TGF-beta 1 attenuates myocardial ischemia-reperfusion injury via inhibition of upregulation of MMP-1

Ischemia-reperfusion (I/R) is thought to upregulate the expression and activity of matrix metalloproteinases (MMPs), which regulate myocardial and vascular remodeling. Previous studies have shown that transforming growth factor-beta(1) (TGF-beta(1)) can attenuate myocardial injury induced by I/R. TGF-beta(1) is also reported to suppress the release of MMPs. To study the modulation of MMP-1 by TGF-beta(1) in I/R myocardium, Sprague-Dawley rats were given saline and subjected to 1 h of myocardial ischemia [total left coronary artery (LCA) ligation] followed by 1 h of reperfusion (n = 9). Parallel groups of rats were pretreated with recombinant TGF-beta(1) (rTGF-beta(1), 1 mg/rat, n = 9) before reperfusion or exposure to sham I/R (control group). I/R caused myocardial necrosis and dysfunction, indicated by decreased first derivative of left ventricular pressure, mean arterial blood pressure, and heart rate (all P < 0.01 vs. sham-operated control group). Simultaneously, I/R upregulated MMP-1 (P < 0.01). Treatment of rats with rTGF-beta(1) reduced the extent of myocardial necrosis and dysfunction despite I/R (all P < 0.01). rTGF-beta(1) treatment also inhibited the upregulation of MMP-1 in the I/R myocardium (P < 0.05). To determine the direct effect of MMP-1 on the myocardium, isolated adult rat myocytes were treated with active MMP-1, which caused injury and death of cultured myocytes, measured as lactate dehydrogenase release and trypan blue staining, in a dose- and time-dependent manner (P < 0.05). Pretreatment with PD-166793, a specific MMP inhibitor, attenuated myocardial injury and death induced by active MMP-1. The present study for the first time shows that MMP-1 can directly cause myocyte injury or death and that attenuation of myocardial I/R injury by TGF-beta(1) may, at least partly, be mediated by the inhibition of upregulation of MMP-1.     

The Protective Role of Interleukin-33 in Myocardial Ischemia and Reperfusion Is Associated with Decreased HMGB1 Expression and Up-Regulation of the P38 MAPK Signaling Pathway

Interleukin-33 (IL-33) plays a protective role in myocardial ischemia and reperfusion (I/R) injury, but the underlying mechanism was not fully elucidated. The present study was designed to investigate whether IL-33 protects against myocardial I/R injury by regulating both P38 mitogen-activated-protein kinase (P38 MAPK), which is involved in one of the downstream signaling pathways of IL-33, and high mobility group box protein 1 (HMGB1), a late pro-inflammatory cytokine. Myocardial I/R injury increased the level of IL-33 and its induced receptor (sST) in myocardial tissue. Compared with the I/R group, the IL-33 group had significantly lower cardiac injury (lower serum creatine kinase (CK), lactate dehydrogenase (LDH), and cTnI levels and myocardial infarct size), a suppressed inflammatory response in myocardial tissue (lower expression of HMGB1, IL-6, TNF-α and INF-γ) and less myocardial apoptosis (much higher Bcl-2/Bax ratio and lower cleaved caspase-3 expression). Moreover, IL-33 activated the P38 MAPK signaling pathway (up-regulating P-P38 expression) in myocardial tissue, and SB230580 partially attenuated the anti-inflammatory and anti-apoptosis effects of IL-33. These findings indicated that IL-33 protects against myocardial I/R injury by inhibiting inflammatory responses and myocardial apoptosis, which may be associated with the HMGB1 and P38 MAPK signaling pathways.     

