ANTI INFLAMMATORY AND ANTIFIBROTIC ACTIONS OF THYMOSIN BETA 4 IN ALD

描述（由申请人提供）：基于公认的ALD双击模型，慢性乙醇(EtOH)/LPS产生的内毒素激活NFκB，上调TNFα和il - 1ß，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Cyp2E1和ADH导致乙醛的代谢生成和活性氧（ROS）的增加，活性氧（ROS）激活静止的HSC到肌成纤维细胞，导致纤维生成基因、血小板衍生生长因子ß-受体（PDGFßr）、α-平滑肌肌动蛋白（αSMA）和细胞外基质蛋白（ECM）、胶原I、III、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，脂肪生成基因、过氧化物酶体增殖物激活受体γ (PPARγ)和甾醇调节元件结合蛋白1c （SREBP1c）被抑制，导致维生素A储存丧失，并从静止的脂质储存表型转分化为活跃的肌成纤维表型。值得注意的是，据报道，胸腺素ß4 （Tß4）是一种生物活性肽，可预防许多肝外组织的炎症和纤维化。基于这些，PI假设Tß4对EtOH/LPS肝损伤的抗炎和抗纤维化作用是通过(i)通过阻断i- κ b的磷酸化和解离来抑制nf - κ b的激活，从而阻止TNFα和il - 1ß（强效的促炎细胞因子）的上调和随后的肝损伤，以及（ii）抑制上调的MeCP2。协同逆转(a)下调的脂肪生成基因和(b)上调的纤维生成基因，从而阻止HSC从脂质储存周细胞向肌成纤维细胞的反分化。我们还假设Tß4通过过表达miR132抑制EtOH/LPS引起的MeCP2过表达而引发上述作用。PI在EtOH/LPS小鼠模型中获得了以下令人鼓舞的初步结果，以确保其新颖探索性方法的可行性：Tß4可防止EtOH/LPS诱导的1。肝损伤中nf - κ b信号级联、pIκB、TNFα、il - 1ß及相应血清标志物的上调2. 上调肝脏MeCP2、PDGFßr、αSMA、Col1α1 &蛋白表达；和3。下调脂肪生成基因PPARγ。因此，我们提出Tß4阻断(i) nf - κ b、tnf - α和炎症级联的激活，以及（ii）静止HSC向纤维化HSC的转分化；然而，Tß4维持肝细胞再生。因此，我们的创新方案的主要目标是实现以下具体目标：Tß4“前后治疗以保护/减轻”EtOH/ lps介导的nf - κ b信号级联、TNFα和il - 1ß的上调，以及随之而来的血清和肝脏标志物对肝损伤的可能作用机制是什么？具体目标2:Tß4“前后处理以保护/缓解”对EtOH/ lps介导的肝纤维化基因及其产物的上调可能的作用机制是什么？(b)下调脂肪生成基因及其产物的调控？肝细胞结构和HSC表型是否有相应的形态学改变？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双击小鼠体内和体外模型。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 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.