The Protective Effect Of Total Flavonoids Of Murraya Paniculata Leaves On Experimental Type Ⅱ Diabetic Nephropathy Rats

Along with social progressing and improvements of people's living condition, the incidence rate of diabetes showed an increasing trend. According to the statistics, about 90% of diabetes patients were type 2 diabetes. Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus and has drawn more and more attention due to its complicated pathogenesis.Total flavonoids of Murraya paniculata leaves (TFMP) were extracted from leaves of Murraya paniculata. The effects of TFMP on blood glucose in T2DM have been proved. Up to now, the protective effects of TFMP on experimental typeⅡdiabetic nephropathy rats have not been reported.Objective:High–sugar high-fat diet with low-dose streptozotocin (STZ) induced type 2 diabetic nephropathy rat model. Studying the protective effects of TFMP on experimental typeⅡdiabetic nephropathy by assaying the values of fasting blood glucose (FBG), lipid metabolism, oxidative stress, cytokines and morphology.Methods:Given high-sugar high-fat diet for 8 weeks, the rats were then injected with low-dose STZ (iv. 30mg/kg) to induce type 2 diabetic nephropathy rats model. Intragastriced with TFMP for 13 weeks. Body weights were recorded every week, blood were gathered from tail vein of the rats every 3 week to assay FBG levels. After 13 weeks'treatment, the rats were placed in metabolic cage and were supplied with no food but were free to drink water, 24h urine were then collected. The rats were anesthetized with 10% chloral hydrate(i.p. 30mg/kg), blood were gathered from abdominal aorta, blood serum were then tested for total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol (LDL-c), interleukin (IL) -6, serum creatinine (Scr) and blood urea nitrogen (BUN). The rats'kidney cortex parts were gathered for the detection of the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA). Collecting all rats'kidney and pancreas for the observation of morphology.Results:1. After feeding with high-fat diet for 8 weeks, then induced with low-dose STZ to form the T2DN model, compared with the normal group, model group's blood glucose values were significantly higher (P <0.01)in the 0,3,6,9,13 weeks after the injection of STZ which meant that T2DN model group has been maintaining a high blood glucose level. Compared with the model group, TFMP 70 mg / kg, 35 mg / kg dose group were significantly lowered in blood glucose levels (P <0.05 or P <0.01) at the 3rd week after firstly given the drugs, and after 13 weeks'treatment, the TFMP groups showed a significantly lowered level of blood glucose. Compared with the control group, model group witnessed a significant rise in GSP (P <0.01). Compared with the model group , in the TFMP70 mg / kg, 35 mg / kg dose group, GSP were significantly decreased (P <0.05), the results have suggested that TFMP could reduce the blood glucose levels significantly, also could reduce the glycosylation reaction .2. Compared with the normal group, model group's TG, TC, LDL-c levels were significantly highered, HDL-c levels decreased significantly (P <0.05 or P <0.01). This showed that dyslipidemia has caused renal injury when type 2 diabetic nephropathy occured. In TFMP 70mg/kg group, TG, TC, LDL-c levels were significantly lowered, HDL-c levels were significantly highered (P <0.05). Compared with model group, in TFMP 35mg / kg group's LDL-c levels were significantly lowered, HDL-c levels were significantly highered (P <0.05), TC, TG level changes were not statistically significant, but there were obviously in a declining trend, indicating that TFMP could improve the dyslipidemia in type 2 diabetic nephropathy.3. Compared with the normal group, the model group's, SOD, GSH-Px activity in renal cortex were significantly decreased (P <0.05 or P <0.01), the content of MDA increased significantly (P <0.01), suggested that oxidative stress has casued injury in the type 2 diabetic nephropathy rats. Compared with model group, in TFMP 70mg/kg dose group, SOD, GSH-Px activity was significantly increased (P<0.05, or P<0.01); compared with model group, in TFMP 35mg/kg dose group, SOD, MDA, GSH-Px changes were not statistically significant, but were obviously bettered to the good trend, indicating that TFMP could increase rats'free radical scavenging ability, and could effectively mitigate the renal oxidative damage.4. Compared with the normal group, the model group's IL-6 level was significantly increased (P<0.01); compared with the model group, TFMP 70mg/kg dose group was significantly decreased in IL-6 level(P<0.05) ;compared with model group, TFMP 35mg/kg dose group of IL-6 levels was not statistically significant, but showed a significant downward trend. Compared with model group, TFMP 70mg/kg, 35mg/kg dose of renal TGF-β1 and CTGF expression were significantly reduced (immunohistochemistry). These results showed that TFMP could delay the development of renal fibrosis by inhibiting the release of IL-6, TGF-β1, CTGF, which were the key factors of renal fibrosis.5. Compared with the control group, in model group, urine protein excretion rate (UAER) and the content of urinary protein (UAlb), BUN, SCr level were significantly increased (P <0.05 or P <0.01), and creatinine clearance rate (CCr) were decreased significantly (P <0.05). Compared with model group, TFMP 70mg/kg, 35mg/kg dose group's UAER, UAlb, BUN, SCr values were significantly reduced (P <0.05 or P <0.01), CCr was significantly higher than that of the model group(P <0.05 or P < 0.01). These results reveal that TFMP can improve the general renal dysfunction in T2DN.6. Morphology: Model group, optical microscopy observation showed that: Parts of glomerular capillary loops were pycnosis and collapsed, glomerular mesangial cells PAS positive substance excessively depositing and there were many protein casts in the kidenry tubules. Electron microscopy observation: segmental thickening of glomerular basement membrane and mesangial cells were hyperplasia. The foot process cells of glomerular were mixed together. All the changes of Model group were alleviated in TFMP group significantly.This study showed that TFMP could improve T2DN renal pathology, and could improve glucose and lipid metabolism metabolism, reduce oxidative damage, inhibit inflammatory factors, etc. The protective effects of TFMP on experimental typeⅡdiabetic nephropathy rats proved to be effective. This research could provide a theoretical and experimental basis for TFMP in the further development and application.