Prevention of graft rejection and graft-versus-host reaction by a novel immunosuppressant, FTY 720, in rat small bowel transplantation

In the present study, we examined the immunosuppressive effect of a new drug, FTY 720, on small bowel transplantation (SBT) in rats. Grafts from (LEW × BN) F 1-to-LEW rats treated with FTY 720 at 0.5 mg/kg from day 0 to 14 post-SBT survived significantly longer than untreated grafts. In addition, the administration of FTY 720 combined with cyclosporin (CyA; 5 mg/kg per day) had a synergistic effect on allograft survival. The graft-versus-host reaction (GVHR) that occurred in the LEW-to-F 1 rats was markedly reduced after the administration of FTY 720. FTY 720 combined with a low dose of CyA completely abrogated GVHR without any adverse reaction. FTY 720 treatment resulted in a significant decrease in the number of lymphocytes in the peripheral blood and the spleen, but the number of peripheral neutrophils was unchanged. Thus, FTY 720 would appear to be an ideal drug to combine with CyA in order to control the immune reaction after SBT. Received: 19 February 1997 Received after revision: 23 May 1997 Accepted: 9 June 1997     

Effect of peritransplant FTY720 alone or in combination with post-transplant tacrolimus in a rat model of cardiac allotransplantation

FTY720 is a recently discovered compound that is derived from the fungus Isaria sinclairii. Using a DA donor-to-LEW recipient rat combination, we assessed the efficacy of peritransplant FTY720 alone or in combination with post-transplant tacrolimus on the survival of cardiac allografts. Peritransplant FTY720 given orally at a dose of 5 mg/kg on days –1 and 0 prolonged graft survival from 5 to 13 days (P < 0.05). Combining peritransplant FTY720 with post-transplant tacrolimus resulted in a further prolongation of allograft survival. The lymphocyte count in transplanted rats decreased within 24 h to 46.6 %. Analysis of lymphocyte subsets by FACS revealed that FTY720 affected the total population of CD3-bearing T cells while the ratio of CD4 to CD8 cells remained unchanged. Kidney and liver biochemistry remained elevated for 2 weeks. In conclusion, FTY720 is a powerful immunosuppressive agent when used as induction therapy and may have an additive effect – perhaps a synergistic one – with post-transplant tacrolimus. Received: 27 October 1997 Received after revision: 23 March 1998 Accepted: 15 April 1998     

A new immunosuppressant, FTY720, in canine kidney transplantation: effect of single-drug, induction and combination treatments

Three different types of treatment were conducted to clarify the properties of a novel immunomodulator, FTY720, in canine kidney allograft models. Survival, biochemical and hematological tests, pharmacokinetics, and histopathology of grafts and extra-renal organs were analyzed. Accompanying a remarkable reduction in circulating lymphocytes, single-drug treatment of FTY720, ranging from 0.05 to 10 mg/kg, exhibited significant prolongation of graft survival without a dose-dependent effect. Short-course induction with FTY720 at 5 mg/kg per day exhibited similar anti-rejection effects as did single-drug treatment but no advantage in rescuing ongoing rejection. In combination with cyclosporine (CsA; 5 mg/kg) or tacrolimus (FK; 0.5 mg/kg), FTY720 had an additive effect. Trough blood concentrations of FTY720 were linearly correlated with dose. No animal showed critical adverse effects at any point. FTY720 holds promise as a candidate in a new category of drugs that can be combined with conventional agents for induction and maintenance immunosuppression in clinical organ transplantation.     

