Activation of ERK or inhibition of JNK ameliorates H(2)O(2) cytotoxicity in mouse renal proximal tubule cells

BACKGROUND: Our previous studies suggest that the balance between the activation of extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal/stress-activated protein kinase (JNK) might determine cell fate following oxidant injury in vivo. METHODS: The mouse proximal tubule cell line (TKPTS) was used to study hydrogen peroxide (H(2)O(2))-induced death and survival. The role of ERK and JNK in this process was studied by using adenoviruses that contain either a constitutively active mitogen-activated protein kinase kinase 1 (MEK1) or a dominant-negative JNK. Acridine orange plus ethidium bromide staining was applied to distinguish between viable, apoptotic, and necrotic cells following H(2)O(2) treatment. We analyzed cell cycle events by fluorescence-activated cell sorter (FACS) analysis and the phosphorylation status of ERK and JNK by Western blotting. RESULTS: TKPTS cells survived a moderate level of oxidative stress (0.5 mM/L H(2)O(2)) via temporary growth arrest, while high dose of H(2)O(2) (1 mM/L) caused extensive necrosis. Survival was associated with activation of both ERK and JNK, while death was associated with JNK activation only. Prior adenovirus-mediated up-regulation of ERK or inhibition of JNK function increased the survival (8- or 7-fold, respectively) of TKPTS cells after 1 mmol/L H(2)O(2) treatment. Interestingly, ERK activation and, thus, survival was associated with growth arrest not proliferation. CONCLUSION: We demonstrate that oxidant injury-induced necrosis could be ameliorated by either up-regulation of endogenous ERK or by inhibition of JNK-related pathways. These results directly demonstrate that the intracellular balance between prosurvival and prodeath mitogen-activated protein kinases (MAPKs) determine proximal tubule cell survival from oxidant injury and reveal possible mediators of survival.     

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway

BACKGROUND: Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). METHODS: Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. CONCLUSIONS: Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.     

Genetic or pharmacologic blockade of EGFR inhibits renal fibrosis

Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-β1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-β1- or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.     

Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity

Cyclosporin A is an immunosuppressant drug widely used in solid organ transplantation, but it has nephrotoxic properties that promote oxidative stress. The JAK2/STAT pathway has been implicated in both cell protection and cell injury; therefore, we determined a role of JAK2 in oxidative stress-mediated renal cell injury using pathophysiologically relevant oxidative challenges. The AG490 JAK2 inhibitor and overexpression of a dominant negative JAK2 protein protected endothelial and renal epithelial cells in culture against peroxide, superoxide anion and cyclosporin A induced cell death while reducing intracellular oxidation in cells challenged with peroxide and cyclosporin A. The decrease in Bcl2 expression and caspase 3 activation, induced by oxidative stress, was prevented by AG490. In mouse models of ischemia/reperfusion and cyclosporin A nephrotoxicity, AG490 decreased peritubular capillary and tubular cell injury. Our study shows that JAK2 inhibition is a promising renoprotective strategy defending endothelial and tubular cells from cyclosporin A- and oxidative stress-induced death.     

Phosphoinositide 3-kinase inhibitors protect mouse kidney cells from cyclosporine-induced cell death

The use of cyclosporine has been restricted by its nephrotoxic effects mediated, in part, by reactive oxygen species (ROS). Phosphoinositide 3-kinase, protein kinase B, and extracellular regulated kinase (ERK) pathways are related to survival and cell death and are activated after ROS generation. In this study, we evaluated the effects of cyclosporine on these pathways and their contribution to cyclosporine-induced toxicity. Viability of cells derived from the proximal tubule of transgenic mice was measured with Trypan Blue, ROS generation by a fluorescent probe, while ERK and phosphoinositide 3-kinase/protein kinase B activation were monitored with phospho-specific antibodies. Cyclosporine decreased cell viability and induced ROS generation and ERK and phosphoinositide 3-kinase activation. Both pathways were activated by the epidermal growth factor receptor (EGFR). Antioxidants blocked ERK activation but failed to inhibit protein kinase B phosphorylation or prevent cyclosporine toxicity. ERK inhibition did not protect from cyclosporine-induced cell death. EGFR or phosphoinositide 3-kinase inhibitors protected from cyclosporine-triggered cell death without decreasing ROS. Small interfering RNA against the catalytic subunit of phosphoinositide 3-kinase decreased protein kinase B phosphorylation but did not prevent cyclosporine-mediated cell death. Our results show that EGFR mediates the cytotoxic effects of cyclosporine through an ROS-independent mechanism. Cyclosporine-induced cell death is triggered by a non-classical phosphoinositide 3-kinase and does not require ERK activation.     