LOX-1在D-葡萄糖诱导人肾小球系膜细胞表达TGF-β1中的作用

目的探讨血凝素样氧化低密度脂蛋白受体1（LOX-1）在D-葡萄糖诱导人肾小球系膜细胞表达转化生长因子β1（TGF-β1）中的作用。方法在体外培养人肾小球系膜细胞,在不同时间加入不同浓度的D-葡萄及LOX-1特异性阻滞剂JTX92,用半定量RT-PCR法检测LOX-1和TGF-β1基因表达的相对含量,用Western blot法检测p38 MAPK蛋白质的相对含量,用酶联免疫吸附法（ELISA）检测细胞培养液中TGF-β1浓度。结果D-葡萄糖以时间和浓度依赖的方式增加细胞内LOX-1和TGF-β1 mRNA表达和培养液中TGF-β1浓度,同时也以时间和浓度依赖的方式增加p38 MAPK的表达,JTX92可以明显抑制LOX-1、TGF-β1和p38 MAPK的表达。结论高浓度D-葡萄糖可能通过上调LOX-1的表达,激活细胞内的p38 MAPK信号传递途径,促使人肾小球系膜细胞合成并分泌大量TGF-β1,参与糖尿病肾病的发生发展。     

cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.     

Efferent vagal nerve stimulation induces tissue inhibitor of metalloproteinase-1 in myocardial ischemia-reperfusion injury in rabbit

Vagal nerve stimulation has been suggested to ameliorate left ventricular (LV) remodeling in heart failure. However, it is not known whether and to what degree vagal nerve stimulation affects matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) in myocardium, which are known to play crucial roles in LV remodeling. We therefore investigated the effects of electrical stimulation of efferent vagal nerve on myocardial expression and activation of MMPs and TIMPs in a rabbit model of myocardial ischemia-reperfusion (I/R) injury. Anesthetized rabbits were subjected to 60 min of left coronary artery occlusion and 180 min of reperfusion with (I/R-VS, n = 8) or without vagal nerve stimulation (I/R, n = 7). Rabbits not subjected to coronary occlusion with (VS, n = 7) or without vagal stimulation (sham, n = 7) were used as controls. Total MMP-9 protein increased significantly after left coronary artery occlusion in I/R-VS and I/R to a similar degree compared with VS and sham values. Endogenous active MMP-9 protein level was significantly lower in I/R-VS compared with I/R. TIMP-1 mRNA expression was significantly increased in I/R-VS compared with the I/R, VS, and sham groups. TIMP-1 protein was significantly increased in I/R-VS and VS compared with the I/R and sham groups. Cardiac microdialysis technique demonstrated that topical perfusion of acetylcholine increased dialysate TIMP-1 protein level, which was suppressed by coperfusion of atropine. Immunohistochemistry demonstrated a strong expression of TIMP-1 protein in cardiomyocytes around the dialysis probe used to perfuse acetylcholine. In conclusion, in a rabbit model of myocardial I/R injury, vagal nerve stimulation induced TIMP-1 expression in cardiomyocytes and reduced active MMP-9.     

Regulation of TGFbeta1-mediated collagen formation by LOX-1: studies based on forced overexpression of TGFbeta1 in wild-type and lox-1 knock-out mouse cardiac fibroblasts

Transforming growth factor beta(1) (TGFbeta(1)) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGFbeta(1)-mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGFbeta(1) was overexpressed in wild-type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGFbeta(1) moiety (AAV/TGFbeta (ACT)(1)). Transfection of WT mouse cardiac fibroblasts with AAV/TGFbeta (ACT)(1) markedly enhanced the expression of NADPH oxidases (p22(phox), p47(phox), and gp91(phox) subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGFbeta(1)-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGFbeta(1)-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGFbeta(1)-reactive oxygen species-LOX-1 cascade.     

Protective effects of taurine against renal ischemia/reperfusion injury in rats by inhibition of gelatinases,MMP-2 and MMP-9, and p38 mitogen-activated protein kinase signaling

