【作者】 唐怡庭；

【导师】 陶立坚；

【作者基本信息】 中南大学 ， 肾脏病学， 2010， 博士

【摘要】 第一章氟非尼酮对肾纤维化炎症反应的影响研究背景：肾纤维化是各种肾脏疾病进展到终末期肾衰竭的共同途径和主要病理基础,其病变程度与慢性肾脏病的预后密切相关,如何防治肾间质纤维化成为目前肾脏病领域的研究热点之一。目前临床上针对抑制肾脏纤维化过程的药物很少,尚缺乏真正有效用于临床治疗的药物。氟非尼酮(AKF-PD)是本研究组合成得到的一种新的吡啶酮类化合物,已获得国家发明专利。在前期的研究中发现AKF-PD可抑制成纤维细胞增殖；对单侧输尿管结扎(UUO)模型大鼠肾脏病变有显著疗效。AKF-PD抗肾纤维化作用的机制尚处于研究之中,目前的研究结果显示AKF-PD能抑制NADPH氧化酶的表达和减少活性氧的产生,抑制肾小管细胞凋亡、转分化及成纤维细胞的增殖,下调致纤维化细胞因子的表达,减少病变肾脏基质胶原合成。尚未观察AKF-PD对肾纤维化早期炎症反应的影响。炎症反应作为肾脏纤维化的始动环节之一,具有非常重要的作用,此时肾单位损伤相对较少,且并无大量肌成纤维细胞的过度增殖,经过有效的修复,可以使受损的细胞恢复自主调节功能,阻止肾病的继续进展。目前国内外学者对肾纤维化炎症反应达成以下共识：(1)炎症反应是肾脏在各种损伤刺激下发生的早期事件(UUO术后4小时已有TNF-α的表达和巨噬细胞的浸润),肾脏炎症反应的迁延持续,最终促使肾纤维化的形成；(2)炎症细胞浸润在肾脏的慢性炎症和纤维化过程中发挥重要作用；(3)趋化因子及炎症因子是肾纤维化形成过程中炎症细胞浸润和慢性炎症持续存在的关键因素。针对肾纤维化的发病环节,国内外学者开展了大量抗肾纤维化药物的研究工作,其中PFD是新型的广谱抗纤维化复合物,能够防止甚止逆转肾纤维化发生,但其抗肾纤维化作用机制也处于研究之中,对肾纤维化早期炎症反应的影响尚未有文献报道；血管紧张素Ⅱ参与促进肾脏损伤后炎症细胞浸润,有报道血管紧张素转化酶抑制剂(ACEI)及血管紧张素受体拮抗剂(ARB)可减轻病变肾组织炎症反应。目的：观察AKF-PD对UUO大鼠肾脏组织炎症细胞浸润及趋化因子、炎症因子表达的影响；探讨AKF-PD对肾纤维化炎症反应的影响,从而进一步了解AKF-PD抗肾纤维化的机制。方法：以单侧输尿管结扎(UUO)方法建立肾纤维化模型。SD大鼠(75只)随机分为假手术组、UUO模型组、AKF-PD治疗组、吡非尼酮治疗组、氯沙坦治疗组；分别于UUO术后3天、7天、14天留取各组大鼠梗阻侧肾脏标本：14天各组肾脏标本分别行HE和MASSON染色观察大鼠肾纤维化成模情况及药物疗效；3天、7天各组肾脏标本分别制备石蜡切片行CD68、CD3标记的炎症细胞免疫组织化学染色,提取总RNA行real-time PCR检测趋化因子(MCP-1,RANTES, IP-10, MIP-1α, MIP-1β)和炎症因子(TNF-α, IL-1β) mRNA表达水平,提取总蛋白以ELISA方法检测上述趋化因子和炎症因子蛋白表达水平。结果：(1)HE和MASSON染色结果显示UUO术后14天模型组大鼠肾组织可见明显间质纤维化病理改变及大量蓝染的胶原纤维,肾间质损伤指数和肾间质胶原相对面积评分较假手术组明显上升,造模成功；与模型组比较,AKF-PD治疗组、PFD治疗组及氯沙坦治疗组肾组织间质纤维化程度减轻,蓝染的胶原纤维减少,肾间质损伤指数和肾间质胶原相对面积评分显著下降(P<0.05)；三种药物作用效果相似。(2)免疫组织化学染色结果显示UUO术后3天模型组可见巨噬细胞及淋巴细胞的浸润,术后7天浸润的巨噬细胞及淋巴细胞增多；AKF-PD、PFD及氯沙坦均能有效抑制UUO术后3天、7天病变肾脏巨噬细胞和淋巴细胞的浸润(P<0.05)。(3)UUO术后3天模型组各趋化因子及炎症因子mRNA和蛋白表达水平明显升高；AKF-PD治疗组、PFD治疗组及氯沙坦治疗组各趋化因子及炎症因子mRNA和蛋白表达水平较模型组显著下降(P<0.01)。且三种药物的作用相似。UUO术后7天模型组与UUO术后3天模型组比较,趋化因子及炎症因子的表达下降；药物对趋化因子及炎症因子的表达的影响有所不同：在mRNA水平,IP-10和MIP-1β组间差异无统计学意义(P>0.05); AKF-PD治疗组RANTES、MCP-1、MIP-1α、TNF-α较模型组显著下降(P<0.05)；PFD治疗组MCP-1、MIP-1α较模型组显著下降(P<0.05)；氯沙坦治疗组MCP-1较模型组显著下降(P<0.05),三种药物对IL-1β作用均不明显(与模型组比较P>0.05)。在蛋白水平,AKF-PD治疗组、PFD治疗组所有趋化因子及炎症因子较模型组显著下降(P<O.05)；氯沙坦治疗组RANTES、MCP-1、IP-10、MIP-1α较模型组显著下降(P<0.05)。结论：(1) AKF-PD具有抗肾间质纤维化的作用。(2) AKF-PD能下调病变肾脏组织趋化因子及炎症因子的表达,从而抑制了炎症细胞的浸润,有效减轻病变肾脏的炎症反应；提示这可能是其抗肾纤维化作用的重要机制之一。(3) AKF-PD、PFD及氯沙坦减轻病变肾脏的炎症反应的作用相似,但对UUO术后7天大鼠肾组织各种趋化因子及炎症因子的表达的影响有所不同。第二章氟非尼酮对内毒素血症小鼠死亡率及炎症因子的影响研究背景：氟非尼酮(AKF-PD)是本研究组合成得到的一种新的吡啶酮类化合物,具有较强的抗肾纤维化作用。目前AKF-PD抗纤维化的机制还不十分确切,前面的研究结果显示AKF-PD能够下调UUO模型大鼠病变肾脏趋化因子及炎症因子的表达,抑制炎症细胞的浸润,提示抑制炎症反应是其抗肾纤维化作用的重要途径之一。肾纤维化中的炎症反应是肾脏组织在各种损伤刺激下,以单核/巨噬细胞系统激活及炎症介质释放为主的缓慢发生、持续存在的局部炎症反应,有别于由感染引起的全身炎症反应综合症,即脓毒血症(sepsis)。脓毒血症的本质是致病原激活炎症相关转录因子,大量下游炎症介质基因过度表达,炎症免疫失控和免疫紊乱。AKF-PD对其影响尚不清楚。当微生物入侵人体,机体免疫防御系统会作出迅速而恰当的反应；免疫防御能力缺陷、反应过高或过低,都可导致脓毒血症的发生和发展。临床脓毒血症患者最常见的是革兰阴性(G-)菌感染。革兰阴性菌内毒素,即脂多糖(LPS)在启动体内免疫系统反应,导致中毒性休克中的重要性已得到普遍认识。LPS介导单核巨噬细胞激活,合成和释放各种炎症因子(如TNF-a和IL-1β),这些炎症因子能促进中性粒细胞与内皮细胞粘附,激活凝血系统,引起全身炎症反应综合征,严重者导致休克、多器官功能衰竭和死亡。炎症反应是损伤与抗损伤的统一过程,现已知机体在启动炎症反应的同时,抗炎症反应也同时发生,产生抗炎性介质等。IL-10是具有抗炎和抑制机体免疫力双重属性的细胞因子,一方面抑制炎性细胞(如单核细胞、淋巴细胞等)的粘附、浸润,另一方面抑制各种促炎性细胞因子的合成与分泌,有助于减轻炎症反应。