Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, China.
Institute of Clinical Pharmacology, Central South University, Changsha, 410078, Hunan, China.
Arch Toxicol. 2019 Jun;93(6):1679-1695. doi: 10.1007/s00204-019-02447-0. Epub 2019 Apr 11.
Low-density lipoprotein receptor-related protein 6 (LRP6) is an important coreceptor in the Wnt/β-catenin upstream signaling pathway. Rs2302685 is a common functional mutation of LRP6 that has been previously associated with reduced alcoholic liver injury among alcoholic liver disease (ALD) patients, and the present research was designed to study the underlying mechanisms of that finding. A total of 107 ALD patients and 138 non-ALD patients were recruited from hospitalized alcoholics in China. Their venous blood samples were collected for DNA extraction and genotyped using Sequenom MassARRAY. We found that the rs2302685 mutation, which impaired the function of LRP6, was present in higher frequency among alcoholics with ALD than those without ALD. We also conducted a mouse model experiment in which LRP6 knockdown mice and LRP6 wild-type mice received daily intragastric doses of ethanol (2.4 g/kg) as well as a larger dose of ethanol (4 g/kg) every 7 days for 28 days. The mouse blood and liver specimens were subsequently collected for laboratory analysis, and cell experiments were performed to compare the inhibition, activation, over-expression, and siRNA of LRP6 in the treatment versus the control HL7702 cells. Expression of the targeted molecules was detected by real-time PCR or western blot analysis. Stably transfected cells with pRL3-CYP2E1 vector were used to further study the underlying mechanisms. The total bile acid (TBA), direct bilirubin, total bilirubin (TBIL), aspartate aminotransferase (AST), mitochondrial aspartate aminotransferase, and AST/ALT values were significantly lower in carriers of the rs2302685 mutation than in the wild-type patients, by 63.4, 60.6, 82.1, 44.8, 45.7, and 21.4%, respectively. Compared to the LRP6 wild-type mice, the LRP6 knockdown mice had lower ALT, TBIL, TBA, and ALB/GLO values, as well reduced liver tissue damage, in accordance with their reduced expressions of LRP6, β-catenin, and CYP2E1. In HL7702 cells exposed to ethanol, AST, ALT, lipid accumulation, and ROS generation decreased in cells that were treated with LRP6 inhibitors or siRNA but increased in cells treated with LRP6 activators or over-expressed LRP6. TCF1 was the transcriptional factor most likely to connect the LRP6-Wnt/β-catenin signaling pathway to the regulation of CYP2E1. We concluded that the LRP6 functional mutation rs2302685 contributes to individual differences in susceptibility to alcoholic liver injury related to the Wnt/β-catenin-TCF1-CYP2E1 signaling pathway.
低密度脂蛋白受体相关蛋白 6（LRP6）是 Wnt/β-连环蛋白上游信号通路中的重要辅助受体。Rs2302685 是 LRP6 的常见功能突变，先前与酒精性肝病（ALD）患者的酒精性肝损伤减少有关，本研究旨在研究该发现的潜在机制。共招募了 107 名 ALD 患者和 138 名非 ALD 患者，他们均来自中国住院的酗酒者。采集他们的静脉血样进行 DNA 提取，并使用 Sequenom MassARRAY 进行基因分型。我们发现，LRP6 功能受损的 rs2302685 突变在患有 ALD 的酗酒者中比不患有 ALD 的酗酒者更为常见。我们还进行了一项小鼠模型实验，LRP6 敲低小鼠和 LRP6 野生型小鼠每天接受 2.4g/kg 的乙醇灌胃，每 7 天接受更大剂量的 4g/kg 的乙醇灌胃，持续 28 天。随后收集小鼠的血液和肝脏标本进行实验室分析，并进行细胞实验，以比较治疗组和对照组 HL7702 细胞中 LRP6 的抑制、激活、过表达和 siRNA。通过实时 PCR 或 Western blot 分析检测靶向分子的表达。使用 pRL3-CYP2E1 载体稳定转染的细胞进一步研究潜在机制。携带 rs2302685 突变的个体的总胆汁酸（TBA）、直接胆红素、总胆红素（TBIL）、天冬氨酸氨基转移酶（AST）、线粒体天冬氨酸氨基转移酶和 AST/ALT 值分别降低了 63.4%、60.6%、82.1%、44.8%、45.7%和 21.4%。与 LRP6 野生型小鼠相比，LRP6 敲低小鼠的 ALT、TBIL、TBA 和 ALB/GLO 值较低，肝组织损伤减轻，相应的 LRP6、β-连环蛋白和 CYP2E1 表达减少。在暴露于乙醇的 HL7702 细胞中，用 LRP6 抑制剂或 siRNA 处理的细胞中 AST、ALT、脂质积累和 ROS 生成减少，而用 LRP6 激活剂或过表达 LRP6 处理的细胞中则增加。TCF1 是最有可能将 LRP6-Wnt/β-连环蛋白信号通路与 CYP2E1 调节联系起来的转录因子。我们得出结论，LRP6 功能突变 rs2302685 导致与 Wnt/β-连环蛋白-TCF1-CYP2E1 信号通路相关的个体对酒精性肝损伤易感性的差异。
