基于HDACs调控的丙戊酸肝毒性机制及其生物标志物研究

Valproic acid (VPA) is an efficacious broad-spectrum antiepileptic drug (AED) commonly used for generalized and focal epilepsies. It potentially causes idiosyncratic liver injury, characterized by the presence of microvesicular steatosis. The mechanistic link of this clinical mystery remains unknown. The formation of toxic metabolites of VPA has been proposed to explain the hepatotoxicity. However, the metabolites potentially related to hepatotoxicity have been detected in our previous studies, but at lower than the minimum toxicity concentrations in patients with liver injury. These data suggests a limited role of toxic metabolites in VPA-induced hepatotoxicity. VPA has recently been shown to inhibit histone deacetylases (HDACs), which can play a central role in the regulation of lipid metabolic pathways in liver. It was found that HDACs knockdown aggravated hepatic steatosis. The new evidences on the importance of HDACs in the regulation of lipid metabolic pathways and the inhibition of HDACs at therapeutic concentrations of VPA open new perspectives in the comprehension of VPA-induced hepatotoxicity. Therefore, firstly, we’ll explore the relationship between liver injury and HDACs activities or lipid metabolism-related gene expressions in animal models. Secondly, we’ll specify the effects of VPA on the subtypes of HDACs with the gene over-expressed of HDACIs profiling in normal hepatocyte. At last, we focuse on the detailed analysis of lipid species in vivo and vitro, and explore the associations of altered lipid profiles, along with the subtype of HDACs, together with the expressions of genes related to lipid metabolism. Our study aimed to provide new insights to deeply understand the pathogenic mechanism of VPA-induced hepatotoxicity and discover novel biomarkers with respect to effective early warning. The information obtained from our study will shed light on the proper use of VPA in clinical settings.

丙戊酸（VPA）是广谱抗癫痫药物，具有潜在肝毒性，表现为肝脂肪变性。关于VPA致肝毒性机制研究尚不清晰，我们发现VPA肝毒性代谢物体内含量低，不能达到体外致毒浓度，可能存在新的作用机制。近年研究表明VPA可抑制组蛋白去乙酰化酶（HDACs），而HDACs可调控肝脏脂代谢，敲除HDACs加重肝脂肪变性。在初步观察VPA体内毒性代谢物水平、HDACs抑制作用及促进脂积累的基础上，本课题拟从VPA对HDACs抑制作用出发，以VPA致肝毒性动物模型为对象，考察HDACs活性、脂代谢相关基因表达谱；人肝细胞过表达不同HDACs，确定VPA肝毒性的HDACs亚型。以动物、细胞模型及肝损伤癫痫患儿为对象，采用脂质组学技术测定生物样本中特定脂质谱，分析肝毒性特异脂质组分与HDACs亚型、脂代谢相关基因相关性。此项研究旨在阐明VPA导致肝毒性新机制，发现可早期预警的生物标志物，为该类药物的研究提供新思路。

丙戊酸（VPA）是广谱抗癫痫药物，具有潜在肝毒性，表现为肝脂肪变性，目前其致肝毒性机制尚不清晰。本项目采用脂质组学及代谢组学技术，以细胞-动物-人体为研究对象，探究VPA致肝毒性机制，并初步完成VPA对不同亚型组蛋白去乙酰化酶（HDACs）的抑制作用及蛋白乙酰化水平的影响。采用脂质组学技术分析VPA致肝毒性癫痫患儿血清及体外细胞的脂质表达谱，发现患儿血清中长碳链甘油三酯（TAG）的增加与VPA致肝毒性呈正相关，溶血磷脂酰胆碱（LPC）、鞘脂（SM）和神经酰胺（Cer）显著降低，均与ALT水平呈较强相关性；细胞实验中，VPA呈剂量依赖性升高肝细胞内长碳链TAG、游离脂肪酸（FFA 20-26）水平，加重高脂状态下TAG累积，筛选出了潜在的VPA致肝毒性生物标志物；机制研究发现VPA可上调PPARγ-CD36/DGAT2等多条脂代谢通路，导致肝脂质代谢紊乱。对VPA致肝毒性癫痫患儿进行非靶向代谢组学分析，发现与肝功正常癫痫患儿相比，VPA致肝毒性癫痫患儿血清代谢模式发生改变，涉及脂肪酸通路、氨基酸代谢、柠檬酸循环、尿素循环和胆汁酸通路等；机制研究首次发现了VPA致肝毒性癫痫患儿血清胆汁酸稳态变化，且FXR的乙酰化水平可能与VPA致肝毒性相关。研究发现VPA可抑制I类HDACs活性，上调组蛋白和非组蛋白的乙酰化水平。研究显示VPA上调CYP2E1表达产生的氧化压力也可以调控CD36及DGAT2的表达，进一步引起肝脏脂质累积。本项目对VPA致肝毒性机制进行了研究，发现可能的预警生物标志物，为该药物的进一步研究提供理论基础。