Dysregulated expression of matrix metalloproteinases (MMPs) is closely associated with the pathogenesis of renal ischemia/reperfusion injury (I/R). The production of excessive reactive oxygen species (ROS) causes tissue damage. Increased ROS production causes activation of p38 mitogen-activated protein kinase (MAPK) signaling, which participates in gene regulation of MMPs, especially MMP-2 and MMP-9 (gelatinases). Taurine (2-aminoethanesulfonic acid) in mammalian cells functions in bile acid conjugation, maintenance of calcium homeostasis, osmoregulation, membrane stabilization, and antioxidation, antiinflammatory, and antiapoptotic action. We investigated the effects of taurine and the possible role of p38 MAPK signaling on regulation of MMP-2 and MMP-9 in a renal I/R injury model in rats. Rats were divided into three groups: sham, I/R, and I/R + taurine treated. After a right nephrectomy, I/R was induced by clamping the left renal pedicle for 1 h followed by 6 h reperfusion. Taurine was administered 45 min prior to induction of ischemia. Renal function was assessed by serum creatinine and blood urea nitrogen (BUN) levels. Tubule injury and structural changes were evaluated by light microscopy. Malondialdehyde (MDA) levels were analyzed by high performance liquid chromatography (HPLC). Superoxide dismutase (SOD) activity levels were measured using a colorimetric kit. mRNA expression of MMP-2 and MMP-9 was determined by real-time polymerase chain reaction. MMP-2 and MMP-9 activities were measured using a fluorimetric kit. Phosphorylated p38 (p-p38) and total p38 MAPK protein expressions were evaluated by western blot. Taurine pretreatment significantly attenuated renal dysfunction and histologic damage, such as renal tubule dilation and loss of brush borders. The pretreatment also decreased the MDA level and attenuated the reduction of SOD activity in the kidney during I/R. Taurine pretreatment also decreased significantly both MMP-2 and MMP-9 mRNA expression and MMP-9 activity induced by I/R. In addition, the activity of p38 MAPK signaling was down-regulated significantly by taurine administration. Inhibition of MMP-2 and MMP-9 expression and MMP-9 activity caused by taurine may be associated with suppression of p38 MAPK activation during I/R induced renal injury in rats. Therefore, taurine administration may prove to be a strategy for attenuating renal I/R injury.     

Cardioprotective effects of rosiglitazone are associated with selective overexpression of type 2 angiotensin receptors and inhibition of p42/44 MAPK

Current evidence points to renin-angiotensin system as a key mediator in ischemia-reperfusion injury. Rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand, has recently been shown to confer cardioprotection against ischemia-reperfusion in animal models. We sought to examine the expression of ANG II receptors during PPAR-gamma-mediated cardioprotection. Male Sprague-Dawley rats (nondiabetic) were fed either regular rat chow (control diet group, n = 9) or rosiglitazone-rich diet (rosiglitazone-rich diet group, n = 9) and were subjected to 1 h of myocardial ischemia followed by 1 h of reperfusion. A third group of rats had only thoracotomy and pericardiotomy and served as a sham control group (n = 9). Hemodynamics, infarct size, and expression of ANG II type 1 and type 2 receptors (AT1 and AT2) were measured in all groups. There was a 58% reduction of infarct size in the rosiglitazone-rich diet group (P < 0.01 vs. control diet group). Increased myocardial expression of AT(1) receptors in the ischemic-reperfused myocardium was attenuated in the rosiglitazone-rich diet group (P < 0.05 vs. control diet group). Importantly, myocardial AT2 mRNA and protein expression were significantly increased (by >100-fold) in the rosiglitazone-rich diet group (P < 0.05). These changes were accompanied by inhibition of p42/44 MAPK in the rosiglitazone-rich diet group, while the Akt1 expression, believed to mediate insulin sensitization, remained similar in all three groups. The cardioprotective effects of rosiglitazone against myocardial ischemia-reperfusion injury are independent of its insulin-sensitizing properties and are associated with significant overexpression of AT2 receptors along with inhibition of p42/44 MAPK.     

