摘要
目的:观察Smad7在梗阻性肾病大鼠肾间质纤维化过程中的表达,研究中药肾络通对肾间质纤维化的防治作用及其作用机制。肾间质纤维化是由各种肾脏损害因素导致以肾小管萎缩、间质成纤维细胞增生及细胞外基质细胞外基质在肾间质内过度沉积为特征的病理改变,是各种肾脏疾病发展到肾衰竭的共同通路和病理基础,其直接影响慢性肾脏疾病的预后。在RIF的各种发生机制中,转化生长因子(TGF-β)为一种强效的致纤维化因子,介导了肾间质纤维化的发生,被认为是肾纤维化发生、发展过程中最重要的作用因子之一。Smads家族蛋白是新近发现的参与TGF-β超家族在细胞内信号传导的一组蛋白质,是TGF-β超家族成员参与生命活动的重要介质。而Smad7作为一种特异性的TGF-β的内源性抑制因子,由TGF-β诱导产生,对TGF-β信号传导起自身负反馈调节的作用,从而抑制细胞增生和细胞外基质沉积,延缓RIF的进程。肾络通是临床上治疗慢性肾炎和早期肾衰竭的有效方剂,具有益气活血化瘀通络作用,动物实验证实该方可以能减轻肾组织的病理损害,减少病变肾组织细胞外基质成分的积聚,延缓间质纤维化的进程,但是否通过Smad7途径发挥作用尚缺乏证据。本实验应用肾络通对单侧输尿管结扎(unilateral ureteral obstruction,UUO)诱导的肾间质纤维化大鼠模型进行干预性治疗,并以血管紧张素受体拮抗剂缬沙坦(Valsartan)为阳性对照,通过观察肾络通对实验动物梗阻侧肾脏病理形态学、Ⅲ型胶原(CollagenⅢ, ColⅢ)以及转化生长因子-β1(TGF-β1)和Smad7的影响,探讨肾络通对肾间质纤维化的拮抗作用及作用机制,从而为肾络通保护肾脏作用提供理论依据。
方法:本实验选用健康雌性SD大鼠40只,适应性饲养一周后,分别置代谢笼里取尿,测定尿蛋白及尿红细胞全部阴性后用于实验。40只小鼠随机分为假手术组(Sham group)、模型组(UUO group)、缬沙坦组(UUOV group)、肾络通组(UUOS group),每组10只。假手术组麻醉后切开腹部,只分离左侧输尿管,不进行结扎,然后缝合。其余各组动物结扎左侧输尿管建立肾间质纤维化模型并在术前一天灌胃给药,假手术组与模型组分别灌入等量的生理盐水。全部动物于手术后第14天断头处死,处死前一天收集24h尿液并称重,之后全部动物断头取血,用于生化指标测定,检测BUN, Scr及24h尿蛋白定量。切取左肾,测定左肾重/体重,观察肾脏病理改变,并分别采用免疫组化及原位杂交方法检测TGF-β1、ColⅢ、Smad7在蛋白及基因水平的表达。
结果:①生化指标:与假手术组比较,模型组大鼠血肌酐和尿素氮显著上升,与模型组相比缬纱坦组、肾络通组大鼠血肌酐和尿素氮显著降低(P<0. 05),而缬沙坦组和肾络通组大鼠相比血肌酐和尿素氮含量无显著性差异。②肾组织HE及Masson染色显示:肾组织HE及Masson染色显示:模型组肾小管基底膜明显增厚,肾间质大量炎细胞浸润和纤维组织增生,肾小管部分萎缩、管腔闭塞或扩张,肾间质明显增宽,肾小球数目明显减少,面积缩小。肾小囊粘连或扩张,小囊周围大量纤维组织增生。假手术组无上述表现,说明肾间质纤维化模型制作成功。其它各治疗组症状比间质可见多少不等的炎细胞浸润,少量纤维组织增生,肾小管轻度扩张,部分肾小球轻度萎缩,小囊扩张。③ColⅢ蛋白的表达、分布及半定量分析:结果显示ColⅢ蛋白主要表达于肾间质。假手术组大鼠肾间质呈弱阳性表达,其余各组表达均显著增强,为条索状或成片块状。模型组与假手术组相比表达呈显著性升高(P<0.05),缬沙坦组、肾络通组与模型组相比,ColⅢ的表达呈显著性下调(P<0.05),提示肾络通、缬沙坦均可抑制梗阻侧肾组织中ColⅢ蛋白的表达,其中,肾络通组优于缬沙坦组,但无显著性差异(P>0.05)。④TGF-β1蛋白及其mRNA的表达、分布及半定量分析结果:假手术组大鼠TGF-β1蛋白及其mRNA的表达均为弱阳性,主要分布在肾小管上皮细胞的胞浆中,少量表达于肾小球系膜细胞、成纤维细胞及巨噬细胞上。其余各组表达均显著增强,见于肾小管上皮细胞、间质细胞及肾小球内。其中,模型组较假手术组TGF-β1的阳性表达明显增强(p<0.05),可见TGF蛋白及其mRNA呈散在的片状分布,以髓质区最为明显,缬沙坦组、肾络通组与模型组相比,TGF-β1及其mRNA的表达均明显下调(P<0.05),仍略高于假手术组,提示肾络通、缬沙坦均可抑制梗阻侧肾组织中的TGF-β1蛋白及及其mRNA的表达。⑤Smad 7蛋白及其mRNA在肾组织中的表达与分布:Smad7蛋白及其mRNA主要表达在肾小管上皮细胞内,Smad7蛋白及其mRNA在假手术组呈中度表达;模型组较假手术组有显著性下调(P<0.05);缬沙坦组、肾络通组与模型组相比,Smad7蛋白及其mRNA表达显著性增强(P<0.05),提示肾络通、缬沙坦均可上调梗阻侧肾组织中的Smad7蛋白及其mRNA的表达。
结论:1肾络通能减轻肾组织的病理损害,减少病变肾组织细胞外基质成分的积聚,延缓间质纤维化的进程。
2肾络通能抑制TGF-β1及mRNA的表达,从而使细胞外基质病变肾组织合成减少。
3肾络通能促进肾组织中Smad7及其mRNA的表达,从而通过其对TGF-β1的负性反馈拮抗肾间质纤维化。
Objective: To observe the expression of Smad7 in tubulointerstitial fibrosis rats and the role of the Shen Luo Tong and its mechanism in tubulointerstitial fibrosis.
