果糖代谢紊乱在丙型肝炎病理损伤中的作用机制研究

Hepatitis C Virus (HCV) infection is a global health threat. The positive-strand genome of HCV encodes a single polyprotein of about 3000 amino acids, which is cleaved by viral and cellular proteases into mature structural proteins (core, E1 and E2) and non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). Recent studies have demonstrated that NS3/4A can cleave Cardif (VISA/MAVS/IPS-1) and TRIF. However, whether NS3/4A is involved in other virus-host interactions through targeting unidenti?ed cellular substrates is largely unknown. In?ammation is a common element in the pathogenesis of most chronic liver diseases that lead to ?brosis and cirrhosis. In?ammasomes are intracellular multiprotein complexes that sense intracellular danger signals via NOD-like receptors (NLR). In?ammasomes play an important role in in?ammation. To date, NLRP3 is the most fully characterized member of the in?ammasome family. It consists of the NOD-like receptor NLRP3, the adaptor molecule ASC, and the effector molecule pro-caspase-1. And lysosomal destabilization triggers NALP3 in?ammasome activation.In our coimmunoprecipitation experiments, we found that ALDOB, a key enzyme involved in the fructose metabolism, was cleaved when co-transfected with NS3/4A in 293T cells. In Huh-7 cells, endogenous ALDOB was also cleaved after JFH-1 infection. ALDOB is an enzyme participating in fructose metabolism that cleaves fructose 1-phosphate (F 1-P) into dihydroxyacetone phosphate (DHAP) and glyceraldehyde. A lack of aldolase B activity results in accumulation of the F 1-P substrate. During passage through the Golgi, terminal mannose residues of lysosomal enzymes are phosphorylated to yield mannose 6-phosphate (M6P), producing a marker specific for lysosomal enzymes. This marker is then recognized by the cation-independent mannose phosphate receptors (CI-MPR). An important feature of the MPR-lysosomal enzyme interaction is that it can be inhibited by quite low concentrations of the monosaccharide F 1-P. So, F 1-P may impede the transport of lysosomal enzymes to the lysosome. As a result, F 1-P can leads to the lysosomal destabilization. The NALP3 in?ammasome senses lysosomal damage as an endogenous "danger"signal. Therefore, it is rational to speculate that F 1-P accumulation by HCV infection, could activate the NALP3 in?ammasome through lysosomal destabilization. We first plan to detect whether intracellular F 1-P accumulate if HCV infect the hepatic parenchymal cells. To determine whether the F 1-P accumulation can result in lysosomal perturbation and NALP3 in?ammasome activation, we will test the IL-1β maturation, caspase-1 activation, and monitor the lysosomal compartment in future experiments.

丙型肝炎病毒（HCV）严重威胁人类健康,全球有1.3-1.7亿HCV慢性感染者,每年至少有三十万人死于HCV感染引起的肝脏疾病。炎症在慢性肝炎演变成纤维化和肝硬化过程中发挥重要作用，研究HCV诱导炎症的分子机制，对于丙肝相关肝病防治具有重要意义。本项目中，我们将在分子和细胞水平，研究HCV感染过程中，NS3/4A与ALDOB相互作用机制；检测HCV感染对果糖代谢的影响；查明ALDOB的作用底物1-磷酸-果糖（F 1-P）对溶酶体、炎性小体功能的调节机制；利用丙型肝炎动物模型，阐述HCV感染的急、慢性期ALDOB分子表达特征和果糖代谢变化规律；检测溶酶体损伤以及炎性小体功能的变化与肝损伤的关系；采用相关抑制剂调节果糖代谢和溶酶体、炎性小体的活性，寻找控制丙型肝炎的策略。最终阐述HCV是否以及如何影响肝细胞果糖代谢，以及果糖代谢紊乱在丙型肝炎病理损伤中的作用机制。

过去几十年里，全世界范围内居民膳食果糖摄入量逐渐增加，果糖的过量摄入加剧了代谢综合症的病理特征。最近的证据表明，代谢因素可能影响慢性丙型肝炎（CHC）患者的自然病史。然而，有关果糖在CHC进展中的作用却鲜见报道。本课题中，我们发现HCV感染阻断果糖糖酵解。利用液相色谱串联质谱（LC-MS/MS）监测细胞内代谢物变化，检测结果显示，HCV感染导致醛缩酶反应的底物果糖-1-磷酸（F-1-P）富集。HCV感染后，细胞内醛缩酶B（ALDOB）的蛋白丰度和酶活性明显降低。ALDOB被HCV感染激活的Caspase-1切割，加入Caspase-1抑制剂可以使ALDOB蛋白水平恢复。ALDOB下调导致的F-1-P的累计加速了HCV感染引起的肝细胞死亡。过表达ALDOB降低F-1-P水平，促进细胞存活。F-1-P通过糖基化反应引发氧化应激而引发肝细胞凋亡。死亡的肝细胞释放炎性小体激活的第二信号，ATP和可溶性尿酸。这些代谢物能快速而短暂的激活免疫细胞的caspase-1炎症小体，并诱导IL-1β分泌。本项目证实，高果糖摄入可能促进HCV相关肝脏炎症反应和病理损伤，为慢性丙肝患者的治疗提供了新的潜在分子靶标。