| Diabetes mellitus (DM) is a serious and complex chronic condition, which is characterized with chronic hyperglycaemia. According to a prediction of the International Diabetes Federation, the worldwide prevalence of diabetes is likely to reach592million by the year of2030. Diabetic nephropathy (DN) is the most serious complication of diabetes and also the main cause of end-stage renal disease. The symptoms of DN are characterized by persistent proteinuria, thickening of basement membrane and decreased glomerular filtration rate. Many studies have shown that inflammation plays critical roles in promoting the development and progression of DN.Lycium barbarum, a Solananceous defoliated shrubbery, has been used as a kind of traditional Chinese herbal medicines for thousands of years. The traditional medicinal part is the fruit, which has a history of use as an ingredient in various soft or alcoholic drinks for its benefits for anti-aging, protecting the kidney and liver. Lycium barbarum polysaccharide (LBP) is the main bioactive component of L. barbarum and composed of arabinose, glucose, galactose, mannose, xylose and rthamnose. Likewise, the researches indicate that LBP has a large variety of bioactivities, such as hypoglycemic, hypolipidemic and antioxidant. In this study, the rabbits are used as experimental object, and the effect of LBP on proteinuria and renal tissue inflammatory injury in diabetic nephropathy are expounded from the levels of animal and molecular. This study aims to discuss whether LBP can delay the appearance of proteinuria, inhibit the renal tissue inflammatory reaction, and delay the onset and progression of DN.Part I The establishment of the rabbits model of diabetic nephropathy and the protection effect of Lycium barbarum polysaccharides on the kidneys inflammatory. injury of diabetic nephropathy rabbitsObjective:To investigate the effect of LBP on the kidneys inflammatory injury of diabetic nephropathy rabbits. Methods:Diabetes was induced by alloxan (ALX) injection combined with high fat diet (4%lard+1%cholesterol). Japanese white rabbits were randomly divided into5groups:normal control group, DN model group, LBP prevention group, Micardis group (positive control group) and LBP treatment group (n=4for each group). LBP (10mg/kg) was given to LBP prevention group after DM model successful for12weeks and LBP treatment group after DN model successful for4weeks, Micardis (3.7mg/kg) was given to Micardis group after DN model successful for4weeks. And the same volume of balanced saline was given to normal group and DN group. Body weight and fasting blood glucose were detected at the end of Ow,2w,4w,6w,8w and12w; blood lipid (TC, TG, LDL-c and HDL-c) was detected at the end of Ow,4w,8w and12w;24h urine protein was detected at the end of4w,6w,8w and12w; serum BUN and SCr were detected at the end of8w and12w, meanwhile to calculate CCr; At the end of the12th week, the rabbits were killed, serum CRP concentration and AGEs of renal cortex were detected; Ang Ⅱ was detected by ELISA. Result:Blood glucose and blood lipid in DN group were significantly increased (P＜0.01), and body weight was significantly decreased (P<0.01).24h urine protein was significantly increased since the fourth week, and the serum BUN and SCr were also significantly increased (P<0.01), meanwhile CCr was decreased (P＜0.01), the serum CRP and the level of AGEs and Ang Ⅱ were increased (P＜0.01). Compared with DN group, blood glucose and lipid in LBP prevention group were significantly decreased (P＜0.01), and body weight was significantly increased (P<0.01).24h urine protein was significantly decreased (P＜0.01) and the appearance of urine protein was delayed. The seum BUN and SCr were decreased meanwhile CCr was increased (P＜0.01). And the serum CRP and the level of AGEs and Ang Ⅱ were decreased (P＜0.01). The condition of the rabbits was also ameliorated in Micardis group and LBP treatment group:the serum BUN and SCr were significantly descreased (P＜0.05),24h uriary protein was significantly decreased (P＜0.05, P<0.01), Micardis group was lower than those of LBP treatment group, but still higher than those of LBP prevention. Conclusion:LBP can alleviate the renal tissue inflammatory injury, improve renal function and glomerular filtration rate of diabetic nephropathy rabbits, and delay the onset of proteinuria. And the prevention effect of LBP is best, the appearance of proteinuria is delayed.Part II Effect of Lycium barbarum polysaccharides on glomerular morphological structure and filtration function damage induced by inflammation of diabetic nephropathy rabbitsObjective:To investigate the effect of LBP on glomerular morphological structure and filtration function of diabetic nephropathy rabbits. Methods:Diabetes was induced by alloxan (ALX) injection combined with high fat diet (4%lard+1%cholesterol). Japanese white rabbits were randomly divided into5groups:normal control group, DN model group, LBP prevention group, Micardis group (positive control group) and LBP treatment group (n=4for each group). LBP (10mg/kg) was given to LBP prevention group after DM model successful for12weeks and LBP