FTY720 Prevents Anti-CD4 mAb-Induced Tolerance but Cannot Reverse Established Tolerance in a Rat Kidney Transplantation Model

FTY720 is highly effective in various models of transplantation and autoimmunity. In order to find drugs that act synergistically with a tolerance-inducing nondepleting anti-CD4 mAb we studied this combination in a strong DA to LEW kidney transplantation model. Rats were treated with 0.3 mg/kg of FTY720 for 14 days and anti-CD4 mAb RIB5/2, alone or in combination. After kidney transplantation serum creatinine and blood lymphocyte counts were monitored. Immunohistology, ELISPOT and TaqMan trade mark -PCR analysis of biopsies were performed. Short-term application of RIB5/2 but not FTY720 induced long-term survival of kidney transplants. Moreover, the combination of FTY720 + RIB5/2 prevented tolerance induction. In the combination group serum creatinine levels increased 1 week after cessation of therapy and all rats died from uremia within 72 days. Intragraft immunohistology, ELISPOT and real-time RT-PCR analysis at day 21 demonstrated an enhanced T-cell infiltration and activation but a diminished up-regulation of protective genes in the grafts from recipients receiving the combination therapy. In contrast, delayed application of FTY720 to RIB5/2-treated rats did not interact with RIB5/2-induced tolerance. In summary, FTY720 is powerful in preventing intragraft infiltration by naive T cells but this might also affect the early development of graft-protecting regulatory T cells and tolerance induction.     

FTY720介导淋巴细胞凋亡抑制小肠移植的移植物抗宿主免疫反应

目的 探讨免疫调节药物FTY720对小肠移植后急性移植物抗宿主病（GVHD）的治疗效果及其作用机制.方法 应用Wistar-Furth（WF）大鼠作为供体,WF和ACI大鼠的子代（F1）作为受体,同种异基因异位全小肠移植的技术方法建立GVHD的动物模型.移植受体分为实验组和对照组,每组6只.实验组从移植手术当日开始予以FTY720治疗,持续14 d;对照组在相同的时间段口服蒸馏水.术后第15天,提取受体靶器官肝脏、小肠及移植物小肠的淋巴细胞,应用免疫组织化学（免疫组化）TUNEL法和流式细胞仪检测两组淋巴细胞凋亡的变化.结果 对照组大鼠术后均死亡于GVHD,平均生存时间（16.0±1.7）d,实验组大鼠均长期成活超过100 d,两组差异具有统计学意义（P＜0.01）.免疫组化TUNEL法检测结果显示,实验组肝脏和移植物小肠黏膜的淋巴细胞凋亡比率均明显高于对照组,差异具有统计学意义（P＜0.05）.流式细胞技术分析结果显示,实验组大鼠移植物小肠黏膜内凋亡的淋巴细胞百分比为19.4%,明显高于对照组的11.8%（P＜0.05）;而肝脏凋亡的淋巴细胞百分比两组差异无统计学意义（P＞0.05）.结论 FTY720可能通过诱导淋巴细胞的凋亡,减少和抑制GVHD对靶器官的损害,改善移植大鼠的预后.     

FTY720诱导大鼠心脏移植物长期存活

目的 观察FTY720对大鼠同种异体心脏移植物存活时间的影响。方法 进行SD Wistar大鼠的腹部异位心脏移植，将受者随机分为对照组、甲泼尼龙(MP)组、环孢素A((SsA)组、FTY720组、FTY720与CsA二药联用组和FTY720、CsA及MP三药联用组，各组按分组要求分别于术前3d至术后14d通过灌胃给予FTY720和CsA，术前1d至术后2d腹腔注射给予MP，观察各组动物术后外周血淋巴细胞数量变化和移植物存活时间。结果 FTY720组、二药联用组和三药联用组的大鼠外周血淋巴细胞在给药后3h开始明显下降，停药后开始回升，至停药14d后恢复正常；移植心脏的存活时间，对照组平均为7．8d，CsA组为16．0d，MP组为27．6d，三药联用组为16．8d，而FTY720组和二药联用组分别超过了150d和124d。结论 FTY720可诱导同种异体大鼠心脏移植物长期存活。     

