| BACKGROUND:Qi and blood are the foundations of the body,whose interaction and association affect the state of physical mechanism of the body.Blood stasis syndrome contains two major types:qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome.Studies have proved that there are similarities and differences between these two types of blood stasis syndrome in terms of pathology,biology and pathogenesis.However,few studies pay enough attention to research on these two types of blood stasis syndrome related to platelets.Based on the previous work done be staff in our laboratory,we found that with the method of irregular sleep deprivation modeling,there appears a pathological transformation process from qi stagnation and blood stasis syndrome in the third week to qi deficiency and blood stasis syndrome in the sixth week.Both of these two types of syndrome are featured with weight loss and yellow,dried-up hair.Besides,differences can also been seen in macroscopic symptoms like pulse,tongue image,pain sensation,hemorheology,and cardiac function.But what are microscopic changes,similarities and differences between qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome?Are there changes in function and morphology of platelets as a key link of blood stasis syndrome?Can platelets be seen as the pivot of interaction between qi and blood in the hypothesis proposed by national research project "973" which holds that "qi and blood interact in pulse"?Can verification and material basis of relevant theories be found in platelets when dealing with different types of blood stasis syndrome with Chinese medicine as common anticoagulant drugs?All of these issues are in need of exploration and discussion.OBJECTIVE:The purposes of this study are to explore changes and differences of platelet function in rat models of qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome,and to probe into regulatory mechanism of Liqi Huoxue recipe and Yiqi Huoxue recipe on platelet function of two types of blood stasis syndrome.SIGNIFICANCE:The significance of the research is that it studies changes of platelets with the model of rats in respects of qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome in a microscopic perspective.Furthermore,we will explore the pharmacodynamics and mechanisms of Liqi Huoxue recipe and Yiqi Huoxue recipe on regulating platelet function as well as similarities and differences between pharmacodynamics and mechanisms of two recipes.We hope that the experimental results can enrich theoretical basis of traditional Chinese medicine and provide evidence for the hypothesis of national "973" project:qi and blood interact in pulse.METHODS:1.The sleep deprivation modeling method with self-made water environment small platform box was used to duplicate the rat models of qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome.Each box contains six small platforms,for five rats modeling,covered with wire mesh.The total deprivation timewas 112 hours per week,with an average of 16 hours per day.The time of making model of qi stagnation and blood stasis syndrome was 3 weeks,and the time of making model of qi deficiency and blood stasis syndrome was 6 weeks.The model was contradicted by Liqi Huoxue recipe and Yiqi Huoxue recipe at the 2nd and 5th weeks respectively.The Liqi Huoxue recipe and Yiqi Huoxue recipe were used for drug counterevidence of the syndromes.At the same time,the pharmacodynamics and mechanism of Liqi Huoxue recipe and Yiqi Huoxue recipe were verified and discussed.2.Evaluations of macroscopic characterizations:The body weight,pulse,tongue image,pain sensation,hemorheology and cardiac function of rats in each group were measured to verify the success of the model and pharmacodynamics.3.Genomic detection:MicroRNA genomics detection was used to evaluate the changes of microRNA in rats of qi stagnation and blood stasis syndrome model group and rats of qi deficiency and blood stasis syndrome model group,and to find the target genes related to platelet activation as well as the possible targets of Liqi Huoxue recipe and Yiqi Huoxue recipe.4.Evaluations of platelet function and morphology:platelet aggregation was induced by three inducers of AA,ADP and RISTO.Flow cytometry was used to detect the expressions of platelet surface glycoproteins CD62p and CD63.The external morphology and internal microstructures of platelets were observed by scanning electron microscope and transmission electron microscope.ELISA was used to detect the content of PEC AM-1 in plasma in each group.RESULTS:1.Macroscopic characterizations:(1)Pulse:The pulse intensity of qi stagnation and blood stasis syndrome model group was significantly higher than the pulse intensity of the normal group at the same time(P<0.01),with the characteristics of higher,steeper,not fluent enough,with small fluctuations and faster rhythm.Compared with qi stagnation and blood stasis syndrome model group,the pulse intensity of Liqi Huoxue recipe group was significantly lower(P<0.01),and the pulse condition alleviated and changed significantly.Compared with the normal group at the same time,the pulse intensity of qi deficiency and blood stasis syndrome model group was significantly lower(P<0.01),with the characteristics of lower,flatter,insufficient fluency and slower rhythm.Compared with qi deficiency and blood stasis syndrome model group,the pulse intensity of Yiqi Huoxue recipe group improved significantly(P<0.01),the pulse pattern was stronger and the rhythm was faster.