Albumin activates ERK via EGF receptor in human renal epithelial cells

Emerging clinical and experimental evidence strongly implicates proteinuria in the progression of kidney disease. One pathway involves the activation of NFkappaB by albumin, and it has been demonstrated that the activation of NFkappaB induced by albumin is dependent on mitogen-activated protein kinase ERK1/ERK2. To study the effect of albumin on gene expression, primary human renal tubular cells were exposed in vitro to albumin (1%) for 6 h, and gene expression profiling was performed with the human oligonucleotide microarray, U133A Affymetrix Gene Chip. In all, 223 genes were differentially regulated by albumin, including marked upregulation of the EGF receptor (EGFR) and IL-8. Accordingly, the authors sought to delineate the signaling pathway linking albumin to the EGFR and activation of ERK1/ERK2. It was found that albumin led to a dose- and time-dependent activation of ERK1/ERK2. Treatment with albumin led to EGFR phosphorylation, but the activation of ERK1/ERK2 was prevented by pretreatment of the cells with AG-1478, the EGFR kinase inhibitor, at a dose that inhibited EGF-induced ERK1/ERK2 activation. Exogenously administered reactive oxygen species (ROS) were found to activate ERK1/ERK2 via the EGFR and src tyrosine kinase activity and pretreatment of cells with the antioxidant N-acetylcysteine (NAC) and the NADPH oxidase inhibitor DPI abrogated albumin-induced activation of ERK1/ERK2. The src tyrosine kinase inhibitor, PP2, also inhibited the albumin-induced activation of ERK1/ERK2. Finally, pretreatment with AG-1478, the MEK inhibitor UO126, and NAC prevented the albumin-induced increase in IL-8 expression. The authors conclude that the EGF receptor plays a central role in the signaling pathway that links albumin to the activation of ERK1/ERK2 and increased expression of IL-8. Gene profiling studies suggest that there may be a positive feedback loop through the EGFR that amplifies the response of the proximal tubule cell to albumin. Taken together, these results suggest that the EGFR may be an important treatment target for kidney disease associated with proteinuria.     

JAK-STAT通路在小鼠单侧输尿管梗阻模型肾间质纤维化中的作用

目的 探讨Janus蛋白酪氨酸激酶-信号转导子和转录激活子(JAK-STAT)通路在小鼠单侧输尿管梗阻(UUO)模型.肾间质纤维化过程中的作用.方法 选用30只雄性Balb/c小鼠建立小鼠UUO模型(n=24)和假手术小鼠(n=6),术后第1、4、7和14天检测JAK-STAT磷酸化情况.另把18只雄性Balb/c小鼠随机分为假手术组、UUO模型组和治疗组,每组各6只.治疗组在建模前2 h开始给予选择性JAK2抑制剂AG490治疗,每天1次;模型组仅注射溶媒.术后第14天处死动物.组织学评估肾小管损伤和.肾间质纤维化程度;免疫组化检测肾脏巨噬细胞浸润和α-SMA表达;RT-PCR检测Ⅲ型胶原和单核细胞趋化蛋白(MCP)1 mRNA表达;Western印迹检测JAK2和STATl磷酸化.结果 JAK2-STAT1在UUO模型中被激活,其磷酸化水平与病情、肾小管组织学损害以及.肾间质纤维化相一致.AG490能显著抑制JAK2和STAT1的磷酸化(P＜0.01).AG490治疗显著减轻肾小管损害[(21.7±1.7)%比(49.4±1.0)%]和肾间质纤维化(1.0±0.1比2.3±0.2)、α-SMA表达(0.9±0.1比2.1±0.2)和巨噬细胞积聚[(13.3±1.6)细胞/HPF比(34.4±1.0)细胞/HPF](均P＜0.01).AG490治疗显著抑制Ⅲ型胶原和MCP-1 mRNA表达.结论 JAK-STAT信号通路在肾小管间质炎性反应和纤维化中发挥重要作用.     

Blockage of JAK/STAT signalling attenuates renal ischaemia-reperfusion injury in rat.