Effect of FTY720 on immunoregulation in concordant xenotransplantation

Abstract The present study was designed to analyze the immunosuppressive activity of FTY720 in concordant xenotransplantation. When T and B lymphocytes of human peripheral blood were incubated with FTY720, the number of viable cells decreased in a dose-dependent manner at doses higher than 4×10^-5 M. DNA fragmentation was observed at doses higher than 4×10 ^-5 M in B cell-rich fractions. These data demonstrate that FTY720 is cytotoxic to B lymphocytes as well as T lymphocytes and apoptosis may play an important role in this cytotoxicity. Golden Syrian hamsters were the donors and Lewis rats the recipients of skin grafts. The recipients were divided into the following four groups; (1) untreated recipients, (2) FTY720 (5mg/kg per day) was administered orally for 8 days (days -1–6), (3) FK 506 (1 mg/kg per day) was injected i. m. for 7 days (days 0–6), and (4) FK506 (1 mg/kg per day) was injected i.m. for 7 days (days 0–6) and FTY720 (5 mg/kg per day) was administered orally for 8 days (days-1–6). The mean graft survival times in groups 1–4 were.7 ± 0.52 days ( n = 6), 12.0 ± 0.71 days ( n = 6), 13.2 ± 1.6 days ( n = 6), and 37.7 ± 4.3 days ( n = 6), respectively. There was a significant difference in the mean survival time between groups one and four. Combined therapy with FTY720 and FK506 is a useful tool for immunoregulation in xenotransplantation.     

FTY720在鼠类异种胰岛移植排斥反应中的作用

目的 在小鼠异种胰岛细胞移植模型上，研究FTY720在控制异种胰岛移植排斥反应中的作用。方法 使用胰管内胶原酶注射和不连续密度梯度纯化法获取大鼠胰岛，建立小鼠肾被膜下移植模型。受体小鼠随机分为3组：对照组，末使用任何免疫抑制药物；实验1组，自移植当日起每日单独喂服FTY720(1.0mg／kg)；实验2组，亦于移植当日起每日联合喂服FTY720(1.0mg／kg)和环孢霉素A(CsA，15mg／kg)，均连续喂饲14d。分别于术后第3，5，7，14天切取移植物，观察并分析排斥反应。结果 对照组和实验1组，植入。肾被膜下的胰岛组织多在1周内完全被排斥，术后第7天胰岛轮廓消失，仅见大量淋巴细胞浸润。实验2组于移植后第7天及第14天肾被膜下仍可见大量完整的胰岛细胞，几乎未见或偶尔可见淋巴细咆浸润。结论 FTY720单独作为免疫抑制剂并不能抑制鼠类异种胰岛移植的排斥反应；联合应用FTY720及CsA则可有效地抑制鼠类异种胰岛移植排斥反应的发生。     

The Effect of a Novel Immunosuppressant, FTY720, in Mice Without Secondary Lymphoid Organs

Purpose FTY720 is a novel immunosuppressive agent that is thought to reduce the number of peripheral blood lymphocytes (PBL) by directing them toward secondary lymphoid organs such as the lymph nodes and Peyer’s patches. We studied the effects of FTY720 on aly/aly mice that do not have either lymph nodes or Peyer’s patches, as well as on splenectomized aly/aly mice.Methods FTY720 was orally administered by gavage (1 mg/kg) to aly/aly mice as well as to aly/+ mice with and without a splenectomy on 14 consecutive days. The number of lymphocytes was then counted using True Cell beads and flow cytometry. The number of B220-, CD3-, and CD4-positive cells was also determined. In addition, skin grafts from C3H donor mice were performed on these mice.Results FTY720 was effective in significantly reducing the total lymphocyte count as well as the B220-, CD3-, and CD4-positive subtypes in the peripheral blood of aly/+ mice as well as in aly/aly mice with and without a splenectomy. While we did observe allograft skin graft rejection in both the aly/+ mice as well as the aly/aly mice recipients and splenectomized aly/aly mice, the graft survival was prolonged in all groups. The skin allografts treated by FTY720 thus demonstrated fewer lymphocytic cells and less infiltration of CD4-positive cells.Conclusions The administration of FTY720 to mice without lymph nodes, Peyer’s patches, or spleens still results in peripheral lymphopenia. In all groups, FTY720 was found to prevent the infiltration of CD4-positive cells in skin allografts while also prolonging skin allograft survival. The fate of these lymphocytes, however, is unclear.     

