LGR4调节低密度脂蛋白胆固醇代谢的作用和机制研究

High levels of circulating low density lipoprotein cholesterol (LDL-c) contribute to the development of CVD and places patients at increased risk of experiencing an adverse cardiovascular event..Our preliminary results showed that LGR4 whole body knockout mice displayed highly reduced serum total cholesterol (TC) and LDL-c when compared with wild type mice. Lgr4 and leptin double mutant mice showed reduced plasma TC and LDL-c levels when compared with ob/ob mice. We also found that knockdown of LGR4 in liver by adenovirus in HFD-fed mice, the disordered TC and LDL-c were significantly corrected. FPLC analysis showed that the decreased cholesterol in LDL was the main reason of reduced total cholesterol after LGR4 knockdown. Consistently, serum proprotein convertase subtilisin/kexin type 9 (PCSK9) levels were significantly decreased in LGR4KO, Lgr4/ob double mutant mice as well as in mice with liver LGR4 knockdown. Interestingly, overexpressing LGR4 in liver by adenovirus in normal mice resulted in elevated serum PCSK9 level and decreased LDLR expression. Plasma TC and LDL-c were significantly enhanced. Our present results suggest that LGR4 plays a key role in the LDL-c metabolism. PCSK9 might be the target of LGR4. In this project, we will elucidate the significant role of LGR4 in regulating LDL-c metabolism in vivo and in vitro by using methods of advanced cell biological and molecular biological. We will also demonstrate the target of LGR4 and dissect the signal pathway of LGR4 in regulating cholesterol metabolism. It will be of great importance to reveal the biological function of LGR4 further and provide a novel therapeutic approach in the management of dyslipidemia.

LDL-c水平长期升高是引发心脑血管疾病的重要危险因素。前期发现，小鼠LGR4全身敲除后血清TC和LDL-c显著降低。LGR4；leptin基因双敲小鼠结果显示，与对照ob/ob鼠相比，双敲小鼠TC和LDL-c也显著降低。另外高脂小鼠通过腺病毒在肝脏沉默LGR4， TC和LDL-c亦显著下降。不论LGR4KO，LGR4；ob双敲小鼠，还是腺病毒介导的肝脏LGR4敲除小鼠，血清PCSK9含量均显著下降；而正常小鼠过表达LGR4后，血清PCSK9浓度明显升高，肝脏LDLR表达显著下降，且小鼠血TC和LDL-c浓度也显著上升。上述提示LGR4在胆固醇代谢中扮演重要角色，PCSK9可能是其靶点。本课题中我们将进一步从体内和体外实验，结合细胞生物学和分子生物学，探究LGR4在LDL-c代谢中调控作用，并将逐步揭示LGR4与其靶点和信号通路在LDL-c代谢过程组成的调控网络，丰富对其生物学功能的认识。

低密度脂蛋白胆固醇(LDL-c)水平升高与脂质代谢紊乱，动脉粥样硬化等息息相关。本研究发现：1、LGR4全身敲除小鼠和LGR4:leptin双基因敲除小鼠体内总胆固醇(TC)及LDL-c水平显著降低；且利用腺病毒介导的肝脏特异性LGR4敲低高脂（HFD）小鼠及瘦素受体缺乏(db/db)小鼠，其血清内TC及LDL-c含量也显著降低。2、快速蛋白液相色谱法（fast performance liquid chromatography，FPLC）分析腺病毒介导的肝脏特异性LGR4敲减的高脂小鼠血清，结果显示，注射ad-shRNA-LGR4的小鼠极低密度脂蛋白（VLDL）水平无明显变化，其对TC的降低主要是由于对LDL的降低；此外，注射ad-sh-LGR4腺病毒的HFD小鼠及db/db小鼠LDLR蛋白水平上升。3、PCSK9能与LDLR结合并介导其在体内的降解，结果显示LGR4表达降低后，不同小鼠模型肝脏PCSK9 mRNA及血清中PCSK9水平均显著下降。4、p-ERK能够调控PCSK9的表达，利用U0126抑制p-ERK表达后，LGR4表达降低引起的PCSK9水平降低的效应被消除。5、HNF1a和HNF1b可能是PCSK9启动子区域的转录调控因子。本课题揭示了LGR4在肝脏脂质代谢中的调节作用和机制研究，更加明确的提出了其作用机制及靶点，为临床上以LGR4为靶点治疗心血管疾病等提供重要参考，具有重要的科研意义及临床价值。