The Role And Mechanism Of Pyk2/MCU Pathway In H₂O₂-induced Endothelial Cells Injury

Objective:Endothelial cells injury is the main pathogenesis of atherosclerosis?Atherosclerosis,AS?.It plays an important role in the formation and development of atherosclerosis through processes such as inflammatory response and oxidative stress,but its specific pathogenesis is still not very clear.The pathological mechanism after endothelial cells injury is a more complex cascade.The change of intracellular calcium ion concentration is one of the key pathological processes of endothelial cell injury.When excessive intracellular calcium ions accumulate,a series of cascade reactions will be triggered,eventually leading to cell damage.The organelles that are closely related to the regulation of intracellular calcium ions are mitochondria.Mitochondrial calcium uniporter?MCU?has been newly identified.It is located on the inner membrane of mitochondria and plays an important role in mitochondrial calcium uptake.Proline-rich tyrosine kinase 2?Pyk2?is a member of the focal adhesion kinase family.It has a certain relationship with changes in intracellular calcium ion concentration and MCU regulation.However,the interaction mechanism between them is still unclear,and the highly complex and precise regulating effect still needs to be further studied.In this study,we used human umbilical vein endothelial cells?HUVECs?as research objects to investigate whether the Pyk2/MCU pathway is involved in endothelial cells damage caused by atherosclerosis through H₂O₂ induced endothelial cells injury,thereby accelerating the formation and development of atherosclerosis.Methods:1. In this study,human umbilical vein endothelial cells?HUVECs?were used to establish an H₂O₂-induced oxidative stress model. The experiment's model was established,and then was divided into control group,H₂O₂group?750?M,24h?,rosuvastatin?2.5?M,5?M and 10?M?+H₂O₂ group?750?M,24h?.The effect of rosuvastatin on cell viability after H₂O₂-induced HUVECs injury was investigated by MTS staining,and Pyk2/MCU mRNA as well as protein expression were detected by real-time reverse transcription-quantitative Polymerase Chain Reaction?RT-qPCR?and western blot respectively.2.The changes of mitochondrial membrane potential,ROS,intracellular calcium ions and apoptosis were detected by flow cytometry.Collecting HUVECs as above,and observing cell morphology and ultrastructure with transmission electron microscopy are needed to detect the ultrastructural changes in cells.The concentration of angiotensin-2?Ang II?was quantitatively determined by ELISA.3.Sh RNA technology made Pyk2 gene lower expression to observe the changes in the experimental group for blank group,blank+H₂O₂ group,NC+We detected cell viability,intracellular Ca²⁺concentration and the change of mitochondrial membrane potential.The expression of Pyk2,MCU and caspase-3 proteins were detected by using western blot technique.Results:1. With the increase of H₂O₂ concentration,the cell viability decreased significantly.Compared with the control group,the cell viability decreased by about 50%when given 750?M H₂O₂ intervention?P<0.05?.HUVECs were treated and cultured for 24 hours.When the concentration of rosuvastatin was2.5,5?M,the survival rate of HUVECs significantly increased,which played a protective role in endothelial cells?P<0.05?.2. Realtime-qPCR results showed that the expression of Pyk2/MCU mRNA in the H₂O₂ model group was significantly higher than that in the control group?P<0.05?.The rosuvastatin 2.5?M and 5?M groups had lower expression than the model group?P<0.05?,of which 5?M rosuvastatin declined most?P<0.05?.In H₂O₂-induced HUVECs injury model,protein expression levels of Pyk2 and MCU were increased?P<0.05?,and rosuvastatin had a significant effect on reversing these changes?P<0.05?.3. The results of flow cytometry showed that H₂O₂ could significantly increase Ca²⁺,decrease mitochondrial membrane potential,and increase reactive oxygen species and apoptosis?P<0.05?;Through transmission electron microscopy,we observed that there was obvious nuclei and nucleoli,intact nuclear membrane,and complete visible organelles such as mitochondria and endoplasmic reticulum in the cytoplasm in the control group.In the H₂O₂ group,mitochondria swelled,mitochondrial ridges disappeared,mitochondrial vacuolation was severe,endoplasmic reticulum was dissolved,nuclear membrane and nucleoli were dissolved.Compared with the H₂O₂group,rosuvastatin-treated cells had less damage,nucleoli were clearer than the model group,and cell membrane,mitochondria,endoplasmic reticulum and other organelles had less damage;by ELISA analysis,we obtained that the changes of Ang II was also found in different treatment groups.4. Sh RNA technology was used to make gene Pyk2 express low in HUVECs,and then the cells were treated differently.Observe and compare how the cells'changes when Pyk2 gene is low expressed.The results showed that compared with the blank+H₂O₂ group,the cell viability of the shRNA+H₂O₂ group was significantly increased?P<0.05?.Similarly,the Rosu+H₂O₂group also protected the cells?P<0.05?.Compared with the H₂O₂ group,the cell proliferation of the NC+H₂O₂ group was not significantly changed.The difference between NC+H₂O₂ group and shRNA+H₂O₂ group was statistically significant?P<0.05?.In addition,the shRNA+H₂O₂+Rosu group had a stronger protective effect than rosuvastatin alone?P<0.05?;in the changes of mitochondrial membrane potential and intracellular free Ca²⁺concentration concentration of the shRNA+H₂O₂ group showed a downward trend,but it was not statistically significant compared with the H₂O₂ group.However,after rosuvastatin treatment,intracellular Ca²⁺decreased significantly?P<0.05?,and the decline was even more pronounced in the shRNA+H₂O₂+Rosu group?P<0.05?.In the detection of mitochondrial membrane potential,we found that the membrane potential of the shRNA+H₂O₂ group was higher than that of the blank+H₂O₂ group,and this trend was more obvious after intervention with rosuvastatin,but the difference was not statistically significant.Then Pyk2,MCU and caspase-3 were detected at the protein level,and the results showed that compared with the blank+H₂O₂ group,the expression of the above three proteins was reduced in the shRNA+H₂O₂ group?P<0.05?,indicating that the low expression of Pyk2 caused downstream gene expression was significantly reduced.When rosuvastatin was administered,the expression of these three proteins also significantly decreased?P<0.05?,suggesting that rosuvastatin can protect endothelial cells from H₂O₂ damage by inhibiting the Pyk2/MCU pathway.At the same time,shRNA and rosuvastatin simultaneously inhibited Pyk2,MCU,and caspase-3?P<0.05?.Conclusions:1.We consider Pyk2/MCU pathway is involved in endothelial cells damage caused by H₂O₂ injury model.2.Rosuvastatin plays a protective role against H₂O₂-induced injury by inhibiting the Pyk2/MCU pathway,suggesting that the Pyk2/MCU pathway may become a new target for atherosclerosis intervention.