益气养阴消癥通络中药防治早期糖尿病肾病的实验研究
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摘要
目的：糖尿病肾病(Diabetic Nephropathy, DN)又称糖尿病肾小球硬化症,是糖尿病(Diabetes Mellitus DM)全身微血管并发症之一。据一般统计DM可由多种途径损害肾脏,并累及肾脏的所有结构,从肾小球到肾小管和肾间质,是导致终末期肾病(end stage renal disease, ESRD)的主要原因。多年来,国内外肾脏病学者都在不断探索糖尿病肾病的发病机理及治疗方法,但目前发病机制尚未十分明了,也未找到有效的治疗方法,因此寻找防止或延缓糖尿病肾病发生发展的有效途径和方法已成为世界范围的重大课题。近十几年来我国中医药界也广泛的开展了中医药防治糖尿病肾病的研究,且逐渐显示出较大的潜力和广阔的应用前景。在前期临床实践中,我们根据中医病因病机学理论,逐步认识到该病的病情虽然复杂,但在早期和中期大多以气阴两虚、瘀血阻络、癥瘕积聚为其基本病机,治疗当以益气养阴、消癥通络为主,为此我们结合多年的临床用药经验,拟定了以益气养阴、消癥通络为主的中药复方制剂,该方在临床上治疗早中期的糖尿病肾病,取得了良好的临床疗效。为了进一步证实该方的治疗作用,探讨其可能的机制,进行了本项研究。即借助糖尿病肾病动物模型,观察其对糖尿病肾病大鼠干预作用及对肾小球足细胞和裂孔膜以及肾小球系膜细胞的影响。
方法：1益气养阴消癥通络中药对糖尿病肾病大鼠干预作用
选用SPF级健康雄性SD大鼠110只,普通饲料适应性喂养一周,分别检测尿蛋白阴性后行左肾摘除术。2周后随机分为单纯肾切组10只,造模组100只。造模组大鼠一次性腹腔注射链脲佐菌素(STZ)40 mg/kg,72小时后测血糖,以三次连续测血糖≥16.7mmol/L为DM模型成模标准。造模组在DM模型成模后,随机分为模型组(Model)、厄贝沙坦组(Irbesatan)、中药低剂量组、中药中剂量组、中药高剂量组,各20只。厄贝沙坦组大鼠给予厄贝沙坦15mg·kg-1·d-1灌胃,中药低、中、高剂量组大鼠分别给予中药5.29g·kg-1·d-1、10.58g·kg-1·d-1、21.16g·kg-1·d-1灌胃,余组给予等剂量生理盐水灌胃,每日给药一次,连续给药6周。于给药第6周末,收集24h尿液,称体重,股动脉采血,处死大鼠,右肾称重,分离皮髓质,检测各组大鼠24 h尿蛋白定量,血糖、糖化血红蛋白、血脂、肌酐、尿素氮。
2益气养阴消癥通络中药对糖尿病大鼠肾脏病理形态学的影响
同上方法复制糖尿病肾病大鼠模型,于注射STZ第6周末,留取肾组织标本,行光镜及电镜超微结构检查。通过观察益气养阴消癥通络中药治疗糖尿病肾病大鼠前后的病理形态学改变情况,探讨该中药复方制剂防治DN的作用。
3益气养阴消癥通络中药对糖尿病大鼠肾小球足细胞和裂孔膜的影响
同上方法复制糖尿病肾病大鼠模型,于注射STZ第6周末,留取肾组织标本,观察益气养阴消癥通络中药对DN大鼠肾组织Nephrin、Podocin、CD2AP、WT-1的影响,进一步探讨其对糖尿病肾病可能的作用机制。
4益气养阴消癥通络中药对高糖联合AngⅡ培养的大鼠肾小球系膜细胞分泌CTGF、TGF-β1及FN、LN影响的实验研究
本研究采用体外细胞培养技术,在高糖环境下,选用AngⅡ诱导系膜细胞(MCs)增殖,给予大鼠的含药血清干预后,进行系膜细胞爬片和胞浆蛋白的提取,用Elisa方法检测培养大鼠肾小球系膜细胞上清液中TGF-β1、纤维粘连蛋白(FN)、层粘连蛋白(LN)的蛋白表达,western blot法检测CTGF的表达。从细胞水平探讨其延缓肾小球硬化的作用机制,为进一步研究其防治肾小球硬化的作用机理提供依据。
结果：
1益气养阴消癥通络中药对糖尿病肾病大鼠的干预作用
1.1各组大鼠24h尿蛋白定量的比较
与单纯肾切组比较：模型组、各治疗组UPro升高(P<0.05)；与模型组比较：厄贝沙坦、中药低、中剂量组UPro下降(P<0.05),中药高剂量组UPro无统计学意义(P>0.05),厄贝沙坦、中药低、中剂量组之间无统计学意义(P>0.05)。
1.2各组大鼠糖化血红蛋白(HbA1C)的比较
与单纯肾切组比较：模型组、各治疗组血糖、糖化血红蛋白升高(P<0.05)；与模型组比较：厄贝沙坦、中药低、中、高剂量组血糖、糖化血红蛋白无统计学意义(P>0.05)。
1.3各组大鼠血脂的比较
与单纯肾切组比较：模型组、各治疗组血清总胆固醇(TC)升高(P<0.01),模型组、中药各治疗组甘油三酯(TG)升高(P<0.05或P<0.01),厄贝沙坦治疗组TG无统计学意义(P>0.05)；与模型组比较：各治疗组TC、TG均降低(P<0.05或P<0.01)；与中药低剂量组比较：厄贝沙坦、中药中、高剂量组TC较高(P<0.01),中药中、高剂量组TG较高(P<0.01),厄贝沙坦治疗组TG无统计学意义(P>0.05)。
1.4各组大鼠肾功能的比较
与单纯肾切组比较：模型组血肌酐、尿素氮值升高(P<0.01),各治疗组血肌酐值无统计学意义(P>0.05),尿素氮值升高(P<0.01)；与模型组比较：各治疗组血肌酐值下降(P<0.05),中药低、高剂量组尿素氮值下降(P<0.05),余组尿素氮值无统计学意义(P>0.05)。
2肾脏病理形态学观察
光镜观察：单纯肾切组大鼠肾脏肾小球未见明显肥大,毛细血管基底膜无增厚,系膜细胞数量与系膜基质分布面积大小正常,肾小管间质无明显病理改变。模型组大鼠肾脏肾小球明显肥大,毛细血管基底膜增厚,系膜区增宽,肾小管上皮细胞空泡变性或颗粒变性。厄贝沙坦、中药各剂量组以上病理改变较模型组为轻,各治疗组间差别不明显。
电镜观察：单纯肾切组大鼠肾脏肾小球基底膜均匀无增厚,足细胞足突无融合,系膜基质无增生。模型组大鼠肾小球毛细血管基底膜呈均质性增厚,足细胞足突融合明显,系膜基质增多。厄贝沙坦、中药各剂量组以上病理改变较模型组为轻,各治疗组间差别不明显。
3益气养阴消癥通络中药对糖尿病大鼠肾小球足细胞和裂孔膜的影响
3.1各组大鼠肾皮质Nephrin, CD2AP, Podocin mRNA表达
3.1.1 Nephrin mRNA表达
与单纯肾切组比较：模型组和各治疗组nephrin mRNA表达明显下降(P<0.05)。与模型组比较：各治疗组nephrin mRNA表达明显上升(P<0.05)；各治疗组比较则无统计学意义(P>0.05)。
3.1.2 CD2AP mRNA表达
与单纯肾切组比较：模型组和各治疗组CD2AP mRNA表达明显下降(P<0.05)。与模型组比较：各治疗组CD2AP mRNA表达明显上升(P<0.01或P<0.05)；各治疗组比较则无统计学意义(P>0.05)。
3.1.3 Podocin mRNA表达
与单纯肾切组比较：模型组和各治疗组Podocin mRNA表达下降(P<0.05)。与模型组比较：各治疗组Podocin mRNA表达上升(P<0.01或P<0.05)；各治疗组间Podocin mRNA表达无统计学意义(P>0.05)。
3.2各组大鼠肾组织Nephrin, CD2AP, WT-1蛋白表达
3.2.1 Nephrin蛋白表达
