【作者】 黄仁发；

【导师】 周巧玲；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 研究背景：急性肾衰竭(acute renal failure,ARF)是一种常见的临床危急重症,常以氮质血症、水电解质和酸碱平衡紊乱及全身各系统并发症为主要临床表现,严重危害人类健康。近年来,尽管ARF的基础和临床研究取得了较大进展,但其发病率和死亡率仍居高不下,其主要原因是ARF发病原因错综复杂,发病机制迄今尚未明确,特别是很多ARF患者早期症状不明显,直致病情发展到一定程度才出现血清肌酐升高,让临床医生失去了适时救治的良好机会。因此,近年来学者们将急性肾损伤(acute kidney injury,AKI)的概念引入肾脏病和急诊医学领域,趋向将ARF更称为急性肾损伤,这样更有利于临床医生早期诊断和治疗AKI,即在肾功能开始下降、甚至肾脏组织学有损伤、生物标志物改变而GFR尚正常的阶段将之识别并及早干预治疗,以避免ARF的发生。如何早期识别和诊断AKI,何种机制和学说能更好的解释AKI的发生发展过程已成为当今肾脏病领域研究的热点。肾缺血-再灌注损伤(Ischemia reperfusion injury, IRI)是AKI的主要发病机制之一,这已被学者们的研究证实。而凋亡和炎症在肾IRI的发生发展中起重要作用。IRI可引起肾小球内皮细胞功能障碍,继而内皮细胞表达细胞间粘附分子增多,介导血流中的有形成分如中性粒细胞、单核细胞粘附至血管内皮细胞,这些细胞都可以通过各种网络信息调控并刺激这些细胞分泌TNF-α、IL-2、IL-6、IL-8等炎症因子和MCP-1等化学趋化因子,通过这些炎性因子再激活炎症的级联反应,导致肾组织炎症反应发生,最终引起AKI。肾小管上皮细胞,作为肾脏的固有细胞,在肾IRI的环境下,其本身既可以因为缺血而受到损伤,也可以在某些因子的影响下转化为炎症细胞,分泌TNF-α、IL-2、IL-6等炎症因子,参与了AKI的发生。实验研究表明,TNF-α、IL-1、IL-6等炎症因子的拮抗剂通过抑制IRI相关的炎症和肾小管上皮细胞凋亡发挥肾保护作用,提示TNF-α、IL-1、IL-6等炎症因子在AKI发生和治疗中起了十分重要的作用。资料表明,AKI发生时启动多种信号通路,Notch信号通路就是其中之一。近年来有研究证明,肾IRI时Notch信号通路被重新激活,参与肾小管上皮细胞损伤后的再生。Notch信号通路是一进化上保守的细胞间信号传导通路,参与细胞的生长、增殖、分化等过程,它不仅在肾脏发育过程中对细胞命运的决定起关键作用,而且在疾病状态下可被重新激活,通过与其他信号通路的交叉对话(across-talking),参与凋亡、再生以及小管-间充质细胞转分化等过程,影响着多种肾脏疾病的发生和发展。而且有学者在呼吸系统和自身免疫性疾病的研究中发现,Notch信号通路通过与NF-κB信号之间的交叉对话,调节Thl和Th2反应而参与了支气管哮喘、类风湿关节炎等炎症过程。但活化的Notch信号通路是否参与调控肾IRI相关的炎症和氧化应激过程,这需要进一步研究证实。本组前期研究已证明冬虫夏草(Cordyceps Sinensis, CS)对糖尿病肾病、高血压肾损伤模型鼠具有良好的肾保护作用,CS是否可以通过影响肾IRI诱导的炎症和氧化应激发挥肾保护作用,其作用靶点是否与抑制Notch信号通路活化有关,目前国内外未见报道。本研究拟以I/R大鼠模型和抗霉素A与肾小管上皮细胞共培养模拟的体外的I/R模型为研究对象,一方面观察I/R损伤时IL-6、TNF-α、MCP-1和HIF-1α的表达状况和Notch2/hes-1信号转导通路是否被激活；另一方面用Notch信号通路关键酶(?)-secretase抑制剂和冬虫夏草进行干预实验,其目的是：1.观察肾I/R是否可上调肾小管上皮细胞IL-6、TNF-α、MCP-1和HIF-1α的表达,从而证实肾IRI存在炎症和氧化应激过程。2.观察肾I/R是否可激活Notch2/hes-1信号通路,活化的Notch2/hes-1信号通是否参与IL-6、TNF-α、MCP-1和HIF-1α的调控,证实Notch2/hes-1信号通路是否参与肾IRI相关的炎症和氧化应激过程。3.观察冬虫夏草是否通过抑制Notch2/hes-1信号通路的活化,继而影响IL-6、TNF-α、MCP-1和HIF-1α的表达,通过抗炎和抗氧化发挥肾保护作用,从而探讨冬虫夏草的作用机制和靶点。实验方法：1.60只健康雄性SD (Sprasue-Dawley)大鼠用10%水合氯醛溶液(3.5mL/kg)腹腔注射局部浸润麻醉后,从腹部正中切口,行右肾切除术,稳定10min后将大鼠随机分成假手术组(sham组)、缺血-再灌注组(I/R)、DAPT组(DAPT)、冬虫夏草(CS)组4组,每组组内再按再灌注时间分为24h、48h、72h 3个时间点。缺血-再灌注组(I/R组)大鼠持续夹闭左肾蒂60min后恢复灌注,予以生理盐水(2 mL/d)灌胃；DAPT组大鼠持续夹闭左肾蒂60min后恢复灌注,术后予以γ-泌肽酶抑制剂DAPT (500μg/100g.d)灌胃；冬虫夏草(CS)组大鼠持续夹闭左肾蒂60min后恢复灌注后,予以冬虫夏草提取液(5g/kg.d)灌胃；假手术组(sham组)大鼠切除右肾后分离左输尿管,但不夹闭左肾蒂,术后予以生理盐水(2 mL/d)灌胃,分别在上述时间点处死大鼠,处死前收集尿液和血液。检测各组大鼠肾功能(BUN、Scr)情况；ELISA法检测各组大鼠TNF-α、IL-6水平；比色法检测各组大鼠尿NAG水平,HE染色观察各组大鼠肾脏病理,并半定量评分法观察各组大鼠肾小管间质损伤；RT-PCR法检测各组大鼠Notch2、hes-1mRNA的表达；免疫激光共聚焦法和western blot法检测各组大鼠Notch2、hes-1、MCP-1、HIF-1α蛋白的表达。2.首先用MTT法观察不同浓度DAPT (0.1μmol/L, 1μmol/L, 10μmol/L, 100μmol/L)和CS提取液(10mg/L,20mg/L,40mg/L, 80mg/L)对NRK52E增殖的影响,确定DAPT工作液和CS工作液的浓度。3.将NRK-52E细胞置于低糖型完全DMEM培养基中培养,分为normal组、缺血-再灌注组(I/R组)、DAPT组和CS组4组。I/R诱导的NRK-52E细胞模型用下法建立：用不含血清的D-Hanks液+抗霉素A(10μmol/L)模拟缺血3h,恢复低糖型完全DMEM培养基模拟再灌注。normal组NRK-52E不作任何处理,正常条件培养；DAPT组NRK-52E造模前24h和再灌注时用含10μmol/L DAPT工作液处理,直至收集细胞,组内按再灌注时间分为24h、48h、72h 3个时间点；CS组(n=15)：按上述方法造模,造模前24h预先用含40mg/L的CS工作液处理,再灌注时用CS工作液培养,直至收集细胞,组内按再灌注时间分为24h、48h、73h 3个时间点。