Cell expression of MMP-1 and TIMP-1 in co-cultures of human gingival fibroblasts and monocytes: the involvement of ICAM-1

Matrix metalloproteinase-1 (MMP-1) plays an important role in the degradation of collagen in inflammatory diseases. The aim of this study was to investigate the cellular expression of MMP-1 and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), in gingival fibroblasts co-cultured with monocytes and the possible mediating role of intercellular adhesion molecule-1 (ICAM-1). In co-cultures, the expression of MMP-1 and TIMP-1 increased in fibroblasts, but not in monocytes, although the number of MMP-1+ and TIMP-1+ adhered monocytes increased. Moreover, ICAM-1 expression in both fibroblasts and adhered monocytes increased. In the presence of an anti-ICAM-1 antibody, the expression of MMP-1 in fibroblasts decreased whereas the number of TIMP-1+ adhered monocytes increased. The p38 MAPK inhibitor SB203580 reduced MMP-1 expression in fibroblasts, as well as ICAM-1 expression in both fibroblasts and adhered monocytes. The results suggest that co-culture with monocytes enhances cellular expression of MMP-1 and TIMP-1 in gingival fibroblasts, and that the increased MMP-1 expression, in contrast to TIMP-1, is partly mediated by the adhesion molecule ICAM-1 and the p38 MAPK signal pathway.     

Inhibition of p38 reduces myocardial infarction injury in the mouse but not pig after ischemia-reperfusion

The MAPK family member p38 is activated in the heart after ischemia-reperfusion (I/R) injury. However, the cardioprotective vs. proapoptotic effects associated with p38 activation in the heart after I/R injury remain unresolved. Another issue to consider is that the majority of past studies have employed the rodent as a model for assessing p38's role in cardiac injury vs. protection, while the potential regulatory role in a large animal model is even more uncertain. Here we performed a parallel study in the mouse and pig to directly compare the extent of cardiac injury after I/R at baseline or with the selective p38 inhibitor SB-239063. Infusion of SB-239063 5 min before ischemia in the mouse prevented ischemia-induced p38 activation, resulting in a 25% reduction of infarct size compared with vehicle-treated animals (27.9 +/- 2.9% vs. 37.5 +/- 2.7%). In the pig, SB-239063 similarly inhibited myocardial p38 activation, but there was no corresponding effect on the degree of infarction injury (43.6 +/- 4.0% vs. 41.4 +/- 4.3%). These data suggest a difference in myocardial responsiveness to I/R between the small animal mouse model and the large animal pig model, such that p38 activation in the mouse contributes to acute cellular injury and death, while the same activation in pig has no causative effect on these parameters.     

Sex differences in endothelial STAT3 mediate sex differences in myocardial inflammation

Recent studies have shown that females have improved myocardial functional recovery, TNF receptor 1 (TNFR1) signaling resistance, and increased STAT3 phosphorylation following acute ischemia/reperfusion (I/R) compared with males. We hypothesized that 1) STAT3 deficiency in endothelial cells (EC) impairs myocardial functional recovery in both sexes, 2) EC STAT3 deficiency equalizes sex differences in functional recovery, and 3) knockout of EC STAT3 decreases activation of myocardial STAT3 and increases p38 MAPK activation following acute I/R. Isolated male and female mouse hearts from WT and EC STAT3 knockout (STAT3KO) were subjected to 20-min ischemia/60-min reperfusion, and +/- dP/dt were continuously recorded. Heart tissue was analyzed for the active forms of STAT3 and p38 MAPK as well as expression of caspase-8 (Western blot) following I/R. EC STATKO had significantly decreased myocardial functional recovery in both sexes (%recovered +dP/dt: male 51.6 +/- 3.1 vs. 32.1 +/- 13.1%, female 79.1 +/- 3.6 vs. 43.6 +/- 9.1%; -dP/dt: male 52.2 +/- 3.3 vs. 28.9 +/- 12%, female 75.2 +/- 4.1 vs. 38.6 +/- 10%). In addition, EC STAT3KO neutralized sex differences in myocardial function, which existed in WT mice. Interestingly, EC STAT3 deficiency decreased myocardial STAT3 activation but increased myocardial p38 MAPK activation in both sexes; however, this was seen to a greater degree in females. We conclude that EC STAT3 deficiency resulted in decreased recovery of myocardial function in both sexes and neutralized sex differences in myocardial functional recovery following I/R. This observation was associated with decreased activation of myocardial STAT3 and increased activation of p38 MAPK in EC STAT3KO heart after I/R.     