与单纯肾切组比较：模型组、各治疗组Nephrin蛋白表达下降(P<0.05)。与模型组比较：厄贝沙坦组和中药低剂量组Nephrin蛋白表达增加(P<0.05),中药中、高剂量组Nephrin蛋白表达无统计学意义(P>0.05)。
3.2.1 CD2AP蛋白表达
与单纯肾切组比较：模型组、各治疗组CD2AP蛋白表达下降(P<0.05)。与模型组比较：厄贝沙坦组和中药低剂量组CD2AP蛋白表达增加(P<0.05),中药中、高剂量组CD2AP蛋白表达无统计学意义(P>0.05)。
3.2.3 WT-1蛋白表达
与单纯肾切组比较：模型组、各治疗组WT-1蛋白表达下降(P<0.05)。与模型组比较：厄贝沙坦组和中药高剂量组WT-1蛋白表达增加(P<0.05),中药中、低剂量组WT-1蛋白表达无统计学意义(P>0.05)。
4益气养阴消癥通络中药对高糖联合AngⅡ培养的大鼠肾小球系膜细胞和TGF-β1、CTGF表达的实验研究
4.1 TGF-β1含量测定
与低糖对照组比较,余组TGF-β1含量均明显升高(P<0.01)；与高糖组比较,高糖+AngⅡ组TGF-β1含量明显升高(P<0.01)；与高糖组+AngⅡ组比较,厄贝沙坦血清、中药血清组TGF-β1含量明显下降(P<0.01)；两药物血清组之间比较无统计学意义(P>0.05)。
4.2 CTGF蛋白表达
与低糖对照组比较,余组CTGF蛋白表达明显升高(P<0.05)；与高糖组比较,高糖+AngⅡ组CTGF蛋白表达明显升高(P<0.05)；与高糖+AngⅡ组比较,厄贝沙坦血清、中药血清组CTGF蛋白表达明显下降(P<0.01)；两药物血清组之间CTGF蛋白表达无统计学意义(P>0.05)。
4.3 FN、LN含量测定
与低糖对照组比较,余组FN、LN含量均明显升高(P<0.01)；与高糖组比较,高糖+AngⅡ组FN、LN含量明显升高(P<0.01)；与高糖组+AngⅡ组比较,厄贝沙坦血清、中药血清组FN、LN含量明显下降(P<0.01)；两药物血清组之间比较无统计学意义(P>0.05)。
结论：
1.益气养阴消癥通络中药能够改善DN大鼠一般状况,减少尿蛋白的排泄及降低血脂,显著改善肾功能。
2.益气养阴消癥通络中药可减轻肾组织病理损害,从而延缓DN的进展。
3.益气养阴消癥通络中药可保护肾小球滤过屏障外层裂孔膜和足细胞的结构与功能,延缓糖尿病肾病的发生发展。
4.益气养阴消癥通络中药可抑制系膜细胞分泌TGF-β1和CTGF,减少细胞外基质聚集,该作用途径可能是益气养阴消癥通络中药延缓DN进展,防止肾小球硬化的重要作用机制之一。
Objective:Diabetic nephropathy (DN) is also known as diabetic glome-rulosclerosis and is one of diabetic microvascular complications in the body. Accordingto General Statistics, DM can damage the kidney through a variety of ways, and all structures kidney are involved, from the glomerulus, renal tubules and blood vessels until the interstitial. DM is the main reason leading to end-stage renal disease(ESRD) and is predicted to become the major cause of ESRD in developing countries. Therefore, the effective prevention of the occurrence and development of DN is important. Over the years, kidney scholars at home and abroad have continued to explore the pathogenesis and treatment of diabetic kidney disease, but the pathogenesis remains unclear and they didn't find effective treatment.so looking for to prevent or delay the development of diabetic nephropathy an effective way to and the method has become a major issue worldwide. Over the last decade, the Chinese medicine sector in China has carried out a wide range of Chinese medicine prevention of diabetic nephropathy, and gradually show greater potential and broad prospects for application. We invented an effective traditional medicine, according to the pathogenesis which is at the early stage of DN and clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and yin, dispersing blood stasis and dredging collateral which was composed of astragali, figwort root, carapax amydae, angle worm, ect. The clinical data showed that the prescription had good effect on the early stage of DN. This study aim to observe the curative effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs and investigate the mechanism in glomerular podocytes, slit membrane and mesangial cells in DN rats.
Motheds:
1 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on the treatment in DN rats:
110 male Sprague-Dauley(SD) rats weighing 250±20 g (SPF level) were carried out left-nephrectomy after making sure the rats'urine protein negative when fed for one week. Two weeks later, dividing the rats randomly into the simple nephrectomy group and the model group which were injected intraperitonally with 40mg·kg-1 streptozotocin. Blood glucose levels were measured for three times continuously(≥16.7 mmol·L-1) on the third day after streptozotocin injection to confirm the development of diabetes. Dividing the model rats randomly into 5 groups:model group, irbesartan group, TCM low dose group, TCM middle dose group and TCM high dose group. Rats in irbesartan group were orally administered with 15 mg·kg-1·d-1 irbesartan, three TCM groups with 5.29 g·kg-1·d-1,10.58 g·kg·d-1,21.16 g·kg-1·d-1 TCM respectively, and the left groups with the same dose of Sodium Chloride everyday for six weeks. At the end of 6th week, collecting 24h urine, body-weighing, obtaining blood samples by puncturing femoral artery, killing rats, right-kidney-weighing, obtaining renal cortex, detecting 24h urine protein (U Pro), blood glucose, glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr) and urea nitrogen (BUN) level.
2 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on renal histopathology and ultrastructure in DN rats:
DM model was duplicated the same as the above. After 6 week of STZ injected, the renal tissue were obtained and observed them by light microscopy and conventional transmission electron microscopy examination. By observing morphological changes before and after treatment, to explore the role of ZY on DN.
3 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs on glomerular podocytes and slit membrane in DN rats:DM model was duplicated the same as the above. After 6 week of STZ injected, the renal tissue were obtained. Nephrin, Podocin, CD2AP and WT-1 were measured by immunohistochemical method and RT-PCR.
4 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on mesangial cells secreteCTGF、TGF-β1 and ECM in rats of high glucose jointing AngⅡ:
In this study by vitro cell culture technology of high glucose environment, to make McS proliferation induced by AngⅡ. After the rats' serum intervention, to the mounting of mesangial cells and extraction of cytoplasmic proteins, By Elisa method to detect mesangial cells, TGF-β1, fibronectin (FN), laminin (LN) of the protein and by western blotting to detect CTGF.From cell level to explore the mechanism of delayed glomerulosclerosis and provide evidence for further study the mechanism of prevention of glomerular sclerosis.
Results:
1 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on the treatment in DN rats:
1.1 The 24 hour urinary protein quantitative in each group
UPro levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. U Pro levels of irbesartan group, TCM low group and TCM middle dose group decreased significantly (P<0.05), compared with that of the model group. But there was no significance statistically between TCM high dose group and model group (P>0.05). There was no significance statistically among irbesartan group, TCM low group and TCM middle dose group(P>0.05).
