Preliminary Studies Of Reactive Oxygen Species Regulate Platelet Apoptosis And Impact Of X-ray On Meg-01Cells

Objective: To study the regulation of Reactive Oxygen Species (ROS) on plateletapoptosis and GPIba shedding which induced by dibucaine or thrombin, the effect ofantioxidant N-caetyl-L-cysteine (NAC), and the impact of X-ray on platelet apoptosis ormegakaryocyte proliferation and differentiation.To explore the possible mechanism.Methods: To detect the generation of reactive oxygen species, expression of plateletGPIba and depolarization of mitochondrial membrane potential with Flow Cytometry.Using the method of western blot detects the activation of pro-apoptotic protein caspase-3,the expression of antiapoptotic protein of Bcl-xL and GPIba shedding fragment GC. UsingCCK-8kit detect the proliferation of Meg-01cells. Flow Cytometry detect the expressionof CD41or GPIba on cell surface. Annexin V–FITC kit detects cell apoptosis.Results: When platelets were incubated with dibucaine or thrombin, the ROSproduction increased by1.64times or1.5times respectively than that of control groups.But the ROS of pre incubated groups with antioxidants NAC were lower than that instimulated groups. Platelet stimulated with dibucaine glycoprotein GPIba express lowerthan that of control group platelet, but shedding fragment GC was more than that of controlin the supernatant after centrifugation. Platelet stimulates with NAC or DTT beforedibucaine, the GPIba expression lever was higher and GC was less in the supernatant thanthat platelet stimulated with dibucaine. The percentage of platelets incubated withdibucaine with normal mitochondrial membrane potential (△Ψm) was (13.75±2.75)%,which group pre incubated with NAC was (96.07±2.47)%; The percentage of plateletsincubated with thrombin with△Ψm was (76.58±5.28)%, which groups pre incubated withNAC was (93.00±3.03)%. Difference were statistically significant (P<0.05). Thus, NACmay inhibite the depolarization of△Ψm caused by dibucaine and thrombin significantly.Platelets were incubated with dibucaine or thrombin, pro-apoptosis protein caspase-3was activated and expression of anti-apoptosis protein Bcl-xL was decreased, which couldinhibite with NAC significantly. When irradiated with2.5Gy~10Gy X-ray, platelet GPIbaand△Ψm have slight fluctuation, but no statistical significance. The proliferation abilityof Meg-01cells which have the potential of evolving into platelets was inhibited when theywere singly irradiated with X-ray dose more than3Gy and cultured24hour. Theexpression lever of CD41and GPIba on irradiated cell surface was higher than that ofcontrol. When cells were irradiated with X-ray dose more than3Gy and cultured48hour,they have little apoptosis, but more necrosis that control.Conclusions: ROS involved in the regulation of the platelet GPIba shedding andapoptosis induced by stimulates in vivo and in vitro. NAC can inhibit platelet apoptosisinduced by dibucaine or thrombin. Platelet irradiated with2.5Gy~10Gy X-ray can not beinduced GPIba shedding or apoptosis. The proliferation ability of Meg-01cells irradiatedwith X-ray dose more than3Gy was inhibited, but the degree of differentiation andmaturity were higher than that of control. The experimental results provide importantguidance for clinical thrombocytopenic diseases or radiation injury of hematopoieticsystem. The research results are expected to be used in the treatment of related diseasesand guide the development of new drugs.