Effects Of Oxidative Stress And PHB On The Autophagy And Activity Of Platelets

Objective:To investigate the effects of oxidative stress on platelet autophagy and platelet activity,and to observe the effects of autophagy on the expression of miR-27a and PHB in platelets and their relationship with platelet activity.Methods:?1?Platelets are obtained from the physical examination personnel in our hospital,include the non-diabetic patients and diabetic patients.The morphology of cells is observed under a microscope.the protein expression levels of PHB and LC3?/?in the platelets are detected by Western Blot.?2?The concentrated platelets are obtained from the central blood station for healthy volunteers,centrifugation,abstersion and resuspension cultule.The normal platelets are treated with Different concentrations?0,0.1,1,10mmol/l?H₂O₂ for 2hours.we detect the activity of platelets by CCK-8 method and ROS expression levels with ROS detection kits,the protein expression levels of PHB and LC3?/?in the platelets are detected by Western Blot.choosing an optimal concentration of H₂O₂?1mmol/l?for next experiment;?3?The plateletsare treated with H₂O₂?1mmol/l?and 3MA?4mmol/l??3-methyladenine,autophagy inhibitor?,divided into 3 groups.Control group:platelets group;H₂O₂ group:platelets+H₂O₂ group;H₂O₂+autophagy inhibition group:platelets+H₂O₂+3MA group,H₂O₂ is added to the treatment group for 2h after pretreating platelets for 1h with 3MA;?4?the protein expression levels of PHB,LC3?/?are detected by Western Blot,and the expression level of miR-27a is detected by RT-PCR.The expression of PAC-1 is detected by Annexin V-FITC/PE flow cytometry.Platelets,function analyzer is used to detect the platelet aggregation rate?PAR?.Results:?1?the platelets are suspended in small particles,without nucleus;?2?the expression of PHB and LC3?/?in the platelets of diabetic patients are significantly higher than that in the control group[?1.33±0.20?vs?0.16±0.02?,?0.14±0.12?vs?0.07±0.01?,p<0.01].?3?after treating platelets 2h with different concentrations H₂O₂?0,0.1,1,10mmol/l?,Compared with the control group,the activity of platelets of 1,10mmol/l H₂O₂ groups are significantly decreased[?88.45±2.72?%vs?100.00±0?%,p<0.05;?71.51±5.34?%vs?100.00±0?%,p<0.01],The ROS level of 1,10mmol/l H₂O₂groups are increased[?8.11±0.77?vs?0.12±0.01?,[?10.54±0.42?vs?0.12±0.01?,p<0.001],There is no significant difference in platelet activity between 0.1mmol/l and 1mmol/l H₂O₂ groups[?97.53+0.83?%vs?88.45+2.72?%,p>0.05],while ROS expression from 1mmol/l H₂O₂ group is higher than 0.1mmol/l H₂O₂ group?p<0.001?.?4?Platelets are cultured with different concentrations H₂O₂?0,0.1,1,10mmol/l?for 2h.Compared with the control group,the protein expression of PHB are significantly increased in 1,10mmol/l H₂O₂ groups[?0.68±0.05?vs?0.26±0.06?,p<0.001;?0.51±0.02?vs?0.26±0.06?,p<0.01],the protein expression of PHB from1mmol/l H₂O₂ group is higher than 0.1mmol/l H₂O₂ group?p<0.001?,and decreased in 10mmol/l H₂O₂ group?p<0.05?.The protein expression of LC3?/?is significantly higher than that in the control group,and increased with the concentration[?3.05±0.76?vs?1.52±0.26?,p<0.05;?3.68±0.59?vs?1.52±0.26?,p<0.01;?6.40±0.50?vs?1.52±0.26?,p<0.001]?There is no significant difference in LC3?/?protein expression between 0.1mmol/l and 1mmol/l H₂O₂ groups?p>0.05?.?5?Platelets are cultured with 1mmol/l H₂O₂ for 2h after pretreating with 3MA for 1h.Compared with the control group,the expression levels of PHB and LC3?/?protein in H₂O₂ group are significantly increased[?0.50±0.03?vs?0.14±0.04?,p<0.01;?0.23±0.03?vs?0.05±0.02?,p<0.001],and miR-27a is decreased[?0.43±0.26?vs?1.00±0?,p<0.05].H₂O₂+3MA group show a significant decrease in the expression of PHB and LC3?/?protein than H₂O₂ group[?0.31±0.04?vs?0.50±0.03?,?0.07±0.01?vs?0.23±0.03?,p<0.01],while miR-27a is increased[?0.97±0.36?vs?0.43±0.26?,p<0.05].?6?Platelets are cultured with 1mmol/l H₂O₂ for 2h after pretreating with 3MA for 1h.Compared with the control group,the PAC-1 expression and PAR of H₂O₂group are significantly increased[?81.21±5.08?%vs?11.86±3.57?%,p<0.001;?52.07±5.69?%vs?28.87±2.82?%,p<0.01],and the expression of PAC-1 and PAR in H₂O₂+3MA group are lower than that in H₂O₂ group[?61.43±5.25?%vs?81.21±5.08?%,p<0.05;?36.37±3.02?%vs?52.07±5.69?%,p<0.05].Conclusion:?1?Platelet basic autophagy is stronger in diabetic patients than in non-diabetic patients,and PHB is involved in this process;?2?In vitro,the oxidative stress caused by H2O2 can stimulate the autophagy of platelets and promote platelet activation.Mir-27a is involved.