ANTI INFLAMMATORY AND ANTIFIBROTIC ACTIONS OF THYMOSIN BETA 4 IN ALD

描述（由申请方提供）：基于广泛接受的ALD两次打击模型，慢性乙醇（EtOH）/LPS产生的内毒素激活NFκB，从而上调TNFα和IL 1，后者是导致肝细胞损伤的强效促炎细胞因子。此外，EtOH诱导的Cyp 2 E1和ADH导致乙醛的代谢产生和活性氧物质（ROS）的增加，其将静止HSC活化为肌成纤维细胞，导致纤维化基因、血小板衍生生长因子β受体（PDGF β r）、α-平滑肌肌动蛋白（αSMA）和细胞外基质蛋白（ECM）、胶原蛋白I、III和纤连蛋白和表观遗传阻遏物基因的上调，甲基-CpG结合蛋白2（MeCP 2）。相反，脂肪形成基因、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（SREBP 1c）受到抑制，导致其维生素A储存丧失，并从静止的脂质储存表型转分化为活跃的成肌纤维细胞表型。值得注意的是，胸腺素β 4（T β 4）是一种生物活性肽，据报道可预防许多肝外组织中的炎症和纤维化。基于这些，PI假设T β 4对EtOH/LPS肝损伤的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NFκB B的活化，从而防止TNFα和IL 1的上调以及随后的肝损伤，和（ii）抑制上调的MeCP 2，其协同逆转（a）下调的脂肪形成基因和（B）上调的纤维形成基因，从而阻止HSC从储存脂质的周细胞转分化为肌成纤维细胞。我们还假设T β 4通过过表达抑制EtOH/LPS引起的MeCP 2过表达的miR 132来发挥其上述作用。PI在EtOH/LPS小鼠模型中具有以下令人鼓舞的初步结果，以保证他的新探索方法的特征：NFκB信号级联、pIκB、TNFα、IL 1 β及相应的肝损伤血清标志物的上调; 2.上调肝MeCP 2、PDGF β r、αSMA、Col 1 α1和蛋白质;和3.成脂基因PPARγ表达下调。因此，我们认为T β 4阻断了（i）NFκB、TNFα和炎症级联反应的激活，以及（ii）静止HSC向纤维化HSC的转分化;然而，T β 4维持肝细胞再生。因此，我们的创新提案的主要目标是实现以下具体目标：具体目标1。T β 4“保护/减轻”EtOH/LPS介导的NFκB信号级联、TNFα和IL 1 β上调的“治疗前和治疗后”可能的作用机制是什么？具体目标二：T β 4“治疗前和治疗后保护/缓解”EtOH/LPS介导的（a）肝纤维化基因及其产物上调的可能作用机制是什么？和（B）成脂基因及其产物的下调？肝细胞结构和HSC表型是否有相应的形态学变化？PI计划利用生物化学、分子生物学、免疫和组织化学技术，使用已建立的EtOH/LPS两次打击小鼠体内和体外模型进行研究。PI有一个强大的分子生物学和生物化学小组，在这个提案的各个方面都有专业知识。这可能导致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 IL1ß 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 ß-receptor (PDGFßr), 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 ß4 (Tß4), a bioactive peptide, is reported to prevent inflammation and fibrosis in many extra-hepatic tissues. Based on these, PI hypothesizes that Tß4'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 IL1ß 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 Tß4 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: Tß4 protects against EtOH/LPS induced 1. Up-regulation of NFκB signaling cascade, pIκB, TNFα, IL1ß and consequent serum markers for liver injury; 2. up regulation of hepatic MeCP2, PDGFßr, αSMA, Col1α1 & proteins; and 3. down-regulation of adipogenic gene, PPARγ. As a result, we propose that Tß4 blocks the (i) activation of NFκB, TNFα and inflammatory cascade and (ii) transdifferentiation of quiescent HSC to fibrogenic HSC; yet, Tß4 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 Tß4 "Pre- and Post-treatment to protect/alleviate" EtOH/LPS-mediated up regulation of NFκB signaling cascade, TNFα & IL1ß, and consequent serum and liver markers for liver injury? Specific Aim 2: What are the possible mechanism/s of action/s of Tß4 "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.