| Objective: We established diabetes mellitus animal model by STZ, andthen lavaged them using traditional Chinese medicine or Irbesartan. Throughobserved the general state of health of rats and their biochemical indicator,pathomorphology change of kidney, the expression of VEGF, we discussed therelation between VEGF and diabetic nephropathy, and looked for the possiblemechanism how traditional Chinese medicine can protect the kidney ofdiabetic rats. We want to provide the experiment foundation for its clinicalapplication.Methods: There were48male Sprague-Dauley rats with the weight of90~120g selected to this experiment. The rats were fed in ordinary ways forone week. Then they were randomly divided into2groups:10in normalgroup and others in model making group. Except the normal group, the rats ofanother group were injected STZ with55mg/kg intraperitoneally. After72hours, the rats whose blood glucose was measured larger than16.7umol/l weresuccess as DM model. Then, they were randomly divided into four groups:11in model group,10in ZY group and10in irbesartan group. One week later,the different drugs started to be lavaged to the rats according their differentgroups every day. Sixteen weeks later,24h urine of all groups was collected todetect24h urinary protein quantitative. After fasting12hour, all rats wereparalysed to measure weight and killed to obtain serum and kidney.Glycosylated hemoglobin, serum albumin and creatinine were assayed by theautomaticbiochemistry analyzer. Weighed the right kidney and calculatedhypertrophy index. Pathomorphology changes of kidney were observed withlight microscope and electron microscope. The expression of VEGF in renaltissue was measured by Immunohistochemistry, Real-Time PCR and Western Blot.Results:1The general state of ratsThe rats of normal group had agility reaction, whose eyes were bright,muscles were chubbiness, and its appetite, dejecta and urine was normal. Afterinjecting STZ, polydipsia, polyphagia, polyuria appeared obviously in modelgroup, ZY group and Irbesartan group. Their body weight decreased comparedwith normal group. Their energy was dispirited; reaction was slow and bodywas languishing. Eight weeks after injecting STZ, they presented manycomplications of diabetes mellitus. The rats of model group were the mostserious in four groups. There were seven rats removed from our experimentbecause two rats had died and others had not met the criterion in later72hours.In the process of experiment, four rats of model group, two of ZY group andtwo of Irbesartan group were died.2The body weight, right kidney weight and hypertrophy index of each groupCompared with normal group, the body weight of the rats in model group,ZY group and Irbesartan group was obviously lighter (P<0.05), there was nosignificant difference among three DN model group (P>0.05). The rightkidney weight of the rats was no significant difference among four groups(P>0.05). The hypertrophy index of the rats in model group, ZY group andIrbesartan group was significantly higher than normal group (P<0.05).Compared with model group, ZY group and Irbesartan group was significantlylower (P<0.05). There was no significant difference between ZY group andIrbesartan group (P>0.05).3Biochemical indicator of each group3.1The24hour urinary protein quantitative of each groupThe24h urinary protein quantitative of the rats in model group, ZY groupand Irbesartan group was significantly higher than that in normal group(P<0.05). Compared with model group, the quantity of ZY group and groupwas significantly lower (P<0.05). There was no statistical difference betweenZY group and Irbesartan group (P>0.05). 3.2The HbA1c of each groupCompared with normal group, the HbA1c of the rats in model group, ZYgroup and Irbesartan group was significantly higher (P<0.05), But there wasno significant difference among model group, ZY group and Irbrsartan group(P>0.05).3.3The serum albumin of each groupCompared with normal group, the serum albumin of the rats in modelgroup was significantly lower (P<0.05). But compared with normal group andmodel group, the administered groups were not statistical difference (P>0.05).There was no significant difference between ZY group and Irbesartan group(P>0.05).3.4The Cr of each groupThere was no statistical difference among four groups (P>0.05).4The pathomorphology changes of kidney4.1Light microscope observationThe normal group showed that: the structure of renal glomerulus wascomplete and the size of that was suitable. Capillary lumen was clear andsuitable. Glomerular basement membrane, mesangium and matrix did notshow abnormal change. The model group showed that: glomerularhypertrophy, glomerular basement membrane thickened, mesangial cellproliferation, mesangial matrix increased, mesangial area broadened. Thepathological changes of the administered groups were lighter than the modelgroup.4.2Electron microscope observationThe normal group showed that: the structure of glomerular basementmembrane was clear, complete and suitable. Foot process, endothelial cell andmesangial area did not show abnormal change. The model group showed that:the glomerular basement membrane was incrassate homogeneously; the footprocess and endothelial cell was extensive fusion. The administered groupsshowed the similar pathological changes, but the degree was lighter thanmodel group. 5Immunohistochemical result of the expression of VEGF in renal cortexVEGF was mainly expressed in nephric tubule of rats in four groups,especially in distal renal tubular and collecting duct. It was also expressed inproximal renal tubular but only few in glomerulus. The result ofsemi-quantitative analysis showed that: compared with normal group, VEGFof others had higher expression (P<0.05); compared with model group, theexpression of administered groups was lower (P<0.05); There was notstatistical difference between two treatment groups (P>0.05).6Real-Time PCR result of the expression of VEGFmRNA in each groupThe expression of model group, ZY group and Irbesartan group wassignificantly higher than normal group (P<0.05). The expression of ZY groupand Irbesartan group was obviously lower than model group (P<0.05). Therewas no statistical difference between two treatment groups (P>0.05).7Western-Blot result of the expression of VEGF in each groupCompared with normal group, all of model group, ZY group andIrbesartan group had higher expression (P<0.05). Compared with model group,the expression of administered groups was lower (P<0.05). There was nostatistical difference between two treatment groups (P>0.05).Conclusions:1The symptoms of early diabetic nephropathy have appeared after sixteenweeks when the rats got diabetes. At this time, the expression of VEGFincreases obviously.2Huoxue-huayu-tongluo recipe can protect kidney from diabetes damaging.3Overexpression of VEGF in renal tissues of DN Rats is inhibited byHuoxue-huayu-tongluo recipe. The effect which is similar to Irbesartan maybe one of the mechanisms to treat diabetic nephropathy. |
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