POSSIBLE MOLECULAR MECHANISMS FOR PORTAL HYPERTENSION AND THE IMPLICATIONS ON ITS TREATMENT

门静脉高压症的特征是内脏器官的高动力循环，以及肝内血管阻力的增加。内皮型一氧化氮合酶(ENOS)和肿瘤坏死因子-α(肿瘤坏死因子-α)的过量产生与全身高动力循环和门脉高压性胃病的发生密切相关。肝内血管阻力的增加与肝星状细胞(HSCs)收缩和肝窦内皮细胞一氧化氮(NO)生成减少有关。肝星状细胞在肝纤维化中也起主要作用，导致门脉高压。然而，这些现象的分子机制仍有待阐明。本研究的目的是确定内脏器官高动力循环和肝内微循环障碍的分子基础。我们的研究首次证明：(1)…中eNOS的激活更多的PHT胃粘膜是由于：(A)eNOS丝氨酸1177位的磷酸化增加；(B)PI 3-K/Akt信号通路的激活；(C)Akt与eNOS的结合增强；(2)肿瘤坏死因子-α中和抗体使PHT胃粘膜eNOS的磷酸化和PI 3-K/Akt信号通路的激活降至正常水平。在PHT胃粘膜中，升高的肿瘤坏死因子-α通过激活PI-3-K/AKT信号通路诱导eNOS在丝氨酸1177位的磷酸化。PHT胃粘膜对酒精、阿司匹林等有害因素的损伤非常敏感，但其机制尚不完全清楚。由于这种粘膜损伤最初是由氧自由基介导的，而且由于MAP激酶(ERK2)保护细胞免受应激并诱导细胞增殖，我们推测氧化应激诱导的ERK2在PHT胃粘膜中的激活是有缺陷的。在这里，我们证明了在PHT胃粘膜中，氧化应激激活的ERK2是受损的。这种损伤是由MAPK磷酸酶-1(MKP-1)的过度表达介导的，MKP-1是由潜在的氧化状态引起的，并通过抑制MKP-1来改善。补充维生素E，一种自由基清除剂，使PHT胃粘膜中MKP-1的表达正常化，并完全逆转对酒精损伤的敏感性增加。我们的发现为PHT胃病的病理生理机制提供了分子机制，并应加快开发有效的非侵入性治疗方法。在二甲基亚硝胺(DMN)诱导的大鼠肝纤维化模型上，口服新型Rho激酶特异性抑制剂法舒地尔可在3周内减轻肝纤维化。肝损伤后HSCs活化，3周后活化的HSCs(_-SMA阳性细胞)数量减少，蛋白表达减少。此外，与单纯DMN相比，口服法舒地尔在1周和3周均可显著降低DMN大鼠的门静脉压力。这项研究表明，口服法舒地尔可减少体内细胞外基质沉积和门脉高压。体外实验表明，1-甲基法舒地尔可诱导HSC凋亡。肝纤维化和门脉高压的减轻可能是通过抑制HSC的激活和凋亡而实现的。本研究在胆管结扎的肝硬变大鼠模型上证实：(1)Rho激酶信号及其dawnstream-SMA蛋白表达上调；(2)法舒地尔长期抑制Rho激酶可导致肝星状细胞收缩减少，eNOS磷酸化上调，阻止微循环障碍，最终改善肝硬变门脉高压。较少

Portal hypertension is characterized by the hyperdynamic circulation in the splanchnic organs as well as by the increased intrahepatic vascular resistance. Excessive nitric oxide production by endothelial nitric oxide synthase (eNOS) and elevated tumor necrosis factor-α (TNF-α) are implicated in the development of the systemic hyperdynamic circulation and portal hypertensive (PHT) gastropathy. The increased intrahepatic vascular resistance is associated with a contraction of hepatic stellate cells (HSCs) and a reduction of nitric oxide (NO) production in sinusoidal endothelial cells. HSCs also plays a major role in hepatic fibrogenesis, leading to portal hypertension. However, the molecular mechanisms for these phenomena remain to be elucidated. The aims of our studies are to determine the molecular basis for the hyperdynamic circulation of splanchnic organs and the intrahepatic microcirculatory disturbance.Our present study demonstrates for the first time that : (1)eNOS activation in … More PHT gastric mucosa is due to : (a)increased phosphorylation of eNOS at serine 1177 ; (b)activation of the PI 3-kinase/Akt signaling pathway ; (c)enhanced binding of Akt to eNOS ; and, (2)TNF-α neutralizing antibody reduces phosphorylation of eNOS and activation of the PI 3-kinase/Akt signaling pathway in PHT gastric mucosa to the normal levels. In PHT gastric mucosa elevated TNF-α induces phosphorylation of eNOS at serine 1177 (required for its activation) via activation of the PI 3-kinase/Akt signaling pathway.PHT gastric mucosa is highly susceptible to injury caused by alcohol, aspirin and other noxious factors, but the mechanism is not fully understood. Since such mucosal injury is initially mediated by oxygen free radicals, and since MAP kinase (ERK2) protects against cellular stress and induces cell proliferation, we postulated that oxidative stress-induced ERK2 activation is defective in PHT gastric mucosa. Here we demonstrated that in PHT gastric mucosa, ERK2 activation by oxidative stress is impaired. This impairment is mediated by overexpression of MAP kinase phosphatase-1 (MKP-1), which results from an underlying oxidative state, and is ameliorated by inhibiting MKP-1. Supplementing vitamin E, a free radical scavenger, normalizes MKP-1 expression in PHT gastric mucosa and completely reverses the increased susceptibility to alcohol injury. Our findings provide the molecular mechanism for the pathophysiology of PHT gastropathy and should expedite developing effective non-invasive therapy.In the rat model of liver fibrosis induced by dimethlnitrosamine(DMN), fasudil (a novel specific inhibitor of Rho kinase) conceptation orally reduced fibrosis of the liver in 3 weeks. HSCs activation induced by liver injuary, as shown by the decreased the number of activated HSCs (_-SMA positive cells) and the protein expression_-SMA was also reduced in 3weeks. Moreover, oral administration of fasudil significantly reduced portal pressure of DMN rats at both 1 and 3 weeks, compared to DMN alone. This study indicates that oral administration of fasudil reduce extracellular matrix deposition and portal hypertension in vivo. In vitro experiments showed 1_M fasudil induced HSC apoptosis. The reduction of liver fibrosis and portal hypertension is likely to be induced by inhibition of HSC activation and apoptosis. These points will impact on the new therapiutic drug for cirrhotic patients.The present study demonstrates in cirrhotic rats by the bile duct ligation that (1)Rho kinase signaling and its dawnstream _-SMA protein is upregulated; and (2)the long-term inhibition of Rho kinase with fasudil leads to the reduced contraction of HSCs and the upregulation of eNOS phosphorylation and prevents the microcirculatory disturbance, finally improving the portal hypertension in liver cirrhosis. Less