BACKGROUND: JAK/STAT signalling is one of the major pathways for cytokine signal transduction. However, the role of JAK/STAT in renal ischaemia/reperfusion (I/R) injury is not clear. The present study investigated the protection against renal I/R injury by in vivo inhibition of JAK2 activation. METHODS: Rats subjected to renal I/R were either treated with daily intraperitoneal injection of selective JAK2 inhibitor tyrphostin AG490 (10 mg/kg) or vehicle alone starting 4 h before, immediately after or until 3 h after I/R. Renal function, histology, infiltration of macrophages, apoptosis, expression of chemokines and adhesion molecules were assessed. RESULTS: AG490 treatment significantly inhibited the phosphorylation of JAK2 and its downstream molecule STAT1 and STAT3. Rats pretreated with AG490 exhibited improved renal function, attenuated histological lesions and reduced apoptosis of tubular epithelial cells. AG490 significantly inhibited renal expression of MCP-1 and ICAM-1 mRNA, as well as the expression of ICAM-1 protein, accompanied by decreased macrophage accumulation in the kidney. Immediate post-ischaemic treatment of AG490 also significantly ameliorated renal injury. However, delayed post-ischaemic treatment until 3 h after I/R failed to attenuate renal damage. CONCLUSIONS: This study demonstrated the involvement of JAK/STAT signalling in the pathogenesis of renal I/R injury, suggesting that JAK/STAT pathway may serve as a potential target for early intervention in ischaemic acute renal failure.     

Janus激酶-信号转导及转录活化因子通路对内毒素/脂多糖体外诱导大鼠腹腔巨噬细胞高迁移率族蛋白B1合成释放的调节作用

目的探讨Janus激酶(JAK)-信号转导及转录活化因子(STAT)通路对内毒素/脂多糖(LPS)诱导大鼠腹腔巨噬细胞高迁移率族蛋白B1(HMGB1)合成释放的调节作用。方法取正常Wistar大鼠腹腔巨噬细胞,培养3d后用LPS刺激,分别于刺激前及刺激10、30、60、120min时观察JAK2、STAT1以及STAT3的活化情况,每时相点重复测定4次,以积分吸光度(IA)值表示;另取细胞分为正常组、LPS刺激组、JAK2抑制组、STAT1抑制组、STAT3抑制组,培养3d后用LPS刺激后4组细胞,刺激前2h,后3组分别加入AG490、氟达拉滨及雷帕霉素,观察各组HMGB1基因表达及蛋白释放情况,每组重复测定4次。结果LPS可诱导大鼠腹腔巨噬细胞JAK2、STAT1及STAT3在短时间(120min)内活化,其中STAT3活化最为迅速,10min即可达到峰值(7.47±0.56)。JAK2抑制组、STAT1抑制组、STAT3抑制组HMGB1基因表达均明显受抑制,其表达量均明显低于LPS刺激组(P<0.01),其中JAK2抑制组明显高于正常组(P<0.01),其余两个抑制组则与正常组相近(P>0·05);但3个抑制组HMGB1蛋白表达量与LPS刺激组基本相同,均明显高于正常组(P<0.01).结论JAK-STAT通路可在LPS刺激下早期活化,部分参与了诱导HMGB1合成的信号调控过程。     

JAK2／STAT3信号通路在IL-6介导肾小管上皮细胞转分化中的作用

目的：研究Janus激酶（JAK2）和信号转导因子和转录活化因子（STAT3）途径的激活在IL-6介导人近端肾小管上皮细胞转分化中的作用。方法：（1）细胞培养及分组：待生长状况良好的HK-2细胞株60%-80%细胞贴壁后,无血清培养基同步24 h,然后根据分组给予相应的刺激。（2）指标检测：采用荧光定量PCR技术检测0 h、24 h、48 h7、2 h不同时间点α-SMA mRNA、E-cadherin mRNA的表达。采用Western blot方法检测25 ng/ml浓度的IL-6刺激24 h、48 h、72 h后α-SMA、E-cadherin蛋白的表达;检测0、5、10、25、50 ng/ml浓度的IL-6刺激30 min,以及IL-6（25 ng/ml）刺激0、15 min、30 min、60 min、120 min后P-STAT3蛋白的表达水平;检测AG490预处理细胞4 h,IL-6（25 ng/ml）分别刺激30 min后及48 h后P-STAT3、P-JAK2和α-SMA、E-cadherin蛋白的表达水平。结果：与刺激前比较,IL-6以时间依赖方式上调HK-2细胞α-SMA mRNA、蛋白的表达,下调E-cadherin mRNA、蛋白的表达;IL-6以时间和剂量依赖方式上调HK-2细胞P-STAT3的表达,高峰发生在30 min;AG490部分抑制STAT3、JAK2的磷酸化,部分抑制IL-6诱导的α-SMA蛋白表达的上调、部分抑制IL-6诱导的E-cadherin蛋白表达的下调。结论：HK-2细胞中,JAK2/STAT3信号通路的激活可能参与了IL-6介导的肾小管上皮细胞转分化的发生。     

Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion

Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X(L) and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.     

