| Purpose：by observing the effect of Yiqi Jiedu Huoluo Chinese Decoction ondiabetic nephropathy rats induced high fat diet and injection of STZ in generalconditions; the pharmacodynamics index changes (body weight, fasting bloodglucose, glycosylated hemoglobin, serum creatinine, blood urea nitrogen, serumβ2microglobulin, urine albumin and Kidney pathological Changes); mechanismof drug action index changes (serum GLUT1and GLUT4, SGLT2, AGEs and its receptorRAGEs, TGF-β1in renal tissue) to investigate the prevention effects of YiqiJiedu Chinese Decoction on early diabetic nephropathy and its effect mechanism,further clarify the molecular mechanism of the traditional Chinese medicinecompound prevention of early diabetic nephropathy.Material and method:1. Experimental materialsTotally healthy90Wistar rats, SPF, there are half male and half female.Effect of Yiqi Jiedu Huoluo Chinese Decoction (decoction of TCM formula granule)composed of Astragalus, polygonatum, giant knotweed rhizome, HerbaLycopi,Coptis, Hirudo powder. Western medicine control drug use Benazepri1Hydroch1oride Tab1ets (Luo Tingxin).2. Modeling methods: high fat diet+STZ method.With the high fat and high sugar diet combined with intraperitoneal injectionof the small dose streptozotocin (STZ), we duplicate the diabetic model, thenaccording to the natural progression of the disease, after2weeks early diabeticnephropathy model is copied.3. Grouping methodThe rats (90, SPF, Wistar) fed for1week, in good health, according torandom weight into normal control group (group A,10rats, half male and halffemale), the rest rats as control group. To be duplicated diabetes model issuccessful, according to the blood sugar value will make module rats again randomly divided into: model control group (group B, half male and half female),western medicine group (C group, half male and half female),the traditionalChinese medicine prevention group (D group, half male and half female),Chinesemedicine high dose group (E1group, half male and half female)and traditionalChinese medicine low dose group (E2group, half male and half female).After thesecond grouping, the rats were divided into6groups.4. Methods of administrationRat dosage according to body (g/kg) and equivalent dose conversion ratioof surface area of rats (by6.3times), group D (traditional Chinese medicineprevention group) and E2group (low dose group) rats daily dosage was12.8g/kg,E1group (high dose TCM group) dosage3times for the E2group; C group (westernmedicine control group) rats daily dose is0.9mg/kg. In the DM model successfullyreproduced, the rats of group D immediate intervention of traditional Chinesemedicine, once a day by intragastric administration for a total of10weeks;C group, E1group and E2group rats were administered orally2weeks later, namelyDN model successfully reproduced, once a day, total drug8week, the A groupand B group rats were given the same amount of physiological saline, once a dayorally. During the experiment, the rats had free access to water, and do notuse any hypoglycemic agents and insulin injection.5. Observing index and measuring methodThrough the detection of the effect of index, to further investigate thecurative effect of Yiqi Jiedu Huoluo Chinese Decoction on early DN in rats andits mechanism.5.1Pharmacodynamics indexThe main observation of Yiqi Jiedu Huoluo Chinese Decoction on the earlydiabetic nephropathy rat in body weight, fasting blood glucose, glycosylatedhemoglobin, serum creatinine, blood urea nitrogen, serum β2microglobulin,urinary microalbumin and renal pathology changes (HE staining and PAS staining).5.2Mechanisms of drug action indexEnzyme linked immunosorbent assay (ELISA) detect the contents of GLUT1and GLUT4in serum of rats.The expression level of mRNA SGLT2, AGEs and its receptor RAGEs, TGF-β1by real-time fluorescent quantitative PCR method to detect the renal tissue inrats.Protein expression levels of SGLT2, AGEs and its receptor RAGEs, TGF-β1were detected by Western-blot in renal tissues of rats.Results:1. The affection on the body weight and the general condition of ratsDuring the experiment, the normal control group (A group) rats were in goodspirits, no death; a blank model group (B group) rats, Sanduo a little symptomof diabetes is obvious, mental apathetic, dull hair withered, reduced activity,3rats died; after drug intervention, symptoms of the intervention group ratswere eased, with traditional Chinese medicine prevention group (D group) ratswere the best, the western medicine group (C group), high dose TCM group (E1)and low dose group (E2) each1rats died. During the experiment, a total of6rats died.2. The affection on blood glucose of rats in the groupsExcept the normal group, the remaining rats blood glucose values weresignificantly higher (>16.7mmol/L). Compared with the normal control group,other groups were statistically significant (P <0.01), the model control groupblood sugar value is the highest; compared with the model group, after drugtreatment, the rats blood glucose values were significantly decreased (P<0.01),but did not reach normal value range, with