The thymus is required for the ability of FTY720 to prolong skin allograft survival across different histocompatibility MHC barriers

The immunosuppressive effect of FTY720 is associated with the reversible sequestration of lymphocytes from the blood and the spleen into secondary lymphoid organs and reduced egress of mature thymocytes from the thymus. This work was designed to dissect the differential effect of FTY720 on CD4 and CD8 T cell-mediated mechanisms of skin graft rejection in the presence (euthymic) or absence (thymectomized) of thymic output. To that end, untreated and FTY720-treated euthymic (Euthy) and thymectomized (ATX) mice received skin allografts across a full, class II or class I major histocompatibility complex (MHC) mismatched (MM) barriers and graft survival was monitored. We demonstrate that a short course of FTY720 treatment significantly augments the survival of full, class I and class II MHC MM skin grafts compared to the nontreated controls. Interestingly, FTY720-treated Euthy recipients showed a significantly prolonged skin allograft survival compared to FTY720-treated ATX mice. These results together show that FTY720 impairs both CD4 and CD8 T cell-mediated mechanisms of rejection and, more importantly, the presence of the thymus is necessary for the ability of FTY720 to modulate skin allograft rejection across different histocompatibility MHC barriers.     

FTV 720A mediates reduction of lymphocyte counts in human renal allograft recipients by an apoptosis‐independent mechanism

Abstract The novel immunosuppressive compound FTY 720A posseses a mode of action which is different from all other immunosuppressive drugs. The most prominent feature is a reversible decrease in peripheral lymphocyte counts observed in animal experiments. We investigated in the first human trial (phase 1) whether FTY 720A induces apoptosis of peripheral blood mononuclear cells (PBMC) in stable renal allograft recipients. Monitoring of lymphocyte counts revealed a significant and dose‐dependent decrease within 6 h post‐FTY 720A dose: placebo 5.1%; 0.25 mg 36.4%; 0.5 mg 40.8%; 0.75 mg 39.4%; 1 mg 45.8%; 2 mg 67.2%; 3.5 mg 64.9%. PBMC apoptosis rates did not change, as determined before intake of FTY 720A and 2 h, 6 h, 24 h and 96 h post‐FTY 720A dose. We detected no significant difference in apoptosis rates between patients who received placebo or FTY 720A. However, in vitro experiments showed that high concentrations of FTY 720 A induced apoptosis in human PBMC.     

FTY720预防大鼠肾脏缺血再灌注损伤的实验研究

目的:研究新型免疫抑制剂FTY720对大鼠肾脏缺血再灌注损伤(IRI)的预防作用.方法:制备大鼠肾脏IRI模型,从下腔静脉注入不同剂量的FTY720,观察术后第1、2、3、5、7 d血清肌酐值(Scr)和术后第2、7 d外周血淋巴细胞数(PLC)的变化,并在术后第2 d取肾脏作组织学检查观察急性肾小管坏死的情况.结果:FTY720处理组动物术后Scr水平显著低于对照组并呈剂量依赖性;FTY720处理组术后第2 d的PLC显著低于对照组;组织学检查显示FTY720处理组肾脏的缺血性损伤轻于对照组.结论:FTY720可以减少PLC,对大鼠肾脏IRI有预防作用.     

Prolongation of canine liver allograft survival by a novel immunosuppressant, FTY720: effect of monotherapy and combined treatment with conventional drugs

BACKGROUND: The immunosuppressive effect and other properties of a novel immunosuppressant, FTY720, have been studied mostly in the experimental transplantation of various extrahepatic organs. In this experiment, we evaluated the antirejection potency and adverse effects of this agent on liver grafts using a canine liver transplantation model. METHODS: Forty-eight orthotopic liver transplantations were performed by the standard technique under a veno-venous bypass. Liver recipients were divided into two studies: a single-dose study with FTY720 at various doses and a combined dose study with conventional immunosuppressants (cyclosporine or tacrolimus) alone and combined with FTY720. Survival, biochemical and hematological tests, blood levels of immunosuppressants, and postmortem histology were determined. RESULTS: The median survival of untreated control animals was 9 days, whereas treatment with FTY720 at a dose of 0.1 mg/kg/day prolonged graft survival to 49.5 days. FTY720 at 1 mg/kg/day showed a slight but insignificant prolongation to 16 days, but when the dose was increased to 5 mg/kg/day, the graft was rejected at 10 days. The combination of FTY720, 0.1 mg/kg/day, with a subtherapeutic dose of cyclosporine, 5 mg/kg/ day, prolonged median animal survival from 40 days with cyclosporine alone to 74 days. A combination of FTY720 (0.1 mg/kg/day) with tacrolimus (0.5 mg/kg/ day) compromised animal survival, reducing survival from 83.5 days with tacrolimus alone to 30.5 days due to infectious complication and emaciation by overimmunosuppression. No evident drug-induced side effects were observed. CONCLUSIONS: FTY720 has a potent immunosuppressive effect when used alone at 0.1 mg/kg/day in canine liver transplantation. FTY720 is a promising candidate for future clinical application in orthotopic liver transplantation.     