Cyclic stretch-induced cPLA2 mediates ERK 1/2 signaling in rabbit proximal tubule cells

BACKGROUND: Recent evidence from this laboratory have demonstrated a critical role of phospholipase A2 (PLA2) and arachidonic acid in angiotensin II type 2 (AT2) receptor-mediated kinase activation in renal epithelium independent of phosphoinositide- specific phospholipase C (PLC) and without the necessity of eicosanoid biosynthesis. In the present study, we investigated whether cyclic stress phosphorylates and activates the mitogen-activated protein kinase (MAPK) pathway and whether PLA2 activation mediates mechanotransduction in renal epithelial cells. The rational for studying kidney epithelial cells relates to their similarity to podocytes, which undergo mechanical stretch related to changes in intraglomerular pressure. METHODS: To produce strain or stretch, primary cultures of rabbit proximal tubular cell cells are grown in tissue culture wells having a collagen-coated Silastic deformable membrane bottoms and applying vacuum to the well to generate alternating cycles of stretch and relaxation (30 cycles/min). RESULTS: We found that cyclic stretching of rabbit proximal tubular cells caused a time- and intensity-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) in proximal tubular cells as detected by its phosphorylation. In addition, mechanical stretch induced PLA2 activation and a subsequent rapid release of arachidonic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphonate ketone (AACOCF3) attenuated both arachidonic acid release and ERK 1/2 activation by cyclic stretch, supporting the importance of PLA2 as a mediator of mechanotransduction in renal proximal tubular cells. A requirement for extracellular Ca2+ and stretch-activated Ca2+ channels was also documented. Complete inhibition of ERK 1/2 by PD98059, a MAPK kinase (MEK) inhibitor, did not suppress stretch- induced PLA2 activation and arachidonic acid release, suggesting the later events were upstream of ERK 1/2. Cyclic stretch also caused rapid phosphorylation of the EGF receptor kinase and c-Src. Furthermore, arachidonic acid itself induced time- and dose-dependent phosphorylation of c-Src. In addition, the c-Src inhibitor PP2 and selective EGF receptor kinase inhibitor AG1478 attenuated both ERK 1/2 and EGF receptor phosphorylation by cyclic stretch. CONCLUSION: PLA2 dependence for ERK 1/2 activation in response to cyclic stretch in proximal tubular epithelial cells was established in this report. In addition, these findings indicate cyclic stretch increased the tyrosine phosphorylation of the EGF receptor and c-Src and that c-Src acts upstream of the EGF receptor to mediate its phosphorylation, whereby both are critical for stretch- induced ERK 1/2 activation in rabbit proximal tubular cells. These observations documents for the first time a mechanism of mechanical stretch-induced kinase activation mediated by stretch activated Ca2+ channels and PLA2-dependent release of arachidonic acid.     

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

To determine the role of epidermal growth factor receptor (EGFR) activation in renal functional and structural recovery from acute kidney injury (AKI), we generated mice with a specific EGFR deletion in the renal proximal tubule (EGFR(ptKO)). Ischemia-reperfusion injury markedly activated EGFR in control littermate mice; however, this was inhibited in either the knockout or wild-type mice given erlotinib, a specific EGFR tyrosine kinase inhibitor. Blood urea nitrogen and serum creatinine increased to a comparable level in EGFR(ptKO) and control mice 24?h after reperfusion, but the subsequent rate of renal function recovery was markedly slowed in the knockout mice. Twenty-four hours after reperfusion, both the knockout and the inhibitor-treated mice had a similar degree of histologic renal injury as control mice, but at day 6 there was minimal evidence of injury in the control mice while both EGFR(ptKO) and erlotinib-treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. Additionally, renal cell proliferation was delayed due to decreased ERK and Akt signaling. Thus, our studies provide both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after acute kidney injury.     