细胞免疫化学荧光法和Western blot法观察各组NRK-52E细胞Notch2、hes-1、MCP-1、HIF-1α蛋白的表达。实验结果：1.体内实验结果显示,肾I/R时大鼠体内血清BUN和Scr升高,尿中NAG排出增多,肾脏病理出现肾小管刷状缘丢失、小管上皮细胞变性坏死、部分小管扩张、管型形成,肾间质大量炎症细胞浸润等病变,提示肾I/R大鼠AKI模型建立成功。肾I/R时大鼠血清TNF-a、IL-6水平增高(P<0.01)；肾组织中Notch2、hes-1、MCP-1、HIF-1α的mRNA和/或蛋白表达上调(P<0.01),并均于再灌注后24h达高峰。而DAPT干预能改善肾小管间质损伤,改善肾功能,降低血清INF-α、IL-6水平(P<0.05或P<0.01),下调Notch2、hes-1、NF-κB2、MCP-1mRNA和/或蛋白表达,上调HIF-1α蛋白表达(P<0.01),提示DAPT可选择性地阻断肾小管上皮细胞Notch2/hes-1信号通路,调控IL-6、TNF-α、MCP-1、HIF-1α等转录,有效地控制肾IRI时所诱导的炎症和氧化应激过程,具有良好的肾保护作用。2.体内实验结果显示,CS干预后可改善I/R大鼠肾小管间质损伤,改善其肾功能,同时降低血清TNF-α、IL-6及尿NAG水平(P<0.01),下调Notch2/hes-1、MCP-1mRNA和/或蛋白表达(P<0.01),上调HIF-1α蛋白表达(P<0.01),提示CS对I/R时的肾保护作用可能是通过抑制Notch信号通路活化,调控IL-6、TNF-α、MCP-1 HIF-1等基因转录而实现的。3.MTT结果显示,与对照组OD值相比较,浓度为0.1～10μmol/L DAPT组和浓度为10-40mg/LCS组NRK52E的OD值差异无统计学意义(P>0.05)；溶度为100μmo1/L的DAPT和80mg/L的CS均能显著抑制NRK-52E的增值(P<0.01)。因此,我们在下面的实验选择DAPT和CS的处理溶度为10μmol/L和40mg/L。4.体外实验结果显示,细胞免疫化学荧光法和western blot结果显示,I/R可诱导NRK-52E的Notch2、hes-1、MCP-1、HIF-1α蛋白表达上调,且Notch2、hes-1、MCP-1和HIF-1α蛋白表达均于再灌注后24h达高峰。然而,DAPT干预可下调Notch2、hes-1、MCP-1蛋白表达(P<0.01),上调HIF-1α蛋白(P<0.01),结果表明I/R能诱导NRK-52E的Notch2/hes-1信号通路活化,从而调控MCP-1和HIF-la的转录。5.体外研究结果显示,经CS (40mg/L)干预后,肾小管上皮细胞Notch2、hes-1、MCP-1蛋白表达下调(P<0.01), HIF-1α蛋白上调(P<0.01),提示CS可能是通过抑制肾IRI时Notch2/hes-1信号通路的活化,影响MCP-1、HIF-1α等炎症因子的转录,而实现肾保护作用。结论：1.肾IRI时IL-6、TNF-α、MCP-1和HIF-1α表达上调,证实肾IRI存在炎症和氧化应激过程。2.肾IRI诱导肾小管上皮细胞Notch2/hes-1信号通路活化,活化的Notch2/hes-1信号通路能调控IL-6、TNF-α、MCP-1和HIF-1α等的转录。3.冬虫夏草能减轻肾IRI时肾脏病理损伤,改善肾功能,有效抑制Notch2/hes-1信号通路的活化,下调IL-6、TNF-α、MCP-1等炎症因子的转录,上调肾小管上皮细胞HIF-1α的表达,证实冬虫夏草提取液通过影响Notch/hes-1信号通路而有效地抗炎、抗氧化作用,这可能是冬虫夏草新的肾保护作用机制。更多还原

【Abstract】 Background:Acute renal failure (ARF)is a common severe disease with an abrupt decease in kidney function that includes the increased blood urea nitrogen and serum creatinine level, disturbance of acid-base balance and water-electrolyte and systemic Complication that threats human health seriously. Although significant advances have been made in basic research along with important technical advances in the recent decades, AKI still keeps high morbidity and mortality due to complex etiopathogenisis and unclear mechanisms hitherto. Eespecially, early symptom in patients with ARF are not obvious untill it developes into some stages that manifestates increased serum creatinine level, which leads to the loss of remedy opportunity for doctor at the best time. Recently, in an attempt to earlierly diagnosis and therapy for ARF, the term "acute kidney injury (AKI) " has been proposed as the replacement of "ARF" in nephrology and emergency so that we could identify and interfere in it at the stage when kidney function begins to discrease, even before kidney tissue damage while biological marker of AKI presents alteratio. It is favourable for avoiding the occurrence of ARF. Recently, schorlars in nephrology are focusing on how to diagnosis