摘要
目的：通过观察益气解毒活络中药复方对链脲佐菌素（STZ）所致早期糖尿病肾病（DN）大鼠一般状况的影响；探讨益气解毒活络中药复方抑制糖尿病模型大鼠肾组织中TGF-β1（转化生长因子）过度表达的具体机制，即研究益气解毒活络中药复方对早期DN大鼠TGF-Smads-UPP（泛素-蛋白水解酶复合体通路）信号通路的干预作用，进一步明确益气解毒活络中药复方防治早期糖尿病肾病的分子机制。
材料与方法：
1.益气解毒活络中药对早期DN大鼠的干预作用
SPF级健康雄性SD大鼠72只，适应性饲养一周，按体重随机分为6组：正常对照组（A）、模型空白组（B）、中药预防组（C）、中药低剂量治疗组（D1）、中药高剂量治疗组（D2）及阳性西药对照组。正常对照组予普通饲料喂养，其余5组予高糖高脂饲料喂养。4周后各治疗组大鼠予尾静脉注射STZ35mg/kg，正常对照组予尾静脉注射等量生理盐水。72小时后测大鼠空腹血糖，≥16.7mmol/L确定为DM模型成模。再根据大鼠血糖值高低对各治疗组进行随机分组。益气解毒活络中药复方选用中药颗粒剂溶于蒸馏水中。各治疗组每日用药量按大鼠-人体（g/kg）的等效剂量6.3倍计算；中药高剂量治疗组的用药量为中药低剂量治疗组的3倍。正常对照组、模型空白组大鼠予2毫升/日生理盐水灌胃。中药预防组在成DM模型后立即给予灌服益气解毒活络中药复方，每日1次，共计给药6周；中药低剂量治疗组、中药高剂量治疗组及阳性西药对照组均在成DM模型2周后，即成DN模型后灌服中药复方，每日1次，共计给药4周。分别在灌服中药2周末、4周末收集大鼠24小时尿液待测尿微量白蛋白；4周后行腹主动脉穿刺取血检测大鼠血糖、糖化血红蛋白、血β2微球蛋白水平；光镜下观察大鼠肾脏组织病理形态学改变。
2.益气解毒活络中药对早期DN大鼠TGF-β1基因及蛋白表达的影响
在DN模型成模4周末，留取肾组织标本，观察益气解毒活络中药对早期DN大鼠肾组织TGF-β1mRNA（RT-PCR法）及蛋白表达（Western-blot法）的影响。
3.益气解毒活络中药对早期DN大鼠TGF-Smads-UPP信号传导通路的影响
在成功复制DN模型4周末留取肾组织标本，观察益气解毒活络中药对早期DN大鼠肾组织Smad3和Smad7mRNA（RT-PCR法）及蛋白表达（Western-blot法）；Smurf1、Smurf2mRNA（RT-PCR法）及蛋白表达（Western-blot法）；肾组织26S蛋白水解复合体酶活性（ELISA法）的影响，进一步探讨其对早期DN可能的作用机制。
结果：
1.益气解毒活络中药对早期DN大鼠的干预作用
1.1各组大鼠体重的比较
在DN模型成模4周末，模型空白组大鼠体重下降明显，与正常对照组比较差异显著（P＜0.01）；经药物干预后各治疗组大鼠体重明显增加，与模型空白组比较有显著差异（P＜0.01），中药预防组效果最为明显。
1.2各组大鼠24小时尿微量白蛋白含量的比较
在DN模型成模4周末，模型空白组大鼠24小时尿微量白蛋白含量明显上升，与正常对照组比较差异显著（P＜0.01）；经药物干预治疗后各治疗组大鼠24小时尿微量白蛋白含量显著降低，与模型空白组比较差异显著（P＜0.01），中药高剂量治疗组和阳性西药对照组效果最为明显。
1.3各组大鼠空腹血糖的比较
在DN模型成模4周末，模型空白组大鼠空腹血糖显著升高，与正常对照组比较差异显著（P＜0.01）；各治疗组大鼠在药物干预治疗后空腹血糖明显降低，与模型空白组比较差异显著（P＜0.01），中药预防组效果最为明显。
1.4各组大鼠糖化血红蛋白水平的比较
在DN模型成模4周末，模型空白组大鼠糖化血红蛋白水平显著增高，与正常对照组比较差异显著（P＜0.01）；各治疗组大鼠糖化血红蛋白水平明显下降，与模型空白组比较差异显著（P＜0.01），其中中药高剂量治疗组效果最为明显。
1.5各组大鼠血β2微球蛋白含量的比较
在DN模型成模4周末，模型空白组大鼠血β2微球蛋白含量显著增高，与正常对照组比较差异显著（P＜0.01）；各治疗组大鼠经药物干预治疗后，血β2微球蛋白含量与模型空白组比较均显著降低（P＜0.01），其中阳性西药对照组效果最为明显。
1.6肾脏病理形态学观察
光镜下正常对照组肾小球结构完整，未见细胞增生，毛细血管基底膜（GBM）、系膜区、细胞外基质（ECM）未见异常改变；模型空白组可见肾小球肥大，GBM增厚，系膜细胞增生、系膜区增宽，部分肾小管上皮空泡变性，肾小管萎缩；各治疗组经药物干预治疗后上述病理改变均有不同程度的改善，与模型空白组比较显著减轻。
2．益气解毒活络中药对早期DN大鼠TGF-β1基因及蛋白表达的影响
正常对照组大鼠4周末肾皮质TGF-β1mRNA仅微弱表达，模型空白组TGF-β1mRNA表达较正常对照组有明显提高（P＜0.01）。与模型空白组比较，各治疗组TGF-β1mRNA表达显著下调（P＜0.05），中药预防组下调最为显著。
检测到正常大鼠肾组织匀浆可见少量TGF-β1蛋白表达，与正常对照组比较，模型空白组大鼠TGF-β1蛋白表达提高显著（P＜0.01）。与模型空白组比较，各治疗组TGF-β1蛋白表达水平下调明显（P＜0.01），中药预防组作用最为明显。
3．益气解毒活络中药对早期DN大鼠TGF-Smads-UPP（泛素-蛋白水解酶复合体通路）信号传导通路的影响
3.1对肾组织Smad3、Smad7mRNA及蛋白表达的影响
