The Effects Of Hydrogen-rich Saline On Platelet Activation In Hypertensive Rats

BackgroundCardiovascular disease accounts for 40%of the mortality in our country.Hypertensi-on is the independent risk factor for ischemic stroke and coronary heart disease.Previous studies have found that platelet activation is related to oxidative stress,and antioxidative stress can inhibit platelet activation.In recent years,it was found that hydrogen can exert antioxidative effects,and it is considered as a kind of antioxidant with potential therapeutic value.AimsThe present study was designed to investigate the effect of hydrogen-rich saline on platelet activation in hypertensive rats.MethodsThe experiment was divided into the in vivo experiment and in vitro experiment.In vivo experiment,male wistar rats were sham-operated or subcutaneously infused with angiotensin II at a rate of 0.7 mg·kg^-1·d^-1 for 2 weeks by osmotic mini-pumps.They were divided into control group,hypertension group,control+hydrogen-rich saline group and hypertension+hydrogen-rich saline group.Control group and hypertensive group were given hydrogen-rich saline solvent(10 ml·kg^-1·d^-1,ip);control+hydrogen-rich saline group and hypertension+hydrogen-rich saline group were given hydrogen-rich saline(10ml·kg^-1·d^-1,ip).Blood pressure was measured by a non-invasive tail-cuff method.Fourteen days after treatment with hydrogen-rich saline or solvent,hemodynamic parameters continuously recorded in consciouse unrestrained rats.Blood pressure（BP）,blood pressure variability（BPV）,heart rate（HR）and heart rate variability（HRV）was calculated.Platelet adhesion on collagen surface was evaluated using a well-defined perfusion chamber at low shear rate(300s^-1)and high shear rate(1080s^-1).The maximum aggregation rate of platelets induced by ADP was determined by turbidimetry.The expression of P-selectin on platelet surface,reactive oxygen species（ROS）,nitric oxide（NO）and Ca²⁺in platelet was measured with flow cytometry.Additionally,levels of malondialdehyde（MDA）,NO,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）and nitrotyrosine（NT）in plasma was detected with radioimmunoassay and UV spectrophotometry.Finally,the p38mitogen-activated protein kinas（MAPK）and endothelial nitric oxide synthase（eNOS）was detected by immuno-blotting（western blot）assay.In vitro experiments,male wistar rats were divided into control group,hypertension group,control+hydrogen-rich saline group,and hypertension+hydrogen-rich saline group.Blood was taken from the heart into heparin tubes.Hydrogen-rich saline（or solvent）was added into blood（or platelet rich plasma）at a ratio of 0.2.Platelet maximum aggregation,platelet adhesion rate,P-selectin express on platelet surface and platelet NO and Ca²⁺level was measured with the above mentioned methods.Results1.Two weeks after subcutaneous infusion of angiotensin II,the systolic BP（SBP）of the hypertensive group was more than 160 mmHg,but the heart rate remained unchanged,suggesting the hypertensive model was successfully produced.2.When compared with the control group,the SBP,DBP,MBP,SBPV,DBPV,and MBPV in the hypertension group were significantly increased.Compared with hypertension group,SBP,DBP,MBP,SBPV,DBPV and MBPV in hypertension+hydrogen-rich saline group didn’t significantly changed.Compared with the control group,no significant change of SBP,DBP,MBP,SBPV,DBPV and MBPV the control+hydrogen-rich saline was found.These results suggest that hydrogen-rich saline n had no effect on significant effect on BP and BPV of hypertensive rats.3.The results showed changes of platelet function in hypertensive rats:（1）Compared with the control group in vivo experiment,P-selectin expression on platelet surface,platelet aggregation,platelet adhesion rate in high shear rate and low shear rate,and the level of ROS,Ca²⁺and p38MAPK in platelets,levels of MDA and NT in serum were elevated significantly in hypertensive group.However the levels of NO,SOD and GSH-Px in serum and eNOS expression and NO level in platelets decreased significantly.（2）Simil-arly,the results showed that in vitro experiment,compared with control group,there was significant increase in P-selectin expression on platelet surface,platelet aggregation rate,platelet adhesion to collagen in low shear rate(300s^-1)and high shear rates(1080s^-1),ROS and Ca²⁺level in platelets increased significantly in hypertension group.Mean fluorescence intensity of NO in platelets decreased significantly in hypertensive group.4.The results showed that:（1）In vivo experiment,compared with the hypertensive group,P-selectin expression on platelet surface,platelet aggregation,platelet adhesion rate in high shear rate and low shear rate,and the level of ROS,Ca²⁺and p38MAPK in platelets,the level of MDA and NT in serum decreased significantly in hypertensive+hydrogen-rich saline group.The levels of NO,SOD and GSH-Px in serum and the eNOS expression and NO level in platelets increased significantly in hypertensive+hydrogen-rich saline group.（2）Similarly,the results showed that in vitro experiment,compared with hypertension group,there was significant decrease in P-selectin expression on platelet surface,platelet aggregation rate,platelet adhesion to collagen in low shear rate(300s^-1)and high shear rates(1080s^-1),ROS and Ca²⁺of platelets in hypertension+hydrogen-rich saline group.Mean fluorescence intensity of NO in platelets significantly increased in hypertension+hydrogen-rich saline group.ConclusionHydrogen-rich saline could inhibit platelet activation in hypertensive rats.This effect may be related to antioxidative stress,p38MAPK and eNOS signals.