Jamuna Sankar, Ashokkumar Rathinavel, Devaraj Sivasitambaram Niranjali
Department of Biochemistry, University of Madras, Guindy campus, Chennai, 600025, India.
Appl Biochem Biotechnol. 2023 Apr;195(4):2664-2686. doi: 10.1007/s12010-021-03792-6. Epub 2022 Mar 31.
C-reactive protein (CRP) is a well-established biochemical marker for atherosclerosis. Modification of LDL inside the artery wall favors the elevation of this acute phase protein. Hence, this mechanism is considered an important factor to trigger the monocyte to macrophages differentiation which results in the formation of foam cells. Therefore, this key event should be targeted and focused on how this complex (OxLDL + CRP) proceeds to endothelial dysfunction. Oligomeric proanthocyanidins (OPC) is a well-known cardioprotective flavon-3-ols. The present study is challenged between the cardioprotective roles of OPC against the deleterious effect of OxLDL + CRP complex upon endothelial cells. Protein-protein docking was carried out between CRP and LOX-1. This docked protein complex was again docked with OPC to show the inhibitory mechanism of CRP binding with LOX-1. OPC showed a promising inhibitory mechanism against OxLDL + CRP complex. Docking studies showed that in the absence of ligands (OPC), binding of CRP and LOX-1 was greater and vice versa in the presence of ligands. Based on these molecular docking results, in vitro studies have been carried out. The monolayer of endothelial cells was incubated with THP-1 monocytes for 48 h, induced with OxLDL (10 μg/ml) + CRP (15 μg/ml) and cotreated with OPC (100 μg/ml). Morphological changes, cell migration assay, and capillary tube forming assay were carried out. Myeloperoxidase levels were estimated to determine the adhesion of monocytes onto EC monolayer. RT-PCR analysis of L-Selectin was also done. The quantification of NO levels and analysis of mRNA expressions of eNOS was to determine the nitric oxide demand caused due to OxLDL + CRP complex. LOX-1, scavenger receptor levels were analyzed by mRNA expression. Proinflammatory markers such as IL-6, MCP-1, and IL-1β were studied. Accumulation of ROS levels was measured fluorimetrically using DCF-DA staining. Mitochondrial membrane potential was determined by JC-1 dye and cell cycle analysis was done by FACS analysis. To emphasis the results, the OPC-treated group showed decreased levels of proinflammatory markers, LOX-1 and L-selectin levels. Endothelial nitric oxide levels were increased upon OPC treatment and reduction in the ROS levels was also observed. Endothelial cells apoptosis was prevented by OPC. To conclude, OxLDL + CRP complex inhibitory effects of OPC could maintain the normal homeostasis.
C反应蛋白（CRP）是一种公认的动脉粥样硬化生化标志物。动脉壁内低密度脂蛋白（LDL）的修饰有利于这种急性期蛋白水平的升高。因此，该机制被认为是触发单核细胞向巨噬细胞分化从而导致泡沫细胞形成的一个重要因素。所以，应该针对这一关键事件，并聚焦于这种复合物（氧化型LDL + CRP）如何导致内皮功能障碍。低聚原花青素（OPC）是一种著名的具有心脏保护作用的黄酮 - 3 - 醇。本研究探讨了OPC对氧化型LDL + CRP复合物对内皮细胞的有害作用的心脏保护作用。对CRP和凝集素样氧化型LDL受体1（LOX - 1）进行了蛋白质 - 蛋白质对接。将这种对接后的蛋白质复合物再与OPC对接，以展示CRP与LOX - 1结合的抑制机制。OPC对氧化型LDL + CRP复合物显示出有前景的抑制机制。对接研究表明，在没有配体（OPC）的情况下，CRP与LOX - 1的结合更强，反之在有配体存在时则较弱。基于这些分子对接结果，进行了体外研究。将内皮细胞单层与THP - 1单核细胞共孵育48小时，用氧化型LDL（10μg/ml）+ CRP（15μg/ml）诱导，并与OPC（100μg/ml）共同处理。进行了形态学变化、细胞迁移试验和毛细血管管形成试验。估计髓过氧化物酶水平以确定单核细胞在内皮细胞单层上的黏附情况。还进行了L - 选择素的逆转录 - 聚合酶链反应（RT - PCR）分析。对一氧化氮（NO）水平进行定量以及对内皮型一氧化氮合酶（eNOS）的mRNA表达进行分析，以确定由氧化型LDL + CRP复合物引起的一氧化氮需求。通过mRNA表达分析LOX - 1、清道夫受体水平。研究了促炎标志物如白细胞介素 - 6（IL - 6）、单核细胞趋化蛋白 - 1（MCP - 1）和白细胞介素 - 1β（IL - 1β）。使用2',7'-二氯二氢荧光素二乙酸酯（DCF - DA）染色通过荧光法测量活性氧（ROS）水平的积累。用JC - 1染料测定线粒体膜电位，并通过荧光激活细胞分选（FACS）分析进行细胞周期分析。为强调结果，OPC处理组显示促炎标志物、LOX - 1和L - 选择素水平降低。OPC处理后内皮一氧化氮水平升高，同时也观察到ROS水平降低。OPC可防止内皮细胞凋亡。总之，OPC对氧化型LDL + CRP复合物的抑制作用可维持正常的内环境稳定。
