Study On The Mechanism Of Tongqiao Huoxue Decoction Against Ferroptosis By Regulating System Xc--GPX4 Pathway In The Treatment Of CIRI

Objective:To observe and compare the neuroprotective effect of Tongqiaohuoxue Decoction（THD）at different doses on cerebral ischaemia-reperfusion injury（CIRI）rats,and to explore its mechanism of antagonizing brain cell ferroptosis based on the system Xc^--glutathione peroxidase 4（GPX4）pathway.Methods:The SPF grade male SD rats were randomly divided into a sham operation group,a model group,low,medium,and high dosage THD groups,and a positive drug control Edaravone（EDA）group.The modified suture method was used to establish the middle cerebral artery occlusion（MCAO）model,and after 2 hours of ischemia,cerebral ischemia reperfusion was performed to replicate the CIRI rat model.Intervention began 24hours after modeling.The THD groups were given THD by gavage at doses of 1.99 g·kg^-1·d^-1（low dose）,3.97 g·kg^-1·d^-1（medium dose）,and 7.95 g·kg^-1·d^-1（high dose）,while the sham operation group and model group were given an equal volume of normal saline by gavage.The EDA group was given an injection of 6mg·kg^-1·d^-1dose by intraperitoneal injection.After continuous treatment for 7 days,the indicators were tested.The m NSS neurological function deficit score was used to evaluate the recovery of rat neurological function.TTC staining was used to detect infarct volume,HE staining was used to observe changes in rat brain tissue morphology,colorimetry was used to detect changes in malondialdehyde（MDA）,glutathione（GSH）and Fe²⁺in ischemic brain tissue,Western blotting was used to detect protein expression of GPX4 and SLC7A11 in ischemic brain tissue,and immunofluorescence multiplex staining was used to detect the co-expression of SLC7A11 and GPX4 with neuronal nuclei（Neu N）,glial fibrillary acidic protein（GFAP）,ionized calcium binding adapter molecule 1（Iba1）,oligodendrocyte lineage transcription factor 2（Olig2）,and platelet-endothelial cell adhesion molecule-1（CD31）in brain tissue.Results:1.Compared to the sham group,the m NSS score was significantly increased（P<0.01）,the infarct volume was significantly increased（P<0.01）,and there was a significant area of cortical necrosis,extensive loss of nerve cells,and degeneration of nerve cells at the edge of the infarct in the model group.The glial cells were proliferated and swollen,and there were missing neurons in the hippocampal area.In the ischemic brain tissue,the levels of MDA and Fe²⁺were significantly increased（P<0.01）,while the GSH content（P<0.01）,GPX4 protein expression（P<0.01）,and SLC7A11 protein expression（P<0.05）were significantly decreased.The co-expression of SLC7A11 with Neu N and GFAP in the hippocampal area of the ischemic side was significantly decreased（P<0.01）,as was the co-expression of SLC7A11 with Olig2（P<0.05）.The co-expression of GPX4 with Neu N and GFAP was also significantly decreased（P<0.05）.2.Compared to the model group,the m NSS score was significantly decreased（P<0.01）,the infarct volume was significantly reduced（P<0.01）,and the area of necrosis was relatively smaller in the THD and EDA groups.The degeneration of nerve cells and the proliferation and swelling of glial cells were improved in these groups.In the ischemic brain tissue,the levels of MDA and Fe²⁺were significantly decreased（P<0.01）in the THD and EDA groups.The GPX4 protein expression was significantly increased in the THD medium-dose（THD-M）group（P<0.01）,the THD high-dose（THD-H）group,and the EDA group（P<0.01）.The SLC7A11 protein expression was significantly increased in the THD-H group and the EDA group（P<0.01）.In the hippocampal area of the ischemic side,the co-expression of SLC7A11 with GFAP was significantly increased in the THD-H group（P<0.05）,and the co-expression of GPX4 with Neu N,Olig2,and GFAP was significantly increased in the THD-H group（P<0.01）.The co-expression of SLC7A11 with Neu N was significantly increased in the THD-H group,and the co-expression of SLC7A11 with GFAP and Olig2 was significantly increased in the THD-H group and the EDA group（P<0.05）.The co-expression of GPX4 with Neu N,GFAP,and Olig2 was significantly increased in the THD-H group,the EDA group,and the THD-H group（P<0.05）.3.When comparing the treatment groups,the THD-H group had a significantly lower m NSS score（P<0.01）and a significantly reduced infarct volume（P<0.01）compared to the THD low-dose group（THD-L）.The THD-H group also had significantly decreased levels of MDA and Fe²⁺（P<0.01）,and significantly increased GSH content（P<0.01）,GPX4protein expression（P<0.05）,and SLC7A11 protein expression（P<0.01）compared to the THD-L group.The THD-H group had a significantly reduced infarct volume（P<0.01）and significantly decreased Fe²⁺levels（P<0.05）compared to the THD-H group.The EDA group had a significantly reduced infarct volume（P<0.01）and significantly decreased Fe²⁺levels（P<0.01）compared to the THD-H group.The THD-H group and the EDA group both had significantly increased GPX4 and SLC7A11 protein expression（P<0.01）compared to the THD-L group.In the hippocampal area of the ischemic side,the THD-H group had significantly increased co-expression of SLC7A11 with GFAP（P<0.05）,and the THD-H group and the EDA group both had significantly increased co-expression of SLC7A11 with Neu N（P<0.01）.The THD-H group had significantly increased co-expression of SLC7A11 with GFAP and Olig2（P<0.05）,and significantly increased co-expression of GPX4 with Neu N,GFAP,and Olig2（P<0.05）compared to the THD-L group.Conclusions:1.THD can significantly improve the nerve function damage of CIRI rats,reduce the volume of cerebral infarction,and promote the repair of ischemic brain tissue morphology,of which the high dose group has the best effect.2.THD can inhibit the Ferroptosis of ischemic brain tissue in CIRI rats,which is closely related to the regulation of System XC^--GPX4 pathway.3.The regulatory effect of THD on the System Xc^--GPX4 pathway in neurons,astrocytes,and oligodendrocytes in the ischemic hippocampal region was significant.