| Stroke is a serious disease that threatens the human health with high mortality,disability,morbidity and relapse rate,and also has been a difficult problem that troubled the medical field for years.Stroke is divided into ischemic stroke and hemorrhagic stroke.Ischemic stroke,also known as cerebral infarction,accounts for 60%-70%in all kinds of strokes.Qi-deficiency and blood-stasis are the main syndromes in Traditional Chinese Medicine theories on stroke.Despite the constant progress made in clinical and experimental studies on cerebral infarction with Qi-deficiency and Blood-stasis syndrome,in recent years,there is still many problems need to be explored.At present,animal models of disease are used to evaluate the therapeutic effect of traditional Chinese medicine mostly,but it is difficult to reflect the characteristic of traditional Chinese medicine objectively.The integrated animal models of disease and syndrome are more accordance with the research of traditional Chinese medicine and requirements of analyzing the occurrence and development regularity.Our previous research discussed and established a stable and reliable rat model of cerebral infarction with Qi-deficiency and Blood-stasis syndrome,studied the changes of characterization and pathophysiology in this model,and found its relationship with energy metabolism disorder,but the mechanism is still not clear.Energy metabolism disorder is the initiating factor of cerebrovascular disease.AMPK,as a receptor and effector of energy metabolism,regulates the body’s energy metabolism,plays an important role in cerebrovascular disease,and may be the biological basis of Qi-deficiency and Blood-stasis;however,there are few related studies and reports in this area.Based on previous work,this study further observed the changes of apparent syndromes,energy metabolism and related factors in rat model of cerebral infarction with Qi-deficiency and Blood-stasis syndrome.Also,we discussed the effects of AMPK-mediated pathway on the model and the intervention of Yiqi Huoxue recipe,so as to provide valuable experimental and theoretical basis for TCM syndrome research and its clinical application.ObjectiveTo establish a rat model of multiple cerebral infarction with Qi-deficiency and blood-stasis syndrome,observe the changes in energy metabolism and related factors and the effect of AMPK-mediated downstream pathways on pathophysiology,syndromes,and energy metabolism changes of the model,as well as study the intervening effect of Yiqi Huoxue Recipe on the model and the correlation of AMPK and its downstream signaling pathways.To clarify the biological basis of The integrated animal models of disease and syndrome,and the scientific connotation of Qi-deficiency and blood-stasis as well as the mechanism of Yiqi Huoxue Recipe,to provide valuable research targets and ideas for clinical applications and further experimental research.MethodsHealthy male SD rats were randomly divided into 4 groups:① Sham group,n=21;②Model group,n=21;③ Yiqi Huoxue Recipe group(26g crude drug/kg),n=21;④Metformin group(250mg/kg),n=21.Rats in sham group were fed normally without any stimulation,and the other three groups were treated with the water environment and small platform induced sleep deprivation for 16 hours every day(16:00 pm to 8:00 am the next day);after one week of sleep deprivation,multiple cerebral infarctions operation were performed and the sleep deprivation were continued for another 28 days.At the same time,rats were intragastrical administered at a dose of 10 ml/kg/day after the operation for 28 days,rats in the sham group and model group were given equal volume of distilled water.Two days(Dosing 2 days)and 28 days(Dosing 28 days)after operation,10 rats were randomly selected from each group for indicators detection.The first part:Rat general state,body weight,neurological deficit score,grasping force,exhausted swimming time,hemorheology,tongue image RGB values,pulse condition,and brain histopathology;The second part:To establish a HPLC method for the content of ATP,ADP,AMP in rats cortex,and calculate AMP/ATP ratio,TAN,EC;The third part:Determination of glucose,lactate,pyruvate,free fatty acid and lactate dehydrogenase activity in rats serum and cortex,calculation lactate/pyruvate ratio;The fourth part:The expression of AMPK,p-AMPK,ACC,p-ACC and CPT-1 protein in rats cerebral cortex was measured by Western Blot,and the expression of p-ACC and CPT-1 protein in brain tissue was measured by immunohistochemistry.ResultsPart Ⅰ:(1)General state Rats in the sham group had good mental status,and the appearance,diet,excretory functions and locomotor activities were all in normal condition;rats in the model group were in poor condition represented as disabled,apathetic,lazy,loose stools,and aggravated with the progression of disease;the general state of rats in the Yiqi Huoxue Recipe group was significantly improved.(2)Syndrome Indexes Compared with the sham group,body weight,grasping force,exhausted swimming time,pulse condition of rats in the model group were significantly reduced 2 days and 28 days after operation(P<0.01);compared with the model group,body weight,grasping force,exhausted swimming time and pulse condition of rats in Yiqi Huoxue Recipe group were significantly increased 28 days after operation(P<0.05 or P<0.01).Compared with sham group,RGB values of tongue image of rats in the model group was significantly decreased 2 days and 28 days after operation(P<0.05 or P<0.01),The whole blood viscosity increased significantly at the shear rate of 5s-1,60s-1,150s-1(P<0.05 or P<0.01);compared with model group,RGB values of tongue image of rats in the Yiqi Huoxue group increased significantly 28 days after operation(P<0.05 or P<0.01),and the whole blood viscosity was significantly reduced at the shear rate of 5s-1 2 days after operation and the shear rate of 5s-1,60s-1 were significantly reduced 28 days after operation(P<0.05 or P<0.01).