参麦注射液对肠缺血/再灌注肺损伤大鼠肺组织p38MAPK及凋亡相关基因表达的影响

目的:观察参麦注射液(SM)对肠缺血/再灌注(I/R)肺损伤大鼠肺组织p38MAPK和凋亡相关基因Bax、Bcl-2蛋白表达的影响,探讨其保护机制。方法:采用夹闭肠系膜上动脉(SMA)方法建立大鼠肠I/R损伤模型。24只SD大鼠随机分为对照组(Control组)、肠缺血/再灌注组(I/R组)、参麦注射液组(SM+I/R组),每组8只。比较各组大鼠肺湿/干比(W/D)、肺表面活性物质主要成分卵磷脂(PC)及总磷脂(TPL)含量的变化;同时免疫组织化学法检测各组大鼠肺组织中p38MAPK、Bax及Bcl-2蛋白的表达水平。结果:与对照组比较,I/R组肺组织W/D明显升高,而PC和TPL的含量显著降低,肺组织p38MAPK、Bcl-2和Bax蛋白表达明显增强(P均＜0.01),其中Bax的增强比Bcl-2的增强更为明显,Bcl-2/Bax比值降低(P＜0.01);与I/R组比较,SM+I/R组大鼠肺组织W/D明显降低,PC和TPL的含量增加,肺组织p38MAPK和Bax蛋白表达下降(P均＜0.01),Bcl-2的表达增强,Bcl-2/Bax比值明显升高(P＜0.01)。相关分析显示,肠I/R时肺组织p38MAPK蛋白表达水平与肺表面活性物质主要功能成分PC含量及凋亡基因Bcl-2/Bax比值呈负相关(r分别为-0.787,-0.731,P均＜0.01)。结论:SM可能通过抑制p38MAPK信号通路的激活,提高Bcl-2/Bax比值来阻抑细胞凋亡,从而减轻肠I/R时的肺损伤。     

15S-Lipoxygenase-2 mediates arachidonic acid-stimulated adhesion of human breast carcinoma cells through the activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.     

在低氧预处理延迟心肌保护中钙网蛋白表达升高

本文分别在整体实验和细胞培养条件下研究钙网蛋白（calreticulin,CRT）在低氧预处理（hypoxic preconditioning,HPC）延迟心肌保护中的表达及其信号转导机制。（1）整体实验时Wistar大鼠随机分为3组：假手术（sham）组、仅结扎冠状动脉的心肌缺血（myocardial infarction,MI）组和HPC后再结扎冠状动脉的HPC＋MI组,分别于术后24h、14d和28d观察HPC对缺血后心功能和梗死区、危险区面积的影响;采用Western blot检测CRT表达以及p38丝裂素活化蛋白激酶（p38mitogenactivated protein kinase,p38MAPK）、应激活化蛋白激酶（stress-activated protein kinase,SAPK）活性。（2）原代培养Sprague—Dawley乳鼠心肌细胞,随机分为6组：低氧,复氧（hypoxia／reoxygenation,H／R）组、HPC组、HPC＋H,R组、p38MAPK抑制剂SB203580＋HPC＋H／R（SB＋HPC＋H／R）组、SAPK抑制剂SP600125＋HPC＋H瓜（SP＋HPC＋H／R）组和正常对照组;采用台盼蓝排斥实验、乳酸脱氢酶（1actate dehydrogenase,LDH）活性检测及流式细胞仪检测各组细胞损伤情况;采用Western blot检测CRT表达及p38MAPK、SAPK的磷酸化水平。主要结果如下：（1）整体动物实验结果表明,HPC改善缺血对心肌左室压力最大上升,下降速度（＋dp／dtmax）的抑制,限制心肌梗死面积;HPC后CRT表达呈动态变化：术后24h时HPC＋MI组CRT表达增高106％（P〈0．05vsMI组）,以危险区最为显著;14d至28d表达逐步降低。相关分析显示,术后24h时CRT表达量与心功能呈正相关（r=0．9867,P〈0．05）,与梗死面积呈负相关（r=-0．9709,P〈0．05）。（2）细胞培养实验结果表明,HPC可减轻H／R诱导的心肌细胞LDH漏出,增加心肌细胞存活率,降低细胞凋亡;单纯HPC可诱导CRT表达轻度增加（222％,P〈0．05vs对照组）,而损伤性H／R诱导CRT过表达（503％,P〈0．05vs对照组）,HPC可降低H／R诱导CRT表达升高的幅度;p38MAPK活性与HPC诱导的CRT表达呈正相关（r=0．9021,P〈0．05）,而SAPK活性与其呈负相关（r=-0．8211,P〈0．05）。由此得出结论：（1）整体实验中HPC可明显改善缺血后心脏的收缩与舒张功能,限制心肌梗死范围,促进危险区心肌恢复;心肌梗死早期,HPC诱导CRT表达上调,参与心肌保护;（2）细胞培养实验中HPC可诱导CRT适度表达,增强原代培养乳鼠心肌细胞对H／R损伤的抵抗力;p38MAPK可能介导HPC诱导的CRT表达,而SAPK激活可能不利于心肌保护。     