1.2 Comparation HbA1C in each group
Blood glucose and HbA1C levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. There was no significance statistically between the treated groups and the model group(P>0.05).
1.3 Comparation of TC and TG in each group
TC levels of the model group and the treated groups increased(P<0.01), and TG levels of the model group and the TCM treated groups also increased (P<0.05 or P<0.01), compared with that of simple nephrectomy group. But there was no significance statistically between irbesartan group and simple nephrectomy group about TG (P>0.05). TC and TG levels of all treated groups decreased significantly(P<0.05 or P<0.01), compared with that of the model group. TC levels of irbesartan group, TCM middle group, and TCM high dose group increased(P<0.01), and TG levels of TCM middle group and TCM high dose group increased(P<0.01), compared with that of the TCM low dose group. There was no significance statistically between irbesartan group and TCM low dose group about TG (P>0.05).
1.4 Comparation Scr and BUN in each group
Scr and BUN levels of the model group and BUN levels of the treated groups increased(P<0.01), compared with that of simple nephrectomy group. There was no significance statistically between the treated groups and the simple nephrectomy group about Scr(P>0.05). Scr levels of the treated groups and BUN levels of TCM low group and TCM high dose group decreased significantly (P<0.05), compared with that of the model group. There was no significance statistically between the other treated groups and model group(P>0.05).
2 Pathomorphology observations on kidney of each group
Light microscope:In simple nephrectomy group, there were no obvious hypertrophy of renal glomerulus, thickening of GBM and the change of the MCs quantity, mesangial area and renal tubulointerstitium. In model group, there were obvious hypertrophy of renal glomerulus, thickening of GBM, expansion of mesangial matrix and vacuolar/granular degeneration of renal tubular cell. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.
Transmission electron microscopy:In simple nephrectomy group, there were no obvious thickening of GBM, confluence of podocyte foot process and expansion of mesangial matrix. In model group, there were obvious homogeneous thickening of GBM, confluence of podocyte foot process and expansion of mesangial matrix. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.
3 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs on glomerular podocytes and slit membrane in DN rats
3.1 The mRNA expression of Nephrin, CD2AP, Podocin in renal cortex
3.1.1 The mRNA expression of Nephrin
Compared with nephrectomy group, the mRNA expression of Nephrin in model group and treatment groups was significantly lower (P<0.05); compared with model group, the mRNA expression of Nephrin in treatment groups was significantly higher (P<0.05); there was no significant difference among treatment groups (P> 0.05).
3.1.2 The mRNA expression of CD2AP Compared with nephrectomy group, the mRNA expression of CD2AP in model group and treatment groups was significantly lower (P<0.05); compared with model group, the mRNA expression of CD2AP in treatment groups was significantly higher (P<0.01 or P<0.05); there was no significant difference among treatment groups (P> 0.05).
3.1.3 The mRNA expression of Podocin
Compared with nephrectomy group, the mRNA expression of Podocin in model group was significantly lower (P<0.05); there was no significant difference among other groups (P> 0.05). Compared with model group, the mRNA expression of Podocin in Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose and middle-dose groups was significantly higher (P<0.01 or P<0.05); there was no significant difference among other treatment groups (P> 0.05).
3.2 The protein expression of Nephrin, CD2AP, WT-1 in renal tissue
3.2.1 The protein expression of Nephrin
Compared with nephrectomy group, the protein expression of Podocin inmodel group was significantly lower (P<0.05). Compared with model group, the protein expression of Podocin Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).
3.2.2 The protein expression of CD2AP
Compared with nephrectomy group, the protein expression of CD2AP in model group was significantly lower (P<0.05). Compared with model group, the protein expression of CD2AP Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and ZY group (P> 0.05).
3.2.3 The protein expression of WT-1
Compared with nephrectomy group, the protein expression of WT-1 in model group was significantly lower (P<0.05). Compared with model group, the protein expression of WT-1 Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).
4 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on mesangial cells secrete CTGF、TGF-β1 in rats of high glucose jointing AngⅡ
4.1 Detection of TGF-p 1 levels
High glucose can stimulate secretion of TGF-β1, compared with the control group, there was a significant difference (P<0.01); compared with high glucose, TGF-β1 levels of high glucose+AngⅡgroup were significantly higher; irbesartan group and the Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group could reduce the secretion of TGF-β1, and compared with high glucose+AngⅡgroup, there was a significant difference (P<0.01). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).
4.2 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on the expression of MCs CTGF protein
The CTGF protein expression of the other groups increased significantly(P<0.01), compared with that of the control group. The CTGF protein expression of the high glucose+AngⅡgroup increased significantly (P<0.01), compared with that of the high glucose group. The CTGF protein expression of the irbesartan blood-serum group and TCM blood-serum group decreased significantly(P<0.01), compared with that of the high glucose+Angll group. There was no significance statistically between the two treated blood-serum groups(P>0.05).
4.3 Detection of FN and LN levels
Compared with the control group, FN and LN levels of high glucose group were significantly higher (P<0.01); compared with high glucose group, FN and LN levels of high glucose+AngⅡgroup were significantly higher; irbesartan group and the Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group could reduce the secretion of FN and LN, and compared with high glucose+AngⅡgroup, there was a significant difference (P<0.01). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P>0.05).