正常对照组大鼠4周末肾皮质Smad3mRNA仅微弱表达，模型空白组Smad3mRNA表达较正常对照组有明显提高（P＜0.01）。与模型空白组比较，各治疗组Smad3mRNA表达显著下调（P＜0.01）。中药预防组、中药低剂量治疗组的Smad3mRNA表达下调最为明显。
正常对照组4周末大鼠肾皮质Smad7mRNA表达显著，模型空白组Smad7mRNA表达较正常对照组有明显降低（P＜0.01）。与模型空白组比较，中药高剂量治疗组、中药低剂量治疗组表达水平均明显上调（P＜0.05），与阳性西药对照组比较无显著差异。治疗组间，中药预防组的Smad7mRNA表达最增强。
模型空白组大鼠肾组织中的Smad3蛋白表达与正常对照组比较明显增强，差异显著（P＜0.01）；模型空白组大鼠肾组织中Smad7蛋白表达水平与正常对照组比较明显下降，差异显著（P＜0.01）；与模型空白组相比较，各治疗组可明显下调肾组织中Smad3蛋白的表达水平（P＜0.01）；上调肾组织中Smad7蛋白的表达水平，其中中药预防组效果最为明显（P＜0.01）。
3.2对肾组织UPP特异信号蛋白Smurf1、Smurf2mRNA及蛋白表达的影响
正常对照组大鼠4周末肾皮质Smurf1、Smurf2mRNA仅微弱表达，模型空白组Smurf1、Smurf2mRNA表达较正常对照组明显上调（P＜0.01）；中药各治疗组和阳性西药对照组与模型空白组比较Smurf1、Smurf2mRNA表达水平均显著下调（P＜0.01）；其中中药预防组的下调作用最为明显。
Western-blot结果显示，大鼠肾组织中可检测到Smurfl、Smurf2的蛋白表达；与正常对照组比较，模型空白组Smurfl、Smurf2的蛋白表达明显增强（P＜0.01）。与模型空白组比较，各治疗组用药4周后Smurfl、Smurf2的蛋白表达水平均明显下调（P＜0.01），其中中药预防组及阳性西药对照组下调幅度大于其他各用药组（P＜0.01）。
3.3对肾组织26S蛋白水解复合体酶活性的影响
4周末正常对照组大鼠肾组织26S蛋白水解复合体酶活性较高，模型空白组较正常对照组比较水平明显降低（P＜0.01）。与模型空白组比较，各治疗组26S蛋白水解复合体酶活性水平升高明显（P＜0.01），中药预防组与各治疗组比较升高最为明显（P＜0.01）。
结论：
1.益气解毒活络中药复方能改善DN大鼠的一般状况，减少尿蛋白的排泄，对早期DN大鼠的体重、24h尿微量白蛋白含量、血β2微球蛋白含量、空腹血糖水平、糖化血红蛋白水平及肾脏组织病理形态学改变均有明显改善，能够有效防治早期大鼠DN。
2.益气解毒活络中药复方防治早期DN大鼠的作用机制是通过抑制TGF-β1mRNA及蛋白表达，调节Smad3、Smad7mRNA及蛋白表达以及UPP特异信号蛋白Smurf1、Smurf2mRNA及蛋白表达，升高26S蛋白水解复合体酶活性而发挥防治效果。
3.早期糖尿病肾病病理机制TGF-β1的过度表达与TGF-Smads-UPP信号传导通路密切相关。
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 DN
72SPF 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 DN
After4weeks 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 DN
1.1The comparison of rats’ weight in each group
After4weeks 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 group
After4weeks 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 group
After4weeks 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 group
After4weeks 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 group
After4weeks 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 observation
Under 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 DN
After4weeks, 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 DN
3.1The effects on renal tissue Smad3，7mRNA and protein expressions
After4weeks, 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 expressions
After4weeks, 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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