Sex differences in the myocardial inflammatory response to ischemia-reperfusion injury

The myocardium generates inflammatory mediators during ischemia-reperfusion (I/R), and these mediators contribute to cardiac functional depression and apoptosis. The great majority of these data have been derived from male animals and humans. Sex has a profound effect over many inflammatory responses; however, it is unknown whether sex affects the cardiac inflammatory response to acute myocardial I/R. We hypothesized the existence of inherent sex differences in myocardial function, expression of inflammatory cytokines, and activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after I/R. Isolated rat hearts from age-matched adult males and females were perfused (Langendorff), and myocardial contractile function was continuously recorded. After I/R, myocardium was assessed for expression of TNF-alpha, IL-1beta, and IL-6 (RT-PCR, ELISA); IL-1alpha and IL-10 mRNA (RT-PCR); and activation of p38 MAPK (Western blot). All indexes of postischemic myocardial function [left ventricular developed pressure, left ventricular end-diastolic pressure, and maximal positive (+dP/dt) and negative (-dP/dt) values of the first derivative of pressure] were significantly improved in females compared with males. Compared with males, females had decreased myocardial TNF-alpha, IL-1beta, and IL-6 (mRNA, protein) and decreased activation of p38 MAPK pathway. These data demonstrate that hearts from age-matched adult females are relatively protected against I/R injury, possibly due to a diminished inflammatory response.     

Modulatory effect of interleukin-1α on expression of structural matrix proteins,MMPs and TIMPs in human cardiac myofibroblasts: Role of p38 MAP kinase

The proinflammatory cytokine interleukin-1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac myofibroblasts (CMF), or the role of p38 mitogen-activated protein kinase (MAPK). We used a focused RT-PCR microarray to investigate the effects of IL-1α on expression of 41 ECM genes in CMF cultured from different patients, and explored regulation by p38 MAPK.IL-1α (10 ng/ml, 6 h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin. However, it induced marked increases in expression of specific ECM proteases, including matrix metalloproteinases MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B) and MMP-10 (stromelysin-2). Conversely, IL-1α reduced mRNA and protein expression of ADAMTS1, a metalloproteinase that suppresses neovascularization. IL-1α increased expression of TIMP-1 slightly, but not TIMP-2. Data for MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and ADAMTS1 were confirmed by quantitative real-time RT-PCR. Tumor necrosis factor-alpha (TNFα), another important myocardial proinflammatory cytokine, did not alter expression of these metalloproteinases. IL-1α strongly activated the p38 MAPK pathway in human CMF. Pharmacological inhibitors of p38-α/β (SB203580) or p38-α/β/γ/δ (BIRB-0796) reduced MMP-3 and ADAMTS1 mRNA expression, but neither inhibitor affected MMP-9 levels. MMP-1 and MMP-10 expression were inhibited by BIRB-0796 but not SB203580, suggesting roles for p38-γ/δ.In summary, IL-1α induces a distinct pattern of ECM protein and protease expression in human CMF, in part regulated by distinct p38 MAPK subtypes, affirming the key role of IL-1α and CMF in post-infarction cardiac remodeling.     