Conclusions:
1. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could decreased the DN rat's proteinuria,improve the lipid metabolism,decrease Scr and BUN
2. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could lessen the damage of nephridial tissue.
3. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could protect the glomerular filtration barrier hole in the outer membrane and podocyte structure and function, reduce proteinuria and delay the development of DN.
4. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could decreased CTGF、TGF-β1 of mesangial cells and reduce extracellular matrix accumulation. This result suggests that mesangial cells can innhibit Mesangial cells to secreteCTGF、TGF-β1 and reducing the accumulation of ECM may be one of the important mechanisms that the herbs delayed glomerulosclerosis.
方法：1益气养阴消癥通络中药对糖尿病肾病大鼠干预作用
选用SPF级健康雄性SD大鼠110只,普通饲料适应性喂养一周,分别检测尿蛋白阴性后行左肾摘除术。2周后随机分为单纯肾切组10只,造模组100只。造模组大鼠一次性腹腔注射链脲佐菌素(STZ)40 mg/kg,72小时后测血糖,以三次连续测血糖≥16.7mmol/L为DM模型成模标准。造模组在DM模型成模后,随机分为模型组(Model)、厄贝沙坦组(Irbesatan)、中药低剂量组、中药中剂量组、中药高剂量组,各20只。厄贝沙坦组大鼠给予厄贝沙坦15mg·kg-1·d-1灌胃,中药低、中、高剂量组大鼠分别给予中药5.29g·kg-1·d-1、10.58g·kg-1·d-1、21.16g·kg-1·d-1灌胃,余组给予等剂量生理盐水灌胃,每日给药一次,连续给药6周。于给药第6周末,收集24h尿液,称体重,股动脉采血,处死大鼠,右肾称重,分离皮髓质,检测各组大鼠24 h尿蛋白定量,血糖、糖化血红蛋白、血脂、肌酐、尿素氮。
2益气养阴消癥通络中药对糖尿病大鼠肾脏病理形态学的影响
同上方法复制糖尿病肾病大鼠模型,于注射STZ第6周末,留取肾组织标本,行光镜及电镜超微结构检查。通过观察益气养阴消癥通络中药治疗糖尿病肾病大鼠前后的病理形态学改变情况,探讨该中药复方制剂防治DN的作用。
3益气养阴消癥通络中药对糖尿病大鼠肾小球足细胞和裂孔膜的影响
同上方法复制糖尿病肾病大鼠模型,于注射STZ第6周末,留取肾组织标本,观察益气养阴消癥通络中药对DN大鼠肾组织Nephrin、Podocin、CD2AP、WT-1的影响,进一步探讨其对糖尿病肾病可能的作用机制。
4益气养阴消癥通络中药对高糖联合AngⅡ培养的大鼠肾小球系膜细胞分泌CTGF、TGF-β1及FN、LN影响的实验研究
本研究采用体外细胞培养技术,在高糖环境下,选用AngⅡ诱导系膜细胞(MCs)增殖,给予大鼠的含药血清干预后,进行系膜细胞爬片和胞浆蛋白的提取,用Elisa方法检测培养大鼠肾小球系膜细胞上清液中TGF-β1、纤维粘连蛋白(FN)、层粘连蛋白(LN)的蛋白表达,western blot法检测CTGF的表达。从细胞水平探讨其延缓肾小球硬化的作用机制,为进一步研究其防治肾小球硬化的作用机理提供依据。
结果：
1益气养阴消癥通络中药对糖尿病肾病大鼠的干预作用
1.1各组大鼠24h尿蛋白定量的比较
与单纯肾切组比较：模型组、各治疗组UPro升高(P<0.05)；与模型组比较：厄贝沙坦、中药低、中剂量组UPro下降(P<0.05),中药高剂量组UPro无统计学意义(P>0.05),厄贝沙坦、中药低、中剂量组之间无统计学意义(P>0.05)。
1.2各组大鼠糖化血红蛋白(HbA1C)的比较
与单纯肾切组比较：模型组、各治疗组血糖、糖化血红蛋白升高(P<0.05)；与模型组比较：厄贝沙坦、中药低、中、高剂量组血糖、糖化血红蛋白无统计学意义(P>0.05)。
1.3各组大鼠血脂的比较
与单纯肾切组比较：模型组、各治疗组血清总胆固醇(TC)升高(P<0.01),模型组、中药各治疗组甘油三酯(TG)升高(P<0.05或P<0.01),厄贝沙坦治疗组TG无统计学意义(P>0.05)；与模型组比较：各治疗组TC、TG均降低(P<0.05或P<0.01)；与中药低剂量组比较：厄贝沙坦、中药中、高剂量组TC较高(P<0.01),中药中、高剂量组TG较高(P<0.01),厄贝沙坦治疗组TG无统计学意义(P>0.05)。
1.4各组大鼠肾功能的比较
与单纯肾切组比较：模型组血肌酐、尿素氮值升高(P<0.01),各治疗组血肌酐值无统计学意义(P>0.05),尿素氮值升高(P<0.01)；与模型组比较：各治疗组血肌酐值下降(P<0.05),中药低、高剂量组尿素氮值下降(P<0.05),余组尿素氮值无统计学意义(P>0.05)。
2肾脏病理形态学观察
光镜观察：单纯肾切组大鼠肾脏肾小球未见明显肥大,毛细血管基底膜无增厚,系膜细胞数量与系膜基质分布面积大小正常,肾小管间质无明显病理改变。模型组大鼠肾脏肾小球明显肥大,毛细血管基底膜增厚,系膜区增宽,肾小管上皮细胞空泡变性或颗粒变性。厄贝沙坦、中药各剂量组以上病理改变较模型组为轻,各治疗组间差别不明显。
电镜观察：单纯肾切组大鼠肾脏肾小球基底膜均匀无增厚,足细胞足突无融合,系膜基质无增生。模型组大鼠肾小球毛细血管基底膜呈均质性增厚,足细胞足突融合明显,系膜基质增多。厄贝沙坦、中药各剂量组以上病理改变较模型组为轻,各治疗组间差别不明显。
3益气养阴消癥通络中药对糖尿病大鼠肾小球足细胞和裂孔膜的影响
3.1各组大鼠肾皮质Nephrin, CD2AP, Podocin mRNA表达
3.1.1 Nephrin mRNA表达
与单纯肾切组比较：模型组和各治疗组nephrin mRNA表达明显下降(P<0.05)。与模型组比较：各治疗组nephrin mRNA表达明显上升(P<0.05)；各治疗组比较则无统计学意义(P>0.05)。
3.1.2 CD2AP mRNA表达
与单纯肾切组比较：模型组和各治疗组CD2AP mRNA表达明显下降(P<0.05)。与模型组比较：各治疗组CD2AP mRNA表达明显上升(P<0.01或P<0.05)；各治疗组比较则无统计学意义(P>0.05)。
3.1.3 Podocin mRNA表达
与单纯肾切组比较：模型组和各治疗组Podocin mRNA表达下降(P<0.05)。与模型组比较：各治疗组Podocin mRNA表达上升(P<0.01或P<0.05)；各治疗组间Podocin mRNA表达无统计学意义(P>0.05)。
3.2各组大鼠肾组织Nephrin, CD2AP, WT-1蛋白表达
3.2.1 Nephrin蛋白表达
与单纯肾切组比较：模型组、各治疗组Nephrin蛋白表达下降(P<0.05)。与模型组比较：厄贝沙坦组和中药低剂量组Nephrin蛋白表达增加(P<0.05),中药中、高剂量组Nephrin蛋白表达无统计学意义(P>0.05)。
3.2.1 CD2AP蛋白表达
与单纯肾切组比较：模型组、各治疗组CD2AP蛋白表达下降(P<0.05)。与模型组比较：厄贝沙坦组和中药低剂量组CD2AP蛋白表达增加(P<0.05),中药中、高剂量组CD2AP蛋白表达无统计学意义(P>0.05)。