(3)TTC staining,disease and pathological indicators 24h after operation,rats were sacrificed and the brain slices were stained with TTC,various sizes of white infarct areas could be found in each group except the sham operation group.There was no neurological deficit of rats in the sham group,and rats in the model group presented severe neurological deficits 2 days and 28 days after operation;compared with model group,neurological deficit of rats was significantly decreased in Yiqi Huoxue Recipe group 28 days after operation(P<0.05).The histopathology of brain tissue showed that the morphological structure of rats in the sham operation group was normal,and the morphological structure of rats in the model group had pathological changes,which aggravated with the development of the disease;The pathological changes of brain tissue were significantly reduced 2 days and 28 days after operation in Yiqi Huoxue Recipe group.Part Ⅱ:Compared with sham group,ATP content and EC value of cerebral cortex in the model group were significantly reduced 2 days and 28days after operation(P<0.01),AMP content and AMP/ATP ratio were significantly increased(P<0.05 or P<0.01);Compared with model group,ATP content and EC value of the cerebral cortex in Yiqi Huoxue Recipe group was significantly increased 2 days and 28 days after operation(P<0.05 or P<0.01),AMP/ATP ratio was also significantly decreased(P<0.05),AMP content in cerebral cortex was significantly reduced 2 days after operation(P<0.05).Part Ⅲ:Compared with the sham group,lactic acid content and lactate/pyruvate ratio in the model group were significantly increased(P<0.05 or P<0.01),and pyruvate content was significantly decreased in serum 2 days and 28 days after operation(P<0.05 or P<0.01),however,there was no obvious difference in glucose,free fatty acid content and lactate dehydrogenase activity;Compared with model group,glucose and pyruvate content increased significantly,free fatty acid content and lactate/pyruvate ratio in serum in the Yiqi Huoxue Recipe group were significantly reduced 2 days and 28 days after operation(P<0.01),lactic acid content and lactate dehydrogenase activity in serum were significantly decreased 2 days after operation(P<0.05).Compared with sham group,lactic acid,free fatty acid content and lactate/pyruvic ratio in cortex in the model group was increased significantly(P<0.05 or P<0.01),and pyruvate content was decreased significantly 2 days and 28 days after operation(P<0.05),lactate dehydrogenase activity in cortex was increased significantly 2 days after operation(P<0.05),and glucose content in cortex was decreased significantly 28 days after operation(P<0.05);Compared with model group,pyruvate content in cortex in the Yiqi Huoxue group was increased significantly(P<0.01),and free fatty acid content and lactate/pyruvate ratio in cortex were decreased significantly 2 days and 28 days after operation(P<0.05 or P<0.01),the content of glucose of cortex was increased significantly(P<0.05),and the content of lactic acid in cortex was decreased significantly 28 days after operation(P<0.01),however,there was no obvious difference in lactate dehydrogenase activity.Part Ⅳ:Western Blot showed that the expression of p-AMPK and p-ACC in cortex in the model group was significantly higher than that in sham group 2 days and 28 days after operation,the expression of CPT-1 was significantly lower than that in sham group(P<0.05 or P<0.01).The expressions of p-AMPK,p-ACC and CPT-1 in cerebral cortex in the Yiqi Huoxue Recipe group 2 days and 28 days after operation were higher than those in the model group(P<0.05).Immunohistochemistry showed that the expression of p-ACC in brain tissue 2 days and 28 days after operation in the model group was significantly higher than that in the sham group(P<0.05 or P<0.01),and the expression of CPT-1 was significantly lower than sham group(P<0.05),the expression of p-ACC and CPT-1 in cerebral cortex in the Yiqi Huoxue Recipe group were higher than that in model group 2 days and 28 days after operation(P<0.05).ConclusionIn the multiple cerebral infarction rat model with Qi-deficiency and blood-stasis syndrome,the ATP production was decreased,AMP/ATP ratio was increased,energy charge was decreased,and energy metabolism was significantly impaired in acute and recovery phases.The energy metabolism disorder of this model may be associated with the tricarboxylic acid cycle and fatty acid β-oxidation disfunction,AMPK and AMPK-mediated ACC and CPT-1 downstream pathways have an impact on the syndrome and energy metabolism of the model.The role of Yiqi Huoxue Recipe in improving the model may be related to regulating energy metabolism,AMPK and its mediated downstream signaling pathways.The results of the study revealed that energy metabolism disorder,AMPK and its mediated downstream signaling pathways may be the biological basis of the cerebral infarction rat model with Qi-deficiency and blood-stasis syndrome during the model formation and development,Qi-deficiency and blood-stasis syndrome,as well as therapeutic methods of replenishing qi to activate blood.This provides valuable research targets and ideas for clinical applications and further experimental studies. |
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