the Chinese traditional medicineprevention group rats blood sugar decreased significantly; compared with thecontrol group of western medicine, differences of Chinese medicine high and lowdose groups were not statistically significant(P>0.05); compared with the lowdose group, high dose group of blood glucose values decreased, the differencewas statistically significant (P<0.05). Above results suggest that the experimentand controlled drugs can reduce the blood sugar of rats, in which the effect of traditional Chinese medicine prevention intervention group blood sugar isbest.3. The affection on glycosylated hemoglobin of rats in the groupsCompared with the normal control group, HbA1C content of other groups weresignificantly increased (P <0.01), the content of model group is the highest;compared with the model group, the HbA1C content of the drug intervention grouprats were lower, and the difference was statistically significant (P <0.01);the difference between the drug intervention groups was not statisticalsignificant (P>0.05). Above results suggest that Chinese traditional medicineprevent group intervention trend of glycosylated hemoglobin of rat for the best.4. The affection on24h micro albumin urine of rats in each groupCompared with the normal control group, the U-mAlb content of rats in eachgroup were significantly increased, and the model control group was the highest,the differences among groups were statistically significant (P<0.01); comparedwith the model group, the U-mAlb content of prevention group and the treatmentgroup rats were significantly decreased, and there is statistical significance(P<0.05); compared among each drug intervention group, the traditional Chinesemedicine prevention group was the lowest, the difference was significant (P<0.01),the western medicine group was lower than Chinese medicine high and low dose groups,and there was significant difference with the low dose group (P<0.05), comparedbetween the Chinese medicine high and low dose groups, the U-mAlb content ofthe high dose group was lower, but the difference was not significant (P>0.05).Above results suggest that the experiment and controlled drugs have lower groupof24h urine trace albumin in rats, which is with Chinese traditional medicineprevent group intervention effect best.5. The affection on serum β2-microglobuli of rats in each groupCompared with the normal control group, the serum β2-MG remaining contentof rats were increased, the difference was statistically significant (P <0.01),and the highest in model group; compared with the model group, after drugintervention treatment the serum β2-MG content of rats in each group decreased significantly, the difference is statistically significant (P<0.01); in the drugintervention groups, differences of control group of Western medicine, Chinesemedicine prevention group and high dose group was not significant (P>0.05),butcompared with the low dose group, the content decreased and the difference wasstatistically significant(P<0.05). This suggests that the experimental drugscompared with the western medicine have lower beta2-MG content in the serumof rats, and to Chinese traditional medicine prevent group, high dose group andcontrol group of western medicine is better than low dose group.6. The affection on serum creatinine and urea nitrogen of ratsThe difference of SCr content of each group rats was not significant, nostatistical significance (P>0.05). Compared with the normal control group, theserum urea nitrogen content of rats in other groups were increased, but therewere only significant differences in model control group and the low dose group(P<0.05), and the highest content in model group; compared with model controlgroup,the contents of each drug intervention group were significantly decreasedthere is a statistically significant difference (P <0.01), and the traditionalChinese medicine prevention group was the lowest; compared among the westernmedicine group, prevention group, Chinese medicine high dose of, and the lowdose group, there was no significant difference (P>0.05). But the best effecton decreasing serum urea nitrogen was that of the traditional Chinese medicineprevention group.7. Effects of GLUT1and GLUT4in serum of rats in each group①Compared with the normal control group, the GLUT1content of the modelgroup, traditional Chinese medicine prevention group and high dose group wasincreased, and that of the model control group was the highest, and there wassignificant difference (P<0.01), and GLUT1content of the traditional Chinesemedicine prevention group and low dose group was close to the normal group, thedifference has no statistical significance (P>0.05); compared with mode group,each drug intervention group values were decreased, and there was statisticalsignificance (P<0.01); compared with the control group of western medicine, the content of the traditional Chinese medicine prevention group and low dose groupwere lower, and the difference was significant (P<0.01),but the content of highdose group increased, but there was no significant difference (P>0.05);comparedwith the traditional Chinese medicine prevention group, content of traditionalChinese medicine high dose group is higher, and the difference was significant(P<0.01), the content of low dose group was decreased, but the difference wasnot significant (P>0.05); compared with the traditional Chinese medicine lowdose group, high dose group were increased, and the difference was significant(P<0.01).