由于操作的方便性,目前常以腹腔注射LPS建立的内毒素血症动物模型来研究脓毒血症的发生发展及其机制,尤其是在药物对脓毒血症的干预与治疗方面。目的：观察AKF-PD对内毒素血症模型小鼠死亡率的影响,探讨AKF-PD对内毒素血症机体的保护作用；在体内外水平观察AKF-PD对内毒素血症中促炎因子(TNF-a、IL-1β)及抗炎因子(IL-10)的影响,了解AKF-PD对内毒素血症影响的机制。方法：以LPS (15mg/kg)腹腔注射建立小鼠内毒素血症模型。Balb/c小鼠(120只)随机分为LPS模型组、AKF-PD (100mg/kg)治疗组、AKF-PD (200mg/kg)治疗组；AKF-PD治疗组采用两种方式给药：AKF-PD和LPS同时腹腔注射或LPS注射2小时后再予以AKF-PD腹腔注射。记录各组小鼠第1-7天存活只数。根据上述实验结果,Balb/c小鼠(30只)随机分为LPS模型组、AKF-PD (200mg/kg)治疗组,于2h、4h、8h后摘眼球取血(每个时间点每组5只),4℃离心取上清,ELISA方法检测血清中TNF-a和IL-1β浓度。获得原代巨噬细胞,设立正常对照组(N), LPS模型组(LPS)、AKF-PD(400μg/ml)组、AKF-PD(600μg/ml)组及AKF-PD(800μg/ml)组,24小时后ELISA方法检测各组细胞上清TNF-α、IL-1β和IL-10浓度；根据实验结果,设立LPS模型组和AKF-PD (800μg/ml)组,分别于不同时间点(3、6、12、16、24小时)收集细胞上清,ELISA方法检测各组TNF-α和IL-1β浓度。结果：在AKF-PD和LPS同时腹腔注射实验及LPS注射后2小时再予以AKF-PD腹腔注射实验中,AKF-PD(200mg/kg)均能降低内毒素血症模型小鼠的死亡率(P<0.05),AKF-PD(100mg/kg)对内毒素血症模型小鼠的死亡率无明显影响(P>0.05); AKF-PD在体内外均可抑制炎症因子TNF-α和IL-1β的表达释放(P<0.05),并且呈剂量和时间依赖性；但对抗炎因子IL-10的作用不明显(P>0.05)。结论：AKF-PD在体内外均可抑制促炎因子TNF-α和IL-1β的释放,降低内毒素血症模型动物死亡率,但对抗炎因子IL-10的作用不明显。提示AKF-PD通过抑制促炎因子TNF-α和IL-1β的释放的释放对内毒素血症模型动物起保护作用。更多还原

【Abstract】 Part 1 The effect of Fluorofenidone on inflammation in renal fibrosisBackground:Renal fibrosis is the common pathway and main pathogenic basis of end-stage of all kinds of renal disease. Moreover, renal fibrosis strongly correlates with progressive loss of renal function. Therefore, development of novel antifibrotic agent is of great importance. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD) is a novel pyridone agent which is developed recently. Our data showed that AKF-PD exerts a strong antifibrotic effect, as shown in experimental renal fibrosis in vivo and in vitro. However, the mechanism underlying the antifibrotic effect of AKF-PD in renal disease remains to be elucidated. Our recent results showed that AKF-PD significantly reduced the expression and activity of NADPH oxidase, and inhibited ROS generation, the apoptosis of renal tubular epithelial cell, as well as the proliferation of renal fibroblast. In addition, AKF-PD significantly downregulated the expression of pro-fibrotic cytokines, and consequent reduced interstitial matrix deposition. In recent year, inflammation is reported to be essential for the development of renal fibrosis. And The effect of Fluorofenidone on inflammation in renal fibrosis is still unkown.At the stage of renal inflammation, the loss of nephrons is relatively rare. And there is no proliferation of fibroblast cells. Effective repair can restore the impaired cells of normal function; therefore prevent the progress of nephritis. The current concepts of inflammation in renal fibrosis are following:(1) Inflammation is the early event in kidney upon any kind of harmful stimulation (The expression of TNF-a and infiltration of Macrophages can be observed in impaired kidney 4 hours after UUO); the persistence of renal inflammation can induce renal fibrosis finally. (2) The infiltration of inflammatory cells plays an important role in the process of chronic renal inflammation and renal