Renal interstitial fibrosis is the common pathological procedure to renal function failure in many kinds of renal diseases. Renal interstitial fibrosis is characterized by atrophy of tubule、proliferation of myofibroblast(Myof) ,accumulation and deposition of extracecullular matrix (ECM). Transforming growth factor-β1 is one of the most important factor in renal fibrosis. It plays the key role in the progression of renal interstitial fibrosis. Smads protein family is a new cluster of proteins which mediates intracellular signal transduction of TGF-β. They translate signals from the cell surface to the nucleus where they down-regulate the expression of TGF-β. Smad7 is a specific endogenous inhibitor of TGF-βand induced by TGF-β.It plays negative feedback regulation role in TGF-βsignal transduction. So it can inhibit cell proliferation and reduce the desposition of ECM in obstructive kidney, prolong the processes of fibrotic.Shen Luo Tong is a basic formula for chronic nephropathy, animal experiments showed that it can alleviate the lesion of renal tissue and reduce the deposition of ECM in obstructive kidney, prolong the process of fibrosis ,but there is no evidence to confirm the role on smad7 for its mechanism .The study was to investigate the effect of Shen Luo Tong and Valsartan (a drug of ARB) in rats with unilateral ureteral obstruction (UUO).The renal morphology, transforming growth factor-β, Collagen III were examined.
Method: Fourty female Sprague-Dawley rats were randomly divided into four groups (n=10), Shame-operation (SHAM), model (UUO), Shen Luo Tong (UUOS) and Valsartan (UUOV).Each animal was ligated left ureter except SHAM group. Drugs were administered from the day before operation but the saline were used in SHAM and UUO groups by oral. Animals were sacrificed after 14 days, the 24-hour urine and the blood were collected to test BUN Scr and 24-hour urinary protein.The left kidneys were harvested for pathological study. Immunohistochemistry staining and in situ hybridization were used for expression of TGFβ1,Col lll,Smad 7.
Result:①Scr and BUN in UUO group was higher than SHAM group markedly and were lowered in treated group(P<0.05). But there in no differences in treated groups. (P> 0.05).
②pathology: The renal tissues were stained with HE and Masson. There was no change in SHAM group. But in UUO group, interstitial macrophage and monocyte infiltration, renal tubular artrophy, emphraxis or expand were shown. The renal capsule adhere or expanded. Fibrous tissues proliferated around the renal capsule. The lesions in treated groups were slighter than in UUO group. It means that Shen Luo Tong and Vslsartan can inhibit fibrosis in obstructive nephropathy.
③The expression of Col lll with Immunohistochemistry has shown a weak expression for Col lll in SHAM group, while in other groups the expression of Col lll increased markedly. Compared with the SHAM group, the expression of Col lll in UUO group was up-regulated markedly(P<0.05).Compared with the UUO group ,the expression of Col lll in UUOS、UUOV groups were down-regulated markedly(p<0.05). It means that Shen Luo Tong and Valsartan can inhibit the expression of Col lll in obstructive kidney. But there in no differences in treated groups(P> 0.05).
④The expression of TGFβ1 with immunohistochemistry and in suit hybridization showed that a weak expression of TGFβ1 in SHAM group ,while in other groups the expression of TGFβ1 in protein level and its mRNA up-regulated markedly. They can be seen in renal tubular epithelial cell, interstitial cells. The expression of TGFβ1 in protein and its mRNA in UUO group was higher than that in SHAM group (p<0.05);Compared with the UUO group, TGFβ1 in UUOS、UUOV groups inhibited markedly(P<0.05). It means that Shen Luo Tong and Valsartan can inhibit the expression of TGFβ1 in protein and mRNA level in obstructive kidney.
⑤The expression of Smad7mRNA with Immunohistochemistry and in suit hybridization: It has shown a stronger expression of Smad7mRNA in SHAM group, they can be seen in renal tubular epithelia cell, compared with SHAM group ,the expression of Smad7mRNA in other groups were down-regulated(P<0.05) .Compared with the UUO group ,the expression of Smad7 in UUOS group increased markedly (p<0.05), it means that Shen Luo Tong can up-regulate the expression of Smad7mRNA in obstructive kidney.
Conclusion:①Shen Luo Tong can alleviate the lesion of renal tissue and reduce the desposition of ECM in obstructive kidney, prolong the processes of fibrotic.
②Shen Luo Tong has the role to inhibit expression of TGFβ1 in protein and mRNA in obstructive kidney, and reduce synthesis of ECM.
③Shen Luo Tong can up-regulate the expression of Smad7 and Smad7mRNA in obstructive kidney, and inhibit the tubulointerstitial fibrosis.
引文
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