②Compared with the normal control group, the content of GLUT4of the othergroups were decreased (P<0.01), and the lowest in model group; compared withmodel control group, the content of each treatment and prevention group wereincreased (P<0.01), and the traditional Chinese medicine prevention group wasthe highest; compared with the control group of Western medicine, the contentof the Chinese medicine prevention group and low dose group increased, and thedifference was significant (P<0.01), and high dose group decreased, but thedifference was not statistically significant (P>0.05); compared with the traditionalChinese medicine prevention group, the contents of the high and low dose groupwere lower, and the difference of high dose group was significantly (P<0.01),but no statistically significant differences in the low dose group (P>0.05),significant difference between the two groups of the high and low dose (P<0.01),and the content of high dose group was higher.By the above results suggest that the experiment and controlled drugs haveintervention in serum glucose transporter protein1and4, and to the best oftraditional Chinese medicine prevention group and low dose group.8. The affection on the gene and protein expression level of SGLT2in kidneytissue of rats in each groupCompared with the normal control group, the gene and protein expression levelof SGLT2in renal tissue of other groups rats were significantly increased andthere was statistical significance (P<0.01), regulation of model group was the most obvious up; compared with the model control group, the SGLT2gene and proteinexpression level of the intervention groups was significantly down regulated,there was statistical significance (P<0.01),the level of the Chinese medicineprevention group lowered obviously; compared with the control group of westernmedicine, the expression level of protein and gene of the Chinese medicineprevention group rats was decreased, there was statisticallysignificant(P<0.05), the gene expression level of the Chinese medicine high doseand low dose group was no significant difference (P>0.05), but the expressionlevel of the high dose group was down, and there was significant difference inthe protein expression level(P<0.01), but the two groups up regulated theexpression; compared with traditional Chinese medicine prevention group, thegene and protein expression level of the TCM high dose and low dose group wassignificantly up-regulated, and there was statistical significance (P<0.01);compared with the traditional Chinese medicine low dose group, the expressionlevel of gene and protein of the high dose group were down regulated, there areno significant difference in the gene level(P>0.05), and the difference of theprotein expression level was significant (P<0.05). Above results suggest thatthe drug can reduce rat renal tissues SGLT2gene and protein expression level,of which the intervention effect of traditional Chinese medicine preventiongroup is the best.9. The affection on the gene and protein expression level of AGEs and its receptorRAGEs in renal tissue of rats in each groupCompared with the normal control group, the expression level of gene andprotein of AGES and RAGES in renal tissue of the other groups rats were significan-tly raised, and there was statistical significance (P<0.01),blank model groupthe most obviously increased; compared with the model group, AGES and RAGES geneand protein expression levels of the rats in drug intervention group were lower,and there was statistical significance (P<0.05), the down level of the Chinesemedicine prevention group was the most obvious(P<0.01);compared with the westernmedicine control group, the protein and gene expression level of the traditional Chinese medicine prevention group rats was decreased (P<0.05), the level of geneand protein expression of traditional Chinese medicine high and low dose groupswere increased, the difference of RAGES protein expression was statisticallysignificant(P<0.05); compared with the traditional Chinese medicine preven-tion group, high dose group and low dose group of gene and protein expressionlevels were significantly increased(P<0.01);compared with traditional Chinesemedicine low dose group, the gene expression level of the high dose group wassignificantly increased, but the difference was not statistically significant(P>0.05), and the protein expression levels were lower, but the difference wasstatistically significant(P<0.05). Results suggest that the drug can reduce ratswhich in the kidney tissues and RAGES gene and protein expression level, of whichthe intervention effect of traditional Chinese medicine prevention group is thebest.10. The affection on the gene and protein expression