and identify AKI earlierly, how to explain the development and progress of AKI by means of perfect theory.Studies have confirmed that ischemia-reperfusion injury (IRI) is one of the major causes of AKI. Both inflammation and apoptosis contribute to the development and progression of renal IRI injury, IRI can lead to endothelial dysfunction, increased endothelial expression of a variety of adhesion molecules that promotes the interactions between endothelial and leukocyte and monocytes, that initiates the inflammatory cascades by inflammatory corpuscle infiltration and cytokine/ chemotactic cytokine production, causes the presention of AKI. Tubular epithelial cells, injuried by hypoxia, also produce TNF-α, IL-2, IL-6, and MCP-1 to partcipate in the initiation of AKI. A growing body of evidences indicated that inhibition of IL-6, IL-1and TNF-αprovided protection against IRI-induced inflammation and apoptosis, which suggests that both inflammation and apoptosis could perhaps become the target of AKI treatment.Molecular pathways, such as Notch signaling, are involved in the course of AKI. Present studies demonstrated that Notch signaling reactivated again in renal IRI is involved in the regerenation of renal tubular. Notch signaling is an evolutionarily conserved and widely used intercellular signaling pathway that influences cellular growth, proliferation and differentiation, it not only plays a key role in the dicision of life and death of cell, but also could be reactivated in the setting of disease. Reactivated notch signaling is involved in the course of apoptosis, regerenation and epithelial-to-mesenchymal transitions by the across-talking with other pathways, participating in the development of many kidney diseases. Studies demonstrated that Notch signaling is invovled in the inflammation in the asthma and arthritis by the across-talking with nucleus transcription factor kappa B pathway through mediating Thl and Th2 response. It needs to be proved whether notch signaling is involved in the course of renal ischemia reperfusion injury associated inflammation and oxidative stress.Our previous works demonstrated that Cordyceps Sinensis plays a nephroprotective role in both diabetic nephropath and hypertension nephropath rat models, there is no study whether CS have nephroprotctive effect by mediating renal IRI associated inflammation and oxidative stress, which is accomplished by repressing Notch signaling activation. Therefore, in our work, we observed the expression of IL-6, TNF-α, MCP-1, HIF-1αfollowing renal IRI and the activation of Notch signaling by means of renal IRI rats models in vivo and ischemia /reperfusion- induced NRK-52E model that was simulated by the incubation with antimycin A in vitro. As well as we use the interventions ofγ-secretase inhibtor DAPT and CS to investigate as following.Objective:1. To observe whether renal IRI up-regulats the expression of IL-6, TNF-a, MCP-1 and HIF-1α, proving the exiting of renal IRI associated inflammation and oxidative stress. 