| Objective: This research establishes DM rat model by high-sugarhigh-fat feed and intraperitoneal injection of a small dose of streptozotocin(STZ), then use Xiezhuojiedu Huoxuetongluo traditional Chinese medicine,irbesartan and the two drugs combined application to intervene the model, byobserving the experimental animal’s biochemical indexes and renalpathological morphological changes and renal cortical tissue Angiopoietin-1(Ang-1)expression level, to explore the relationship between DN and Ang-1and Xiezhuojiedu Huoxuetongluo traditional Chinese medicine’ s protectiveeffect on DN rat renal and possible mechanism, in order to provideexperimental basis for clinical treatment of DN.Methods:85male Sprague-Dauley rats, weighing170±10g, adaptivebreeding one week, after determination of blood sugar and urinary proteinwere negative, randomly selected15into the normal group(ZG), only to giveordinary feed. The rest of the rats were given high-sugar high-fat feed(formula for containing18.6%protein, fat22.3%, sugar44.4%, water10%,the rest is minerals and vitamins, etc.) After12weeks gave one-time STZ35mg/kg for intraperitoneal injection, and normal group rats were injected bythe same volume of0.1mol/l citrate buffer. After72hours, measure bloodsugar, when caudal vein blood sugar≥16.7mmol/l, the model of diabetes wassuccessfully built, two rats died in the process of building, and one rat’s bloodsugar did not reach16.7mmol/l, so they were rejected. The model rats wererandomly divided into four groups: model group (MG), traditional Chinesemedicine group (ZG), western medicine group (JG), Chinese medicine andwestern medicine group (JG), Chinese medicine and western medicine group16,17in remaining groups. Once per day, the treatment group rats were given4ml per rat/d drug solution, the normal group and model group rats wereadministered equal volume of physiological saline, dosing16weeks. Animalsin normal group were in common feed, the model rats were fed by high-sugar high-fat feed, free drinking water, eating, room temperature22-25℃, relativehumidity40%-60%,12hours alternate lighting. During the dosing days of4weeks,8weeks,12weeks monitored body weight, blood glucose and urinaryprotein. At the16th week of administration, fasted for12hours, but water,specimens24-hour urine of each rat, for the quantitative determination of24hours urinary protein, weigh them,10%chloral hydrate anesthetized theanimal and specimens femoral artery blood, picked the right kidney andweighing, calculating hypertrophy index, observed kidney pathologicalmorphological changes under light microscope and electron microscope.Extracted renal cortex RNA, using real-time fluorescent quantitative PCRmethod to detect the contents of Ang-1mRNA, and usingimmunohistochemical staining to observe the expression of Ang-1at the sametime.Results:1Body weight, Kidney weight, Kidney Hypertrophy Index (kidneyweight/body weight)Compared with normal group, the rats’ weight in model group and eachtreatment group reduced significantly (P<0.05); Weight between model groupand treatment group and among each treatment group had no significantdifference (P>0.05). Compared with normal group, the kidney hypertrophyindex in model group and treatment group increased significantly (P<0.05);Comparison between model group and treatment group had no significantdifference (P>0.05). The right kidney weight in different group had nostatistical significance (P>0.05).224-hour urinary total protein (24h UTP) in different groupsCompared with normal group, the UTP of model group and treatmentgroup increased (P<0.05); Compared with model group, the UTP of thetreatment group decreased (P<0.05);Compared with the traditional Chinesemedicine group and western medicine group, the UTP of the Chinese medicineand western medicine group decreased (P<0.05);The UTP of Chinese traditional medicine group and western medicine grouphave no obvious difference (P>0.05).3Biochemical indicators in different groupsCompared with normal group, serum total protein (TP) ane albumin (ALB) in model group and treatment group were lower (P<0.05）；Comparedwith model group, TP and ALB in each treatment group were higher (P<0.05);Compared with the traditional Chinese medicine group and western medicinegroup, TP and ALB in Chinese medicine and western medicine groupincreased (P<0.05); Between the traditional Chinese medicine group andwestern medicine group there was no significant difference (P>0.05),Compared with normal group, total cholesterol (TC) and triglyceride (TG) inmodel group and treatment group were significantly increased (P<0.05);Compared with model group, in treatment group TC and TG were lower(P<0.05); Compared with the traditional