level of TGF-β1in kidneytissue of rats in each groupCompared with the normal control group, the TGF-β1mRNA and protein expressi-on level in renal tissue of the other group rats were significantly increased,and there was statistical significance (P<0.01), and the level of the blank modelgroup the most obviously increased; compared with the model group, the expressionlevel of TGF-β1gene and protein of the rats in drug intervention group weresignificantly reduced, and there was statistical significance (P<0.01),theChinese medicine prevention group the most obviously reduced (P<0.01); comparedwith the control group of western medicine, the expression level of protein andgene of the Chinese medicine prevention group rats were significantly decreased(P<0.01), there is statistical significance, the gene expression levels of theChinese medicine high dose group and low dose group were not statisticallysignificant(P>0.05), and the protein expression level increased, the differencewas statistically significant (P<0.05); compared with the traditional Chinesemedicine prevention group, the expression level of gene and protein of Chinesemedicine high dose group and low dose group were significantly up-regulated, differences was statistically significant (P<0.01); compared with the low dosegroup, the level of gene and protein expression in high dose group were downregulated,but the gene expression level was no significant difference (P>0.05),and the difference of the protein expression level was significantly (P<0.01),there was statistical significance. This result suggests that the drug can reducerat kidney tissues of TGF-beta1gene and protein expression level, and theintervention effect of traditional Chinese medicine prevention group is thebest.11. The pathologic changes in kidney tissue of the ratsHE staining: the nucleus renal tissues showed blue purple, and cytoplasm,the glomerular basement membrane and collagen fiber showed pink. To observe thechange of rats in normal control group, there was no obvious pathology of therenal tubular and lomerular esangial matrix at low magnification; the glomerularsclerosis, mesangial cells and matrix hyperplasia, vacuolar degeneration ofrenal tubular epithelial cell in kidney tissues of the model control group rats;after drug intervention, the pathological changes of the kidney in rats werelighter than the model group. Compared with the normal group, the glomeruli andtubules of traditional Chinese medicine prevention group rats increased slightly,while the glomerular volume of the western medicine group rats and high dosegroup rats have no significant difference, but were higher than that of the normalgroup rats.PAS staining: the obvious pathological changes in kidney tissues of the ratsin the normal group were no observed; there was a wide range of PAS stainingpositive substances in the central part of renal lobule of model group rats,and the glomerular basement membrane thickening, mesangial matrix increasedobviously, the glomeruli showed diffuse lesions, the renal arteriole hyalinechanges, glomerular internal occasionally nodules (tapered, rounded or oval),individual glomerular obsolescence; after drug intervention, the PAS stainingpositive substances of the rats kidney in each group were weakly positive,western medicine control group showed diffuse mesangial sclerosis, mesangial matrix and ECM obviously increased, globular atrophy; the traditional Chinesemedicine prevention group showed mild increase in mesangial matrix, the GBM andrenal tubule without disease; the kidney tissues of the traditional Chinesemedicine high dose group showed mesangial matrix and mesangial cells increased,GBM and tubular no lesion; the kidney tissues of traditional Chinese medicinelow dose group rats showed segmental glomerular sclerosis, mesangial matrixinand thickening of GBM.Conclusion:1Effect of Yiqi Jiedu Huoluo Chinese Decoction can significantly improve theearly DN rat general state and the pathological changes, and can effectivelyreduce the blood glucose, glycosylated hemoglobin, blood β2-microglobulin,urinary albumin and urea nitrogen level, can effectively delay the developmentof early diabetic nephropathy.2Effect of Yiqi Jiedu Huoluo Chinese Decoction can effectively reduce the GLUT1content and increase the GLUT4content in serum of early DN rats;3Effect of Yiqi Jiedu Huoluo Chinese Decoction can reduce the mRNA and proteinexpression level of SGLT2, AGEs and its receptor RAGEs, TGF-β1in the tissuesof early DN rats.4The pathogenesis of early diabetic nephropathy may be related to abnormalexpression of the glucose transporter protein, non enzymatic glycosylation andthe TGF-β1.5The adjustable mechanism of Yiqi Jiedu Huoluo Chinese Decoction on early DNrats probably by regulating the abnormal expression of glucose transporter,reduced glycosylation and cut the over expression of TGF-β1, which play therole of the prevention and treatment of DN, protect renal injury.6Early application of Chinese medicine compound intervention DN, its curativeeffect is remarkable.
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