2. To observe whether renal IRI activates Notch signaling that mediates the transcription of IL-6, TNF-α, MCP-1 and HIF-1α,proving whether Notch signaling regulates renal IRI associated inflammation and oxidative stress.3. To observe whether CS represses the activation of Notch signaling, through which mediates the transcription of IL-6, TNF-α, MCP-1 and HIF-1α, plays a nephroprotective role by the way of anti-inflammation and anti- oxidative stress, investigating the mechanism and target of CS.Method:1. After 60 healthy male Sprague-Dawley(SD) rats were anesthetized with intraperitoneal hydral (0.35ml/100g), Unilateral flank incisions were made, and right kidneys were excised and collected as the normal group kidneys after 10 min stabilization period, the 60 healthy male Sprague-Dawley(SD) rats were randomly divided into four groups: sham group(sham, n=15), ischemia-reperfusion (I/R)group(I/R, n=15), DAPT treatment group(DAPT, n=15), and CS treatment group(CS, n=15), and the rats in each group were divided into 24h,48h,72h time point respectivly. The left kidney in the I/R group was subjected to 60min of ischemia with atraumatic vascular clamp, then the clamps were removed to anastate reperfusion, Rats in the I/R group received NS (an equivalent volume with DAPT group and CS)by intraperitoneal injection. The rats in the DAPT treatment group received DAPT (500μg.100g-1) intragastric administration. The rats in the CS treatment group received CS (5g/kg.d)by intragastric administration. Sham group rats, subjected to the same surgical procedure without clamping the left renal vessels, received the same volume of NS. Both blood and urine in all the rats were collected before sacrificed at the time point aboved. Kidney function was evaluated by measurement of Scr and BUN level with a Biochemical Autoanalyzer, and the concentrations of serum TNF-αand IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA) and urine NAG level was measured with Microplate colorimetric assay, Serial sections were stained with hematoxylin and eosin (HE) for histopathological analysis, and the degree of tubular damage was valuated in terms of semi-quantitative score, the mRNA expression of Notch2 and hes-1 were detected with with methods of reverse transcription-Polymerase chain reaction(RT-PCR). and the protein expression of Notch2, hes-1, MCP-1and HIF-αwere measured by means of immune confocal laser microscope and western blot.2. Observ the effect of different concentration DAPT (0.1μmol/L, 1μmol/L, 10μmol/L,100μmol/L) and CS(10mg/L,20mg/L,40mg/L, 80mg/L) on the proliferation of NRK-52E by means of MTT so that we can identify the saturation of DAPT fluid and CS fluid in the next experiment.3. Rat renal tubular epithelial cells (normal rat kidney