3.2.3 WT-1蛋白表达
与单纯肾切组比较：模型组、各治疗组WT-1蛋白表达下降(P<0.05)。与模型组比较：厄贝沙坦组和中药高剂量组WT-1蛋白表达增加(P<0.05),中药中、低剂量组WT-1蛋白表达无统计学意义(P>0.05)。
4益气养阴消癥通络中药对高糖联合AngⅡ培养的大鼠肾小球系膜细胞和TGF-β1、CTGF表达的实验研究
4.1 TGF-β1含量测定
与低糖对照组比较,余组TGF-β1含量均明显升高(P<0.01)；与高糖组比较,高糖+AngⅡ组TGF-β1含量明显升高(P<0.01)；与高糖组+AngⅡ组比较,厄贝沙坦血清、中药血清组TGF-β1含量明显下降(P<0.01)；两药物血清组之间比较无统计学意义(P>0.05)。
4.2 CTGF蛋白表达
与低糖对照组比较,余组CTGF蛋白表达明显升高(P<0.05)；与高糖组比较,高糖+AngⅡ组CTGF蛋白表达明显升高(P<0.05)；与高糖+AngⅡ组比较,厄贝沙坦血清、中药血清组CTGF蛋白表达明显下降(P<0.01)；两药物血清组之间CTGF蛋白表达无统计学意义(P>0.05)。
4.3 FN、LN含量测定
与低糖对照组比较,余组FN、LN含量均明显升高(P<0.01)；与高糖组比较,高糖+AngⅡ组FN、LN含量明显升高(P<0.01)；与高糖组+AngⅡ组比较,厄贝沙坦血清、中药血清组FN、LN含量明显下降(P<0.01)；两药物血清组之间比较无统计学意义(P>0.05)。
结论：
1.益气养阴消癥通络中药能够改善DN大鼠一般状况,减少尿蛋白的排泄及降低血脂,显著改善肾功能。
2.益气养阴消癥通络中药可减轻肾组织病理损害,从而延缓DN的进展。
3.益气养阴消癥通络中药可保护肾小球滤过屏障外层裂孔膜和足细胞的结构与功能,延缓糖尿病肾病的发生发展。
4.益气养阴消癥通络中药可抑制系膜细胞分泌TGF-β1和CTGF,减少细胞外基质聚集,该作用途径可能是益气养阴消癥通络中药延缓DN进展,防止肾小球硬化的重要作用机制之一。
Objective:Diabetic nephropathy (DN) is also known as diabetic glome-rulosclerosis and is one of diabetic microvascular complications in the body. Accordingto General Statistics, DM can damage the kidney through a variety of ways, and all structures kidney are involved, from the glomerulus, renal tubules and blood vessels until the interstitial. DM is the main reason leading to end-stage renal disease(ESRD) and is predicted to become the major cause of ESRD in developing countries. Therefore, the effective prevention of the occurrence and development of DN is important. Over the years, kidney scholars at home and abroad have continued to explore the pathogenesis and treatment of diabetic kidney disease, but the pathogenesis remains unclear and they didn't find effective treatment.so looking for to prevent or delay the development of diabetic nephropathy an effective way to and the method has become a major issue worldwide. Over the last decade, the Chinese medicine sector in China has carried out a wide range of Chinese medicine prevention of diabetic nephropathy, and gradually show greater potential and broad prospects for application. We invented an effective traditional medicine, according to the pathogenesis which is at the early stage of DN and clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and yin, dispersing blood stasis and dredging collateral which was composed of astragali, figwort root, carapax amydae, angle worm, ect. The clinical data showed that the prescription had good effect on the early stage of DN. This study aim to observe the curative effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs and investigate the mechanism in glomerular podocytes, slit membrane and mesangial cells in DN rats.
Motheds:
1 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on the treatment in DN rats:
110 male Sprague-Dauley(SD) rats weighing 250±20 g (SPF level) were carried out left-nephrectomy after making sure the rats'urine protein negative when fed for one week. Two weeks later, dividing the rats randomly into the simple nephrectomy group and the model group which were injected intraperitonally with 40mg·kg-1 streptozotocin. Blood glucose levels were measured for three times continuously(≥16.7 mmol·L-1) on the third day after streptozotocin injection to confirm the development of diabetes. Dividing the model rats randomly into 5 groups:model group, irbesartan group, TCM low dose group, TCM middle dose group and TCM high dose group. Rats in irbesartan group were orally administered with 15 mg·kg-1·d-1 irbesartan, three TCM groups with 5.29 g·kg-1·d-1,10.58 g·kg·d-1,21.16 g·kg-1·d-1 TCM respectively, and the left groups with the same dose of Sodium Chloride everyday for six weeks. At the end of 6th week, collecting 24h urine, body-weighing, obtaining blood samples by puncturing femoral artery, killing rats, right-kidney-weighing, obtaining renal cortex, detecting 24h urine protein (U Pro), blood glucose, glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr) and urea nitrogen (BUN) level.
