The Study On The Effect Of Shenkang Injection In Treating Diabetic Nephropathy And It’s Mechanism

Objective:1. Consulting the literature related to the DN model and then explored the methodsto establish stable and reliable DN models in rats for further research.2. In order to explore the effect of SKI on DN and it’s possible mechanism weestablished DN rat models and DN cell model; the ultimate aim is to provide theoreticalbasis for the clinical application.3. To establish a HPLC method for simultaneous determination of Aloeemodin，Rhein，Emodin，Chrysophanol and Physcion in Shenkang Injection and then explore theeffet of Rhein and Emodin on DN cell models.Methods:Firstly, the explore of how to make DN models：1. In order to make type I DN model we used STZ by intraperitoneal injection andexplore the dosage of STZ.2. In order to make type II DN model we used high-fat diet combine with STZ30mg/Kg and explored the feed cycle of high-fat diet.3. In order to establish a feasible method to make DN model which was made by themethod of operation, we used unilateral renal resection or unilateral ligation and combinedSTZ30mg/kg.Secondly, the effect of SKI on DN and it’s mechanism： 1. Using STZ65mg/kg, intraperitoneal injection, to establish type I DN model andhigh-fat feed for4weeks and then combination with intraperitoneal injection of STZ30mg/kg to make type II DN model. The established DN model then randomly divided intoSKI high dosage group (SKI10mL/kg,2times/d, i.p.); SKI middle dosage group (SKI5mL/kg,2times/d, i.p.); SKI low dosage group (SKI2.5mL/kg,2times/d, i.p.); Insulingroup (10U/kg,2times/d, im., for type I model and4U/kg,2times/d, im., for type IImodel) and DN model group (normal saline,5mL/kg,2times/d, i.p.). Set normal groupgive saline5mL/kg,2times/d, i.p. After adminating for2,4,8weeks, collected bloodfrom eyeground vein plexus to dectect serum creatinine, blood urea nitrogen, bloodtriglycerides, glycosylated hemoglobin, low density lipoprotein cholesterol, high-densitylipoprotein cholesterol and other biochemical indicators. At the eighth week, collectedserum to detect T-SOD, CAT, NO, NOS and MDA; Weighed the left kidney andhistopathological examination.2. we made DN cell model by high glucose (25mmol/L glucose) cultivating ratmesabagial cell. And then divided them into SKI high dosage group (SKI100mg/L); SKImiddle dosage group (SKI50mg/L); SKI low dosage group (SKI25mg/L). In theexperiment we set low glocuse group (5.6mmol/L glucose) and mannitol group (5.6mmol/L glucose and19.4mmol/L mannitol). In this research we focuse on the inhibitioneffect of SKI on rat messagial cells.Thirdly, the detection of the five anthraquinones of SKI and the effect of Emodin andRhein on rat RMC：1. For the analysis of the five anthraquinones of SKI: Trichloromethane was used toabstract SKI，and HPLC was performed on Stamsil-BP C18（250mm×4.6，5μm）column and the mobile phase was consisted of methanol-0.2%Phosphoric acid bygradient elution. The flow rate was1.0mL/min， the temperature of column was30℃，and the wave length of detection was440nm.2. For the exploring of the effect of Rhein and Emodin on rat massagial cells: wemade DN cell model by high glucose (25mmol/L glucose) cultivating rat mesabagial cell.And then divided them into Rhein high dosage group (Rhein-80μmmol/L); Rhein middledosage group (Rhein-40μmmol/L); Rhein low dosage group (Rhein-20μmmol/L);Emodin high dosage group (Emodin-80μmmol/L); Emodin middle dosage group(Emodin-40μmmol/L); Emodin low dosage group (Emodin-20μmmol/L). In the experiment we set low glocuse group (5.6mmol/L glucose) and mannitol group (5.6mmol/L glucose and19.4mmol/L mannitol). In this research we focuse on the inhibitioneffect of Rhein and Emodin on rat messagial cells.Result and discussion：1. STZ65mg/kg, intraperitoneal injection,is a best method to make I type DNmodel with a high success rate and little animal injury.2. High-fat diet for4weeks and combined intraperitoneal injection of STZ30mg/kg is a suitable method to make DN model with a high success rat， more economicand has a short experiment term.3. Unilateral kidney ligation combined intraperitoneal injection of STZ30mg/kg isbetter than unilateral renal resection then ombined intraperitoneal injection of STZ30mg/kg, for it has a low animal mortality.4. SKI can significantly decrease the serum creatinine, blood urea nitrogen,triglyceride, low density lipoprotein and increase the concentration of high densitylipoprotein and it can inhibit renal hypertrophy (P＜0.05).5. SKI can notably increase the activity of T-SOD and CAT; significantly decreasethe concentration of NO, NOS and MDA in serum (P＜0.05).6. SKI can improve the kidney damage induced by high glucose, alleviateglomerular sclerosis and interstitial fibrosis.7. SKI can significantly inhibit mesangial cell proliferation, promote the mesangialcell apoptosis and affect DNA synthesis in the mesangial cell cycle.8. We established a method to detect five anthraquinones of SKI with a good linearrelationship for each component within its concentration range and a good recoveries,respectively; and reveal the inhibiting effect of Rhein and Emodin on rat massagical cellswhich may related to the cell cycle arrest at G1and induce mesangial cell apoptosis; andup-regulating the proteins of bax, caspase-3, caspase-6, caspase-8.