不同剂量寻骨风致大鼠慢性肾间质纤维化初探

目的:观察马兜铃科中药寻骨风水煎剂引起大鼠慢性肾间质纤维化与剂量的关系并探讨其发病机制。方法:将雌性Wister大鼠随机分5组:(1)正常对照组(NC组,n=8):给予蒸馏水2 ml/d灌胃;(2)马兜铃酸对照组(AA组,n=16):给予马兜铃酸制剂5 mg.kg-1.d-1腹腔注射;(3)寻骨风高剂量组(XGFH组,n=16):给予寻骨风水煎液27 g.kg-1.d-1灌胃;(4)寻骨风中剂量组(XGFM组,n=16):给予寻骨风水煎液13.5 g.kg-1.d-1灌胃;(5)寻骨风低剂量组(XGFL组,n=16):给予寻骨风水煎液2.7 g.kg-1.d-1灌胃。各组在45 d、90 d时分别处死8只大鼠,处死前测体重、采血、留尿,做肾功能,尿潜血、MA/Cr、TF/Cr及尿NAG/Cr检查,处死后测肾重,留取肾组织行HE、PAS染色,做LN、FN、TGF-β1、MMP-2、TIMP-2免疫组化检查。结果:90 d后AA组、XGFH组、XGFM组大鼠体重和肾重较NC组均有下降(P<0.01或P<0.05),AA组、XGFH组、XGFM组MA/Cr、TF/Cr、NAG/Cr、Scr和BUN明显高于NC组(P<0.05或P<0.01)。光镜检查XGFH组用药45 d后,出现肾小管上皮细胞肿胀,管腔缩小,小管间质增生;AA组、XGFM组肾小管上皮细胞发生空泡变性;XGFH组用药90 d后,肾小管上皮细胞坏死,肾小管萎缩,肾间质出现灶性纤维化。AA组、XGFM组肾脏近曲小管上皮细胞明显肿胀,管腔变小,小管间质增生,部分发生纤维化;XGFL组肾组织未见明显损害。免疫组化显示AA组、XGFH组、XGFM组肾小管和间质上MMP-2的表达下调,而TGF-β1、TIMP-2、LN、FN的阳性表达较NC组明显上调(P<0.05)。结论:XGFH组、XGFM组和AA组均导致了实验大鼠肾小管间质的损害,而且病变的程度与用药剂量相关,XGFH组肾小管间质损害程度最严重,90 d内造成了大鼠肾间质纤维化,同时我们证实了慢性马兜铃酸肾病大鼠肾间质纤维化的发生同致纤维化因子TGF-β1和抑制细胞外基质降解因子TIMP-2的过度表达导致FN、LN胶原纤维在肾间质的过度增殖有关。

Experimental Study on Chronic Renal Interstitial Fibrosis Induced by Aristolochia Acid in Aristolochiae Mollissimae Herba in Rats

Objective:To observe the relationship between chronic renal interstitial fibrosis induced by Aristolochiae Mollissimae Herba decoction and its dosage and to investigate the pathogenesis. Methods:Female Wister rats were randomly divided into five groups.(1)Normal control group(n=8) was given distilled water 2 ml/d.(2)Aristolochic acid group(AA,n=16) was given aristolochic acid 5 mg·kg-1·d-1,intraperitoneal injection.(3)High dosage group(XGFH,n=16) was given aristolochiae mollissimae herba decoction 27 g·kg-1·d-1.(4)Moderate dosage group(XGFM,n=16)was given aristolochiae mollissimae herba decoction 13.5 g·kg-1·d-1.(5)Low dosage group(XGFL,n=16) was given aristolochiae mollissimae herba decoction 2.7 g·kg-1·d-1.Beside the normal group,the eight rats randomly chosen in other groups were killed at 45 dayss and 90 days.Before the animals were killed,the body weight of rats,specimens of blood and urine was taken in order to test regularly the renal function,red blood cell in urine,urinary microtransferrin/urinary creatinine(TF/Cr),urinary microalbumin/urinary creatinine(MA/Cr) and urinary N acetyl β D glucosaminidase/urinary creatinine(NAG/Cr).After the animals were killed,the renal weight of rats was taken.Pathological examination,including HE and PAS dyeing,was taken.The expression of LN,FN,TGF-β1,MMP-2 and TIMP-2 was detected by the immunohistochemical method. Results:In 90 days the body weight and renal weight of rats in AA group,XGFH and XGFM dosage group became lower than those of normal control group(P<0.01 or P<0.05).The levels of MA/Cr,TF/Cr,NAG/Cr,Scr and BUN in them increased significantly than those of normal control group(P<0.05 or P<0.01).In 45 days Optical microscopy showed renal tubular epithelial cell swelling,tubule atrophy and interstitium hyperplasia in XGFH dosage group.Partial focal vacuolus and granular degeneration were shown in AA control group and XGFM dosage group.In 90 days renal tubular epithelial cells were shown severe degeneration and necrosis in XGFH dosage group.Renal tubular atrophy and multifocal renal interstitial fibrosis were also shown.In AA control group and XGFM group renal tubular epithelial cells were shown swelling,tubule diminishing and interstitium hyperplasia and partial fibrosis.However renal interstitium showed no visible pathological changes in XGFL dosage group.The positive expression of TGF-β1、TIMP-2、LN、FN in renal interstitium and tubule was significantly upregulated by the immunohistochemical method compared to that in normal control group(P<0.05).Inversely,the positive expression of MMP was downregulated. Conclusion:The AA group,XGFH and XGFM dosage group could induce the tubular interstitial damage in experimental rats,then the degree of pathological changes was related with its using dosage.The renal tubular interstitial damage was the most serious in XGFH dosage group.In 90 days the renal interstitial fibrosis was shown in it.Meanwhile,It was proved that renal interstitial fibrosis in chronic aristolochic acid nephropathy was related with overexpression of fibrogenic factors TGF-β1 and extracelluar matrix degradation inhibitors TIMP-II may induce propagation of collagen fibers of FN and LN.