2 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on renal histopathology and ultrastructure in DN rats:
DM model was duplicated the same as the above. After 6 week of STZ injected, the renal tissue were obtained and observed them by light microscopy and conventional transmission electron microscopy examination. By observing morphological changes before and after treatment, to explore the role of ZY on DN.
3 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs on glomerular podocytes and slit membrane in DN rats:DM model was duplicated the same as the above. After 6 week of STZ injected, the renal tissue were obtained. Nephrin, Podocin, CD2AP and WT-1 were measured by immunohistochemical method and RT-PCR.
4 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on mesangial cells secreteCTGF、TGF-β1 and ECM in rats of high glucose jointing AngⅡ:
In this study by vitro cell culture technology of high glucose environment, to make McS proliferation induced by AngⅡ. After the rats' serum intervention, to the mounting of mesangial cells and extraction of cytoplasmic proteins, By Elisa method to detect mesangial cells, TGF-β1, fibronectin (FN), laminin (LN) of the protein and by western blotting to detect CTGF.From cell level to explore the mechanism of delayed glomerulosclerosis and provide evidence for further study the mechanism of prevention of glomerular sclerosis.
Results:
1 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on the treatment in DN rats:
1.1 The 24 hour urinary protein quantitative in each group
UPro levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. U Pro levels of irbesartan group, TCM low group and TCM middle dose group decreased significantly (P<0.05), compared with that of the model group. But there was no significance statistically between TCM high dose group and model group (P>0.05). There was no significance statistically among irbesartan group, TCM low group and TCM middle dose group(P>0.05).
1.2 Comparation HbA1C in each group
Blood glucose and HbA1C levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. There was no significance statistically between the treated groups and the model group(P>0.05).
1.3 Comparation of TC and TG in each group
TC levels of the model group and the treated groups increased(P<0.01), and TG levels of the model group and the TCM treated groups also increased (P<0.05 or P<0.01), compared with that of simple nephrectomy group. But there was no significance statistically between irbesartan group and simple nephrectomy group about TG (P>0.05). TC and TG levels of all treated groups decreased significantly(P<0.05 or P<0.01), compared with that of the model group. TC levels of irbesartan group, TCM middle group, and TCM high dose group increased(P<0.01), and TG levels of TCM middle group and TCM high dose group increased(P<0.01), compared with that of the TCM low dose group. There was no significance statistically between irbesartan group and TCM low dose group about TG (P>0.05).
