| Objective: This experiment adopted high fat diet combination with smalldose of streptozotocin (STZ, streptozotocin) intraperitoneal injection todevelop DN model, Huayu Tongluo traditional Chinese medicine was used tointervented, by observing the general situation, the biochemical index of therats, hemorrheology, quantitative measurement of24hours urinary protein,pathological changes of kidney, TGF-beta1/Smad signaling pathways of renaltissue to study Huayu Tongluo traditional Chinese medicine role, and toexplore its possible mechanism.Method: Sixty-five healthy male Sprague-Dauley rats, weight of100g±10g, fed adaptability for one week. Randomly selected10rats as normalcontrol group (CG) gived ordinary diet, remaining55rats fed with high fatdiet. Four weeks later, randomly selected10rats from high fat group (HG), theremaining45rats were made into diabetic model. After fasting12hours,40mg/kg STZ was given by intraperitoneal injection. Random blood glucose ofrats′tail vein was measured, which≥16.7mmol/L after72hours as successfulDM model, and continued high fat diet. Then the DM model rats wererandomly divided into3groups again:12rats for model group(MG),13ratsfor Huayu Tongluo group(ZG),13rats for Irbesartan group(IG). Each givecorresponding drug lavage, once a day, for16weeks. At the end of16th week,collected24hours′urine for24-hour urinary protein quantitative determination,measured24-hour urine, wate rintake, and food intake. Rats fasting12hours,measured body weight, anesthesiaed rats, opened abdominal cavity, blood wascollected from abdominal aorta for testing index of blood; Picked right renaltissue for western blotting, Real-time PCR, immunohistochemistry.Pathomorphology changes of kidney were observed with light microscope and electron microscope.Results:1The general condition of the ratsNormal control group and high fat group rats were generally in goodcondition; Building groups of rats were significantly increased water intake,diet intake and urine output, with the model group performance was moreoutstanding. At the end of8th week, one rat in each group was eliminatedbecause its blood glucose not up to the standard. In M group,3rats died, andin I group,3rats died, in Z group2rats died.2The body weight,the right kidney hypertrophy index comparisonCompared with normal control group, model group and the two treatmentgroups significantly reduced body weight and significantly higher hypertrophyindex(P<0.05); compared with the model group, body weight increasedsignificantly in both treatment groups hypertrophy index and decreasedsignificantly (P<0.05).3Comparison of diet intake, water intake, urine outputCompared with normal control group, diet intake, water intake, urineoutput of every model group was significantly increased (P<0.05), high-fatgroup and the normal group, water intake, urine output was significantlyreduced (P<0.05); Compared with model group, the two treatment groupsdecreased urine output (P<0.05), TCM group compared with model group,significantly reduced water intake (P<0.05), irbesartan group and the modelgroup showed no significant difference in water intake (P>0.05).4The24hour urinary protein quantity in each group(24h UTP)Compared with normal control group,24hUTP of every model groupsignificantly increased (P<0.05); compared with the model group,24hUTP ofthe two treatment groups were significantly reduced (P<0.05);24hUTP ofTCM group significantly reduced compared with irbesartan group (P<0.05).5Comparison of blood biochemical parameters in each group5.1Comparison of fasting blood glucose (FBG)Compared with the normal control group, FBG of high-fat group and model groups was significantly elevated (P<0.05); and compared with high-fatgroup, FBG of every model group was significantly higher (P<0.05); FBGof every model group showed no significant difference (P>0.05).5.2Comparison of total cholesterol(TC), triglyceride(TG) and low densitylipoprotein(LDL)The level of TC, TG and LDL in model group and the two treatmentgroups was significantly higher than the normal group (P<0.05). Comparedwith normal group, the level of TC, LDL in high-fat group was significantlyhigher (P<0.05); Compared with the model group,the level of TG and LDL inthe two treatment groups decreased significantly(P<0.05), the level of inirbesartan group decreased significantly (P<0.05), comparing TC nosignificant difference (P>0.05) TCM group and model group; TCM group andirbesartan group, TC, LDL were significantly higher (P<0.05),TG decreasedsignificantly (P<0.05).5.3Comparison of rats′Scr and BUNCompared with normal control group, model group was significantlyhigher in Scr level(P<0.05); BUN model group and the treatment groupcompared to the control group was significantly higher (P<0.05), TCM groupcompared with the model group BUN decreased significantly (P<0.05),irbesartan group had no significant difference compared with the model group(P>0.05).6The comparison of rats′hemorrheologyCompared with normal control group, model group and the two treatmentgroups plasma viscosity and whole blood viscosity increased significantly(P<0.05); compared with the high-fat group, model group plasma viscosityand whole blood viscosity were significantly increased (P<0.05); comparedwith the model group, the two treatment groups plasma viscosity and wholeblood viscosity decreased significantly (P<0.05); there was no significantdifference between the two treatment groups (P>0.05).7The pathomorphologic changes of kidney7.1Light microscope observation Normal control group rats had clear and normal renal tissue structure;model group rats′glomerular hypertrophy, renal capsule was slit-like evenballoon adhesions, thickening of the basement membrane, mesangial cellproliferation, mesangial matrix accumulation; compared with the model group,two treatment groups′pathological changes were significantly improved.7.2Electron microscope observationNormal control group rats had clear and normal renal tissue structure;model group rats heterogeneity of glomerular basement membrane thickening,increased mesangial matrix, podocyte foot processes were widely fusion;compared with the model group, the pathological changes in the two treatmentgroups significantly improved.8Immunohistochemical result of TGF-β1in kidneyThe results showed that, TGF-β1was mainly expressed in glomeruli andtubular epithelial cells. Compared with the normal group, the expression wassignificantly increased in model group (P<0.05); compared with the modelgroup, the expression was significantly decreased in TCM group andirbesartan group (P<0.05).9Real-time PCR detection of renal tissue TGF-β1, Smad2, Smad7mRNAexpressionCompared with the normal group, modeling group TGF-β1mRNAexpression was significantly increased (P<0.05), the expression of the modelgroup and irbesartan group Smad2mRNA was significantly increased(P<0.05); compared with the model group, the two treatment groups ofTGF-β1, Smad2mRNA expression were significantly reduced (P<0.05). Fatgroup and each modeling group Smad7mRNA expression was significantlyreduced compared with the normal group (P<0.05); the expression of Smad7mRNA of two treatment groups was significantly increased compared with themodel group (P<0.05).10Western-blotting to detect the expression of TGF-β1in renal tissueCompared with the normal group, the expression of TGF-β1protein wassignificantly increased in model group(P<0.05); compared with the model group, TGF-β1protein of the traditional Chinese medicine group andirbesartan group was significantly decreased (P<0.05).Conclusions:1Huayu-Tongluo medicine can improve the general situation of DN rats,decrease total24-hour urine protein, improve lipid metabolism disorder anddecrease Scr and BUN level, improve blood high viscosity state, reduce therenal pathological change, show a protective effect in the DN rats kidney.2Huayu-Tongluo medicine can reduce the expression of TGF-β1, Smad2,increase the expression of Smad7, which indicate that the protective effect ofTCM on DN rats′renal tissue may interfere the signal transduction pathway ofTGF-β1/Samd. |
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