| Purpose： Through observing the general effects of compound Chinese medicineYiqi Jiedu activating method on rats with early diabetic nephropathy (DN) causedby streptozotocin (STZ), to probe into the specific mechanism of activeinhibition on over expression of TGF-β1(transforming growth factor) of YiqiJiedu activating method in diabetic model rats’ renal tissue, namely to researchthe intervention effects of Yiqi Jiedu activating method on TGF-Smads-UPP(ubiquitin proteolytic enzyme complex pathways) signal pathway of rats withearly DN, and to further clarify the molecular mechanism of Yiqi Jiedu activatingmethod in treating early diabetic nephropathy.Material and method：1. The intervention effects of compound Chinesemedicine Yiqi Jiedu activating method on rats with early DN72SPF healthy male SD rats were adaptively fed for a week and randomlydivided into6groups by weight: normal group(A), model group (B), traditionalChinese medicine prevention group (C), low dose group of traditional Chinesemedicine(D1), high dose group of traditional Chinese medicine (D2) and westernmedicine group (E). The normal group was given common feed, and the remaining5groups were given high-sucrose and high-fat feed. After4weeks, the rats ofeach treatment group were injected STZ35mg/kg by tail vein, while those ofnormal group were injected saline of the same amount. After72hours, rats’fasting blood glucose was measured, and≥16.7mmol/L was identified as DM model.Then, according to the blood glucose level in each treatment group, the ratswere randomly divided again. The traditional Chinese medicine granules dissolvedin distilled water. In each treatment group, daily volume of medication wascomputed by6.3times of the equivalent dose of rat-human body (g/kg); the dosageof the high dose group of traditional Chinese medicine was3times of low dosegroup.2ml saline lavage was poured into rats’ stomach every day in the normalgroup and the model group. After DN model taking shape, the traditional Chinese medicine prevention group was fed traditional Chinese medicine immediately;after DN model taking shape for2weeks, low and high dose groups of traditionalChinese medicine and western medicine group were fed Chinese herbal compound,once every day for4weeks. On the2nd and4thweekends, rats’24-hour urinewas collected to observe urinary albumin; after4weeks, blood was drawn throughabdominal aortic puncture to determine blood glucose, glycosylated hemoglobin,and serum β2microglobulin; the pathological changes of rats’ kidney tissuewas observed by light microscopy.2. The effects of Yiqi Jiedu activating herbs on TGF-β1genes and proteinexpressions of rats with early DNAfter4weeks of DN model taking shape, the kidney tissue specimens weretaken to observe the effects of Yiqi Jiedu activating herbs on renal tissue TGF-β1mRNA (RT-PCR) and protein (Western-blot) expressions of rats with earlyDN.3. The effects of Yiqi Jiedu activating herbs on TGF-Smads-UPP signaltransduction pathway of rats with early DN.On the4th weekend after successfully replicating DN model, renal tissue sampleswere taken to observe the expressions of renal tissue Smad3and Smad7mRNA(RT-PCR) and protein (Western-blot), the expressions of Smurf1and Smurf2mRNA (RT-PCR) and protein (Western-blot method), and the effects on renaltissue26S proteolytic complex enzyme activity (ELISA), to further explorethe possible effective mechanisms of early DN.Results：1. Yiqi Jiedu activating method had intervention effects on rats with early DN1.1The comparison of rats’ weight in each groupAfter4weeks of DN model taking shape, the weight of rats in the model groupdecreased, which had significant difference compared with the normal group (P<0.01); after drug intervention, the weight of rats in each treatment groupremarkably increased, which had significant difference compared with the model group (P <0.01), and the traditional Chinese medicine prevention group had thebest effect.1.2The comparison of rats’24h urine micro albumin content in each groupAfter4weeks of DN model taking shape, the24h urine micro albumin contentof rats in the model group obviously increased, which had significant differencecompared with the normal group (P <0.01); after drug intervention, the24h urinemicro albumin content of rats in each treatment group significantly decreased,which had significant difference compared with the model group (P <0.01), andthe traditional Chinese medicine high dose group and western medicine group hadthe best effect.1.3The comparison of rats’ fasting glucose in each groupAfter4weeks of DN model taking shape, the fasting glucose of rats in themodel group increased, which had significant difference compared with the normalgroup (P <0.01); after drug intervention, the fasting glucose of rats in eachtreatment group significantly decreased, which had significant differencecompared with the model group (P <0.01), and the traditional Chinese medicineprevention group had the best effect.1.4The comparison of rats’ glycosylated hemoglobin level in each groupAfter4weeks of DN model taking shape, the glycosylated hemoglobin levelof rats in the model group increased, which had significant difference comparedwith the normal group (P <0.01); after drug intervention, the glycosylatedhemoglobin level of rats in each treatment group obviously decreased, which hadsignificant difference compared with the model group (P <0.01), and thetraditional Chinese medicine high dose group had the best effect.1.5The comparison of rats’ serumβ2micro globulin content in each groupAfter4weeks of DN model taking shape, the serumβ2micro globulin contentof rats in the model group obviously increased, with significant differencecompared with the