消瘀泄浊饮防治糖尿病肾病小鼠肾纤维化的作用研究

Study on Effect of Xiaoyu Xiezhuo Decoction in Preventing and Treating Renal Fibrosis in Diabetic Nephropathy Mice

关键词:  糖尿病肾病  肾纤维化  消瘀泄浊饮  DNA 甲基转移酶 1  转化生长因子 β1

Key words:  diabetic nephropathy  renal fibrosis  Xiaoyu Xiezhuo Decoction  DNMT1  TGF-β1

摘要:

目的 探讨消瘀泄浊饮防治糖尿病肾病(diabetic nephropathy,DN)小鼠肾纤维化的潜在作用机制。方法 60只db/db小鼠制备DN模型,随机分为模型组(等体积生理盐水),阳性组(16 mg·kg^-1·d^-1厄贝沙坦),消瘀泄浊饮低、中、高剂量组(7.5,15,30 g·kg^-1·d^-1消瘀泄浊饮)5组,同时选取12只同周龄db/m小鼠设为对照组(等体积生理盐水),定时灌胃给药,持续8周。检测各组小鼠空腹血糖(fasting blood glucose,FBG)浓度、尿肌酐(urine creatinine,Ucr)、尿微量白蛋白(microalbuminuria,mAlb)、血尿素氮(blood urea nitrogen,BUN)、甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)水平及肾脏指数的差异。HE、PAS、Masson染色分别检测小鼠肾脏组织病理学改变及肾纤维化程度。Western blotting检测小鼠肾组织α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、转化生长因子β1(transforming growth factor β1,TGF-β1)、DNA甲基转移酶1(DNA methylation transferase1,DNMT1)蛋白表达水平,免疫组化检测肾组织α-SMA、TGF-β1表达。结果 与对照组相比,模型组DN小鼠的血样和尿样中FBG、BUN、mAlb、TG、TC水平显著升高(P<0.01),Ucr、肾脏指数显著降低(P<0.01)。与模型组相比,一定剂量的消瘀泄浊饮干预处理,可显著降低小鼠FBG、BUN、mAlb、TG、TC水平,升高Ucr水平。肾组织HE、PAS、Masson染色结果显示除消瘀泄浊饮低剂量组外,其余给药组均可减小肾小球体积,降低基底膜厚度,减轻纤维组织增生症状和病理损伤,改善程度从高到低依次为阳性组、消瘀泄浊饮高剂量组及消瘀泄浊饮中剂量组。此外,中、高剂量的消瘀泄浊饮可显著下调DN小鼠肾组织中α-SMA、TGF-β1、DNMT1蛋白表达水平及α-SMA、TGF-β1免疫组化表达。结论 消瘀泄浊饮可抑制DNMT1及α-SMA、TGF-β1等肾纤维化相关因子表达,起到抗肾纤维化的作用。

Abstract:

OBJECTIVE To explore the potential mechanism of Xiaoyu Xiezhuo Decoction in preventing and treating renal fibrosis in diabetic nephropathy(DN) mice.METHODS The 60 db/db mice were used to prepare DN model and randomly divided into five groups including model group(equal volume saline), positive group(16 mg·kg^-1·d^-1 irbesartan), low dose, medium dose, and high dose of Xiaoyu Xiezhuo Decoction groups(7.5, 15, 30 g·kgm^-1·d^-1 Xiaoyu Xiezhuo Decoction), and 12 db/m mice of the same week age were selected as the control group(equal volume saline), the drug was administered by timed gavage for 8 weeks. The levels of fasting blood glucose(FBG), urinary creatinine(Ucr), microalbuminuria(mAlb), blood urea nitrogen(BUN), triglyceride(TG), total cholesterol(TC) and renal index of mice in each group were detected. HE, PAS and Masson staining were used to detect the pathological changes and the degree of renal fibrosis. Western blotting was used to detect the expression of α-smooth muscle actin(α-SMA), transforming growth factor β1(TGF-β1) and DNA methylation transferase1(DNMT1) in renal tissue, and immunohistochemistry was used to detect the expression of α-SMA and TGF-β1. RESULTS Compared with the control group, the levels of FBG, BUN, mAlb, TG and TC in blood and urine samples of DN mice in model group were significantly increased(P<0.01), while Ucr and renal index were significantly decreased(P<0.01). Compared with the model group, a certain dose of Xiaoyu Xiezhuo Decoction could significantly reduce the levels of FBG, BUN, mAlb, TG and TC, and increase the level of Ucr. The results of HE, PAS and Masson of kidney tissue staining showed that except for the low dose group of Xiaoyu Xiezhuo Decoction, the other treatment groups could reduce the glomerular volume, reduce the basement membrane thickness, alleviate the fibrous tissue hyperplasia symptoms and pathological damage. The improvement degree from high to low was positive group, high dose group and medium dose group of Xiaoyu Xiezhuo Decoction. In addition, medium and high dose of Xiaoyu Xiezhuo Decoction could significantly down regulate the protein expression levels of α-SMA, TGF-β1 and DNMT1 in the kidney tissues of DN mice, as well as the immunohistochemical expression of α-SMA and TGF-β1.CONCLUSION Xiaoyu Xiezhuo Decoction can inhibit the expression of DNMT1 and other renal fibrosis related factors, such as α-SMA and TGF-β1, and play an anti renal fibrosis role.