Mechanical Stretch Stress And OxLDL Additively Promote The Activition Of MAPKs And Proliferation/apoptosis Of Macrophages

Objective Hypertension or hyperlipaemia will eventually develop into atherosclerosis(AS),However, when the two coexist, progress of atherosclerosis developes quicklly, the underling mechanism is unknown. Foam cells in the atherosclerotic plaque are primarily derived from vascular smooth muscle cells(VSMCs) and macrophages.There have been numerous studies on VSMC in Hypertensive vasculopathy. However, the role of macrophage in this disease is not well understood.In this study, RAW264.7 cells were treated by oxidized low density lipoprotein(oxLDL) and/or mechanical stretch stress to study the effects of oxLDL and/or stretch stress on activation of MAPKs,oxidative stress and Ki67/Tunel positive ratio and DNA methylation, which would explain pathogenesis about accelerating vascular remodeling in hyperlipaemia with hypertension from a new perspective.Methods 1. Low-density lipoprotein(LDL) was isolated from normal human plasma by high-speed centrifugation. And thenLDL was treated by copper sulfate to obtain oxidated low-density lipoprotein(oxLDL). Oil Red staining and MTT assay were used to identified whether LDL was successfully oxidated. 2. RAW264.7 cells were treated by oxLDL or/and stretch stress,activation of MAPKs in RAW264.7 cells was detected. 3. RAW264.7 cells pre-treated by simvastatin were treated by oxLDL and/or stretch stress, activation of MAPKs was detected by Western blot. 4. RAW264.7 cells were treated by oxLDL,stretch stress and simvastatin, and then Ki67/TUNEL positive cells were detected by immunofluorescent staining. 5. RAW264.7 cells pre-treated by simvastatin were treated by oxLDL and/or stretch stress, Expression of ROS was detected by immunofluorescent staining. 6. RAW264.7 cells pre-treated by NADPH oxidase inhibitor(Apocynum Ning) were treated by oxLDL and/or stretch stress, Expression of ROS was detected by immunofluorescent staining. 7. VSMCs were isolated from mice aorta and cultured in vitro. The cells were identified by their morphology with optical phase contrast microscope. Smooth muscle-specific antigen α-actin was detected via Western blot and immunofluorescent staining. 8. RAW264.7 cells were treated by oxLDL,stretch stress and simvastatin, and then expression of DNA methylation was detected.Results 1.LDL was successfully isolated from plasma. And the concentration of LDL was to meet the requirement of experiments. Macrophages in oxLDL group significantly swallowed a larger number of lipid droplets than ones in nLDL group via Oil red O staining. MTT data analysis shows nLDL or oxLDL could significantly induce the proliferation of Raw264.7 cells. and oxLDL could more significantly induce the proliferation than nLDL. Simvastatin can inhibit proliferation of Raw264.7 cell induced by treatment of oxLDL or nLDL. 2.Western blot showed that oxLDL could induce the phosphorylation of MAPKs individually(time and dose dependent), but the effects of oxLDL on MAPKs activation were higher than nLDL in the same dose. Stretch stress could induce phosphorylation of MAPKs in Raw264.7 cells with time dependently. Cotreatment of Raw264.7 cells with stretch stress and oxLDL had additive effects on the phosphorylation of MAPKs representing higher levels of MAPK phosphorylation than individual treatment with oxLDL or stretch stress. 3.Simvastatin inhibited the activation of MAPK induced by treatment of oxLDL or/and stretch stress. 4.Either stretch stress or oxLDL could increase expression of ki67 and TUNEL, while combined stimulation with both could result in additive effects. Simvastatin inhibited the increase in ki67 and TUNEL expression induced by treatment of oxLDL or/and stretch stress. 5.Stretch stress or oxLDL alone could increase expression of ROS,while combined stimulation with both could result in additive effects. Simvastatin inhibited increase of expression of ROS induced by treatment of oxLDL or/and stretch stress. 6. NADPH oxidase inhibitor(Apocynum Ning)inhibited increase of expression of ROS induced by treatment of oxLDL or/and stretch stress. 7.VSMCs were successfully isolated from mice aorta and cultured in vitro.The cells were identified by their morphology with optical phase contrast microscope. Smooth muscle-specific antigen α-actin was detected via Western blot and Immunofluorescence. 8.Stretch stress and oxLDL alone could significantly induce the decrease of methylation level in VSMCs.when the two work together, methylation level was significantly lower than any single factor stimulation. Simvastatin inhibited the processes.Conclusions 1. Stretch stress and oxLDL can induce the phosphorylation of MAPKs and increase of expression of ROS, ki67 and TUNEL, respectively, while combined stimulation with both could result in additive effects.Simvastatin can inhibit these effects. 2. NADPH oxidase inhibitor(Apocynum Ning) can result in the decrease of expression of ROS induced by treatment of oxLDL or/and stretch stress. 3. Stretch stress and oxLDL alone can lead to the decrease of methylation level in VSMCs. while combined stimulation with both could result in additive effects. Simvastatin can inhibit these effects. 4. These results would provide important experimental basis for prevention and treatment of hypertension-related diseases.