The Absorption Effect On Hematoma Of Brain Tissue And Mechanism Of Tongqiao Huoxue Decoction In Mice With Traumatic Brain Injury

Objective:The formation of hematoma after Traumatic brain injury(TBI)is an important cause of secondary brain injury.Previous studies have confirmed that Tongqiao Huoxue Decoction(TQHXD)has strong anti-inflammatory and antioxidant properties,which can be a potential drug for the treatment of TBI.This study aims to explore the therapeutic effect of TQHXD on TBI mice and the mechanism of promoting microglial absorption of intracranial hematoma in TBI mice.Methods:The method of modified free fall of Feeney was used to establish the TBI model.neurological deficient scores(NDS),wire hanging test(WHT),forelimb placing test(FPT),Y maze test,brain lesion volume and brain water content were used to measure the neuroprotection and the absorption of intracranial hematoma of TQHXD and to select optimal therapeutic dose.BV-2 was scratched to establish TBI model in vitro and cultured with red blood cells for 6 hours to observe the ability of microglia to phagocytic red blood cells.UPLC-MS/MS was used to detect the main absorbed components in the blood and brain tissue of mice after TQHXD intervention.Proteomics was applied to determine the changes of the protein profile in the brain tissue of mice in each group and to screen out the pathways and proteins related to the action of TQHXD.Subsequently,the differentiated pathways and proteins were further verified with western blot(WB)and immunofluorescence(IF).CX3CR1 genetically engineered mice and the CD36 inhibitor sulfosuccinimidyl oleate(SSO)were used to further validate the pathway dependent on TQHXD uptake in microglia hematoma after TBI in vivo and in vitro.~2Results:The optimal therapeutic dose for TQHXD to exert its neuroprotective effect after TBI was 3g/kg.TQHXD could play a role in the absorption of hematoma via improving the behavior of TBI mice and reducing the volume of brain injury and brain water content.Compounds were widely screened through comparison with the mz Cloud and mz Vault databases.Qualitative analysis of compounds in peripheral blood and brain tissue of TQHXD was conducted,and 336 compounds were found in both peripheral blood and brain tissue.The seven compounds with the highest ratio with the database were selected and UPLC-MS/MS was used to further determine the main absorption components of TQHXD in blood and brain tissue.It was found that the main absorbed components in peripheral blood and brain tissue were catechin,hydroxy-safflower yellow pigment A,ferulic acid,paeoniflorin,baicalin,amygdalin and kaempferol.Proteomic showed that intervention of TQHXD could change the protein profile of brain tissue of in mice with TBI,and the differential proteins were enrichment in the ferroptosis pathway.The expression of CD36 and CX3CR1 of microglia promoted the absorption of hematoma.Knocking out CX3CR1 eliminated the role of TQHXD in promoting microglia to absorb hematoma.CX3CR1 knockout and CD36 inhibition could aggravate ferroptosis in brain tissue of TBI mice and further promote cerebral hematoma.Conclutions:TQHXD has a neuroprotective effect on TBI mice,and this neuroprotective effect depends on the absorption of hematoma in brain tissue after TBI.The possible pharmacodynamic substances was catechin,hydroxy-safflower yellow pigment A,ferulic acid,paeoniflorin,baicalin,amygdalin and kaempferol.The mechanism of TQHXD promoting the absorption of hematoma in TBI mice is related to the inhibition of ferroptosis around the lesion of brain tissue in TBI mice and the elevation of the expression of CX3CR1 and CD36 on the surface of microglia cells.