PDCD4 Mediated Pathological Shear Stress On Autophagy And Inflammatory Corpuscles Of Vascular Endothelial Cells

BackgroundAtherosclerosis(AS)is one of the most common diseases in the cardiovascular and cerebrovascular system,and its pathogenesis has been the focus of research,and many related theories have emerged.In recent years,by analyzing the location of atherosclerosis and the hemodynamics at these locations,it is found that there is a common hemodynamic characteristic in the atherosclerotic areas,that is,the change of blood flow shear force.Normal laminar flow shear force can protect endothelial cells,promote the secretion of vasodilating active substances,and inhibit the development of atherosclerotic plaques.Abnormal pathological shear stress can enhance the oxidative stress and inflammatory response of endothelial cells,promote the secretion of vasoconstrictor and inflammatory mediators,and promote the occurrence and development of atherosclerotic plaques.In the 90 s,Shibahara discovered a new species of apoptosis related gene,or Programmed Cell Death factor 4(Programmed Cell Death 4,PDCD4).With the development of research,PDCD4 has been found to regulate the proliferation,invasion and migration of a variety of tumor cells,and participate in the process of a variety of immune,metabol;ic and inflammatory diseases.Previous studies have found that PDCD4 is closely related to cardiovascular disease,promoting apoptosis of vascular smooth muscle cells,inhibiting proliferation of vascular extracellular fibroblasts,increasing atherosclerotic plaque instability,and accelerating the occurrence and development of atherosclerosis,but the specific mechanism remains unclear.Autophagy is an ancient biological phenomenon widely existing in eukaryotic cells.It is also a new way of programmed cell death,which maintains homeostasis by degrading the metabolic wastes in cells and renewing organelles.Autophagy and blood flow shear stress are important factors affecting the function of vascular endothelial cells and are involved in multiple pathological processes of atherosclerosis.However,it is not clear how oscillatory shear stress regulates autophagy in vascular endothelial cells and whether PDCD4 is involved in this process.Objectives1.To observe the effect of oscillatory shear stress on autophagy and inflammatory corpuscles of vascular endothelial cells.2.To observe whether PDCD4 is involved in the regulation of autophagy and inflammatory corpuscule of vascular endothelial cells by oscillatory shear stress.Methods1.In vitro extraction and culture of HUVECsThe ultra-clean table was irradiated with ultraviolet light,and the surgical instrument was high-pressure sterilized to ensure the aseptic conditions.Neonatal umbilical cords with a length of about 15-30cm were obtained in good condition.The lumen was washed with preheated sterile saline,and then the preheated trypsin digestion solution was injected to digest at room temperature.After centrifugation,discard the supernatant,add the cell culture medium,mix the cells,transfer the cell suspension into the culture bottle,put it in the cell culture box,change the culture medium after 24 hours and observe the state of the cells,and then change the culture medium every other day.2.In vitro shear stress interventionType I rat tail collagen was spread on autoclaved glass slides,dried,and uniformly inoculated with 4-7 generations of HUVECs in good culture state in vitro for shear force intervention experiment.When the cell density reached 90%,0,1,3,6,9,12,24 hours of oscillatory shear stress(size of 0±4 dyne/cm2,frequency of 1Hz)was given.To observe the effect of pathological shock shear force on PDCD4 in HUVECsExpression of the effect,and to find the appropriate time for the stimulation of follow-up experimental research.3.Cell TransfectionPDCD4 siRNA transfected into cells and negative control siRNA were purchased from Shanghai Gemma gene company.The specific steps were followed in accordance with the instructions of Lipo2000 reagent,and the transfection was changed 6-8 hours later,followed by corresponding experimental stimulation and detection.4.Western BlotUsing protein extraction liquid extraction,parallel SDS-page electrophoresis,transfer film,closed room temperature milk,4 ? incubation,corresponding to the second incubation resistance,chemical resistance enhancement,protein stripe grey value,statistical analysis.5.Real-time fluorescence quantitative PCR(RT-PCR)HUVECs after different