cells, NRK-52E) cultured in complete low-glucose DMEM was randomly divided into four groups:normal group (n=5), renal ischemia and reperfusion model group (I/R, n=15), treatment group with DAPT (DAPT group, n=15); treatment group with Cordyceps Sinensis group (CS, n=15). The I/R-induced NRK-52E models were established as following:the NRK-52E were cultured in no serum- D-Hanks fluid and antimycin A (10μmol/L) for three hour to simulate ischemia. There was no treatment in NRK-52E cells in normal group cultured under noral condition; NRK-52E cells in DAPT group were treated with DAPT fluid(10μmol/L) at 24h before operation and following reperfusion; NRK-52E cells in DAPT group were treated with CS fluid(40mg/L) at 24h before operation and following reperfusion; In additon to normal group, NRK-52E cells in the other groups were collected at 24h,48h 72h, after reperfusion respectively. Protein expressions of Notch2, hes-1, MCP-1, HIF-1αin the NRK-52E in all the groups were detected by means of cell immunofluorescence and Western blot.Results:1. Our results in vivo showed that, renal I/R induced the increase of serum Scr and BUN level, the elevation of urinary NAG, and tubulointerstitial injury including focal areas of proximal tubular dilation and distal tubular casts, effacement and loss of proximal tubule brush border, and interstitial inflammatory cell infiltration, which suggests an successful renal IRI associated AKI model. Renal I/R induced the increase of the concentrations of serum TNF-a, IL-6 (P<0.01), the up-regulation in mRNA and/or protein expression of Notch2, hes-1, MCP-1 and HIF-1α(P<0.01). Our work also indicates that expression of Notch2, hes-1, MCP-1 and HIF-1αmRNA and/or protein expression reaches a peak at 24h after reperfusion, a time point that coincides with the peak of renal tubular injury, urinary NAG levels, BUN, Scr, serum TNF-αand IL-6 and semi-quantitative score of renal tubular cells damage. Inhibtion of the protein expression of Notch2 and hes-1 by DAPT treatment could attenuate the severity of renal tubule damage, improve kidney function, lower the concentration of pre-inflammation factors(TNF-a and IL-6), and down-regulate protein expression of MCP-1 (P<0.01), up-regulate the protein expression of HIF-1α(P<0.01), which suggests renal I/R may induce the activation of Notch2/hes-1 signaling in the renal tubular cells that mediates the transcription of IL-6, TNF-a, MCP-1 and HIF-1α. Notch2/hes-1 signaling maybe is involved in the course of renal IRI associated inflammtion and oxidative stress. DAPT plays a nephronprotective role, and Notch signaling may be considered as a promising pharmacological target for AKI.2.Our results in vivo showed that, CS treatment could attenuate the severity of renal tubule damage, improve kidney function, lower the concentration of pre-inflammation factors(TNF-αand IL-6), down-regulate the protein