【作者】 王晓玲；

【导师】 傅淑霞；

【作者基本信息】 河北医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:肾间质纤维化是各种原因引起的肾脏疾病进展至终末期肾衰竭的共同途径,肾间质纤维化的程度与肾功能损害程度密切相关;小管间质积聚的巨噬细胞(macrophage,MP)可直接或间接参与间质纤维化过程,与肾脏疾病的进展尤为相关[1],是不可逆性慢性肾损伤的标志。调节正常T细胞表达和分泌的细胞因子(regulated on activation of normal T cell expressed and secreted,RANTES)是趋化因子家族中的一员,可趋化并激活MP、淋巴细胞和嗜酸粒细胞,引导炎症细胞浸润到肾脏并诱导多种炎症因子的大量表达。巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)是诱导局部MP增殖的关键调控因子[2],单侧输尿管梗阻(unilateral ureteral obstruction,UUO)大鼠肾组织中M-CSF的增加与局部MP的增殖显著正相关[3]。核因子-κB(nuclear factor-κB,NF-κB)调控多种基因表达,参与了许多炎症介质的信号传导过程。转化生长因子-β(transforming growth factor-β,TGF-β)是重要的促纤维化因子,介导了细胞损伤、细胞外基质积聚等诸多过程。川芎嗪为我国常用的活血化瘀中药,其肾保护作用包括抗细胞增殖、抗炎、抗纤维化、免疫调节等多个角度,但其是否有下调RANTES、M-CSF的表达从而使MP浸润减少而抑制肾间质纤维化尚未见报道。本课题拟通过UUO模型,观察川芎嗪对肾间质RANTES、M-CSF、TGF-β1、NF-κB的表达,单核/MP浸润及肾间质纤维化程度的影响,从细胞因子水平探讨川芎嗪对肾间质炎症反应及纤维化的影响。方法:选择清洁级健康雄性SD大鼠45只,体重180～220克,随机分为三组:假手术组(A组)、UUO组(B组)、川芎嗪治疗组(C组)各15只。将各组大鼠以10%水合氯醛(3.0 ml/kg)腹腔注射麻醉后,分离左侧输尿管并在中上1/3处用4号线上下结扎两处,从中剪断输尿管,以防逆行性感染。假手术组除不结扎和剪断输尿管外,其他手术过程同前。C组大鼠以川芎嗪混悬液40mg/kg/d腹腔注射,术前1天开始给药,1次/日。A组和B组用同体积生理盐水腹腔注射。分别于术后第3、7、14天每组每次随机取5只,快速取出左肾,并处死大鼠。光镜HE、Masson染色观察肾间质病理学改变;免疫组织化学染色测定肾间质RANTES、M-CSF、NF-κB、TGF-β1、CD68+细胞的表达,并对以上实验结果通过图像分析系统进行半定量分析;应用实时荧光定量PCR(RT-PCR)技术检测RANTES mRNA的表达。所有数据用均数±标准差表示,用SPSS11.5统计软件进行分析,P<0.05为有统计学意义。结果:①体视学研究:A组大鼠肾脏外观正常。梗阻大鼠的肾体积随梗阻时间的延长而逐渐增大,增大的肾脏内可见明显积水,积液清亮透明,肾实质随梗阻时间延长而逐渐变薄,肾盂扩张,肾盏乳头受压。②组织病理学检查:A组肾组织未发现异常。B组术后第3天肾间质水肿,肾小管扩张,小管间质区域出现单核细胞、淋巴细胞浸润。术后第7天上述变化更加明显:间质明显增宽,扩张的肾小管有不同程度的萎缩和坏死,上皮细胞变性、水肿、甚至坏死。术后第14天可见肾间质区域较多炎细胞浸润及肾间质纤维化进行性加重。C组与B组平行相比肾小管间质病变程度明显减轻。③川芎嗪抑制肾间质单核/MP的浸润:A组各时期偶见CD68+细胞浸润(0.034±0.044～0.042±0.043,P>0.05)。B组术后3天可见CD68+细胞在肾间质浸润,并随时间延长而增加(0.268±0.030～0.472±0.048,P<0.05)。C组与B组同时相点相比表达明显减少(0.192±0.068～0.386±0.061,P<0.05),炎症反应减轻。④川芎嗪下调肾组织RANTES蛋白及RANTES mRNA表达:A组RANTES蛋白仅微量表达于肾小球或肾间质(0.047±0.020～0.061±0.017,P>0.05)。B组RANTES主要表达于肾小球及肾间质,血管、近曲小管少量表达,且随时间全部呈进行性上升,至第14天达峰值(0.221±0.044～0.453±0.040 , P均<0.05)。C组RANTES蛋白在各时相点均明显下调(0.172±0.020～0.374±0.03,P<0.05)。RANTES mRNA水平变化与其蛋白质水平的变化一致,呈明显正相关关系(r=0.520,P<0.05)。⑤川芎嗪下调M-CSF的表达:A组肾组织M-CSF微量表达于肾小球或肾间质(0.039±0.032～0.048±0.034,P>0.05)。B组M-CSF主要分布于肾小球及肾间质,近曲小管仅有少量表达,且随着时间延长表达量逐渐增加(0.332±0.051～0.501±0.084,P<0.05)。C组较B组相比表达明显下调(0.242±0.044～0.409±0.029,P<0.05),但仍高于A组。⑥川芎嗪可下调促纤维化因子TGF-β1、NF-κB的表达:TGF-β1和NF-κB梗阻术后3天即表达上调,且随着时间延长表达量逐渐增加,至第14天达峰值(分别为0.266±0.004～0.545±0.070,0.270±0.028～0.476±0.054;P均<0.05),较A组(分别为0.025±0.005～0.027±0.001,0.015±0.003～0.017±0.007;P均>0.05)有显著统计学意义(P<0.05)。C组各因子的表达均显著低于同时期B组的表达(分别为0.186±0.006～0.424±0.020,0.217±0.014～0.409±0.062;P均<0.05),但仍高于A组(P<0.05)。⑦RANTES、M-CSF、TGF-β1的表达与CD68呈正相关关系(r=0.857、0.565、0.876,P均<0.05),亦与NF-κB的表达呈正相关关系(r=0.832、0.550、0.813,P均<0.05)。结论:川芎嗪可有效抑制UUO肾组织前炎症因子RANTES、M-CSF及促纤维化因子TGF-β1、NF-κB的过度表达,在早期抑制单核/MP趋化与增殖,从而抑制肾组织炎症反应,减轻肾间质的纤维化。更多还原

【Abstract】 Objective: Renal interstitial fibrosis is a common way of kidney disease, induced by various causes, developing into final renal failure. The degree of renal interstitial fibrosis has correlation with the extent of renal function damage. Tubulointerstitial macrophage (MP) accumulation in particular correlates with kidney disease progression, which may directly or indirectly be involved in tubulointerstitial fibrosis[1], and it is the hallmark of irreversible chronic kidney injury. Regulated on activation of normal T cell expressed and secreted (RANTES) is a member belonging to chemotatic factor’s