Based On The Regulation Of Ferroptosis By GPX4 Pathway To Explore The Mechanism Of Huoxuejiedu Method In The Treatment Of Cerebral Hemorrhage With Fire Poison Syndrome

[Objectives]1.To verify the anti-oxidative and curative effects of Xingnaojing injection as a blood circulation-promoting and detoxifying therapy for patients with acute intracerebral hemorrhage(AICH)based on a clinical study comparing and analyzing the size of hematoma,traditional Chinese(TCM)syndrome score,the Barthel index(BI),the NIH Stroke Score(NIHSS),and serum levels of oxidative stress factors such as superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),and glutathione peroxidase(GSH-Px)before and after treatment.2.To study the effects of the blood circulation-promoting and detoxifying Xingnaojing injection on nerve injury,brain iron metabolism,brain oxidative stress markers,and GPX4-related gene and protein expression in SD rat models with intracerebral hemorrhage(ICH),thereby revealing the mechanism of action of Xingnaojing injection in treating ICH by modulating ferroptosis,providing experimental support for the clinical use of Xingnaojing injection and guiding the clinical application of the blood circulation-promoting and detoxifying therapy and the theory of fire toxin-induced damage to brain collaterals from the perspective of modern biology.[Methods]1.Clinical study:Thirty patients who were diagnosed with ICH manifested by the clinical symptoms of the fire-toxin syndrome according to TCM syndrome differentiation and admitted by our hospital for nonoperative treatment were enrolled and provided with basic treatment and the blood circulation-promoting and detoxifying therapy(Xingnaojing injection)for 2 weeks.The size of hematoma,TCM syndrome score,BI value,and NIHSS in each patient were determined before and after treatment to evaluate the treatment outcomes.Additionally,the pre-and post-treatment levels of serum SOD,MDA,and GSH-Px were measured for comparative analysis.2.Animal experiment:SD rat models with AICH were established via autologous blood injection,and the AICH SD rat models were randomized into a model group,a sham group,and three treatment groups(XI-H,XI-M,XI-L groups)separately administrated with high-,medium-and low-dose Xingnaojing injection for 5 days using a random number tableIn each AICH SD rat,the Longa Score Scale(LSS)was employed to assess the severity of neurological disorders;the H&E staining method was used to give an overview of the damage to the perihematomal brain tissue;transmission electron microscopy(TEM)was performed to investigate the ultramicroscopic changes in the perihematomal brain tissue;Fe2+in the perihematomal brain tissue was determined;a western blot(WB)experiment was conducted to measure TFR,IRP-2.and Fpn-1 protein expression in the perihematomal brain tissue;real-time PCR was implemented to detect TFR,IRP-2,and Fpn-1 mRNA expression in the perihematomal brain tissue;It was carried out to measure the levels of SOD,MDA,GSH,and GPX4 in the perihematomal brain tissue;the WB method was used for determining the cystine/glutamate antiporter system(system Xc-)protein expression in the perihematomal brain tissue;real-time PCR was performed to confirm system Xc-,and GPX4 mRNA expression in the perihematomal brain tissue;WB analysis was conducted to evaluate the level of Nrf2 protein expression in the focal brain tissue;real-time PCR was used to test Nrf2,HO-1,and NQO1 mRNA expression in the focal brain tissue3.To ensure data accuracy,relevant data were separately entered by two individuals into an MS Access database.The software SPSS25.0 was used for data analysis.[Results]1.Clinical study:This study included 30 AICH patients,including 19 males and 11 females at the mean age of(58.42 ± 8.93)years and(59.91 ± 11.18)years,respectively,showing no significant difference between the two groups(P>0.05).The post-treatment BI value was substantially elevated compared with the pre-treatment level,and the post-treatment NIHSS was markedly reduced,both suggesting a difference of statistical significance(P<0.05,respectively).Following the treatment course,the size of hematoma in the brain was significantly reduced when compared with that before treatment(P<0.05).After treatment,the serum levels of SOD and GPX were dramatically increased,with the pre-and post-treatment differences demonstrating statistical significance(P<0.05),and serum MDA was significantly reduced in comparison with the pre-treatment level(P<0.05).Compared with the pre-treatment level,the TCM syndrome score was markedly reduced after treatment,and the difference was statistically significant(P<0.05)2.Animal experiment:The treatment groups had reduced Longa score,Bederson score,limb symmetry index,and beam test score compared with the model group,and the differences were statistically significant(P<0.05,P<0.01);H&E staining of the brain tissue showed substantial alleviation of pathological changes in the treatment groups in comparison with the model group.In the treatment groups,the focal brain tissue had a reduced Fe2+level,indicating a significant difference between the treatment groups and the model group(P<0.01);after treatment,the TFR and IRP-2 mRNA expression levels were significantly reduced,and the Fpn-1 mRNA expression level was significantly increased in the focal brain tissue(P<0.01,respectively);the TFR and IRP-2 protein expression levels were significantly decreased,and the Fpn-1 protein expression level was significantly elevated in the focal brain tissue(P<0.01,respectively).[Conclusions]1.Xingnaojing injection can markedly improve oxidative stress in ICH patients with the fire-toxin syndrome and support neurological recovery.2.With its blood circulation-promoting and detoxifying properties,Xingnaojing injection can ameliorate neurological impairment in ICH rats and modulate ICH-induced iron(Fe2+)metabolism disorder in the brain tissue by regulating TFR,RP-2,and Fpn-1 gene and protein expression during iron metabolism in the brain tissue.3.As a blood circulation-promoting and detoxifying therapy,Xingnaojing injection has inhibitory effects on lipid peroxidation and ferroptosis in the brain tissue of ICH rats probably because of its regulatory role in the process of system Xc-and GPX4 expression.4.Xingnaojing injection produces the anti-lipid peroxidation effect to inhibit ferroptosis by upregulating the expression of Nrf2,as well as Nrf2-activated antioxidant molecules such as HO-1 and NQO1.