FTY720,40-氧-(2-羟乙基)-雷帕霉素和环孢素A延长小鼠心脏移植物存活时间的对比研究

目的 FTY720和40-氧-(2-羟乙基)-雷帕霉素(RAD)是两种新型免疫抑制剂。为评估这两种药物的效果,我们以小鼠心脏移植为模型,对比观察了这两种药物和环孢素A(CsA)对移植物存活时间的影响。方法 供体为BALB/C小鼠,受体为C57BL/6小鼠,受鼠随机分为4组,每组6只:A组空白对照;B组每日管饲CsA 10 mg/kg体重;C组每日管饲RAD 3 mg/kg体重;D组每日管饲FTY 3 mg/kg体重。各服药组均由移植当日开始喂药,至移植心停跳或术后14 d停药。结果 各组的存活天数分别为:A组6、7、8、8、9、9;B组9、9、10、11、12;C组10、12、13、13、14;D组10、12、16、16、17、18。B、C组各有1例带心死亡。所有移植心都出现程度不同的急性排斥反应征象。结论 单独使用RAD或FTY可显著延长小鼠心脏移植物的存活时间。在各所选剂量上,CsA和RAD差异无显著性(P>0.05),FTY的效果明显优于CsA和RAD。FTY和RAD是较有前途的新型免疫抑制剂。     

Immunosuppressive activity of FTY720, sphingosine 1-phosphate receptor agonist: I. Prevention of allograft rejection in rats and dogs by FTY720 and FTY720-phosphate

FTY720, a new class of immunomodulator, induces lymphopenia by sequestration of circulating lymphocytes into secondary lymphoid tissues. FTY720 at 0.1 to 1 mg/kg significantly prolonged the allograft survival in a dose-dependent manner and showed a marked synergistic effect in combination with cyclosporine (CsA) in rat skin and cardiac allograft models. In addition, the canine renal allograft survival was significantly prolonged by combination therapy with FTY720 at 0.03 to 1 mg/kg and CsA at 10 mg/kg as compared with monotherapy of FTY720 or CsA. By contrast, the combination therapy with CsA and azathioprine or CsA and mycophenolate mofetil resulted in only an additive effect in rat skin allograft. When FTY720 was administered to rats, FTY720 was metabolized by omega-oxidation of the octyl side chain, and beta-oxidation subsequently, or phosphorylated by sphingosine kinase. Omega- and beta-oxidized 4 metabolities of FTY720 at 10 mg/kg i.v. showed neither lymphopenia nor immunosuppressive activity in rat skin allograft. On the other hand, (S)-enantiomer of FTY720-phosphate at 0.1 and 1 mg/kg intravenously induced a marked lymphopenia and significantly prolonged the allograft survival in the rat allotransplantation. From these results, it is suggested the lymphopenia and the immunosuppression induced by FTY720 administration is due to the agonistic activity against SIP receptors of the active metabolite, (S)-FTY720-phosphate.     