family, which can chemotatic and activates MP, leukomonocyte and eosinophils, and can lead to the infiltration of inflammatory cells to the kidney, inducing the substantial expression of various inflammatory factors. Macrophage colony-stimulating factor (M-CSF) is the critical factor that induces strong local MP proliferation[2]. In rat’s kidney, M-CSF increased in association with local MP proliferation of unilateral ureteral obstruction (UUO)[3]. Nuclear factor-κB (NF-κB) can regulates expression of a wide range of genes, and participates the process of much inflammatory mediator’s signal transducer. Transforming growth factor-β(TGF-β) is an important factor to promote fibrosis, mediating to many processes, such as accumulate cellular damage and extracellular matrix. Ligustrazine is a Chinese herbs with the functions of activating the blood circulation and removing the blood stasis. It’s effect of protect kidney include anti-cell proliferation, anti-inflammatory, anti-fibrosis, immuno- regulation and so on. But it’s unknown that whether ligustrazine can inhibit the expression of RANTES, M-CSF, thereby reduced the renal infiltrating of MP and interstitial fibrosis. Through UUO, this study is about the effect of Ligustrazine makes on the expressions of RANTES, M-CSF, TGF-β1, NF-κB, and Monocyte/MP infiltration, the degree of the affected renal interstitial fibrosis. We will have a discussion of Ligustrazine on renal interstitial inflammation and fibrosis from the levels of cytokines.Methods: Forty-five male Sprague-Dawly rats weighing 180~220g were randomly divided into sham operated group (group A), unilateral ureteric obstructive operated group (group B) and unilateral ureteric obstructive operation with ligustrazine treatment group (group C), (n=15). After giving intraperitoneal injection of anesthesia with 10% chloral hydrate (3.0ml/kg) to each of the groups of rats, separate the left ureter, ligate at two points with 4.0 silk in the middle and upper 1/3 and cut between the ligatures in order to prevent retrograde urinary tract infection. Rats in group A had their ureters manipulated but not be ligated. Ligustrazine (40mg/Kg/d) began to intraperitoneal injection one day before surgery to the rats in group C. A and B groups received Saline of equal volume by daily peritoneal injection. Rats were killed in the 3, 7, 14 day after surgery. Immunohisto-chemistry was used to measure the level of expression of RANTES, M-CSF, NF-κB, TGF-β1 and CD68-positive cells. The changes of renal tissue were also observed by HE and Masson stain. The results were analyzed semi-quantitatively by the pathological image analysis system. RANTES mRNA was detected by Quantitative real-time PCR (RT-PCR). All the data were analyzed by SPSS 11.5 statistics software, and P value< 0.05 was considered to have statistical significance.Results: 1. Stereology research: Kidneys of the rats in group A had normal appearance. While the kidneys of the obstructed rats became bigger as time went on. Clear water could be found in the bigger kidneys, renal cortex became thinner, renal pelvis expanded and minor calyx oppressed.2. The changes of histopathology: The group A did not found change. The situation among rats in group B that renal interstitial edema was formed, tubular expanded, monocyte came up in tubulointerstitial regions