of DN treatment is to control blood glucose,lower blood pressure and control hyperlipidemia.Existing treatments may slow down the process,but they cannot stop ESRD from occurring and causing side effects.Therefore,it is of great theoretical and practical significance to develop novel therapeutic agents that target the major pathological mechanisms of DN.Plant-derived bioactive peptides in foodstuffs are widely used in many fields due to their potential pharmaceutical and nutraceutical benefits.Major biological effects of plant-derived peptides include lowering cholesterol,inhibiting angiotensin I,antibiosis,immunoregulation and antioxidant.However,this kind of peptide has not yet been studied in renal fibrosis.Previous studies indicated that the peptide RAP(the sequence is YWDHNNPQIR),a natural peptide derived from rapeseed protein,has anti-oxidative stress effect.The oxidative stress is believed to be associated with diabetic nephropathy.The aim of this study was to evaluate the pharmacological effects of RAP against renal fibrosis of DN and high glucose(HG)-induced mesangial dysfunction.Methods:(1)In vivo experiments,eight-week-old male C57BL/6 mice were induced by injecting STZ at dosage of 45 mg/kg for 5 consecutive days,and the same dose of citric acid buffer was injected in the normal group.The blood glucose level was measured 72 hours after STZ injection.Mice with fasting blood glucose levels above 11.1 mM were considered diabetic.Next,mice were randomly assigned to the four experimental groups: normal control(control,n=6),diabetic model(DM,n=8),diabetic + low dose RAP(RAP-L,0.1 mg/kg/day,n=8)and diabetic + high dose RAP(RAP-H,0.5 mg/kg/day,n=8).Treatment was continued until the 12 th week and with free access to HFD.In the RAP treatment group,mice were administrated with RAP every two days.The same volume of PBS was given to the mice in both the normal control group and diabetic group.After 12 weeks of treatment,all animals were housed in metabolic cages to allow 24-hour urine collection and measurement of albuminuria.The serum creatinine,blood urea nitrogen,triglyceride and serum TGF-?1 were detected after blood collection;the m RNA and protein levels of TGF-?1,CTGF,FN and ?-SMA in the mouse kidneys were measured by western blot,immunohistochemical and RT-PCR analysis.The phosphorylation levels of ERK1/2,p38 and p65 in the mouse kidneys were detected by western blot and immunohistochemical analysis.In vitro experiments,GMC were induced by high glucose,and the MTT assay was used to measure cell proliferation and cytotoxicity to selected RAP effective concentrations(50 and 100?M)in our subsequent experiments.The mRNA and protein levels of TGF-?1,CTGF,FN and ?-SMA induced by HG in GMC were detected.The phosphorylation levels of ERK1/2,p38 and p65 in GMC induced by HG were measured.In addition,we measured the levels of FN and COL4 in the supernatant of GMC using ELISA analysis.Results:(1)After 12-weeek treatment,RAP improved the diabetic syndromes and renal fibrosis compared with DN mice,including: Improves renal function indexes BUN,Scr and TG;Ameliorates oxidative stress parameters SOD and MDA;Inhibited the expression of TGF-?1,CTGF,FN and ?-SMA in DN mice;Reduced collagen deposition in the mouse kidneys;Decreased glomerular hypertrophy and thickening of basement membrane;Inhibited the MAPK and NF-?B signaling pathways in DN mouse kidneys.(2)Compared with high glucose group,RAP treatment inhibited the levels of FN and COL4 in the supernatant of GMC.Meanwhile,on m RNA and protein levels,the expression of TGF-?1,CTGF,FN and ?-SMA were reduced by RAP.In addition,RAP treatment restrained the MAPK and NF-?B signaling pathways in HG-induced GMC.Conclusions: RAP can attenuate fibrosis in vivo and in vitro by antagonizing the MAPK and NF-?B pathways.