expression of Notch2, hes-1 and MCP-1 (P<0.01), up-regulate the protein expression of HIF-1α(P<0.01) in the rats with renal IRI, which suggests that CS could protect against renal IRI by the inhibition of Notch2/hes-1 signaling activation, then represse the transcription of inflammation factors such as IL-6, TNF-α, MCP-1, upregulate the transcription of HIF-1α, inhibiting of the course of renal IRI associated inflammation and oxidative stress, Notch signaling maybe is its target.3. MTT examination showed that there was no significant difference in the integrated optical density (IOD) between normal group and DAPT group (from 0.1μmol/L to 10μmol/L in concentration) and CS group (from 10 mg/L to 40mg/L in concentration). However, DAPT (100μmol/L) and CS (100μmol/L) could significantly inhibt the proliferation of NRK-52E(P<0.01). Therefore, we choosed 10μmol/L and 40mg/L as treatment concentration of DAPT and CS respectively in the next experiment.4. Cell immunofluorescence and Western blot results in vitro showed that, I/R could induce the up-regulation in protein expression of Notch2, hes-1, MCP-1 and HIF-1αthat reached a peak at 24h after reperfusion, then gradually decreased at 48h and 72h. However, DAPT treatment could down-regulate the protein expression of Notch2, hes-1 and MCP-1 (P<0.01), up-regulate the protein expression of HIF-la(P<0.01), which suggests that I/R may induce the activation of Notch2/hes-1 signaling in NRK-52E cells that mediated the transcription of inflammation factors such as MCP-1 HIF-1α. Notch2/hes-1 signaling maybe is involved in the course of renal IRI associated inflammation and oxidative stress.5.Our results in vitro showed that, CS treatment could down-regulate the protein expression of Notch2, hes-1, NF-κB2 and MCP-1 (P<0.01), up-regulate the protein expression of HIF-la(P<0.01), which suggests that CS could inhibit the activation of Notch2/hes-1 signaling, then represse the transcription of inflammation factors such as MCP-1, Notch2/hes-1 signaling could be its target, and its nephroprotective mechanism is associated with the inhibition of inflammation and oxidative stress.Conclusion:1. Renal IRI up-regulates the expression of IL-6, TNF-α, MCP-1 and HIF-1α. Our work confirms that there are inflammation and oxidative stress following renal IRI.2. Renal I/R may induce the activation of Notch2/hes-1 signaling in the renal tubular cells that mediates the transcription of IL-6, TNF-α, MCP-1 and HIF-1α.3. CS treatment could attenuate the severity of renal tubule damage and kidney function, inhibit the activation of Notch2/hes-1 signaling down-regulate the transcription of inflammation factors such as IL-6, TNF-a, MCP-1, upregulate the transcription of HIF-la. Our work also confirms CS could provide a protection against renal IRI by anti-inflammation and anti- oxidative stress, and Notch signaling may be considered as its target.更多还原