Protective Effect And Mechanism Of Taohong Siwu Decoction On Inflammatory Injury In Ischemic Stroke Mice Based On Microbiota-Gut-Brain Axis

OBJECTIVE:Based on the microbia-gut-brain axis theory,the intestinal flora disorder after ischemic stroke was taken as the entry point to explore the role of Taohong Siwu decoction(THSWD)in interfering with the inflammatory response of ischemic stroke by regulating intestinal flora and repairing intestinal barrier.METHODS:Fifty SPF male C57 mice were randomly divided into sham group,MCAO group,THSWD low-dose(6.5g/kg),medium(13g/kg)and high-dose(26g/kg)groups,with 10 mice in each group.A mouse model of permanent middle cerebral artery occlusion was prepared by thread embolism.THSWD low-dose,medium-dose and high-dose groups were given 6.5g/kg,13g/kg,26g/kg THSWD by intragastric administration,while sham operation group and model group were given the same amount of normal saline by intragastric administration.After 7 days of continuous administration,neurological function loss was detected.Hematoxylin-eosin staining staining(HE)was used to detect pathological injury of brain tissue.The expression of Claudin-5 and ZO-1 protein in brain tissue and colon were detected by immunohistochemistry.The concentrations of LPS,DAO and D-lactic acid in serum of mice were determined by enzyme-linked immunosorbent assay(ELISA).The levels of IL-1β and TNF-α in brain tissue were determined by ELISA.16 Sr DNA amplicon sequencing was used to detect intestinal flora.The expression of i NOS and Arg-1 in brain tissue was detected by Immunofluorescence.Western Blotting(WB)and q RT-PCR were used to detect the expression of TLR-4 and NF-k B proteins and genes in brain tissue.RESULT:1.Protective effect of THSWD on brain injury in mice with ischemic strokeCompared with the sham group,the neural function score of the MCAO group was significantly increased,the structure of neurons in the ischemic area of the hippocampus was severely damaged,a large number of neurons were lost,the cytoplasm was heavily staining with eosinophilic acid,and the cytoplasm was pyretosis.The expression of claudin-5 and ZO-1 in brain tissue were decreased.Compared with MCAO group,neurological function scores were significantly decreased and brain histomorphology was improved in different degrees after treatment with THSWD at different doses,and the expression of claudin-5 and ZO-1 were increased.2.Effects of THSWD on intestinal flora disturbance and intestinal barrier destruction in mice with ischemic strokeCompared with the sham group,the Alpha diversity and Beta diversity in the MCAO group were significantly decreased.The abundance of Actinobacteridae and Firmicutes,uncultured,Parabacteroides,Acinetobacter,Ralstonia,Allobaculum,Dubosiella,Corynebacterium and Aquabacterium was decreased;The abundance of Proteobacteria,Achromobacter,Comamonas,Brevibacillus and Paenibacillus was increased.The expression levels of claudin-5 and ZO-1 in colon tissue were lower than those in the sham group.The contents of LPS,DAO and D-lactate in serum were significantly increased(P<0.01);It is suggested that ischemic stroke destroys intestinal barrier,leading to intestinal microbial imbalance and translocation.THSWD increased Alpha diversity and Beta diversity in ischemic stroke mice,and had a certain reversal effect on different flora.In addition,claudin-5 and ZO-1 expressions were increased in THSWD medium dose group and high dose group,and serum LPS,DAO and D-lactic acid levels were decreased significantly(P<0.01,P<0.05).It is suggested that THSWD can regulate intestinal flora disturbance and intestinal barrier destruction in mice with ischemic stroke.3.Anti-inflammatory effect and mechanism of THSWD on ischemic stroke miceCompared with sham group,the contents of IL-1β and TNF-α in ischemic lateral brain tissue of MCAO group were significantly increased(P<0.01).The number of M1-type microglia marker i NOS positive cells increased,while the number of M2-type microglia marker Arg-1 positive cells decreased.The expression levels of TLR-4,NF-κB protein and gene were significantly increased(P< 0.01).Compared with MCAO group,the contents of IL-1β and TNF-α in THSWD medium dose and high dose groups were significantly decreased(P<0.01,P<0.05);The number of i NOS positive cells was decreased,the number of Arg-1 positive cells was increased,and The levels of TLR-4protein in THSWD groups was significantly decreased(P< 0.01,P< 0.05);The levels of TLR-4 gene in THSWD medium dose group and high dose group were significantly decreased(P< 0.01).The levels of NF-κB protein and gene in medium dose group and high dose group of Taohong Siwu decoction were significantly decreased(P< 0.01).CONCLUSIONS:Taken together,our findings suggest that THSWD may influence ischemic stroke prognosis by regulating post ischemic stroke intestinal flora disturbance and intestinal barrier disruption to inhibit inflammatory responses.The results of this study will provide a new idea for clinical treatment of ischemic stroke and prevention of intestinal dysfunction after ischemic stroke.