| Currently, diabetes mellitus(DM) has become a global public health problem because of the increasing incidence. High glucose on long-term diabetes can cause a variety of individuals with chronic diabetes complications, including diabetic nephropathy(DN). DN is the most common and most serious diabetic complication. Currently, DN has become the primary cause of end-stage renal disease (ESRD) and the resulting dialysis and renal transplantation treatment costs to patients and cause great financial burden on society.The pathogenesis of DN has not yet fully aware and effective treatment of DN is lack. Now, an unified mechanism in diabetic complications is widely accepted. The relationship between oxidative stress, nitrosative stress, inflammation and DN has become a new issues in DN study field. Oxidative stress is an imbalance between the generation of reactive oxygen species (ROS) and antioxidant defense system. In DM state, high glucose, non-enzymatic glycation and lipid oxidation process can lead to ROS production increased. ROS can interfere with normal glucose metabolism, participate in glomerular hemodynamics, also kidney tissue extracellular matrix metabolism, and to participate in extracellular matrix degrading enzyme system of regulation. In addition, ROS is closely related to cell transmembrane signaling pathway, kidney inflammation and less cellular damage. A large number of animal experiments and clinical studies have shown that antioxidant treatment can effectively delay the occurrence and development of DN.Recent studies have shown, peroxynitrite (ONOO-) is the reaction products of NO and O2 , which is another important cause of body injury. In pathological conditions, ONOO- generate increased, leading to lipid peroxidation, DNA breakage, protein damage and loss of small molecule antioxidants, which caused body injury in a variety of ways. It is one of the important mechanisms of disease. Nitration of protein tyrosine residues by ONOO- can produce 3-nitrotyrosine (3-NT), which is specific markers of ONOO- content in vivo. The damage of nitrosylation stress is far greater than oxidative stress. Studies have shown that ONOO- is a stronger oxidant and nitration agent even more than the oxidation of H2O2. Currently the role of body injury by ONOO- has caused widespread concern, but there is few report is about the relationship between nitrosative stress and DN.In addition, more and more studies suggest that hemodynamic disturbances, hyperglycemia, advanced glycation end products (AGEs), renin-angiotensin, oxidative stress and other factors may induce inflammation process of DN alone or in combination. DN is considered an inflammatory disease because of metabolic disorder. Continuous renal micro-inflammatory state may be an important reason that it is difficult to control DN currently. In-depth study of these mechanisms in the pathogenesis of DN, especially research and development for effective drug to control DN have great significance, which can help to clarify the pathogenesis of DN, effectively prevent the occurrence and development of DN and thoroughly cured DN.Chinese traditional medicine has a unique advantage in prevention and treatment of DM and its complications.Tannin acid, which is recorded in the Chinese Pharmacopoeia, has a wide range of physiological activity. Particularly, it has significant anti-inflammatory antioxidant, hypoglycemic lipid-lowering and anti-tumor effects. It is rich of natural resources extraction of tannic acid preparation in China. Chinese herbal medicine for more than 70% and a variety of fruits and trees contain such tanning substance. But Few studies is about the effect of tannic acid on DN.This study used animals vivo experiment to observe the improvement effects of tannic acid on diabetic nephropathy, studied the mechanism of tannic acid how to control DN. The experiments were divided into three parts:1. Animal experiments: STZ-diabetic rats were used for diabetes model, biochemical, immunohistochemical, ELISA, molecular biology and other experimental techniques and methods were used, this study investigated the change of metabolism, renal morphology and function, oxidase activity, oxidative stress marker(8-OHdG), markers of nitrosative stress(3-NT) of diabetic rats and the expression of gene and protein of inflammatory factors in order to clarify the effect of tannic acid on the progression of DN.2. Inhibition of non-enzymatic glycation: through establishment of non-enzymatic glycation system, this study observed the effect of tannic acid on inhibition of non-enzymatic glycation.3. Cell culture experiments: high glucose and AGEs were used to stimulate the mesangial cells. In Diabetes simulation environment, this study observed the effects of tannic acid on mesangial cell proliferation, extracellular matrix secretion, the level of intracellular oxidative stress, nitrosative stress and inflammatory factor expression, studied the mechanism of tannic acid how to control DN from the cellular level.The main findings are as follows:1. Tannic acid treatment improved the general state of DM rats, reduced blood sugar levels, decreased serum creatinine and urea nitrogen, uric acid levels and urinary protein excretion in the DM rats, improved renal hypertrophy and renal lesions in DM.2. Antioxidant capacity of diabetic rats decreased, 8-OHdG and 3-NT in kidney were increased. The expression of inflammatory cytokines was increased. Tannic acid lowered MDA content in serum and renal tissue, decreased the level of renal 8-OHdG, improved GSH-Px, SOD and CAT activities in serum and renal tissue.3. The expression of ICAM-1, MCP-1, TNF-α, PAI-1 and CRP protein and ICAM-1, MCP-1 and IL-8 mRNA in the kidney were decreased by tannic acid. Tannic acid also reduced serum CRP levels in diabetic rats.4. The level of 3-NT in the kidney was reduced by tannic acid.5. The GMC proliferation and the expresion of collagen typeⅣstimulated by high glucose and AGEs were inhibited by tannic acid.6. A certain concentration of tannic acid effectively enhanced GSH-Px, SOD, CAT activity and lowered MDA content in supernatant of GMC stimulated by high glucose and AGEs.7. The expression of ICAM-1 protein in GMC stimulated by high glucose and AGEs, and the expression of MCP-1 and IL-8 mRNA in GMC stimulated by high glucose were decreased by tannic acid.8. The content of 3-NT in supernatant of GMC stimulated by high glucose and AGEs was reduced by tannic acid.9. In vitro, tannic acid inhibited non-enzymatic glycosylation of protein in a concentration dependent.The results showed that tannic acid had obvious role in anti-inflammatory, antioxidant, anti-nitrosylation, and inhibition of protein non-enzymatic glycation. Tannic acid treatment reduced the renal oxidative stress, nitrosative stress and micro-inflammation, ameliorated renal function and structure in diabetic rats.The main innovation of this study is that the mechanism of tannic acid improving diabetic kidney diseasefor was fully explored form the whole animal to cellular level for the first time. The present studies had shown that tannic acid reduced lipid peroxidation, increased the serum antioxidant enzyme activity in the serum and renal tissue of DM rats, also in supernatant of GMC stimulated by high glucose and AGEs. The GMC proliferation and expresion of collagen typeⅣstimulated by high glucose and AGEs were inhibited by tannic acid. The protein and gene expression of some micro-inflammatory in diabetic rats' kidney and glomerular mesangial cells were decreased by tannic acid. Tannic acid reduced the content of 3-NT in renal tissue of diabetic rats and supernatant of GMC stimulated by high glucose and AGEs. In addition, This study proved that tannic acid has the effect of inhibiting protein non-enzymatic glycosylation in vitro.
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