fibrosis. (3) Chemokine and proinflammatory factor is the key factor, which mediates the infiltration of inflammatory cells and maintains the persistent state of chronic inflammation during the early stage of renal fibrosis.Focusing the pathology of renal fibrosis, scholars investigated extensively on the anti-fibrogenic drugs. PFD is a new broad-spectrum anti-fibrogenic compound, which can prevent and even reverse the renal fibrosis. However, the mechanism underlying its anti-fibrogenic effect is yet to be found. And whether PFD has an effect on inflammation of early stage of renal fibrosis is unknown. Angiotensin II is involved in promoting infiltration of inflammatory cells in injured kidney. And it is reported that angiotensin-converting enzyme inhibitor (ACEI) as well as angiotensin receptor blockade (ARB) can ameliorate inflammatory response in tissue of injured kidney.Objective:To investigate whether AKF-PD can ameliorate the infiltration of inflammatory cells and the expression of chemokine and proinflammatory factor after ureteral obstruction; to investigate the effect of AKF-PD on renal inflammation, in order to get the better understandings of the mechanism underlying the anti-fibrogenic effect of AKF-PD.Methods:(1) SD rats were submitted to unilateral ureteral obstruction (UUO) and studied after 3,7 or 14 days. Renal tissue after ureteral obstruction at day 14 was dyed by HE and Masson staining for general histology and collagen detection. For renal tissue after ureteral obstruction at day 3 and 7, immunostaining was used to assess the infiltration of macrophage (CD68) and lymphocyte (CD3). And ELISA and Real-time PCR were done to assess the expression of chemokines (MCP-1, RANTAS, IP-10, MIP-1α, MIP-1β) and proinflammatory factors (TNF-αand IL-1β).Result:(1) HE and MASSON staining revealed that there is significant interstitial pathological change in rat injured kidney, and huge amount of collagen fiber deposited in renal tissue was seen. The Tubulointerstitial damage index and relative area of renal interstitial collagen significantly increased in UUO model 14 days after operation. All of above observations indicate that the establishing of the UUO model is successful. Compared with UUO group, treatment of AKF-PD, PFD and losartan significantly ameliorated renal interstitial fibrosis, and significantly reduced the deposit of collagen fiber, renal interstitial injury index, as well as the relative area of renal interstitial collagen (P<0.05).The therapeutic effects of these three drugs are indistinguishable.(2) AKF-PD, PFD and losartan treatment significantly attenuated macrophage and lymphocyte infiltration in kidney after ureteral obstruction at day 3 and day 7 after ureteral obstruction. All three treatments have the similar inhibitory effect.