and lymphocytic infiltration existed at the 3th after the survey. That change became obviously at the 7th: Interstitial obviously widened, expansion had various degrees of tubular atrophy and necrosis, epithelial cell degenerated, had oedema and some even died. On the 14th day, substantial inflammatory cell infiltrated and renal interstitial fibrosis aggravated.3. Ligustrazine inhibits the infiltration of monocyte/MP: In group A, mainly no CD68-positive cells expression in nephridial tissue(0.034±0.044~0.042±0.043,P>0.05). In group B, CD68-positive cells were found in renal interstitium at the 3th after the survey. With the time went on, CD68-positive cells were highly expressed (0.268±0.030~0.472±0.048, P<0.05). In group C, the level of CD68-positive cells were lower than that of group B at the same timepoint (0.192±0.068~0.386±0.061, P<0.05), the renal inflammation was released.4. Ligustrazine inhibits the expression of RANTES and RANTES mRNA in nephridial tissue: In group A, the expression of RANTES in renal glomerulus or renal interstitial were trace (0.047±0.020~0.061±0.017, P>0.05). RANTES was mainly expressed in renal glomerulus and renal interstitial, and had a small quantity of expression in blood vesse and proximal convoluted tubule in group B. The area of RANTES increased and reached to the bottom on the 14th day (0.221±0.044~0.453±0.040, P<0.05). The level of RANTES in group C were lower than that of group B at the same timepoint (0.172±0.020~0.374±0.030, P<0.05). The changes of the RANTES mRNA level were consistent with the protein, and closely related to RANTES protein (r=0.520,P<0.05).5. Ligustrazine inhibits the expression of M-CSF: In group A, the expression of M-CSF had a rare quantity in renal glomerulus or renal interstitial (0.039±0.032~0.048±0.034, P>0.05). M-CSF was mainly expressed in renal glomerulus and renal interstitial, and had a small quantity of expression in proximal convoluted tubule in group B and the area of M-CSF increased significantly (0.332±0.051~0.501±0.084, P<0.05); In group C, the expression of M-CSF reduced more significantly than that of group B (0.242±0.044~0.409±0.029,P<0.05). But it was still higher than the level of group A (P<0.05).6. Ligustrazine inhibits the expression of promote fibrosis factors (TGF-β1, NF-κB): From the 3th day, the expressions of TGF-β1 and NF-κB were upregulated in group B, and increased with time went on, reached to the bottom on the 14th day (0.266±0.004~ 0.545±0.070 and 0.270±0.028~0.476±0.054, P<0.05), had statistical significance compared with group A (0.025±0.005~ 0.027±0.001 and 0.015±0.003~0.017±0.007). In group C, both of them were decreased than that in group B at the same timepoint (0.186±0.006~0.424±0.020 and 0.217±0.014~0.409±0.062,P<0.05), but they were still higher than the level of group A (P<0.05).7. RANTES, M-CSF, TGF-β1 were both closely related to CD68 (r=0.857,0.565,0.876,P<0.05), and also positively related to NF-κB (r=0.832,0.550,0.813, P<0.05).Conclusion: Treatment with Ligustrazine in UUO rats markedly limits the renal overexpression of inflammatory mediators like pro-inflammatory factors RANTES, M-CSF and factors of pro-fibrosis TGF-β1, NF-κB. This pathway inhibits the infiltration of monocyte/MP in the earlier period, thereby reduced renal interstitial inflammation and interstitial fibrosis.更多还原