目的 探究组织因子途径抑制物（TFPI）对大鼠心肌缺血再灌注（I/R）及心肌细胞缺氧复氧（H/R）损伤的影响，并从心肌细胞凋亡的变化探索其机制。方法 在体内实验中，通过SD大鼠心脏原位结扎法可逆阻断前降支建立大鼠心肌I/R模型。将大鼠随机分为对照组、I/R组和I/R+rTFPI组，再灌注后3天采用HE染色观察大鼠心肌组织形态学变化，TTC染色法评估心肌梗死区范围，扫描透射电镜观察心肌超微结构损伤情况，Western-Blot法检测各组大鼠心肌组织中Bcl-2、Bax和cleaved-caspase-3蛋白的表达。在体外实验中，采用胰酶消化法及差速贴壁法培养SD乳鼠原代心肌细胞，用MIC101系统模拟心肌细胞I/R损伤，缺氧2小时、复氧12小时后建立体外心肌细胞缺氧/复氧（H/R）模型。将心肌细胞分为对照组、H/R组和H/R+rTFPI（10μg/L）组，用CCK8法检测心肌细胞活力，TUNEL法检测心肌细胞凋亡率，Western-blot方法检测心肌细胞中Bax、Bcl-2及cleaved-caspase-3蛋白的表达水平。结果 体内实验中，成功建立大鼠在体心肌I/R模型。HE染色结果显示I/R组较对照组心肌细胞坏死程度加重，I/R+rTFPI组较I/R组心肌细胞坏死程度减低；TTC染色示I/R+rTFPI组较I/R组心肌梗死范围减少了39.76%（P＜0.05）；扫描透射电镜观察显示I/R组凋亡及损伤程度较对照组加重，I/R+rTFPI组凋亡及损伤较I/R组减轻；Western-Blot结果示，再灌注3天后I/R组心肌组织Bcl-2的表达较对照组降低了53.43%（P＜0.05）、Bax和cleaved-caspase-3（P＜0.05）的表达较对照组分别增加了29.05%和73.25%（P＜0.05），而I/R+rTFPI组Bcl-2的表达水平较I/R组升高了55.01%（P＜0.05），Bax和cleaved-caspase-3的表达水平较I/R组分别降低了13.77%和24.25%（P＜0.05）。在体外实验中，CCK8检测结果显示H/R组细胞活力较对照组下降了29.70%（P＜0.05），H/R+rTFPI组细胞活力较H/R组升高了19.77%（P＜0.05）。TUNEL结果显示H/R组较对照组凋亡率增加了56.76%，H/R+rTFPI组细胞凋亡率较H/R组降低了24.55%（P＜0.05）。Western-blot结果示：H/R组细胞Bcl-2表达较对照组降低了46.92%，Bax表达较对照组增加了41.90%（P＜0.05），cleaved-caspase-3表达较对照组升高了2.68倍（P＜0.05）。H/R+rTFPI组Bcl-2表达较H/R组增加了28.24%（P＜0.05），Bax及cleaved-caspase-3表达较H/R组分别降低了26.34%和57.60%（P＜0.05）。结论 TFPI可显著拮抗心肌I/R和心肌细胞H/R损伤，此效应与其抑制心肌细胞凋亡有关。

Autophagy plays various roles at different stages of ischemia reperfusion (I/R) injury in cardiomyocytes. It has been reported that tissue factor pathway inhibitor (TFPI) has a protective effect on I/R injury. This study aimed to determine the roles of TFPI in autophagy during the I/R injury process in cardiomyocytes and the possible mechanisms. An isolated hypoxia/reoxygenation (H/R) pattern of cardiomyocytes was established by the MIC101 system. The cell viability and oxidative stress of cardiomyocytes were detected by an MTT assay and ROS assay, respectively. The autophagy level was measured by Ad-mCherry-GFP-LC3B and MDC. We detected the expression levels of autophagy-related proteins by western blotting. After 2 h of hypoxia and 12 h of reoxygenation, the cardiomyocyte viability in the H/R group was significantly lower than that in the control group (p < 0.05) than in the H/R group. According to intracellular ROS production, the fluorescence intensity in the H/R group was enhanced compared with that in the negative control group, and it was weaker in the H/R + rTFPI group compared with the H/R group. The level of autophagy and the expression levels of autophagy-related proteins (LC3-II/LC3-I, Beclin-1 and PI3K) were markedly increased in the H/R group compared to the control group (p < 0.05) whereas the levels were markedly decreased in the H/R + rTFPI group compared to the H/R group (p < 0.05). TFPI could relieve cardiomyocyte injury by inhibiting the Class III PI3K/Beclin-1 pathway and oxidative stress; thus, TFPI decreased autophagy and protected cardiomyocytes induced by H/R injury. In conclusion, TFPI may be a new direction for the prevention of myocardial I/R injury.

组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)是一种在生理条件下血液中天然存在的抗凝物质,通过作用于依赖组织因子(tissue factor,TF)的外源性凝血途径对体内凝血系统起调节作用.近年来研究发现,TFPI可对多种疾病的诊断与治疗发挥作用,其中对于冠心病(coronary heart disease,CHD)、动脉粥样硬化(atherosclerosis,As)、心房颤动(atrial fibrillation,AF)、妊娠期高血压等心血管疾病的作用尤为突出.本文将从TFPI的结构、功能特性以及其对心血管疾病的诊疗作用进行综述.