Chinese medicine group and westernmedicine group, TC and TG reduced in the traditional Chinese medicine andwestern medicine group (P<0.05); Between the traditional Chinese medicinegroup and western medicine group,there was no significant difference(P>0.05). There were no statistical significance about serum creatinine andurea nitrogen among the normal group, model group and each treatment group(P>0.05).4The comparison of glycated hemoglobin (HbA1c)Compared with normal group, HbA1c in model group and treatmentgroup significantly increased (P<0.05);Compared with model group andwestern medicine group, HbA1c in Chinese medicine and western medicinegroup and Chinese medicine group were significantly lower (P<0.05); HbA1cof the western medicine group were lower than model group but withoutstatistical significance (P>0.05); HbA1c of traditional Chinese medicine andwestern medicine group were lower than Chinese medicine group but withoutstatistical significance (P>0.05).5Renal pathological morphology observation5.1Light microscopy observationThe size of rats’ renal glomerular in normal group was normal and thestructure was clear and we did not see the glomerular becoming hypertrophyor atrophy, basement basement membrane thickening and the mesangialmatrix’s distribution area was normal. The size of rats’ renal glomerular werehypertrophy in model group, basement membrane were thickening andmesangial matrix’s area widened significantly. The above pathologicalchanges in each treatment group were lighter than that in model group, and there were no significant difference between each treatment group.5.2Electron microscopy observationThe glomerular endothelial cells in normal group were not seenabnormalities, the thickness of basement membrane were uniform and thestructure were integrated. The glomerular podocyte were not seen fusion ordeficiency; The glomerular endothelial cell in model group were seenproliferation and obviously prominent, basement membrane thickening,podocyte widely fusion; Each treatment group also has different degree ofpathological changes, but were lighter than that in the model group, thedifference between groups was not obvious.6The expression of Ang-1in rat renal cortical6.1Real-Time PCR detection of renal tissue Ang-1mRNA expressionCompared with normal group, Ang-1mRNA expression amount in modelgroup and treatment group were significantly increased (P<0.05); Comparedwith model group, Ang-1mRNA expression quantity of the treatment groupwere significantly increased (P<0.05); Compared with western medicinegroup and Chinese medicine group, Chinese medicine and western medicinegroup Ang-1mRNA expression was significantly increased (P<0.05);Therewas no statistically significant difference between traditional Chinesemedicine group with western medicine group (P>0.05).6.2Immune histochemical method to detect the expression of renal tissueAng-1There was a small amount of expression of glomerular podocyte Ang-1innormal group; Compared with normal group, the expression of renal tissueAng-1in the building module increased significantly (P<0.05); highlighted inthe podocyte; Compared with model group, the Ang-1expression in treatmentgroup increased significantly (P<0.05); Comparison between each treatmentgroup, the kidney tissues express the strongest Ang-1in Chinese traditionalmedicine and western medicine group, compared with the traditional Chinesemedicine group and western medicine group with significant difference(P<0.05); There were no significant difference between the traditional Chinesemedicine group and western medicine group (P>0.05)Conclusion:1Xiezhuojiedu Huoxuetongluo traditional Chinese medicine can reduce urinary protein’s excretion, increase serum total protein and albumin, reduceblood sugar and blood fat and delay the progress of DN.2Xiezhuojiedu Huoxuetongluo traditional Chinese medicine caneffectively inhibit the proliferation of mesangial matrix, thickening ofbasement membrane, the proliferation of endothelial cells and glomerularpodocyte’s fusion, delay the renal pathological progress.3Xiezhuojiedu Huoxuetongluo traditional Chinese medicine can increasethe expression of Ang-1in the DN rat renal tissue, suggesting the party canreduce the leakage of the blood vessels, reduce urinary protein’s excretion topromote blood vessels maturation and stability to achieve the protective effectof DN rat kidney.4Xiezhuojiedu Huoxuetongluo traditional Chinese medicine andirbesartan in combination is better than simply using traditional Chinesemedicine or western medicine in reducing proteinuria, blood lipid lowering,elevated serum protein, regulating the expression of Ang-1, etc. |
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