四妙勇安汤对脑出血后神经元内血红素清除作用的机理研究

Intracerebral hemorrhage (ICH) is a disease causing high disability in survivals. Following the hemorrhage, excessive free hemin is accumulated in the brain. In our preliminary study, we found that heme can enter neurons rapidly, and directly injure the brain function and neurons, but can not damage brain capillary endothelial cells (BCEC) and astrocytes. We also found that neurons lack of expression of heme exporter proteins (HEP) including breast cancer resistant protein (BCRP) and feline leukemia virus，subgroup C, receptor (FLVCR), while both BCRP and FLVCR are highly expressed in BCECs and astrocytes. These findings can explain the reason that heme remains in the neurons and cause neuronal injury and death. According to our clinical observation that decoction of Simiao Yongan (SMYA) could improve the neurological functions of ICH patients, we proposed this decoction may have the effects on expelling free hemin from the brain and neurons. Therefore, we will examine the effects of SMYA on BCRP and FLVCR expressions, hemin content and cellular survival rate in cultured neurons, hemin efflux rate from the brain- blood barrier (BBB) model in vitro, hemin content and BCRP/FLVCR expressions in the brain and neurons, pro-inflammation cytokines in the blood and brain, and hemopexin (Hx) in the blood and liver in rat ICH model. We will also determine the effects of SMYA on decreasing iron content in the brain and improving neuronal functions of ICH patients in clinic. The results from this study will be helpful to elucidate the pharmacological mechanism of SMYA in treating ICH and the importance of expelling heme from the brain to neuronal survival.

脑出血所致中枢神经功能损伤是全球性治疗难题。研究证实，组织内出血时，游离血红素迅速从红细胞逸出进入周围组织引起细胞氧化损伤和炎症损伤。细胞可通过血红素氧合酶降解少量游离血红素、通过血红素排出蛋白（HEP）如乳腺癌抵抗蛋白（BCRP）和猫白血病病毒（FLVCR）排出大量游离血红素以减轻损伤。但我们最近发现神经元缺乏BCRP和FLVCR表达，所以易被游离血红素损伤。我们已发现四妙勇安汤对脑出血具有良好治疗作用，推猜该方清热祛瘀的作用可能有助于清除脑内血红素。因此，我们拟在原代培养神经元、体外血脑屏障模型、动物脑出血模型上观察该方对HEP表达、脑内和神经元内血红素浓度、神经元死亡率、体内炎性细胞因子、血红素结合蛋白等的影响，以期阐明其对脑出血后减轻神经元损伤的药理作用机制。

研究背景：脑出血（ICH）所致中枢神经功能损伤的病理机制主要在于出血后脑内游离血红素和其代谢产物铁离子所引起的神经元细胞损伤，属于中医学“瘀证”与“瘀阻化热”范围，本项目旨在阐明四妙勇安汤对脑出血后脑内与神经元内血红素的清除作用及其机理，为临床运用四妙勇安汤或同类方药治疗ICH提供依据。.研究内容和方法：分别通过建立体外原代培养神经元的血红素损伤模型、体外血脑屏障模型、大鼠脑出血动物模型，观察四妙勇安汤对神经元内血红素、动物脑内血红素与铁离子含量的变化，相应检查神神经元存活率、经元内羟自由基（•OH）含量、两种血红素排出蛋白（ HEP）即乳腺癌抵抗蛋白（BCRP）和猫白血病病毒（FLVCR）表达水平、动物体内炎症细胞因子、血红素结合蛋白（HPX）水平等。.研究结果：1. 四妙勇安汤提高血红素损伤神经元的存活率达48.3%，降低神经元内血红素浓度达68.27%， 降低羟自由基（•OH）含量达78.26%，分别提高神经元内BCRP蛋白表达水平92.10%、mRNA水平83.33%，FLVCR蛋白93.55%、mRNA水平92.30%，降低受损神经元内IL-1、IL-6、TNF-蛋白水平分别为94.68%，97.90%，95.17%。 .2. 四妙勇安汤促进血红素和锡中卟啉（SnMP）跨模拟血脑屏障的转运，在24小时内能降低大脑侧血红素69.57%、SnMP52.94%，降低IL-1、IL-6、TNF-水平，提高血红素转运效率。.3. 在动物模型上，四妙勇安汤降低脑出血动物神经缺损积分（mNSS）和动物体温，降低脑内血红素和铁含量，使脑毛细血管内BCRP和FLVCR 蛋白表达水平显著增高，脑内和血中炎性细胞因子表达水平显著降低，血液中HPX蛋白水平显著提高。.结论与意义：四妙勇安汤能降低脑出血后脑内和神经元内游离血红素和铁离子水平，提高细胞膜上BCRP和FLVCR表达、降低炎症细胞因子水平、提高血液HPX含量。提示四妙勇安汤有通过提高细胞膜上BCRP和FLVCR表达、降低炎症细胞因子水平、提高血液HPX来含量促进游离血红素从神经元细胞及脑组织内排出作用。研究成果在理论上创新和丰富了清热解毒、活血通脉方药治疗ICH的学术内容，具有良好的推广前景和应用意义。