normal group (P <0.01); after drug intervention, comparedwith the model group, the serumβ2micro globulin content of rats in eachtreatment group significantly decreased (P <0.01), and the western medicine group had the best effect.1.6renal pathomorphologic observationUnder the light microscope, glomerular structure of the normal group wasintact, with no cell proliferation and no abnormal changes in glomerular basementmembrane (GBM), mesangial area, and extracellular matrix (ECM); it was shownin the model group that glomeruli hypertrophied, GBM thickened, mesangial cellsproliferated, mesangial area widened, portions of the tubular epithelialvacuolation degenerated, and renal tubular withered; through pharmacologicalintervention, the lesions in each treatment group improved to different degreesand significantly reduced compared with the model group.2. The effects of Yiqi Jiedu activating herbs on the TGFβ1genes and proteinexpressions of rats with early DNAfter4weeks, the renal cortex TGFβ1mRNA of rats in the normal group onlyweakly expressed compared with normal group, and the mRNA expression of modelgroup significantly improved (P <0.01). Compared with the model group, the mRNAexpression of each treatment group obviously decreased (P <0.05), among whichtraditional Chinese medicine prevention group was the most significant.It was detected that normal rats’ kidney tissue homogenate had a bit ofTGFβ1expression, and compared with the normal group, the protein expressionof rats in the model group increased significantly (P <0.01). Compared withthe model group, the protein expression levels of each treatment group obviouslydecreased (P <0.01), among which traditional Chinese medicine prevention groupwas the most evident.3. The effects of Yiqi Jiedu activating herbs on TGF-Smads-UPP (ubiquitinproteolytic enzyme complex pathways) signal transduction pathway of rats withearly DN3.1The effects on renal tissue Smad3，7mRNA and protein expressionsAfter4weeks, the renal cortical Smad3mRNA of rats in the normal grouponly showed weak expression, and compared with the normal group, the mRNAexpression of the model group improved remarkably (P <0.01). Compared with the model group, the mRNA expression of each treatment group significantly decreased(P <0.01), among which the Chinese medicine prevention group and the low dosegroup of traditional Chinese medicine were the most obvious.After4weeks, rats’ renal cortical Smad7expression in the normal groupwas remarkable, and compared with the normal group, the mRNA expression of themodel group significantly decreased (P <0.01). The expression of high and lowdose groups of Chinese medicine remarkably increased (P <0.05) compared withthe model group, and there was no significant difference compared with thewestern medicine group. Among these treatment groups, the mRNA expression ofChinese medicine prevention group increased the most.Compared with the normal group, the Smad3protein expression of rats’ kidneytissue in the model group remarkably enhanced with significant difference (P<0.01); the Smad7protein expression obviously decreased with significantdifference (P <0.01) compared with the normal group; the treatment group candown-regulate Smad3protein expression level in renal tissue (P <0.01) comparedwith the model group; the Chinese medicine prevention group had the most notableeffects on up-regulating Smad7protein expression level in renal tissue (P <0.01).3.2The effects on renal tissue UPP specific signal proteins Smurf1, Smurf2mRNAand protein expressionsAfter4weeks, renal cortical Smurf1, Smurf2normal of the rats in the normalgroups only expressed weakly, and compared with the normal group, the expressionof the model group significantly increased (P <0.01); compared with the modelgroup, traditional Chinese medicine groups and western medicine group obviouslydecreased (P <0.01); Chinese medicine prevention group was the most remarkable.Western-blot results showed that Smurfl and Smurf2protein expression canbe detected in rats’ renal tissue; compared with the normal group, proteinexpression of the model group obviously increased (P <0.01). After4weeks,compared with the model group, the protein expression level of each treatmentgroup significantly decreased (P <0.01), among which the reducing degree of traditional Chinese medicine prevention group and western medicine group wasgreater than other medication groups (P <0.01).3.3The effects on26S proteolytic complex enzyme activity of renal tissueAfter4weeks, renal tissue26S proteolytic complex enzyme activity of the ratsin the normal group was higher than that of the model group (P <0.01). Comparedwith the model group, other treatment groups increased significantly (P <0.01),while Chinese medicine prevention group decreased more obviously than othertreatment groups (P <0.01).Conclusion：1. Yiqi Jiedu compound Chinese medicine can effectively prevent rats’ earlyDN by reducing urinary protein excretion and improving DN rats’ generalcondition, such as the weight of rats with early DN,24h urine micro albumincontent, serum β2microglobulin content, fasting blood glucose, glycosylatedhemoglobin level and pathological changes of renal tissue.2. Yiqi Jiedu compound Chinese medicine can prevent rats’ early DN throughinhibiting the expressions of TGF-β1mRNA and protein, regulating Smad3,7mRNAand protein expressions, and UPP specific signaling proteins, Smurf1,Smurf2mRNA and protein expressions, increasing26S protein hydrolysate complexenzyme.3. The over expression of TGF-β1in the pathological mechanism of early diabeticnephropathy is closely related to the TGF-Smads-UPP signal transduction pathway. |
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