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 LC3Ⅱ/Ⅰin 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 LC3Ⅱ/Ⅰin 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,LC3Ⅱ/Ⅰare 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 LC3Ⅱ/Ⅰin 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 LC3Ⅱ/Ⅰis 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 LC3Ⅱ/Ⅰprotein 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 LC3Ⅱ/Ⅰprotein 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 LC3Ⅱ/Ⅰprotein 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.