Preoperative administration of FTY720 prolonged renal allograft survival

BACKGROUND: FTY729 is an immunomodulator obtained by chemical modification of Myriocin(ISI-1) which exists in the culture filtrate of an ascomycete, Isaria sinclairii. It has been reported that postoperative administration of FTY720 prolonged survival of various kinds of transplanted organs. In the present study, we evaluated the effect of 2-day preoperative administration of FTY 720 on graft survival. MATERIALS AND METHODS: We used a rat renal transplantation model in which Wistar King Aptekman Hokkaido (WKAH, RT1K) served as the organ donor and Lewis (LEW, RTl) as the recipient. FTY720 was given to the recipients consecutively 2 days (day-2, day-1) before transplantation at the doses of 1, 3 or 5 mg/kg/day. Renal allograft survivals, hematological parameters of recipient blood and phenotypic analysis of recipient splenic cells and graft infiltrate were evaluated. RESULTS: Consecutive 2-day preoperative oral administration of FTY 720 at the doses of 1, 3 or 5 mg/kg/day significantly prolonged WKAH allograft survivals compared with those of the untreated recipients. The number of peripheral blood lymphocytes was markedly decreased in the recipients treated with FTY720 at the doses of 3 mg/kg/day or 5 mg/kg/day on the 5th postoperative day. Preoperative FTY 720 administration significantly decreased the number of CD4 positive cells and the percentage of interleukin 2 receptor (IL-2 R) positive cells infiltrating both spleen and allograft at the dose of 3 mg/kg/day or 5 mg/kg/day. CONCLUSION: FTY 720 could act as a safe and potent immunomodulator by decreasing the number of peripheral lymphocytes, especially CD4 positive cells and IL-2R positive cells when it is given to the recipient preoperatively.     

转化生长因子-β1转基因联合FTY720对大鼠心脏移植物缺血-再灌注损伤的影响

目的 探讨经冠脉灌注转化生长因子(TGF)-β1基因联合FTY720对移植心脏缺血-再灌注损伤(IRI)的影响及其机制.方法 实验分为空白对照组、空载体组、FTY720组、转基因组和转基因+FTY720组.心脏移植8 h后切取移植心脏,免疫组织化学检测mTGF-β1、ICAM-1、核转录因子(NF)-κB表达;逆转录-聚合酶链反应(RT-PCR)检测mTGF-β1 mRNA转录强度;测定超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、髓过氧化物酶(MPO)活性.结果 mTGF-β1成功转到心肌细胞,转基因组和转基因+FIY720组mTGF-β1的表达强度分别是(1.08±0.24)和(1.16±0.22),明显高于其他3组(P<0.01);而两组ICAM-1的表达积分分别是(2.43±0.46)和(1.90±0.20),NF-κB的表达积分分别是(9.80±1.85)和(10.10±2.27),较其他3组显著下调(P<0.01);FRY720组、转基因组和转基因+FTY720组心肌细胞凋亡指数分别是(9.20±1.12)、(5.90±1.09)和(5.40±0.77),显著减少(P<0.01);FTY720组、转基因组和转基因+FTY720组SOD活性分别是(51.03±5.54)、(55.91±6.66)和(73.42±6.42)U/mg,明显升高(P<0.01),MDA含量(10.90±1.93)、(11.02±2.45)和(9.28±1.64)U/g,明显下降(P<0.01);而MPO活性分别是(4.38±1.43)、(4.63±1.04)和(3.16±0.64)U/g,亦明显下降(P<0.01);转基因和FTY720两因素对减轻心肌细胞凋亡以及对SOD、MDA和MPO的影响存在交互作用(P<0.05).结论 TGF-β1和FTY720均具有减轻移植心脏缺血-再灌注损伤的作用,机制可能与抑制心肌细胞凋亡、下调ICAM-1、NF-κB表达、增高SOD活性等因素有关;两者联合应用在减轻缺血-再灌注损伤方面有协同作用.     