(3) The expression of all the chemokines and proinflammatory factors is also significantly reduced by the treatment of AKF-PD, PFD and losartan at day 3 (P<0.05). All three treatments have the similar inhibitory effect.However, expression of chemokines and proinflammatory factors in rat kidney at day 7 after ureteral obstruction is lower than that of at day 3; AKF-PD, PFD and losartan differentially affect the expression of chemokines and proinflammatory factors in rat kidney at day 7 after ureteral obstruction:there is no significant difference in the mRNA lever of IP-10 and MIP-1βbetween the groups (P>0.05); AKF-PD treatment significantly reduced the mRNA lever of RANTES、MCP-1、MIP-1α、TNF-a(P<0.05); PFD treatment significantly reduced the mRNA lever of MCP-1、MIP-1α(P<0.05); whereas losartan treatment significantly reduced the mRNA lever of MCP-1 (P<0.05); all three drugs did not have significant effect on the mRNA expression of IL-1β(P>0.05). However, AKF-PD and PFD treatment decrease the protein lever of all kinds of chemokines and proinflammatory factors (P<0.05); whereas losartan treatment selectively decrease the protein lever of RANTES、MCP-1 IP-10、MIP-1α(P<0.05).Conclusion:(1) AKF-PD ameliorats renal fibrosis. (2) AKF-PD significantly attenuated macrophage and lymphocyte infiltration in kidney as well as the expression of chemokines and proinflammatory factors in unilateral ureteral obstruction (UUO) model, indicating that the beneficial effect of AKF-PD in renal interstitial fibrosis is attributable, at least in part, to an anti-inflammatory action.(3) AKF-PD, PFD and losartan have the similar anti-inflammation effect in renal fibrosis, but the effect on chemokines and proinflammatory factors in rat kidney at day 7 after ureteral obstruction is bit of different.Part 2 The effect of Fluorofenidone on lethal endotoxemia and inflammatory factorBackground:Fluorofenidone(1-(3-fluorophenyl)-5-methyl-2-(1 H)-pyridone, AKF-PD) is a novel pyridone agent which is developed recently. Our data showed that AKF-PD exerts a strong antifibrotic effect, as shown in experimental renal fibrosis in vivo and in vitro. However, the mechanism underlying the antifibrotic effect of AKF-PD in renal disease remains to be elucidated. Our results showed that AKF-PD significantly attenuated macrophage and lymphocyte infiltration in kidney as well as the expression of chemokines and proinflammatory factors in unilateral ureteral obstruction (UUO) model. However, the inflammation which results in renal fibrosis, is different from that of in sepsis.And The effect of Fluorofenidone on sepsis and inflammatory factor is unkown.Sepsis is the systemic inflammatory response syndrome (SIRS) induced by infection. The host defence system will response immediately and correctly to the presence of micro-organisms or tissue invasion by microorganisms. Immunodeficiency, immunological response too strong or two weak can result in the genesis and progress of sepsis. The most common cause of sepsis in clinic is the infection of gram negative bacteria. And It is the common concept that gram negative bacterial endotoxin (also referred to