PGIS Regulation Of Prostacyclin Synthesis In Vibration Induced Vascular Endothelial Cell Early Damage

Objectives In this study,we combined animal and cell experiments to investigate the changes of metabolites and enzymes related to arachidonic acid（AA）metabolic pathway in vascular endothelial injury induced by vibration and the possible mechanism.Furthermore,the role of prostacyclin synthase gene（PGIS）regulating prostacyclin（PGI₂）synthesis in the damage of vascular endothelial cells（VEC）induced by vibration was investigated and verified by gene silencing techniques.To explore the early injury mechanism of arm vibration syndrome（HAVS）at the molecular level,and to provide a scientific basis for the search of biomarkers for early diagnosis of HAVS.Methods（1）Animal experiment:50 SPF male SD rats,weighing（178.3±6.1）g,aged 7-8 weeks,were randomly divided into 5 groups:control group,1 day exposure to vibration,3 days exposure to vibration,and 7 days exposure to vibration,14 days exposure to vibration,consisted of 10 rats in each group.The vibration exposure conditions were as follows:each rat was exposed to vibration for 4 hours/day,the vibration frequency was 125Hz,the acceleration was 5.9m/s²,and the vibration direction was linear and vertical in the upper and lower directions.The control rats were completely the same as the experimental rats in the whole experimental process except that they were not exposed to vibration exposure.The rats in each group were anesthetized after the completion of the exposure to vibration,and the abdominal cavity was opened to collect blood from the abdominal aorta.Blood samples were collected from the middle caudal and caudal arteries for the preparation of pathological light microscope and electron microscope sections.The blood vessels of the middle caudal artery of rats were observed under inverted microscope and transmission electron microscope,including histopathological observation,cell and cell substructure observation Enzyme-linked immunosorbent assay（ELISA）was used to determine the expression levels of seven key sites in the arachidonic acid（AA）metabolic pathway:AA,PGG₂,PGH₂,COX,PGIS,and PGI₂,6-Keto-PGF_1αwhich were selected based on the results of the previous metabolomics study in rat plasma.（2）Cell experiment:primary human umbilical vein endothelial cells（HUVEC）were used to establish a cell line and construct an experimental model of cell vibration exposure.Cell vibration exposure experiment was carried out.Cell vibration exposure experiment was divided into:control group,1 day exposure to vibration and 2 days exposure to vibration.The vibration exposure was 0.5 h/day,the vibration frequency was 125Hz,the acceleration was 5.9m/s²,and the direction of vibration was vertical up and down.During the whole experiment,the control group was completely the same as the experimental group except that the HUVEC was not exposed to vibration,and the cell morphology of HUVEC in each group was observed before and after vibration exposure with an inverted microscope.Cell samples from each HUVEC group were collected after the vibration exposure experiment,and the expression level of AA,PGG₂,PGH₂,PGIS,PGI₂,6-Keto-PGF_1α,in the samples was determined by ELISA.The PGIS genes responsible for regulating the synthesis of PGIS in the AA metabolic pathway were knocked down by siRNA transfection.The HUVEC and negative control group（NC）after silencing were used for cell vibration exposure experiment.The same experimental grouping and vibration exposure conditions were kept as before,and cell samples were collected from each group.The regulation of PGI2 by PGIS in siRNA-silenced HUVEC in NC group was determined by q RT-PCR.The cell morphology of NC group and gene knockdown group before and after vibration was observed under inverted microscope.Morphological observation results of normal HUVEC vibration experiment were compared.Statistical software SPSS 23.0 and Graph Pad Prism 8 were used for statistical analysis of the experimental data.Results（1）The results of pathological section and ultrastructural observation of electron microscope showed that the vascular tissue structure of the control group was basically normal,the elastic fascial layer of the middle artery was complete and smooth,VEC and VSMC were arranged neatly without swelling and hyperplasia,and the cell structure was not vacuolated.In the 1 day and 3 days vibration group,the elastic fascial layer of the arteries was basically normal,some of the elastic fascial layers were slightly interrupted and disappeared,the VEC arrangement showed a slight tendency of swelling,disorder and degeneration,and a few small vacuoles appeared in the VSMC.VEC cells in the 7-day vibration group showed more vacuolation,mitochondria swelling and nuclear abnormality.There were large vacuoles in the cytoplasm of VSMC,and the elastic fascia was obviously thinned and interrupted.VECs in the 14-day vibration group showed extensive and massive vacuolization with obvious swelling,nuclear atomorphism,large vacuoles and incompact cell arrangement in the cytoplasm of VSMC in the multilayer media,and most of the elastic fascial layer thinning and interrupted to disappear.The results of rat plasma ELISA assay showed that the average expression level of AA,PGG₂,PGH₂,Cox,PGI₂,6-Keto-PGF_1αin rat plasma was not completely the same among the five groups.The expression level was the lowest in the control group,and the highest in the 14-day exposure to vibration group,with statistical difference（P<0.05）.Compared with the control group,the difference in the overall mean expression level of PGIS among the five groups was statistically significant（P<0.05）,the highest expression level was found in the 3-day vibration exposure group,and the lowest expression level was found in the 7-day vibration exposure group（P<0.05）.（2）The HUVEC ELISA results showed that the overall mean expression levels of AA,PGG₂,PGH₂,PGIS,PGI₂,6-Keto-PGF_1αamong the three groups were not identical to the overall mean expression levels of HUVEC.The expression level was the lowest in the control group,and the highest in the2-day exposure to vibration group,with statistically significant differences（P<0.05）.siRNA transfection could change the expression of PGIS at the transcriptional level in HUVEC cells,and siRNA transfection could decrease the transcription level of PGIS and increase PGIS mRNA with the prolonging of vibration exposure time.Microscopic observation showed that the cells in NC group were normal in morphology and intact in structure without damage.Pathological damage was observed in the PGIS gene knockdown group for 1 day and in the PGIS gene knockdown group for 2 days.Compared with the group without siRNA treatment,the pathological lesions such as vacuolation of cells were increased in the group with 1 day exposure to vibration.The damage was more serious in the PGIS silencing treatment group than that in the untreated group.Conclusions（1）The results of pathological section and ultramicro morphology observation of rat tail vibration exposure showed that the degree of vascular pathological damage was different with different duration of vibration exposure.The longer the vibration time is,the more serious the vascular damage is.It is true that local vibration will cause damage to the peripheral vascular tissue,including vascular endothelial cells.Combining the results of metabonomics analysis with the ELISA results of animal plasma samples,it was found that The expression levels of key sites and metabolites in the AA metabolic pathway,such as AA PGG₂,PGH₂,Cox,PGIS,PGI₂6-Keto-PGF_1α,are closely related to the damage of vascular endothelial cells and surrounding vascular tissue caused by local vibration.（2）The results of cell vibration exposure experiment showed that the AA metabolic pathway and the PGI₂synthesis process regulated by PGIS played a certain role in the vibration to vascular endothelial cell injury.The change of PGIS mRNA expression under the condition of vibration exposure may be an important link in the process of damage of vascular endothelial cells caused by vibration exposure,and play a role in the process of damage of vascular endothelial cells and perivascular tissues caused by vibration exposure.