(2)Weight:Compared with the normal group at the same time,the weight of rats in qi stagnation and blood stasis syndrome model group and qi deficiency and blood stasis syndrome model group decreased significantly(P<0.01).Compared with qi stagnation and blood stasis syndrome model group,there was no significant difference in body weight between Liqi Huoxue recipe group and qi deficiency and blood stasis syndrome model group.There was no significant difference in body weight between Yiqi Huoxue recipe group and qi deficiency and blood stasis syndrome model group.(3)Tongue image:Compared with the normal group at the same time,tongue images were red and dark,the R,G and B values of the tongue images in the qi stagnation and blood stasis syndrome model group were significantly lower(P<0.01,P<0.05).Compared with the qi stagnation and blood stasis syndrome model group,the tongue images were pale red,the R and G values of the tongue images in the Liqi Huoxue recipe group improved significantly(P<0.05).Compared with the normal group at the same time,the R,G and B values of the tongue images in the qi deficiency and blood stasis syndrome model group were significantly lower(P<0.01,P<0.05).Compared with the qi deficiency and blood stasis syndrome model group,the R,G and B values of the tongue images in the Yiqi Huoxue recipe group improved significantly,with statistical significance(P<0.01,P<0.05).(4)Pain sensation:Compared with the normal group at the same time,the time of pain response in qi stagnation and blood stasis syndrome model group was significantly shorter(P<0.01),and the time of pain response in Liqi Huoxue recipe group was significantly longer than that in qi stagnation and blood stasis syndrome model group(P<0.01).Compared with the normal group at the same time,the pain response time of rats in qi deficiency and blood stasis syndrome model group was significantly longer(P<0.01).Compared with the qi deficiency and blood stasis syndrome model group,the pain response time of rats in Yiqi Huoxue recipe group was significantly shorter(P<0.01).(5)Rat echocardiography:Compared with the normal group at the same time,the left ventricular diastolic volume of rats in qi stagnation and blood stasis syndrome model group reduced significantly(P<0.05),the stroke volume reduced significantly(P<0.05),and the cardiac output reduced significantly(P<0.01);compared with the qi stagnation and blood stasis syndrome model group,the parameters of cardiac function of Liqi Huoxue recipe group had no significant changes.Compared with the normal group at the same time,the left ventricular diastolic diameter,left ventricular diastolic volume,stroke volume and cardiac output in the qi deficiency and blood stasis syndrome model group decreased significantly(P<0.05);compared with the qi deficiency and blood stasis syndrome model group,the heart rate and left ventricular diastolic diameter in the Yiqi Huoxue recipe group increased significantly(P<0.05),cardiac output increased significantly(P<0.01),diastolic volume and stroke output increased,but there was no statistical significance.(6)Hemorheology:Compared with the normal group at the same time,the blood viscosity of qi stagnation and blood stasis syndrome model group increased significantly under the conditions of low shear,middle shear and high shear(P<0.05);compared with qi stagnation and blood stasis syndrome model group,the blood viscosity under the condition of low shear of Liqi Huoxue recipe group decreased significantly(P<0.05),the blood viscosity under the conditions of middle shear and high shear decreased significantly(P<0.01).Compared with the normal group at the same time,the blood viscosity of qi deficiency and blood stasis syndrome model group increased significantly under the conditions of low shear,middle shear and high shear(P<0.01);compared with qi deficiency and blood stasis syndrome model group,the blood viscosity under the conditions of low shear of Yiqi Huoxue recipe group tended to decrease,but there was no statistical difference,the blood viscosity under the conditions of middle shear decreased significantly(P<0.05),and the blood viscosity under the conditions of high shear decreased significantly(P<0.01).2.Genomics detection:(1)Compared with the normal group at the same time,the expression of multiple microRNAs in qi stagnation and blood stasis syndrome model group and qi deficiency and blood stasis syndrome model group were different,suggesting that platelet activation may be regulated by related genes.(2)There were two microRNAs expressed differently in the two model groups:rno-mir-344a-5p and rno-mir-762.The expression of rno-mir-344a-5p was closely related to the expressions of CD63,P-selectin,Gplb and GpV.(3)The mechanisms of Liqi Huoxue recipe and Yiqi Huoxue recipe in treating qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome may be related to the regulation of microRNAs.3.Study of platelet function and morphology:(1)Platelet aggregometry experiment:Compared with the normal group at the same time,the maximum aggregation rate(MAR)of platelet and average aggregation rate(AAR)of platelet in qi stagnation and blood stasis syndrome model group induced by AA,ADP and RISTO increased significantly(P<0.01).Compared with qi stagnation and blood stasis syndrome model group,AAR of platelet in Liqi Huoxue recipe group induced by AA was significantly lower(P<0.05);compared with qi stagnation and blood stasis syndrome model group,MAR and AAR of platelet in Liqi Huoxue recipe group induced by ADP were significantly lower(P<0.05,P<0.01);compared with qi stagnation and blood stasis syndrome model group,MAR and AAR of platelet in Liqi Huoxue recipe group induced by RISTO were significantly lower(P<0.01).Compared with the normal group at the same time,AAR of platelet in qi deficiency and blood stasis syndrome model group induced by AA was significantly higher(P<0.01,P<0.05);compared with the normal group at the same time,the maximum platelet aggregation rate(MAR)and average aggregation rate(AAR)of rats in qi deficiency and blood stasis syndrome model group induced by RISTO were significantly higher(P<0.01).Compared with the model group of qi deficiency and blood stasis syndrome,the AAR of platelets in the Yiqi Huoxue recipe group induced by AA was significantly lower(P<0.05);compared with the model group of qi deficiency and blood stasis syndrome,the MAR and AAR of platelets in the Yiqi Huoxue recipe group induced by RISTO were significantly lower(P<0.01,P<0.05).