Sphingosine 1-phosphate receptor modulators: a new class of immunosuppressants

In this review, we summarize how FTY720 came from the lab bench to the bedside by examining its structural similarities to natural occurring sphingosine analogues, the mechanism of action, and clinical applicability to not only transplantation but also autoimmune, oncological, and neurobiological fields. FTY720, a sphingosine 1-phosphate (S1P) analogue, promotes the survival of human and animal allografts by sequestering T lymphocytes within peripheral lymphoid tissue. The mechanism of sequestration is three-fold: (1) T lymphocytes are driven into peripheral lymph nodes in a chemokine dependent manner by FTY720; (2) FTY720 downregulates sphingosine 1-phosphate receptors (S1PRs) on the T lymphocyte surface, rendering it unable to migrate along a S1P gradient; and (3) FTY720 closes stromal gates on the abluminal side of the lymphatic endothelium. Future areas of investigation include developing S1P analogues that have specific agonist binding to S1PRs avoiding side effects seen in non-specific binding.     

FTY720联合雷帕霉素抗胰腺癌细胞增殖作用的体外研究

目的 探讨FTY720和雷帕霉素联合用药体外抗胰腺癌细胞增殖的相互作用.方法 采用Panc-1和AsPc-1胰腺癌细胞株作为体外研究模型.以不含FTY720或雷帕霉素培养液处理的细胞株为对照组,FTY720单独用药的浓度范围为1～15 μmol/L;雷帕霉素单独用药的浓度范围为0.002～200 μmol/L,联合用药方案采用两组浓度的雷帕霉素联合7组不同浓度的FTY720;或者采用两组浓度的FTY720联合5组不同浓度的雷帕霉素.采用MTT法评估药物对细胞增殖的抑制率,采用双变量相关性分析法统计药物剂量与细胞增殖抑制率之间的相关性.结果 单独用药作用下,FTY720或雷帕霉素用药剂量与胰腺癌细胞增殖的抑制率呈现剂量依赖性（FTY720＋AsPc-1：r=0.887,P=0.000;雷帕霉素＋AsPc-1：r=0.822,P=0.000 ;FTY720＋Panc-1：r=0.796,P=0.000;雷帕霉素＋Panc-1：r=0.786,P=0.000）.当10μmol/L FTY720和0.002 μmol/L雷帕霉素联用时,对AsPc-1细胞增殖的抑制率达50%（P=0.000）,对Panc-1细胞增殖的抑制率达40%（P=0.000）,两药联用具有协同作用.结论 FTY720及雷帕霉素在体外均能呈剂量依赖性地抑制胰腺癌细胞增殖,联合用药后能协同抑制胰腺癌细胞的增殖.     

FTY720 attenuates tubulointerstitial inflammation and fibrosis in subtotally nephrectomized rats

Abstract

Tubulointerstitial fibrosis is a common pathway that leads to kidney failure, and persistent tubulointerstitial inflammation is a key event in the development of tubulointerstitial fibrosis. The new immunosuppressive drug FTY720 modifies lymphocyte migration into injured tissues by sequestering lymphocytes within secondary lymphoid organs. However, its therapeutic effect on tubulointerstitial inflammation and fibrosis had not been well understood. This study was designed to explore the effect of FTY720 on tubulointerstitial inflammation and fibrosis in subtotally nephrectomized (SNX) rats. In total, 24 male Sprague–Dawley rats were used. Seven days after 5/6 nephrectomy, rats were randomized to FTY720 (1?mg/kg/d) and placebo-treated groups. Sham-operated rats served as controls. FTY720 significantly attenuated the rise in proteinuria, serum creatinine, urea nitrogen and N-acetyl-β-D-glucosaminidase activity in SNX rats, and reduced the count of peripheral white blood cells and lymphocytes in SNX rats. Morphological analysis revealed that there was severe tubulointerstitial inflammation and fibrosis in SNX group and much more tubulointerstitial infiltrating inflammatory cells with high expression of CD3, CD4, CD8, CD20, CD68, CD163 and CCR-7 in SNX group, as compared with the controls, but the lesions were attenuated significantly by treatment with FTY720. Furthermore, the expressions of proinflammatory molecules (IL-6, TNF-α and MCP-1), profibrotic molecule (TGF-β1) and production of extracellular matrix proteins such as fibronectin and types I and III collagens were upregulated in SNX rats. FTY720 administration significantly reduced these abnormalities. In summary, FTY720 exerts therapeutic effects on tubulointerstitial fibrosis in SNX rats by inhibiting the tubulointerstitial inflammatory response.