三明治培养人源永生化肝细胞中Aβ分布的体外研究

In Vitro Study on the Distribution of Aβ in Sandwich Cultured Human Immortalized Liver Cells

摘要: 阿尔茨海默病(Alzheimers Disease, AD)是一种神经退行性疾病，发病率高且疾病负担重。已有研究发现PXR激动剂是AD的潜在治疗药物，肝实质细胞中P-gp和LRP1在Aβ₄₂的快速清除中发挥了重要作用，本研究旨在探索PXR激动剂是否通过影响肝脏中P-gp和LRP1的表达发挥AD治疗作用。首先选择HSEC细胞进行CCK-8实验确定HYP给药浓度，通过免疫印迹检测HepG2细胞中LRP1、P-gp蛋白量，最后通过Transwell模型检测对FAM-Aβ及罗丹明123 (Rho123)转运能力。实验结果证明，选择HYP (0.5 μM)进行免疫印迹实验证明了HYP能够上调P-gp蛋白表达，但对LRP1有轻微下调影响。用Rho123流出实验表明了HYP增强HepG2细胞中P-gp的外排能力。最后研究了HepG2细胞对Aβ₄₂转运能力影响，其显示HYP能够增强HSEC和HepG2细胞双层膜屏障对Aβ₄₂转运，并且通过P-gp抑制维拉帕米验证HYP是通过增强hCMEC/D3中P-gp活性导致Aβ₄₂转运增多。结果论证激动PXR可能通过上调HepG2细胞中P-gp的表达增加并肝脏对外周Aβ₄₂的快速清除能力发挥治疗 AD的作用。经过科学严谨的研究，我们发现HYP在未来有望作为一种新型AD治疗候选药物，其潜在疗效将为HYP的临床应用提供坚实科学支撑。

Abstract: Alzheimer’s Disease (AD) is a neurodegenerative disorder with high incidence and significant disease burden. Previous studies have suggested that PXR agonists may be potential theraputic agents for AD treatment. Additionally, P-glycoprotein (P-gp) and LRP1 play crucial roles in the rapid clearance of Aβ₄₂. PXR agonists have been shown to increase the expression of P-gp protein, This study aims to further investigate whether PXR agonists exert their therapeutic effects by affecting the expression of P-gp and LRP1 in liver. In the cellular experiments, we choose HSCE cells CCK-8 assay was performed to determine the concentrations of HYP. The protein levels of LRP1, P-gp in HepG2 cells were detected by immunoblotting experiments. Transwell assay was conducted to evauate the transport capacity of FAM-Aβ42 and Rhodamine 123 by HSEC and HepG2 cell bilayer membrane barrie.The results showed that HYP (0.5 μM) was found to upregulate P-gp protein expression in a concentration-dependent manner but little effect on LRP1. Rho123 efflux assay showed that HYP enhanced the efflux capacity of P-gp in HSEC and HepG2 cell bilayer membrane barrie. Furthermore, HYP enhanced the barrier function of HSEC and HepG2 cell bilayer membrane barrie. for Aβ₄₂ transport in normal, this effect was confirmed to be mediated by increasing P-gp activity through verapamil inhibition. The study suggests that PXR agonists may exert therapeutic effects on AD by upregulating the expression of P-gp and LRP1 and increasing the rapid clearance of Aβ₄₂ in the liver. These findings provide scientific evidence for the future clinical application of HYP, which may become a promising candidate drug for AD treatment.