The Effect Of Shenkangwan On The NF-κB And MCP-1 In Diabetic Nephropathy

As the life habit and the structure of diet changes,the incidence of Diabetes mellitus (DM) rapidly rising in global. Diabetic nephropathy is one of the commonest and serioust systemic microvascular complications in the DM patients. At present, the pathogenesis of DN is unclear,and also lack of effective treatment methods, therefore, effective treatment and prevention DN become an important problem of clinical medical field.The occurrence of DN is believed that is the results of varieties of comprehensive reasons. Recent research reveals the occurrence of a DN is in the basis of metabolic disorder and hemodynamic changes, inflammation mechanism is another important factor. In the study of DN, inflammatory cytokine Nuclear factor-KB (Nuclear factor-kappa B, NF-κB) and monocytes Monocyte chemotactic protein-1 (chemoattractant protein-1, MCP-1) play an important role of mediated.NF-KB is an important nuclear transcription factors, usually exists in cytoplasm in the form of an inactie, and it can be caused to activate by infection,stress,cytokines,oxygen free radicals, glucolipid metabolism disorders, insulin resistance and so on, then it can induce the transcription of many factors, such as a tumor necrosis factor (TNF-a); Interleukin system; Chemotactic factors such as MCP-1,etc.High expression inflammation factors can stimulate NF-κB, form the positive feedback of inflammatory signals, and injure the kidney. MCP-1 is a specific chemotactic factors, it can make mononuclear cells gathered from the peripheral circulation migrated to the inflammation place, then damage the kidney tissue through various mechanisms. So the studies of NF-κB and MCP-1 in DN will help us to study the pathogenesis of it and provide theoretical basis for clinical drug development and new treatments.Now the treatment of the DN in western medicine mainly aims at its symptom which include controlling the intensive blood glueose,reducing the albuminuria colltrol the hypertension,and so on.But so far neither one medicine can prevent the progression of the DN.so it is important to Prevent and treat DN by exerting superiority and characteristic of traditional chinese medicine(TCM) in treating DN and explore actively effective treatment from TCM. Shenkangwan has been used to treating DN Patients in clinic for many years and has a assured curative effect.But its molecular mechanism in treating DN is not clear and need farther research. so we carried out the experiment on animal and in vitro to explore the molecular and genic mechanism of Shenkangwan in treating DN from NF-κB and MCP-1 to provide labo-atorial evidence for its clinic application.The details were as follows:Chart I Laboratory study on the Protective effect of Shenkangwan on the kidney of the DN ratObjective:To observe the effect of Shenkangwan in treating rats of DN model and protecting their kidneys and provide the laboratorial foundation for further studying its therapeutic mechanism.Methods:Firstly,we established the DM rat models by intraperitoneal injection of streptozotocin(STZ) according to the dose of 55mg·kg-1 body weight. In the following two weeks, we detected the blood glucose, the 24 hour urine protein and the urine volume. The rats, which value of blood glucose exceeding 16.6mmol·L-1 and 24 hour urine protein exceeding 30mg/kg/24h, urine volume 1.5 times as baseline were taken for the successful DM rat models and calculated the rate of them. Then they were randomly divided into 3 groups:model control group, micardis group, Shenkangwan group. Other eight normal rats were used as normal control group. All rats were treated with corresponding drugs for 8 weeks. During and after the treatment, the general state, blood and uric glucose levels of the rats in every group were observed. After treated in the last time, all rats were put into the metabolizing cage to be measured the volume of their 24 hours urine and drinking water. Then we hocused all the rats with 10% chloral hydrate,collected the blood via ventral artery,gathered the serum by centrifuge and detected the content of the blood glucose and the glucosylated hemoglobin(GHb),the excretion of the 24 hour urine protein(Upro),the levels of serum creatinine(SCr),blood urea nitrogen(BUN),total