利用组织芯片技术研究酪氨酸蛋白激酶在肝癌组织中的表达及意义

目的 观察酪氨酸蛋白激酶ERK、P38、C-jun、JAK2、STAT3、STAT5在肝癌组织中的表达及相互关系,探讨酪氨酸蛋白激酶表达与肝癌病人临床特征之间的关系.方法 收集原发性肝癌手术切除标本30例,制作组织芯片,以肝硬化组织作对照,采用免疫组化SP法研究酪氨酸蛋白激酶ERK、P38、C-jun、JAK2、STAT3、STAT5在肝癌组织与肝硬化组织中的表达.结果 ERK、P38、C-jun、JAK2、STAT3、STAT5在肝癌组织中的表达平均光密度值分别为(0.220±0.033,0.174±0.024,0.183±0.064,0.192±0.044.0.197±0.078,0.181±0.066),显著高于肝硬化组(0.065±0.028,0.058±0.028,0.042±0.016,0.070±0.030,0.052±0.024,0.052±0.023,P＜0.01).ERK与C-jun、JAK2、STAT3、STAT5在肝癌组织中表达呈显著正相关,(P＜0.01或P＜0.05),与P38呈显著负相关(r=0.404,P＜0.05).JAK2的过度表达与肝癌组织中细胞的分化程度有关,在低分化肝癌组织中表达显著率高于高中分化肝癌组织.结论 MAPK和JAK-STAT通路的过度活化在肝癌发生发展过程中起重要作用,细胞信号转导系统失去正常的协调平衡可能是导致肿瘤发生的重要原因.     

IL-6在前列腺增生基质细胞上诱导APRE的研究

目的 研究IL-6在前列腺增生(benign prostatic hyperplasia,BPH)的基质细胞上诱导急性期蛋白反应元件(APRE)的活化及STAT3的敲除抑制其活化,推测STAT3介导的JAK/STAT信号及其所激活的ARPE可能作为BPH治疗的靶点.方法 建立IL-6刺激的前列腺基质细胞的体外模型,使用商品化的siRNA,利用Fugene HD进行siRNA转染来敲除STAT3,通过WB检测STAT3表达情况和APRE的活化程度.结果 使用IL-6刺激前列腺基质细胞后,荧光素酶的表达增加水平到9倍,JAK2和STAT3的磷酸化显著性增加.STAT3敲除后,通过荧光素酶的活性检测结果可知在IL-6刺激的情况下,APRE的活性从大约8倍的水平下降到不到2倍.这证实了IL-6介导的JAK/STAT信号通路被严重抑制.结论 JAK2和STAT3的活化能激活APRE的表达,STAT3所介导的JAK/STAT信号及其所激活的ARPE可能作为BPH治疗的靶点.     

JAK/STAT通路在金属蛋白酶1组织抑制剂抑制肾小球系膜细胞凋亡中的作用

目的 探讨金属蛋白酶1组织抑制剂（TIMP-1）抑制大鼠肾小球系膜细胞（RMC）凋亡与Janus激酶/信号转导和转录激活子（JAK/STAT）通路的关系。 方法 用无血清培养基体外培养pcDNA3空载体、人正义、反义TIMP-1基因重组真核表达载体转染RMC。根据是否加JAK2特异性抑制剂AG490刺激24 h,将细胞分为未转染组、未转染＋AG490组、空载体组、空载体＋AG490组、正义组、正义＋AG490组、反义组和反义＋AG490组。另外设正常培养条件下的RMC作为正常对照组。应用流式细胞技术检测各组RMC的凋亡率。RT-PCR检测TIMP-1、bcl-xl、cyclin D1、p27kip1和JAK2 mRNA的表达。Western印迹检测胞质中JAK2、STAT3、STAT5及其相应磷酸化蛋白(p-JAK2、p-STAT3、p-STAT5)的表达。 结果 未转染组、正义组及反义组RMC凋亡率分别为（10.59±0.96）％、（7.08±0.43）％和（21.91±0.25）％，各组间差异有统计学意义(P < 0.05或P < 0.01)。在未加AG490的各组RMC中,bcl-xl和cyclin D1 mRNA在正义组中表达最高，反义组中最低，p27kip1 mRNA在反义组中表达最高。加入AG490后，各组细胞的凋亡率均显著增加（P < 0.01）；TIMP-1、bcl-xl和cyclin D1 mRNA表达均减少；p27kip1 mRNA表达均增加。在未加AG490的各组细胞中，p-JAK2、p-STAT3和p-STAT5在正义组中表达最高，反义组中最低。加入AG490后上述蛋白表达均减少，且正义＋AG490组最高，反义＋AG490组最低。 结论 TIMP-1表达受JAK/STAT信号通路调控，后者可通过上调前者的表达抑制RMC凋亡；TIMP-1通过JAK/STAT信号通路抑制RMC凋亡。 bcl-xl、cyclin D1和p27kip1参与了上述过程。