treatment group after DN model successful for4weeks, Micardis (3.7mg/kg) was given to Micardis group after DN model successful for4weeks. And the same volume of balanced saline was given to normal group and DN group. At the end of the12th week, the rabbits were killed and bilateral kidneys were taken out. The kidney weight index (KWI) was estimated by calculating the ratio of the both kidneys weight to body weight. And the morphological changes occurred to renal tissue was observed by light microscope and electron microscope. The expression of Nephrin mRNA was detected by RT-PCR. Result:KWI of the rabbits was increased in DN group (P<0.01). Under light microscope, the renal tissue of the rabbits in DN group showed markedly severe destruction in glomerular lesions, such as glomerular volume and glycogen deposition increase, mesangial expansion, extracellular matrix accumulation and basement membrane thickening. Under electron microscope, fusion of foot process and thickness of basement membrane were observed. Meanwhile, the expression of Nephrin mRNA was decreased (P＜0.01). These pathological changes were remarkably ameliorated in the LBP prevention group, Micardis group and LBP treatment group. Glomerular volume was decreased, basement membrane thickening and glycogen deposition were reduced. And the expression of Nephrin mRNA was increased (P＜0.01). Conclusion:LBP can ameliorate the pathological changes of renal tissue, and delay the onset of proteinuria and DN.Part III Effect of Lycium barbarum polysaccharides on inflammatory associated cytokine expression in of diabetic nephropathy rabbitsObjective:To investigate the effect of LBP on inflammatory associated cytokine expression of diabetic nephropathy rabbits. Methods:Diabetes was induced by alloxan (ALX) injection combined with high fat diet (4%lard+1%cholesterol). Japanese white rabbits were randomly divided into5groups:normal control group, DN model group, LBP prevention group, Micardis group (positive control group) and LBP treatment group (n=4for each group). LBP (10mg/kg) was given to LBP prevention group after DM model successful for12weeks and LBP treatment group after DN model successful for4weeks, Micardis (3.7mg/kg) was given to Micardis group after DN model successful for4weeks. And the same volume of balanced saline was given to normal group and DN group. At the end of the12th week, the rabbits were killed. And the protein expression of MCP-1, ICAM-1, TGF-β and TNF-a in renal cortex were detected by immunohistichemical staining, at the same time, the expression of MCP-1mRNA and ICAM-1mRNA were detected by RT-PCR. Result:The serum CRP was significantly increased in DN group (P<0.01). And the protein expression of MCP-1, ICAM-1, TGF-p and TNF-a were increased, meanwhile the expression of MCP-1mRAN and ICAM-1mRNA were also increased (P<0.01). The protein expression of MCP-1, ICAM-1, TGF-β and TNF-a were decreased in LBP prevention group, Micardis group and LBP treatment group (P<0.01), meanwhile the expression of MCP-1mRAN and ICAM-1mRNA were decreased (P<0.01). Conclusion:LBP can down-regulate the expression of inflammatory-associated cytokine, and inhibit inflammatory reaction in the kidneys of diabetic nephropathy rabbits, alleviate the glomerular filtration function damage and delay the onset of proteinuria and DN.Part IV Effect of Lycium barbarum polysaccharides on oxidative stress and the expression of NF-κB signal pathway in renal cortex of diabetic nephropathy rabbitsObjective:To investigate the effect of LBP on oxidative stress and the expression of NF-κB signal pathway in renal cortex of diabetic nephropathy rabbits. Methods:Diabetes was induced by alloxan (ALX) injection combined with high fat diet (4%lard+1%cholesterol). Japanese white rabbits were randomly divided into5groups:normal control group, DN model group, LBP prevention group, Micardis group (positive control group) and LBP treatment group(n=4for each group). LBP (10mg/kg) was given to LBP prevention group after DM model successful for12weeks and LBP treatment group after DN model successful for4weeks, Micardis (3.7mg/kg) was given to Micardis group after DN model successful for4weeks. And the same volume of balanced saline was given to normal group and DN group. At the end of the12th week, the rabbits were killed, and SOD, MDA, GSH, GSH-Px and CAT of renal cortex were detected.8-OHdG was detected by ELISA. The mRNA and protein expression of NF-κB were detected by RT-PCR and Western blot respectively. Result:The activity of SOD, GSH-Px and CAT were significantly reduced (P＜0.01) meanwhile the level of8-OHdG, MDA and GSH were increased (P＜0.01). The mRNA and protein expression of NF-κB were significantly increased (p<0.01). The antioxidant capacity was higher than those of DN group (P<0.01). Meanwhile the mRNA and protein expression of NF-κB were significantly down-regulated (P＜0.01). Conclusion:LBP can improve the antioxidant capacity and inhibit NF-κB activation, down-regulate the expression of inflammatory cytokine and alleviate inflammatory reaction, finally delay the development of DN. |
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