Study On Protective And Mechanism Of Tangshen Formula On Type 2 Diabetic Nephropathy And NAFLD

Diabetes mellitus is a metabolic disorder that results from a reduction of insulin available for normal function of many cells in the body. The effects of DM include long-term damage, dysfunction, and failure of various organs, especially the kidneys, livers, hearts, blood vessels, nerves and Skeletal muscles. Diabetic nephropathy (DN) and diabetic non-alcoholic fatty liver (NAFLD) are common progressive diabetic complications. DN is the most common cause of chronic kidney disease, which is characterized by a gradual increase of proteinuria and glomerular filtration rate, eventually leading to end-stage renal disease. DN is a serious and progressive complication of diabetes and occurs in 30%-40% of diabetic patients. Although there are many methods of treatment with diabetic nephropathy, including agents blocking the renin-angiotensin axis, etc., the prevalence of diabetic nephropathy was still significantly increased. Along with rising living standards, the prevalences of NAFLD and T2DM have increased globally. In 2003, the American Association of Clinical Endocrinologists recommended NAFLD as one of the diagnostic factors for metabolic syndrome (MS). NAFLD not only increases the risk of type 2 diabetes, glycemic control interference, but also promotes the occurrence of major chronic complications of diabetes. Insulin resistance and metabolic syndrome associated with progression of NAFLD, which can cause excessive inflow of fatty liver, oxidative stress and other symptoms. In the present study oxidative stress in diabetic NAFLD is being more and more attention.ObjectiveTraditional Chinese medicine emphasizes the whole concept of syndrome.Patients take medicine for individual treatment. Its multi-organ, multi-target, systemic treatments regulate the endocrine metabolism of DN. In previous clinical basis of our experience on the treatment of diabetic nephropathy developed traditional Chinese medicine Tangshen Formula. Tangshen Formula includes astragalus, habitat, rhubarb and other 4 herbs. We study the mechanism of action of Tangshen formula on DN rats and db/db mice, observed therapeutic effects of Tangshen and provide theory evidence for clinical application.Methods1. Rats were taken to establish the rat model of T2DM with the method of high-sucrose-fat diet in combination with low dose STZ. Rats were randomly divided into control group, model group and Tangshen Formula group. General condition, body weight, blood glucose, urinary albumin/creatinine were observed Every four weeks.The rats were sacrificed at the end of the 20th week. We examined ALT, AST, CHO, TG, LDL-C in serum, evaluated mesangial metrix and tubular injury index in kidneys. SOD, CAT, VE, NO, NOS and TG in liver tissues were determined. Collagen I、collagen IV,MCP-1,IL-1β、were detected by Immunohistochemistry, the mRNA expression of collagen I, collagen IV,MCP-1,IL-1β,LOX,ABCA1,RXR-aand RXR-β mRNA were measured by RT-PCR. The proteins of Collagen I and collagen IV were measured by Western Blot.2.9 db/m mice were taken as db/m group (n=9),17 fueling animal model db/db mice with DN were randomly divided into db/db group (n=9) and db/db+TSF group(n=8).General condition, body weight, blood glucose, urinary albumin/ creatinine of mice were observed Every four weeks. The mice were sacrificed at the end of the 12th week. We examined ALT, AST, CHO, LDL-C in serum, evaluated mesangial metrix in kidneys. Pathology of kidney tissues and liver tissues were observed under the microscope. SOD, MDA, CAT, GSH, GSH-Px, VC, CHO and HL in liver tissues were determined.Results1. Effects of Tangshen formula on type 2 DN rats induced by high-sucrose-fat diet in combination with STZ.General state and weight of model group was significantly worse than that of control group, and the difference was more significant followed by the progress of the experiment, and the difference was peaking in 20th week. The general state of rats of Tangshen group was better than that of model group. Body weight in rats of TSF group was the same as that of model group. Urine protein/creatinine of the rats of model group was signicantly increased from 4th week. Urine protein/creatinine of rats in TSF group group were signicantly lower than that of model group from 4th week. Blood glucose, cholesterol, mesangial metrix and tubular injury index of model group were higher significantly than that of control group. Cholesterol, mesangial metrix and tubular injury index of TSF group were lower than that of model group. Blood glucose in rats of TSF group was the same as that of model group. The result of immunohistochemistry shows that the expression of renal tissue of model group was higher significantly than that of control group. Collagen I, collagen IV, MCP-1, IL-1βof TSF group were lower than that of model group. Experiment of RT-PCR shows that expression of mRNA of collagen I, collagen IV,MCP-1,IL-1β,LOX of renal tissue of TSF group were lower significantly than that of model group and expression of mRNA of ABCA1,RXR-αand RXR-βwere higher significantly than that of model group. Experiment of Western Blot shows that expression of Collagen I and collagen IV of TSF group were lower than that of model group.2. Effects of Tangshen formula on diabetes NAFLD rats induced by high-sucrose-fat diet in combination with STZ.Serum ALT and AST in the model group was significantly higher than that of control group, and ALT and AST in TSF group were significantly lower than that of model group. Compared with the control group, SOD, CAT, VE in liver tissues of TSF group were significantly higher and NO, NOS were lower than that of model group. Liver weight and liver weight index in model group was significantly lower than that of normal group, TSF group increased liver weight and liver weight index than that of model group. HE staining and oil red O staining found that significantly TSF alleviated hepatic steatosis and relieved inflammation compared with the model group.3. Effects of Tangshen formula on db/db rats with type 2 DN.General state and weight of model group was significantly worse than that of control group, and the difference was more significant followed by the progress of the experiment, and the difference was peaking in 12th week. The general state of rats of Tangshen group was better than that of model group. Body weight of Tangshen group was increased from 6th week and continued until the end of the experiment.24-hour urine protein of the rats of model group was signicantly increased from 4th week. 24-hour urine protein of rats in TSF group group were signicantly lower than that of model group from 8th week. Blood glucose, cholesterol, mesangial metrix of model group were higher significantly than that of control group. Cholesterol, mesangial metrix of TSF group were lower than that of model group. Blood glucose in rats of TSF group was the same as that of model group.4. Effects of Tangshen formula on db/db mice with diabetes NAFLD.Serum ALT and AST in the db/db group was significantly higher than that of control group, and ALT and AST in db/db+TSF group were significantly lower than that of db/db group. Compared with db/m group, SOD, CAT, GSH, GSH-Px, VC in liver tissues of TSF group were significantly higher and MDA, CHO, HL were lower than that of model group. Liver weight and liver weight index in model group was significantly lower than that of normal group, TSF group increased liver weight and liver weight index than that of model group. HE staining and oil red O staining found that significantly TSF alleviated hepatic steatosis and relieved inflammation compared with the model group.Conclusion1. Tangshen formula can reduce urinary protein excretion in mice, correcting lipid metabolism, improve renal pathological injury in DN rats induced by high-sucrose-fat diet in combination with STZ and db/db mice, which has a better therapeutic effect in type 2 diabetes nephropathy.2. The mechanism of the protective effect of Tangshen formula with type 2 diabetic nephropathy rats may be associated with reduced renal tissue MCP-1 and IL-1 mRNA and protein, reduced LOX-1 mRNA, increased ABCA1, RXRα and RXRβ mRNA, reduced collagen type Ⅰ, Ⅳ collagen mRNA and proteins.3. Tangshen formula significantly improved db/db mice and rats with type 2 diabetes NAFLD, lowered cholesterol, improved liver pathological damage, reduced type 2 diabetes NAFLD disease, which may be related to ameliorated oxidative stress injury and regulated lipid metabolism.