lipopolysaccharide (LPS)) plays an important role in initiation of immune response and pathogenesis of septic shock. LPS can stimulate monocyte/macrophage to express and release all kinds of proinflammatory factors (such as TNF-a and IL-1β). These proinflammatory factors can promote adhesion of neutrophils and endothelial cells, activate coagulant system, induce systemic inflammatory response syndrome, and even result in shock, multiple organ failure and death.At the same time, the anti-inflammatory response is induced, which result in the anti-inflammatory factors such as IL-10. IL-10 can inhibit macrophage/lymphocyte adhesion and infiltration as well as the expression of proinflammatory factors, which facilitate sepsis.Objective:To investigate whether AKF-PD can promote survival in animal model of established endotoxemia, and whether it can reduce the level of TNF-a, IL-1βand IL-10 in vivo and in vitro, which we can get a better understanding of the anti-inflammation effect of AKF-PD.Methods:Balb/c mice were injected i.p. with LPS (15mg/kg); Different dose of AKF-PD (100mg/kg or 200mg/kg) was administered i.p. concurrently with LPS injection or 2 hours after LPS injection. And animal survival was monitored for up to 7 days. Then mice were injected i.p. with LPS (15mg/kg), AKF-PD (200mg/kg) was administered i.p. concurrently with LPS injection; At dedicated time points(2h、4h、8h), serum was obtained and was assayed for TNF-a and IL-1βproduction by ELISA.In vitro experiments, primary mouse peritoneal macrophage were stimulated with LPS (0.5μg/ml) in the presence or absence of AKF-PD with a concentration range of 400-800μg/ml.24h after stimulation, supernatant was collected and the levels of TNF-α, IL-1βand IL-10 were determined by ELISA. In another set of experiment, primary mouse peritoneal macrophage were stimulated with LPS (0.5μg/ml) in the presence or absence of AKF-PD (800μg/ml). The supernatant was collected at the indicated time(3、6、12、16、24h) and the levels of TNF-a and IL-1βwere determined by ELISA.Result:In the model of murine endotoxemia, treatment of AKF-PD (200mg/kg) both at the beginning of LPS infusion and after the onset of endotoxemia(2h after LPS infusion) prevented the lethality of endotoxemia (P<0.05). A lower dose of AKF-PD (100mg/kg, i.p.) provided partial protection (P>0.05). Besides, AKF-PD treatment (200mg/kg, i.p.) reduced circulating levels of TNF-a and IL-1βin a time-dependent manner (P<0.05).In vitro, AKF-PD significantly inhibited TNF-a and IL-1βrelease in a dose-and time-dependent manner (P<0.05), with the maximal effect at concentration of 800μg/ml. However, AKF-PD did not affect IL-10 release (P>0.05).Conclusion:AKF-PD significantly inhibited the release of the proinflammatory cytokine (TNF-a and IL-1β) but not anti inflammatory cytokine (IL-10), and improves survival during lethal endotoxemia, indicating that this new pyridone agent may be a novel candidate for the treatment of sepsis.更多还原