ANTI INFLAMMATORY AND ANTIFIBROTIC ACTIONS OF THYMOSIN BETA 4 IN ALD

描述(申请人提供)：基于公认的急性酒精性肝病两次打击模型，慢性乙醇/脂多糖产生的内毒素激活核因子κB，上调肿瘤坏死因子α，以及白介素1β，这是导致肝细胞损伤的强有力的促炎细胞因子。此外，乙醇诱导的细胞色素P450 1和脱氢酶导致乙醛的代谢生成和活性氧自由基(ROS)的增加，激活静止的肝星状细胞形成肌成纤维细胞，导致纤维化基因、血小板衍生生长因子β受体(PDGFRβr)、α平滑肌肌动蛋白(αSMA)和细胞外基质蛋白(ECM)、I型和III型胶原、纤维连接蛋白和表观遗传抑制基因甲基CpG结合蛋白2(MeCP2)的上调。相反，成脂基因、过氧化物酶体增殖物激活受体γ(PPARγ)和固醇调节元件结合蛋白1c(SREBP1c)被抑制，导致其维生素A储备的丧失和从静止的脂肪储存表型向活跃的肌成纤维细胞表型的转分化。值得注意的是，胸腺肽β4(Tβ4)是一种生物活性多肽，据报道在许多肝外组织中可以预防炎症和纤维化。在此基础上，PI假设Tβ4‘κ抗炎和抗纤维化作用是通过(I)通过阻断IκB的磷酸化和解离来抑制NFα的激活，从而阻止强有力的促炎细胞因子IL1β的上调而导致肝损伤；(Ii)抑制上调的MeCP2，从而协同逆转(A)下调的成脂基因和(B)上调的纤维化基因，从而阻止肝干细胞从储脂周细胞向肌成纤维细胞的反式分化。我们还假设Tβ4通过过度表达miR132抑制乙醇/脂多糖引起的MeCP2过表达而产生上述作用。PI在乙醇/脂多糖小鼠模型上取得了以下令人鼓舞的初步结果：Tβ4对乙醇/脂多糖诱导的新方法的可行性具有保护作用：1.上调NFκB信号级联、PIκB、肿瘤坏死因子α、IL 1β和随后的血清肝损伤标志物；2.上调肝脏MeCP2、PDGFβr、αSMA、COL1α1和蛋白；3.下调脂肪生成基因PPARγ。因此，我们认为Tβ4可阻断(1)活化的核因子κB、肿瘤坏死因子α和炎性级联反应，以及(2)静止的肝星状细胞向纤维化的肝星状细胞的转分化，但Tβ4维持肝细胞的再生。因此，我们的创新方案的主要目标是实现以下特定目标：特定目标1.可能的机制/Tβ4的S/S“治疗前后保护/缓解”乙醇/脂多糖介导的NF-κB信号级联、肿瘤坏死因子α和白介素1β以及随后的血清和肝损伤标志物的上调？具体目标2：乙醇/脂多糖介导的(A)肝纤维化基因及其产物上调的可能机制/Tβ4的S/S“治疗前后保护/缓解”？以及(B)对成脂基因及其产物的下调？在肝细胞结构和HSC表型上是否有相应的形态变化？PI计划利用生物化学、分子生物学、免疫和组织化学技术建立Etoh/LPS二次打击小鼠的体内和体外模型。PI拥有一个强大的分子生物学和生化小组，在这项提议的所有方面都拥有专业知识。这可能会导致ALD的新疗法。

DESCRIPTION (provided by applicant): Based on the well accepted two-hit model for ALD, Chronic Ethanol (EtOH)/LPS generated endotoxins activate NFκB that up regulates TNFα, and IL1β the potent proinflammatory cytokines causing hepatocyte injury. Further, EtOH induced Cyp2E1 and ADH lead to metabolic generation of acetaldehyde and increased reactive oxygen species (ROS), which activate quiescent HSC to myofibroblasts resulting in up regulation of fibrogenic genes, platelet derived growth factor β-receptor (PDGFβr), α-smooth muscle actin (αSMA) and extracellular matrix proteins (ECM), collagen I, III, and fibronectin and epigenetic repressor gene, methyl-CpG binding protein 2 (MeCP2). In contrast, adipogenic genes, peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element-binding protein 1c (SREBP1c) are suppressed resulting in the loss of their vitamin A stores and their transdifferentiation from quiescent lipid storing phenotype to active myofibroblastic phenotype. Significantly, thymosin β4 (Tβ4), a bioactive peptide, is reported to prevent inflammation and fibrosis in many extra-hepatic tissues. Based on these, PI hypothesizes that Tβ4's anti-inflammatory and anti-fibrogenic actions against EtOH/LPS liver injury are mediated by (i) inhibiting the activation of NFκB by blocking the phosphorylation and dissociation of IκB and thereby prevent the up regulation of TNFα, and IL1β the potent proinflammatory cytokines and consequent liver injury, and (ii) suppressing the up regulated MeCP2, that coordinately reverses (a) the down regulated adipogenic genes and (b) up regulated fibrogenic genes and thereby prevent the trans-differentiation of HSC from lipid-storing pericytes to myofibroblasts. We also hypothesize that Tβ4 elicits its above actions by overexpressing miR132 that suppresses MeCP2 overexpression caused by EtOH/LPS. PI has the following encouraging preliminary results in the EtOH/LPS mouse model to reassure the feasibilities of his novel exploratory approaches: Tβ4 protects against EtOH/LPS induced 1. Up-regulation of NFκB signaling cascade, pIκB, TNFα, IL1β and consequent serum markers for liver injury; 2. up regulation of hepatic MeCP2, PDGFβr, αSMA, Col1α1 & proteins; and 3. down-regulation of adipogenic gene, PPARγ. As a result, we propose that Tβ4 blocks the (i) activation of NFκB, TNFα and inflammatory cascade and (ii) transdifferentiation of quiescent HSC to fibrogenic HSC; yet, Tβ4 maintains hepatocyte regeneration. Thus, the major goals of our innovative proposal are to accomplish the following specific aims: Specific Aim 1. What are the possible mechanism/s of action/s of Tβ4 "Pre- and Post-treatment to protect/alleviate" EtOH/LPS-mediated up regulation of NFκB signaling cascade, TNFα & IL1β, and consequent serum and liver markers for liver injury? Specific Aim 2: What are the possible mechanism/s of action/s of Tβ4 "Pre- and Post-treatment to protect/alleviate" against EtOH/LPS-mediated (a) up regulation of hepatic fibrogenic genes and their products? and (b) down regulation of adipogenic genes and their products? Are there corresponding morphological changes in liver cell architecture as well as in HSC phenotype? PI plans to approach using established EtOH/LPS two-hit mouse in vivo & in vitro model utilizing biochemistry, molecular biology, immuno- & histo-chemistry techniques. PI has a strong molecular biology and biochemical group that has the expertise in all aspects of this proposal. This may lead to novel therapy for ALD.