| Ischemic stroke is characterized by high incidence and disability rate,and has become one of the major diseases threatening people’s health.Although clinical observations have shown that Guhong injection is effective in the treatment of cerebral infarction and can improve the indexes of hemorheology,the research on its pharmacological effects and mechanism of action is still not deep enough,and there is a lack of quality evaluation methods related to its biological effects.Zebrafish,a model animal,which has the advantages of rapid reproduction,large number of embryos and easy observation,is increasingly used in the evaluation of the efficacy and safety of Chinese medicine.In this thesis,two zebrafish models were used to evaluate the antiischemic stroke efficacy of Guhong injection,to investigate its mechanism of action,to identify the main active substances,and to explore the use of related models for quality evaluation.The main researches of this thesis are as follows.1.The pharmacological effects of Guhong injection on anti-ischemia and recovery of motor function were defined in stroke related mice and zebrafish models.Using a mouse model of middle cerebral artery occlusion,it was found that Guhong injection significantly attenuated neurological deficits caused by cerebral ischemia-reperfusion injury,restored motor ability,reduced the area of cerebral infarction,and improved the pathological morphology of brain tissue.A zebrafish brain ischemia model induced by Ponatinib was established.Through hemoglobin staining and behavioral observations,it was found that Guhong injection effectively inhibited cerebral ischemia,and increased the movement distance,mean speed and cumulative time of the injured zebrafish.qRTPCR results showed that Guhong injection inhibited the expression of inflammationrelated genes such as nuclear factor-κB(NF-κB),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α).Both safflower-derived components and acetylglutamide from Guhong injection could restore motor function in cerebral ischemic zebrafish to different degrees.Using the ponatinib model,it was found that acetylglutamide,chlorogenic acid and gallic acid were the main active components in improving motor dysfunction after cerebral ischemia.2.A model of Phenyl hydrazine(PHZ)induced thrombosis in zebrafish was established.It was found that Guhong injection and its main components had significant anti-thrombotic effects.The hemorheology of zebrafish was observed by hemoglobin staining combined with dynamic observation of green fluorescent zebrafish labeled erythrocytes.qRT-PCR showed that Guhong injection significantly decreased the mRNA expression of genes related to coagulation cascade(F2 and F7)and inflammation(NFκB and IL-6)in zebrafish.The antithrombotic active compounds in Guhong injection mainly included baicalein,gallic acid and rutin from safflower,while acetylglutamide had no significant effect on zebrafish hemodynamics.3.Based on the above zebrafish model,a quality evaluation method based on bioeffect detection of Guhong injection was established and successfully used for consistency evaluation among different batches of products.Using the PHZ-induced zebrafish thrombosis model,the hemoglobin staining and dynamic observation of blood flow revealed that different batches of Guhong injection or safflower extract had antithrombotic activity,and the quality consistency was maintained between the preparations before and after the change of plant site.In conclusion,this thesis identified the anti-ischemic and anti-thrombotic effects of Guhong injection on animal models,and its mechanism of action may be related to antiinflammation and the inhibition of coagulation cascade.Baicalein,chlorogenic acid,gallic acid and rutin,which are derived from safflower,and acetylglutamide are the main active compounds of Guhong Injection,and the biological effect evaluation based on zebrafish model can be used for the quality consistency evaluation of Guhong Injection. |
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