Experimental Study On The Effect Of Yiqi Jiedu Active Recipe On Pathological Mechanism Of "Poisoning Renal Collateral" In Early Diabetic Nephropathy Rats

Purpose：1. To observe the influence of Yiqi Jiedu Huoluo Formula on pharmacodynamicindexes of DN rats，including fasting blood glucose, glycated hemoglobin,creatinine, blood urea nitrogen, serum β2-microglobulin,24h urinary albuminand renal pathology，in order to discuss the effect of this formula.2. To observe the influence of Yiqi Jiedu Huoluo Formula on VEGF, Ang-2, Tie2and PAI-1in serum and kidney tissue of DN rats in order to discuss its mechanism.Material and method：90Wistar SPF rats，weighted (280±20) g，were randomly divided into normaland model groups by random number method. The normal group was fed common dietand the other groups were given high fat and high glucose diet combined withinjection of STZ（42mg/kg.bw）in the abdomen to establish the DM rats. Aftersuccessful modeling of DM，according to the blood glucose level(more than16.7mmol/L)，the model rats were randomly divided into6groups：normal group，model group，Western medicine group，TCM prevention group，low and high doseof TCM formula groups. The TCM prevention group was given medicine immediatelyafter modeling success.2weeks later，the microalbuminuria levels were detectedand the levels more than30-300mg/24h indicated the successful model of DN. modelgroup，Western medicine group and low and high dose of TCM formula groups weregiven the corresponding interventions，lasting for8week. During the experiment，rats drunk water freely and were not given insulin or other medicines for loweringblood glucose. In the end，the24h urine was collected to measure the urinaryalbumin levels. By abdominal aortic puncture，the fasting blood glucose,glycated hemoglobin, serum β2-microglobulin, creatinine, blood urea nitrogenin groups were detected. After killing， the kidney was fixed by4%paraformaldehyde solution. By HE and PAS staining，the pathological changes ofkidney were observed. By ELISA method，contents of VEGF，Ang-2，Tie2and PAI-1 in kidney were detected.Results：1. Influence on common condition of ratsDuring the experiment，the normal group rats were in a good condition withoutdeath. The model group rats were lack of vigor and had loss of fur and increasewater intake. There were6rats who didn’t met the diagnosis criteria andexpelled.8rats diet during the experiment.2. Influence on fasting blood glucoseCompared with normal group，the FBG in other groups was obviously increased（＞16.7mmol/L）with significant difference（P<0.01） and the FBG in the modelgroup was the highest. Compared with model group，the intervention groups’ FBGlevels were decreased with significant difference（P<0.01）. The FBG level inTCM prevention group was the lowest，which compared with that of Western medicinegroup and low and high dose of TCM formula groups had significant difference（P<0.01，P<0.05）. Compared with Western medicine group，low and high dose ofTCM formula groups had no significant difference and there was significantdifference between low and high dose of TCM formula groups（P<0.05）.3. Influence on glycated hemoglobin levelsCompared with normal group，the glycated hemoglobin levels in other groupswere obviously increased with significant difference（p＜0.01）. Compared withmodel group，the glycated hemoglobin levels in medicine intervention groups wereall decreased with significant difference（p＜0.01）. And there were nosignificant difference among the medicine intervention groups（p＞0.05）.4. Influence on24h microalbuminuriaCompared with normal group，24h microalbuminuria levels in other groupswere obviously increased with significant difference（P<0.01）and the level inmodel group was the highest. Compared with model group，the levels in medicineintervention groups were all decreased with significant difference（P<0.01，P<0.05）. Among the medicine intervention groups，the level in TCM prevention group was the lowest and had significant difference compared with that of othermedicine intervention groups（P<0.01）. Western medicine and TCM high dose grouphad no significant difference（P>0.05）. However，TCM low and high dose groupshad significant difference（P<0.05） and the level in Western medicine was lowerthan that of TCM low and high dose groups and there was no significant differencebetween TCM high and low dose groups（P>0.05）.5. Influence of β2-microglobulin levels in serumCompared with normal group， the levels in other groups were