interventions were collected and total RNA was extracted by TRIzol method.Reverse transcription reaction was performed using the kit of TaKaRa company in Japan,and the reaction system was prepared according to the instructions.The reaction system was prepared according to the instructions with the SYBR kit from TaKaRa company in Japan.Real-time fluorescence quantitative PCR detection was performed and statistical analysis was performed on the data.6.Statistical analysisSPSS 16.0 software was used to analyze the experimental data,which were all expressed as mean standard error.Unpaired t test was used for the comparison between the two groups,and ANOVA was used for the analysis of multiple samples.P<0.05 was considered to be statistically significant.Results1.Oillatory shear stress up-regulated the expression of PDCD4 in HUVECsCompared with the static control group,the protein expression level of PDCD4 was up-regulated with the extension of time under the intervention of oscillatory shear stress,and the mechanical intervention was statistically significant for 6 to 24 hours(P<0.05).The expression of PDCD4 mRNA increased significantly at 6 and 9 h after pathological shock shear force intervention(P<0.05),and reached the peak at 6 h.These results suggest that pathological shock shear force may up-regulate PDCD4 mRNA in HUVECs and promote the expression of PDCD4 protein in HUVECs at the transcription level.2.PDCD4 is involved in regulating autophagy of vascular endothelial cellsAt rest,PDCD4,LC3B?/?,ATG5,free ATG12,conj ATG12,P62,Beclin expression were down-regulated after siRNA transfection compared with negative control group(P<0.05),while ATG3 and ATG7 expression were not significantly changed.It was suggested that PDCD4 was involved in the regulation of autophagy in vascular endothelial cells.3.Oscillatory shear stress is involved in regulating autophagy of vascular endothelial cellsCompared with the static control group,the up-regulation of LC3B?/?expression at 3,6,and 9 h after mechanical action was statistically significant(P<0.05)(figure 3.a),and reached the peak at 6 h.HUVECs were cultured in vitro with pathological shock shear force for 6 hours,and the expression of autophagic molecular proteins in HUVECs was detected by Western Blot.The results showed that in the negative control group,oscillatory shear stress promoted the expression of ATG5,free ATG12,conj ATG12,ATG3,ATG7,P62,Beclin protein compared with the static control group(P<0.05).It suggests that oscillatory shear stress is involved in regulating autophagy of vascular endothelial cells.4.PDCD4 is involved in the regulation of autophagy of vascular endothelial cells by oscillatory shear stress.Compared with the negative control group,silencing PDCD4 blocked the promotion of pathological shock shear force on LC3B?/?,ATG5,free ATG12,conj ATG12,P62 and Beclin(P<0.05),but had no significant effect on the expression of ATG3 and ATG7(P<0.05)'It was suggested that PDCD4 was involved in the process of oscillatory shear stress regulating autophagy in vascular endothelial cells.5.PDCD4 is involved in the regulation of inflammatory corpuscles in vascular endothelial cellsThe mRNA and protein expressions of silenced PDCD4,NLRP3 and ASC were down-regulated under static conditions compared with negative control group(P<0.05).It is suggested that PDCD4 is involved in the regulation of inflammatory corpuscles in vascular endothelial cells.6.Oscillatory shear stress is involved in the regulation of vascular endothelial cell inflammatory corpuscles.In the negative control group cells,oscillatory shear stress promoted the expression of NLRP3,ASC mRNA and protein compared with the static control group(P<0.05).It suggests that oscillatory shear stress promotes the expression of inflammatory corpuscles in vascular endothelial cells.7.PDCD4 is involved in the regulation of inflammatory corpuscles in vascular endothelial cells by oscillatory shear stress.Compared with the negative control group,silencing PDCD4 blocked the promotion of NLRP3,ASC mRNA and protein by pathological shock shear force(P<0.05).It was suggested that PDCD4 was involved in the regulation of inflammatory corpuscles in vascular endothelial cells by oscillatory shear stress.Conclusions1.Oscillatory shear stress regulates autophagy and inflammatory corpuscles of vascular endothelial cells.2.PDCD4 is involved in the regulation of autophagy and inflammatory corpuscles in vascular endothelial cells by oscillatory shear stress.