serum cholesterol(TC),triglyceride(TG),high density lipoprotein (HDL-c),low density lipoprotein(LDL-c).And we took out the kidney of the rats, observed the size and volume of them and measured the kidney weight and relative kidney weight. Then we investigated their renal pathological changes by optical microscope with the coloration method of Hematoxylin and Eosin(HE).Results:46 rats were used to established the DM models and the rate of successful models was 78.9%(30/38).And the death rate of DM model rats was 4.67%(2/30). During the treating eight weeks,The model rats blood glucose were all over 16.7mmol·L-1,the uric glucose positive experiment were exceeded+++.And they appeared the symptom of the DM such as the quantum of drinking, eating and urinating increased. Compare with the normal rats,their following indexes including GHb,24hUpro, Scr, BUN, TC, TG, LDL-c were increased distinctly (P<0.05);and HDL-c is decreased distinctly (P<0.05).Their kidney weight and relative kidney weight increased distinctly and came forth about the 3 stages renal pathological lesions according to the stages of Mogensen. Compare with the rats in model group, Shenkangwan could ameliorate the rat general state, amelioration the above indexes markedly and mitigate the renal pathological lesion.Conclusions:Shenkangwan could treat DN rats and had a certain protective effect on the kidney of DN rats.Chart II The effect of Shenkangwan on NF-κB and MCP-1 in the kidneys of DN ratsObjective:To explore the effect of the Shenkangwan on NF-κB and MCP-1 in the kidneys of the DN rats.Methods:The kidneys used of this study were from the rats in chart I.We detected the protein expression of NF-κB and MCP-1 with the method of immune histochemistry, and observed the expression of NF-κB and MCP-1mRNA in the renal tissue of the rats in every group with the method of the quantitative real-time PCR.Results:The renal tissue expression of NF-κB and MCP-1 mRNA in the rats of DN model group were increased markedly than those in the rats of normal control group. And the expressions of NF-κB and MCP-1 protein were increased distinctly in these rats' kidneys too.Compare with those in the rats of the DN model group,the expression of NF-κB and MCP-1 in the rats of Shenkangwan and micardis group were decreased markedly.Conclusions:Shenkangwan could restrain the activation of NF-κB and MCP-1 expression in the renal tissue of DN rats and reduced the inflammatory reaction,which would conduce to staying the course of the DN. ChartⅢThe effect of Shenkangwan on NF-κB and MCP-1 in the rat mesangial cells controlled by high glucoseObjective:To explore the effect of the Shenkangwan on NF-κB and MCP-1 in the rat mesangial cells controlled by high glucose.Methods:The rat mesangial cells used of this study were bought from Wuhan University.We detected the protein expression of NF-κB and MCP-1 with the method of Enzyme-linked immunosorbent assay, and observed the expression of NF-κB and MCP-1mRNA in the rat mesangial cells in every group with the method of the quantitative real-time PCR.Results:The mesangial cells expression of NF-κB and MCP-1mRNA in the mesangial cells of high glucose model group were increased markedly than those in mesangial cells of normal control group. And the expressions of NF-κB and MCP-1 protein were increased distinctly in these mesangial cells too(P<0.05). Compare with those in the mesangial cells of the high glucose model group,the expression of NF-κB and MCP-1 in the the mesangial cells of Shenkangwan and Micardis group were decreased markedly,and the Shenkanwan group of high doszge is the best.Conclusions:Shenkangwan could restrain the activation of NF-κB and MCP-1 expression in the rat mesangial cells controlled by high glucose and reduced the inflammatory reaction,which would conduce to staying the course of the DN and delay the development of kidney diseaseConclusions of the whole paper:We carried out the experiment on animal and in vitro proved again that Shenkangwan could treat DN and had a certain protective effect on the kidney of DN. Its therapeutic mechanism was related to restraining the activation of NF-κB and MCP-1, then reducing the inflammatory reaction. And those provided the molecular and genie therapeutic mechanism and laboratorial evidence for its clinic treatment on DN.