Chemokines may promote the formation and instability of atherosclerotic plaque, which is the most common cause of acute coronary syndrome. The aim of this study was to clarify the function of monocyte chemotactic protein-3 (MCP-3) in the stability of atherosclerotic plaque, to determine the role of tissue factor pathway inhibitor (TFPI) on the development and stability of atherosclerotic plaques, and to further elucidate the anti-atherosclerotic mechanism of TFPI with the emphasis on chemokine MCP-3. We constructed an adenovirus-mediated shRNA against mouse MCP-3 (Ad-MCP-3-shRNA) and an adenovirus-containing TFPI (Ad-TFPI), and tranferred them in a model of vulnerable plaque in ApoE-/- mice respectively. Here, we reported that MCP-3-shRNA and TFPI could both reduce the plaque area and decrease the content of lipids and macrophages, on the contrary, the fibrous cap thickness and content of collagen and smooth muscle cells were increased. In addition, the expression of MCP-3 and CC chemokine receptor 2 (CCR2) was decreased by TFPI transfer. These data provide the first in vivo evidence that MCP-3 is a major contributor to the unstability of atherosclerotic plaque and TFPI may exert its anti-atherosclerotic effects and promote stabilisation of plaque at least partly through inhibiting MCP-3/CCR2 pathway, which may be a new therapeutic method for atherosclerosis.

细胞凋亡是机体的一种适应性反应,生理性的细胞凋亡对于细胞生长、生存及维持人体内环境稳定至关重要.然而过度凋亡可能促进冠状动脉粥样硬化、心肌缺血/再灌注损伤以及缺血后心脏重塑等.组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)除了可以抗凝、抗栓,还具有诱导细胞凋亡的作用.因此,探讨冠心病中TFPI对细胞凋亡的影响,可为冠心病的治疗提供新的途径.

Background: The present study was designed to examine whether cortistatin (CORT) could protect rats from myocardial injury induced by subcutaneously injecting isoproterenol (ISO) and to clarify the possible mechanisms. Methods: Male Sprague-Dawley (SD) rats were placed at random into four groups: the control group, the ISO group, the ISO + CORT 25 mu g/(kg.d) group, and the ISO + CORT 50 mu g/(kg.d) group. Rat models of myocardial injury were established with the subcutaneous (s.c.) injections of 85 mg/kg ISO for 2 days. In the ISO + CORT 25 mu g/(kg.d) group and ISO + CORT 50 mu g/(kg.d) group, rats were given s.c. injections of CORT 25 mu g/(kg.d) and CORT 50 mu g/(kg.d) on the day before ISO, 3 days, respectively. Serum malondialdehyde (MDA) content, lactate dehydrogenase (LDH) activity, and creatine kinase isoenzyme (CK-MB) activity were measured by corresponding test kits. Western blot was applied to evaluate the expression of endoplasmic reticulum stress-related protein glucose regulatory protein 78 (GRP78), enhancer-binding protein homologous protein (CHOP), cysteinyl aspartate specific proteinase-12 (caspase-12), LC3-II, Beclin-1, and p62 in the rat myocardium. Results: CORT alleviated the increased enzyme activities of serum LDH and CK-MB, and content of MDA (a typical marker of lipid peroxidation) in rats induced by ISO. CORT also prevented pathological myocardial injury in rats induced by ISO. Moreover, CORT attenuated the increased protein levels of GRP78, CHOP, and caspase-12, and reduced the increase of LC3-II, LC3-II/I, Beclin-1, and p62 in rats induced by ISO. Conclusions: These data demonstrate that CORT can attenuate ISO-induced acute myocardial injury in rats likely by reducing lipid peroxidation, and inhibiting endoplasmic reticulum stress and autophagy. This supports CORT as a potentially being a new target for preventing and treating myocardial injury and its related disease.

Endothelium (EC) dysfunction plays an important role in vascular diseases, such as arteriosclerosis and hypoxia/reoxygenation (H/R) injury. Tissue factor pathway inhibitor (TFPI) is the only physiological inhibitor of the TF/FVIIa complex in vivo. This experiment aimed to determine the effect of TFPIα on H/R-induced EC injury and the possible mechanisms. The MIC101 hypoxia system was used to establish an EC H/R injury model in vitro. Our results showed that 6 h after reoxygenation, the EC injury in H/R group was higher than that in the control group, whereas after adding TFPIα, the EC injury was alleviate than that in H/R group. The level of ROS was higher in the H/R group than in the control group, while it was apparently lower in the H/R+TFPIα group than in the H/R group. After H/R, the number of autophagosomes and the autophagic flux were significantly increased, whereas TFPIα could decrease the autophagy level after H/R. The expressions of LC3-II/LC3-I, Beclin-1 and PI3K were obviously higher after H/R and lower after adding TFPIα. In conclusion, autophagy contributes to EC injury during the H/R period. TFPIα could decrease autophagy in ECs, and the mechanism might be class III PI3K/Beclin-1 pathway regulation.