Molecular Mechanisms Of Reactive Oxygen Species Generation In Macrophages Cells Initiated By Mechanical Stretch Stress And Oxidized Low Density Lipoprotein

Objective:There are two main sources which are important components of atherosclerotic plaque lesions foam cells:vascular smooth muscle cells and macrophages. In previous studies, we have demonstrated that hypertension resulting mechanical strain(SS) and/ or hyperglycemia resulting advanced glycation end products(AGEs) can activate MAPK pathway and oxidative stress ROS pathway of vascular smooth muscle cells;date also pointed out, SS and/or oxidized low density lipoprotein(oxLDL) can activate ERK1/2signaling pathway of macrophages; But the SS and/or oxLDL induce the ROS oxidative stress pathway of macrophages which is the important source of foam cells is not clear;these two risk factors on ROS generation of macrophages with ROS oxidation stress upstream molecular pathway, NADPH oxidase(mainly Nox1) relationship is unclear;there were reports that atherosclerosis commonly used lipid regulating agent simvastatin can slow the progression of vascular remodeling, the relationship with this effect and oxidative stress of macrophages and the specific mechanism are also unclear.There are issues this paper focuses primarily caused by vascular remodeling mechanisms to investigate further added “non-specific activation of the mechanical force hypertension multiple signaling pathways”, the group proposed the development of the theory before,in-depth SS and oxLDL, looking for the new target of hypertensive patients who with high cholesterol's clinical prevention to provide experimental data.Methods:(1) Macrophages that bought from ATCC were observed under inverted phase contrast microscope, and cultured with DMEM medium.(2) oxLDL was extract from human plasma, then was identified through the color change of its appearance and Agarose gel electrophoresis.(3) With Hoechst 33342 and H2 DCFDA fluorescent probe, we detected the fluorescence intensity of ROS caused by oxLDL stimulation and/or Stretch Stress.(4) After pretreating macrophages with NADPH oxidase inhibitor(Apocynum Ning /apocynin), experimental procedure(3) was repeated.(5) After pretreating macrophages with simvastatin, experimental procedure(3) was repeated.(6) Nox1 expression in macrophages was detected using Western Blot after the pretreat of simvastatin.(7)After pretreating with simvastatin,we detected the MAPK's expression of macrophages which caused by oxLDL stimulation and/or Stretch Stress.Results:(1) macrophages were brought from ATCC, presented a round shape and half adherent growth..(2) Successful production of oxLDL presented as a transparent liquid without precipitation, in agarose gel electrophoresis,the electrophoretic mobility of oxLDL was higher than that of n LDL.(3) oxLDL and stretch stress could increase the intracellular ROS of macrophages respectively, and both showed a time or concentration(strength) dependent trend.(4) The combined effect of oxLDL and stretch stress could further increase the ROS level,which was higher than any single stimulated effect, showing a synergistic effect between the dual factors.(5) After pretreating macrophages with apocynin, these oxLDL and/or stretch stress induced ROS was partially reversed, suggesting that the intracellular ROS of macrophages which caused by oxLDLand/or stretch stress was related with the activation of Nox1.(6) After pretreating macrophages with simvastatin, these oxLDL and/or stretch stress induced ROS was reversed significantly, suggesting that simvastatin participate the production of the intracellular ROS of macrophages which caused by oxLDL and/or stretch stress.(7)After pretreating with simvastatin, the MAPK's expression of macrophages which caused by oxLDL stimulation and/or Stretch Stress was reversed significantly.Conclusions:(1) oxLDL and hypertension induced mechanical stress could increase the intracellular ROS of macrophages respectively, the combined effect of them could further elevate the ROS level, showing a synergistic effect, resulting in a large accumulation of ROS and thus triggered oxidative stress response which may accelerated the vascular remodeling.(2) Nox1 as an important intracellular signaling molecule was involved in the regulation of theroduction of ROS which was under the single or combined effects of oxLDL and stretch stress, leading to the reduce of ROS. However,simvastatin could suppress the production of ROS by reduce the expression of Nox1. Furthermore,simvastatin can suppress the MAPK's expression of macrophages which caused by oxLDL stimulation and/or Stretch Stress.(3) This study preliminary explored the molecular mechanisms of ROS generation in macrophages induced by oxLDL and hypertension induced mechanical stress alone or combined. Besides, we further complement our previously doctrine-hypertension-induced abnormal mechanical stress could triggers signal transduction via various membrane receptors in macrophages, enriching the mechanisms of vascular remodeling caused by hypertension induced mechanical stress and oxLDL and providing new data for seeking novel targets to prevent and cure the vascular diseases resulting from hypertension with hyperlipidemia.