一氧化碳减轻脂多糖诱导的大鼠小肠损伤与p38 MAPK磷酸化水平的关系

目的：观察低浓度一氧化碳（CO）吸入和腹腔给予对脂多糖（LPS）诱导大鼠小肠损伤的作用及作用过程中丝裂原活化蛋白激酶p38（p38 MAPK）磷酸化水平的变化。方法：6组SD大鼠静脉注入5mg／kg体质量IPS或等容量生理盐水;1h后,对照及LPS注入组吸入室内空气,CO吸入及LPS注入＋CO吸入组吸入体积分数为2．5×10^-4CO．CO腹腔及LPS注入＋CO腹腔组腹腔通入体积分数为2．5×10^-4CO。观察1、3、6h后放血处死,取回盲部上小肠,酶联免疫吸附法测定血小板活化因子（PAV）及细胞间黏附分子-1（ICAM-1）水平;光镜观察组织形态学变化;蛋白印迹法测定p38 MAPK磷酸化水平。结果：LPS注入组PAF、ICAM-1及p38 MAPK磷酸化水平显著高于相应时间点的对照、CO吸入及CO腹腔组（P均〈0．01）;组内各时间点比较,差异无统计学意义。与相应时间点的LPS注入组比较,LPS注入＋CO吸入及LPS注入＋CP腹腔组的PAF和ICAM-1明显降低（P均〈0．05）,但p38 MAPK磷酸化水平进一步增高（P均〈0．05）;此两组间及两组内各时间点比较,差异无统计学意义。结论：低浓度CO吸入和腹腔给予以非时间依赖方式下调LPS诱导的大鼠小肠PAF、ICAM-1表达而起相似的保护作用;p38 MAPK信号转导通路可能参与了这一过程。     

A peptide inhibitor of c-Jun NH2-terminal kinase reduces myocardial ischemia-reperfusion injury and infarct size in vivo

The c-Jun NH(2)-terminal kinase (JNK) pathway of the mitogen-activated protein kinase (MAPK) signaling cascade regulates cell function and survival after stress stimulation. Equally robust studies reported dichotomous results suggesting both protective and detrimental effects of JNK during myocardial ischemia-reperfusion (I/R). The lack of a highly specific JNK inhibitor contributed to this controversy. We recently developed a cell-penetrating, protease-resistant peptide inhibitor of JNK, d-JNKI-1. Here we report on the effects of d-JNKI-1 in myocardial I/R. d-JNKI-1 was tested in isolated-perfused adult rat hearts. Increased activation of JNK, p38-MAPK, and extracellular signal-regulated kinase-1/2 (ERK1/2), as assessed by kinase assays and Western blotting, occurred during I/R. d-JNKI-1 delivered before onset of ischemia prevented the increase in JNK activity while not affecting ERK1/2 and p38-MAPK activation. JNK inhibition reduced ischemic injury, as manifested by increased time to contracture (P < 0.05) and decreased left ventricular end-diastolic pressure during ischemia (P < 0.01), and enhanced posthypoxic recovery of systolic and diastolic function (P < 0.01). d-JNKI-1 reduced mitochondrial cytochrome-c release, caspase-3 activation, and the number of apoptotic cells determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (P < 0.05), indicating suppression of the mitochondrial machinery of apoptosis. d-JNKI-1 delivered at the time of reperfusion did not improve functional recovery but still prevented apoptosis. In vivo, d-JNKI-1 reduced infarct size after coronary artery occlusion and reperfusion by approximately 50% (P < 0.01). In conclusion, d-JNKI-1 is an important compound that can be used in preclinical models to investigate the role of JNK signaling in vivo. Inhibition of JNK during I/R is cardioprotective in anesthetized rats in vivo.