1.4 Comparation Scr and BUN in each group
Scr and BUN levels of the model group and BUN levels of the treated groups increased(P<0.01), compared with that of simple nephrectomy group. There was no significance statistically between the treated groups and the simple nephrectomy group about Scr(P>0.05). Scr levels of the treated groups and BUN levels of TCM low group and TCM high dose group decreased significantly (P<0.05), compared with that of the model group. There was no significance statistically between the other treated groups and model group(P>0.05).
2 Pathomorphology observations on kidney of each group
Light microscope:In simple nephrectomy group, there were no obvious hypertrophy of renal glomerulus, thickening of GBM and the change of the MCs quantity, mesangial area and renal tubulointerstitium. In model group, there were obvious hypertrophy of renal glomerulus, thickening of GBM, expansion of mesangial matrix and vacuolar/granular degeneration of renal tubular cell. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.
Transmission electron microscopy:In simple nephrectomy group, there were no obvious thickening of GBM, confluence of podocyte foot process and expansion of mesangial matrix. In model group, there were obvious homogeneous thickening of GBM, confluence of podocyte foot process and expansion of mesangial matrix. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.
3 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs on glomerular podocytes and slit membrane in DN rats
3.1 The mRNA expression of Nephrin, CD2AP, Podocin in renal cortex
3.1.1 The mRNA expression of Nephrin
Compared with nephrectomy group, the mRNA expression of Nephrin in model group and treatment groups was significantly lower (P<0.05); compared with model group, the mRNA expression of Nephrin in treatment groups was significantly higher (P<0.05); there was no significant difference among treatment groups (P> 0.05).
3.1.2 The mRNA expression of CD2AP Compared with nephrectomy group, the mRNA expression of CD2AP in model group and treatment groups was significantly lower (P<0.05); compared with model group, the mRNA expression of CD2AP in treatment groups was significantly higher (P<0.01 or P<0.05); there was no significant difference among treatment groups (P> 0.05).
3.1.3 The mRNA expression of Podocin
Compared with nephrectomy group, the mRNA expression of Podocin in model group was significantly lower (P<0.05); there was no significant difference among other groups (P> 0.05). Compared with model group, the mRNA expression of Podocin in Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose and middle-dose groups was significantly higher (P<0.01 or P<0.05); there was no significant difference among other treatment groups (P> 0.05).
3.2 The protein expression of Nephrin, CD2AP, WT-1 in renal tissue
3.2.1 The protein expression of Nephrin
Compared with nephrectomy group, the protein expression of Podocin inmodel group was significantly lower (P<0.05). Compared with model group, the protein expression of Podocin Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).
3.2.2 The protein expression of CD2AP
Compared with nephrectomy group, the protein expression of CD2AP in model group was significantly lower (P<0.05). Compared with model group, the protein expression of CD2AP Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and ZY group (P> 0.05).
3.2.3 The protein expression of WT-1
Compared with nephrectomy group, the protein expression of WT-1 in model group was significantly lower (P<0.05). Compared with model group, the protein expression of WT-1 Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).
4 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on mesangial cells secrete CTGF、TGF-β1 in rats of high glucose jointing AngⅡ
4.1 Detection of TGF-p 1 levels
High glucose can stimulate secretion of TGF-β1, compared with the control group, there was a significant difference (P<0.01); compared with high glucose, TGF-β1 levels of high glucose+AngⅡgroup were significantly higher; irbesartan group and the Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group could reduce the secretion of TGF-β1, and compared with high glucose+AngⅡgroup, there was a significant difference (P<0.01). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).
4.2 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on the expression of MCs CTGF protein
The CTGF protein expression of the other groups increased significantly(P<0.01), compared with that of the control group. The CTGF protein expression of the high glucose+AngⅡgroup increased significantly (P<0.01), compared with that of the high glucose group. The CTGF protein expression of the irbesartan blood-serum group and TCM blood-serum group decreased significantly(P<0.01), compared with that of the high glucose+Angll group. There was no significance statistically between the two treated blood-serum groups(P>0.05).
4.3 Detection of FN and LN levels
Compared with the control group, FN and LN levels of high glucose group were significantly higher (P<0.01); compared with high glucose group, FN and LN levels of high glucose+AngⅡgroup were significantly higher; irbesartan group and the Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group could reduce the secretion of FN and LN, and compared with high glucose+AngⅡgroup, there was a significant difference (P<0.01). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P>0.05).
Conclusions:
1. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could decreased the DN rat's proteinuria,improve the lipid metabolism,decrease Scr and BUN
2. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could lessen the damage of nephridial tissue.
3. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could protect the glomerular filtration barrier hole in the outer membrane and podocyte structure and function, reduce proteinuria and delay the development of DN.
4. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could decreased CTGF、TGF-β1 of mesangial cells and reduce extracellular matrix accumulation. This result suggests that mesangial cells can innhibit Mesangial cells to secreteCTGF、TGF-β1 and reducing the accumulation of ECM may be one of the important mechanisms that the herbs delayed glomerulosclerosis.
引文
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