Wnt通路增强谷氨酰胺代谢调控肝星状细胞活化与肝纤维化的分子机制

Hepatic stellate cell (HSC) activation is the pivotal event during the pathogenesis of liver fibrosis. It is also a process highly dependent on substance and energy metabolism. Latest evidence suggests that glutaminolysis is a more powerful force to drive and maintain HSC activation, but little is known about the underlying mechanisms. The applicant previously found that Wnt signaling could increase the intracellular glutaminolysis level in HSCs and significantly upregulated the protein expression of ASCT2, a key membrane transport carrier of glutamine. Based on these, the applicant proposes that Wnt signaling could enhance ASCT2-mediated glutaminolysis to promote HSC activation and hepatic fibrogenesis. To validate this hypothesis, the applicant will use hepatic LRP5 (a co-receptor of Wnt signaling) conditional knockout mice and various animal models of liver fibrosis and cell culture systems to carry out in vivo and in vitro experiments. First, the applicant will validate that regulation of glutaminolysis is involved in Wnt promotion of HSC activation and liver fibrosis. Then, the roles and regulatory pathways of ASCT2-mediated glutaminolysis and β-catenin-mediated transactivation will be investigated and elucidated. Finally, all the discovered mechanisms will be validated in clinical human liver samples with liver fibrosis. These investigations will comprehensively elucidate the specific mechanisms underlying Wnt signaling regulation of glutaminolysis in HSCs, and uncover novel molecular mechanisms of HSC activation and liver fibrosis.

肝星状细胞（HSC）活化是肝纤维化病理机制的核心环节，也是一个高度依赖物质与能量代谢的过程。新近发现，谷氨酰胺代谢是推动和维持HSC活化更强有力的因素，但其中的调控机制不明。申请者前期发现Wnt通路能够增强HSC谷氨酰胺代谢水平，并上调谷氨酰胺关键转运载体ASCT2的表达。据此提出Wnt通路通过促进ASCT2介导的谷氨酰胺代谢，推动HSC活化与肝纤维化的科学假说。为此拟利用肝脏LRP5（Wnt通路的膜受体）特异性敲除小鼠及多种肝纤维化动物模型与细胞模型，首先明确Wnt通路能够通过调控谷氨酰胺代谢促进HSC活化与肝纤维化；然后探究ASCT2介导的谷氨酰胺转运、β-catenin介导的转录激活在其中的关键作用与调控机制；最后利用人临床肝纤维化样本进行验证。以此阐明Wnt通路调控HSC谷氨酰胺代谢的特异性途径，揭示HSC活化与肝纤维化的病理新机制。

肝纤维化是多种慢性肝病的共同病理过程，肝星状细胞（HSC）活化是肝纤维化发生发展的核心环节。新近发现，HSC活化过程中发生谷氨酰胺代谢重编程，但其调控机制以及在药物干预肝纤维化中的作用还知之甚少。本项目以谷氨酰胺代谢为核心，深入研究了Wnt信号通路对HSC谷氨酰胺代谢的调控机制、谷氨酰胺转运蛋白ASCT2对HSC衰老及衰老相关分泌表型（SASP）的调控机制、天然化合物白术内酯III和大黄素通过调控HSC谷氨酰胺代谢抗肝纤维化的分子机制。本研究通过体内外实验相结合，在人临床样本、整体动物、细胞及分子水平发现：（1）Wnt/Lrp6通路通过β-Catenin与m6A RNA甲基化酶ZC3H13的mRNA直接结合，减少ZC3H13的表达，进而抑制ASCT2的m6A RNA甲基化，导致ASCT2表达上调及HSC谷氨酰胺代谢的增强。（2）抑制ASCT2可以通过阻断谷氨酰胺代谢诱导HSC发生细胞衰老，通过阻断IL-1/NF-κB信号途径抑制促炎性的SASP表达，这一机制显著不同于传统的细胞衰老诱导剂依托泊苷的作用；白术内酯III通过与ASCT2蛋白的Asn230氨基酸残基相结合阻断ASCT2的功能及谷氨酰胺代谢，进而诱导HSC衰老发挥体内外抗肝纤维化作用。（3）大黄素通过促进核受体Nur77在HSC核中聚集并与DNA甲基转移酶DNMT3b相互作用，促进谷氨酰胺酶GLS1的DNA甲基化，进而抑制GLS1的表达并阻断谷氨酰胺代谢发挥抑制HSC活化的作用；体内靶向HSC的大黄素维生素A脂质体在小鼠肝纤维化模型中，通过Nur77更加有效地抑制HSC谷氨酰胺代谢并诱导其衰老，进而减轻肝纤维化。上述研究结果均为本项目创新发现，揭示了HSC谷氨酰胺代谢重编程调控肝纤维化的上游机制与下游效应，明确了HSC谷氨酰胺代谢可以成为药物干预肝纤维化的有效靶标并筛选获得2个潜在的抗肝纤维化天然产物，为肝纤维化的有效防治提供了新的策略与科学依据。本项目研究建立起了以HSC物质与能量代谢为基础，多学科交叉融合的慢性肝病病理学与药理学研究技术体系，为项目负责人在此领域的后续研究提供了创新启示和坚实基础。