increasedsignificantly（P<0.01） and the model group’s was the highest. Compared withmodel group，the β2-MG levels in medicine intervention groups were decreasedsignficantly（P<0.01）. Among the medicine intervention groups，compared withTCM prevention group，Western medicine and TCM high dose groups had no signficance（P>0.05） and TCM low dose group had significance（P<0.05）. The level in TCMprevention group was lower than that of TCM low dose group. Compare with Westernmedicine group，TCM high dose group had no significance（P>0.05）and TCM lowdose group had signficance（P<0.05）. The β2-MG level in Western medicine groupwas lower than that of TCM low dose group. There was significance between TCMlow and high dose groups（P<0.05）and the level in TCM high dose group was lowerthan that of TCM low dose grou.6. Influence on Scr levelsThere was no significant differences among these groups（p＞0.05）.7. Influence on BUN levelsThe BUN level in normal group was the lowest and in model group was the highest.Compared with normal group，model group had significant difference（p＜0.01）and TCM low dose group had signficance（p＜0.05）. Compared with model group，BUN levels in medicine intervention groups were all decreased significantly（p＜0.01）. Among medicine intervention groups，the level in TCM preventiongroup was the lowest without significance（p＞0.05）.8. Pathological changes of ratsHE staining：under the high microscopic view，there was glomerulosclerosis， mesangial cells and mesangial matrix proliferation，vacuolar degeneration oftubular epithelial cells in model group. The pathological changes in medicineintervention groups were obviously light than those of model group. Glomerularand tubular in TCM prevention were enlarged slightly than those of normal group.And those of Western medicine group and TCM high dose group had no obviousdifference，and those were both larger than that of normal group.PAS staining：there was no positive substance in normal group and glomerular,tubular and mesangial matrix had no obvious pathological changes. In model group，there were positive substance in the center of kidney tissues and thickenedglomerularbasementmembrane and increased mesangial matrix. There was vitreousdegeneration of mini-arteries. In medicine intervention groups，the PAS stainingwas lightly positive. There was diffuse glomerulosclerosis，increased mesangialmatrix and extracellular matrix in Western medicine group. Tubular atrophy：there was slight increase of mesangial matrix in TCM prevention group andglomerular and tubular basement membrane had no pathological changes. There wasincreased mesangial matrix and mesangial cells in TCM high dose group andglomerular basement membrane as well as renal tubules had no pathological changes.There was segmental glomerulosclerosis，increased mesangial matrix and thickenedbasement membrane.9. Influence on VEGF in serum and kidney tissueCompared with normal group，the serum levels in other groups were all higherwith significant difference（P <0.01） and that of model group was the highest.Compared with the model group，VEGF levels in medicine groups were all decreasedwith significant difference与（P <0.01）. Among medicine intervention groups，the level in TCM prevention group was the lowest and in Western medicine groupwas the highest. Compared with the TCM prevention group，the levels in Westernmedicine and TCM high dose groups had significant difference（P<0.01，P <0.05）and TCM low dose group had no significant difference（P>0.05）. Compared withWestern medicine group，TCM high dose group had no significance（P>0.05）but TCM low dose group had significant differnece（P <0.01）. There was significant difference between TCM low and high dose groups（P <0.05） and thatof TCM low dose group was lower than that of Western medicine and TCM high dosegroup.Compared with normal group，VEGF levels in kidney tissues in other groupswere all higher than that of normal group significantly（P <0.01） and thatof model group was the highest. Compared with model group，VEGF levels in medicineintervention groups were all decreased significantly（P <0.01）. And there wasno significant difference among medicine intervention groups（P>0.05）.10. Influence on Ang-2in serum and kidney tissueCompared with normal group，Ang-2levels in serum in other groups were allhigher significantly（P <0.01）and that of model group was the highest. Comparedwith model group，Ang-2levels in medicine intervention groups were all decreasedsignificantly（P <0.01）. Compared with Western medicine group，Ang-2levelsin serum of TCM prevention group，TCM high and low dose groups had significantdifference（P <0.01）. TCM high and low dose groups had no significant differencecompared with TCM prevention group（P>0.05）. There was TCM high and low dosegroups had significant difference（P <0.05）and the level in TCM low dose groupwas higher than that of TCM high dose group.Compared with normal group，Ang-2levels in kidney tissue in other groupswer all higher than that of other groups significantly（P <0.01） and that ofmodel group was the highest. Compared with model group，Ang-2levels in kidneytissue in medicine intervention groups were all decreased significantly（P <0.01）. Compared with Western medicine group，the level in TCM prevention andTCM low dose groups were lower than that of Western medicine group withsignificant difference（P <0.01，P <0.05）. TCM high dose group had no significantdifference（P>0.05）. Compared with TCM prevention group，TCM high dose grouphad significant difference（P <0.05） and TCM low dose group had no difference（P>0.05）. There was significance between TCM high and low dose groups（P<0.05） and the level in TCM low dose group was lower than that of TCM highdose group. 11. Influence on Tie2in serum and kidney tissueCompared with normal group，Tie2levels in serum in other groups were allhigher significantly（P <0.01）and the model group’s level was the highest.Compared with model group，Tie2levels in serum in medicine intervention groupswere all decreased significantly（P <0.01）. Compared with Western medicinegroup，TCM high and low dose groups and TCM prevention group had no significance（P>0.05）. Compared with TCM prevention group，TCM high and low dose groupshad significance（P <0.05，P <0.01）. TCM low and high dose groups had nosignificance（P>0.05）.Compared with normal group，Tie2levels in kidney tissue in other groupswere all higher significantly(P <0.01）and the level in model group was thehighest. Compared with model group，Tie2levels in kidney tissue in medicineintervention groups were all decreased significantly（P <0.01）. Compared withWestern medicine group，TCM prevention and TCM low dose groups had significantdifference（P <0.01）and the levels in these two groups were both lower thanthat of Western medicine group. TCM high dose group had no significance（P>0.05）. Compared with TCM prevention group，TCM high and low dose groups hadno difference（P>0.05）.12. Influence on PAI-1levels in serum and kidney tissueCompared with normal group，the PAI-1levels in plasma in other groups wereall higher significantly（P <0.01）and the model group’s level was the highest.Compared with the model group，the PAI-1levels in plasma in medicine interventiongroups were all decreased significantly（P <0.01）. Compared with Westernmedicine group，TCM prevention group and TCM low dose group had significance（P <0.05，P <0.01）. Compared with TCM prevention group，TCM high and lowdose groups had no significance（P>0.05）. There was obvious significance betweenTCM high and low dose groups（P <0.01）and the level in TCM low dose group waslower than that of TCM high dose group.Compared with normal group，the PAI-1levels in kidney in other groups wereall higher significantly（P <0.01）and the model group’s level was the highest. Compared with the model group，the PAI-1levels in serum in medicine interventiongroups were all decreased significantly（P <0.01）. Compared with Westernmedicine group，TCM prevention group and TCM low dose group had significance（P <0.01）and TCM high dose group had no significance（P>0.05）. Comparedwith TCM prevention group，TCM high and low dose groups had no significance（P>0.05）. There was no significance between TCM high and low dose groups（P>0.05）.Conclusion：1. Yiqi Jiedu Huoluo Formula can reduce the levels of glucose, glycosylatedhemoglobin,24h urinary albumin, serum β2-microglobulin, urea nitrogen ofDN rats，improve the pathological changes of kidney to prevent the kidney damage.2. Yiqi Jiedu Huoluo Formula can reduce the VEGF、Ang-2and Tie2contents inserum and kidney tissue of DN rats.3. Yiqi Jiedu Huoluo Formula can reduce PAI-1content in plasma and kidney tissueof DN rats.4. One of the pathological mechanisms of early DN “Evil damaging kidneycollateral”may be related to abnormal expression of VEGF、Ang-2、Tie2andPAI-1.The protect mechanism of Yiqi Jiedu Huoluo Formula for kidney damage ofearly DN may be related to down-regulating VEGF，Ang-2，Tie2and PAI-1contents.