(2)The expressions of platelet membrane glycoproteins CD62p and CD63:Compared with the normal group at the same time,the expression of CD63 in qi stagnation and blood stasis syndrome model group increased significantly(P<0.01).Compared with qi stagnation and blood stasis syndrome model group,the expression of CD63 in Liqi Huoxue recipe group was significantly lower(P<0.01).Compared with the normal group at the same time,the expression of CD62p in qi deficiency and blood stasis syndrome model rats increased significantly(P<0.01),while the expression of CD63 increased significantly(P<0.05).Compared with qi deficiency and blood stasis syndrome model group,the expression of CD62p in Yiqi Huoxue recipe group was significantly lower(P<0.01),while the expression of CD63 decreased significantly(P<0.05).(3)Scanning electron microscopic observation of platelets:In normal group,platelets were scattered,non-aggregated,less or less activated.Compared with the normal group at the same time,the platelet activation in the qi stagnation and blood stasis syndrome model group was obvious,the dilated platelet increased,the pseudopodia extended obviously,the surface of cell membrane was uneven,the platelet had aggregation phenomenon,and the phenomenon of intercellular membrane fusion existed.Compared with the qi stagnation and blood stasis syndrome model group,the platelet aggregation was significantly improved,the cell membrane recovered smooth,and the pseudopodia extension reduced in the Liqi Huoxue recipe group.Compared with the normal group at the same time,the platelet aggregation and microthrombosis in the qi deficiency and blood stasis syndrome model group increased significantly.Compared with the qi deficiency and blood stasis syndrome model group,the platelet aggregation in Yiqi Huoxue recipe group lessened significantly,the platelet microthrombosis depleted,and the platelet cell membrane recovered smooth and intact.(4)Transmission electron microscopy observation of platelets:In normal group,the boundary of platelet cell membrane was clear,the granules in platelet were clearly visible,and the cell structures were complete.Compared with the normal group at the same time,the platelet cell membrane of the qi stagnation and blood stasis syndrome model group was obviously damaged.It can be seen that the platelet granular membrane fused with the cell membrane,the granular membrane in the platelet ruptured obviously,the granular membrane fused with each other,the cell structures damaged,the shape of the platelet changed,and the tendency of pseudopodia extension was observed.Compared with the qi stagnation and blood stasis syndrome model group,the membrane boundary of platelet cells in Liqi Huoxue recipe group was clear,the destruction of granules in cells lessened obviously,and the shape of platelet cells improved.Compared with the normal group at the same time,the platelet membrane fusion was obvious in the qi deficiency and blood stasis syndrome model group,the platelet connected into pieces,there were a large number of giant granules or even vacuoles,the granules severely damaged,the mitochondrial cristae broken,and the cell structures damaged severely.Compared with qi deficiency and blood stasis syndrome model group,platelets in Yiqi Huoxue recipe group were round-like,cell membrane was clear and complete,granular membrane was clear and complete,and mitochondrial structure was complete.(5)Content of PEC AM-1 in plasma:Compared with the normal group at the same time,the content of PEC AM-1 in plasma of qi stagnation and blood stasis syndrome model group was significantly higher(P<0.01);compared with qi stagnation and blood stasis syndrome model group,the content of PECAM-1 in plasma of Liqi Huoxue recipe rats was significantly lower(P<0.01).Compared with the normal group at the same time,the content of PECAM-1 in plasma of the qi deficiency and blood stasis syndrome model group was significantly higher(P<0.01),while compared with the qi deficiency and blood stasis syndrome model group,that in the Yiqi Huoxue recipe group was significantly lower(P<0.01).CONCLUSIONS:1.Rat models of qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome induced by sleep deprivation were successfully established.The models were stable and easy to be established,which accorded with the pathogenesis of traditional Chinese medicine.2.In the model groups of qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome,the platelet activity increased,the morphology of platelets changed.Besides,there was membrane fusion between platelets,cell membrane was damaged,granule release increased and structure was damaged.3.The platelet activation of the model group of qi stagnation and blood stasis syndrome is more related to dense granules.The platelet activation of the model groups of qi deficiency and blood stasis syndrome is characterized by microthrombosis formation and more serious structural damage.4.Both Liqi Huoxue recipe and Yiqi Huoxue recipe can treat qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome respectively by inhibiting platelet activation,and their mechanisms may be related to the regulation of microRNAs. |
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