The Effect And Mechanism Of Supplementing QI And Activating Blood Circulation Method On Hematoma Absorption In Intracerebral Hemorrhage

Objective To explore the mechanism and material basis of mechanism of supplementing QI and activating blood circulation method representing Naoxueshu oral liquid in treating cerebral hemorrhage in rats.Combined with in vivo experiments to study the effect of Naoxueshu oral liquid on collagenase-induced intracerebral hemorrhage model,to elucidate the mechanism and partial material basis of Naoxueshu oral liquid promoting hematoma absorption in intracerebral hemorrhage rats.Methods Part 1:Intracerebral hemorrhage model was established by intracerebroventricular injection of collagenase.50 SD rats were randomly divided into sham operation group,model group,low dose of Naoxueshu oral liquid group,medium dose of Naoxueshu oral liquid group and high dose of Naoxueshu oral liquid group.The drug was administered once daily for 3 consecutive days.The neurological function of rats was assessed by modified neurological deficit score at 1 and 3 days after the model was induced.On the 3rd day,the brains were perfused by the heart.The hematoma volume and bleeding volume of rats were counted by coronal section method and hemoglobin spectrophotometry,respectively.2.Intracerebral hemorrhage model was established by intracerebroventricular injection of collagenase.24 SD rats were divided into sham operation group,model group and Naoxueshu oral liquid group.The drug was administered once daily for 3 consecutive days.Western blot was used to detect the expression levels of CD36,CD206,and YM-1 protein around the hematoma,and immunofluorescence was used to detect the expression levels of CD36 around the hematoma.3.TCMSP database was used to search Astragalus membranaceus,Hirudo,Cortex Moutan,Rhubarb,Acorus calamus,Achyranthes bidentata and Rhizoma Chuanxiong,and the active components and corresponding targets were collected,Network Analyzer was used for topological analysis to obtain the key active components in Naoxueshu oral liquid.Part 2:1.Intracerebral hemorrhage model was established by intracerebroventricular injection of collagenase.50 SD rats were divided into sham operation group,model group,vehicle group,astragaloside IV low dose group and astragaloside Ⅳ high dose group.The drug was administered once daily for 3 consecutive days.On the third day,the brain was collected by cardiac perfusion,and the hematoma volume and bleeding volume were counted by coronal section method and hemoglobin spectrophotometry,respectively.2.Network pharmacology and molecular docking were used to predict the potential mechanism of astragaloside IV in promoting hematoma absorption in rats with intracerebral hemorrhage.3.Intracerebral hemorrhage model was established by intraventricular injection of collagenase and 18 SD rats were divided into sham operation group,model group and astragaloside Ⅳgroup.The drug was administered once daily for 3 consecutive days.Western blot assay was used to detect PI3K,AKT,CD36 protein expression levels around the hematoma.Results Part:1:1.On the first day after intracerebral hemorrhage,compared with the model group,the low,medium,and high dose groups of Naoxueshu Oral Liquid tended to reduce the neurological deficit score of rats.On the third day after intracerebral hemorrhage,compared with the model group,the low,medium,and high dose groups of Naoxueshu Oral Liquid significantly reduced the modified neurological deficit score of intracerebral hemorrhage rats.Compared with the model group,the hematoma volume of the low,medium,and high dose groups of Naoxueshu Oral Liquid was significantly reduced.2.The results of Western blot showed that CD36,CD206 and YM-1 around the hematoma were significantly increased in the Naoxueshu oral liquid group compared with the model group.Immunofluorescence experiments showed that CD36 around the hematoma was significantly increased in rats in the Naoxueshu oral liquid group compared with the model group.3.Network pharmacology results showed that there were 18 active ingredients of Astragalus membranaceus,4 active ingredients of Acorus calamus,17 active ingredients of Achyranthes bidentata,6 active ingredients of Cortex Moutan,10 active ingredients of Rhubarb and 6 active ingredients of Rhizoma Chuanxiong in Naoxueshu Oral Liquid.Through drug-component-target mapping and topological analysis,the active ingredients were determined to be astragaloside Ⅳ,kaempferol,myricetin,wogonin,isorhamnetin,formonetin,astragaloside isofosanoside,baicalin,β-sitosterol and 8-Isopentenyl-kaempferol,and astragaloside Ⅳ was selected according to the previous experiments of the research group to carry out its effect and mechanism on rats with intracerebral hemorrhage.Part:2:1.Compared with the vehicle group,the low-dose astragaloside Ⅳ group and high-dose astragaloside Ⅳ group could significantly reduce the modified neurological deficit score and the number of turns to the left after 3 days in rats with intracerebral hemorrhage.Compared with vehicle group,Astragaloside Ⅳ low dose group and Astragaloside Ⅳ high dose group could increase forelimb grip strength in rats with intracerebral hemorrhage after 3 days.Compared with vehicle group,Astragaloside Ⅳ low dose group and Astragaloside Ⅳhigh dose group significantly decreased hematoma volume and bleeding volume in rats with intracerebral hemorrhage.2.Network pharmacological analysis showed that the target of astragaloside Ⅳ in the treatment of cerebral hemorrhage was concentrated in the PI3K/ATK pathway.Molecular docking experiments showed that the binding energies of astragaloside Ⅳto PI3K and AKT were-12.6 kcal/mol and-12.2 kcal/mol,respectively.3.Western blot results showed that astragaloside Ⅳ could inhibit the expression of PI3K,AKT and increase the expression of CD36 compared with the model group.Conclusion 1.Naoxueshu oral liquid can promote hematoma absorption and neurological improvement in rats with intracerebral hemorrhage after 3 days.2.Naoxueshu Oral Liquid promotes hematoma absorption in rats with intracerebral hemorrhage by increasing the expression of CD36,CD206 and YM-1.3.Combined with network pharmacology and experimental verification,it is clear that astragaloside Ⅳ in Naoxueshu oral liquid can promote hematoma absorption and neurological function improvement in rats with intracerebral hemorrhage after 3 days;astragaloside Ⅳ promotes CD36 expression through PI3K/AKT pathway to accelerate hematoma absorption in rats with intracerebral hemorrhage,and elucidates some material basis of Naoxueshu oral liquid in the treatment of intracerebral hemorrhage in rats.