Study On The Effect And Mechanism Of Blood-heat Recipe On The Inflammatory Response Of Ischemic Stroke Based On Gut Microbiota

Ischemic stroke,a disease characterized by vascular occlusion caused by embolus or thrombosis,has become the second leading cause of death worldwide.Although the time window for recanalization treatment of ischemic stroke continues to expand,reperfusion injury and hemorrhagic transformation are still unavoidable.Neuroinflammatory response plays an important role in neuronal damage after cerebral ischemia,which can induce the release of large amounts of pro-inflammatory factors and worsen the outcome of stroke.Studies have confirmed that intestinal microorganisms are a key regulator of the neuroinflammatory response after cerebral ischemia.Acute brain injury causes intestinal microbial imbalance and increased intestinal mucosal permeability,changes intestinal microorganisms and their products,and leads to endotoxins and bacteria.The translocation is conducted to the ischemic side brain tissue through signaling pathways,aggravating the inflammatory response and outcome after brain injury.Therefore,the intestinal microbiota is expected to become a potential therapeutic target for ischemic stroke.Previous studies on Blood-heat Recipe have confirmed its effectiveness and related mechanisms in inflammatory response after stroke,but the upstream targets that regulate this mechanism are still unclear.This topic intends to use the perspective of intestinal flora and its products as an entry point to further explore the upstream targets of Blood-heat Recipe in treating post-stroke inflammatory response,and to elucidate the possible signaling pathways.This topic is mainly divided into two parts:clinical research and experimental research on how Blood-heat Recipe improves neuroinflammatory response by regulating gut microbiota.Clinical ResearchObjective:Study the neurological deficit score(NIHSS),traditional Chinese medicine syndrome scale score,intestinal flora and its products and serum related factors(LPS,Zonulin,TLR4,NF-κB,IL-1β,IL-6,TNF-α)concentration levels.Methods:Select 60 patients with acute ischemic stroke syndrome of blood stasis,heat and orifice obstruction who were admitted to the Department of Neurology,Jiangsu Provincial Hospital of Integrated Traditional Chinese and Western Medicine and Shangnan County Hospital,Shangluo City,Shanxi Province from August 2022 to August 2023 and met the inclusion criteria,and were randomly divided into The control group and the treatment group included 30 cases each.Both groups were given conventional treatment(anticoagulation,antiplatelet,lipid-lowering,neuroprotection,blood sugar and blood pressure control,etc.).The treatment group was additionally given Blood-heat Recipe water decoction,which was taken orally for 7 days to compare the syndrome scores and efficacy of Blood-heat Recipe before and after treatment.NIHSS score,intestinal microbiota and its products,and serum related factors(LPS,Zonulin,TLR4,NF-κB,IL-1β,IL-6,TNF-α)concentration changes.Results:1.There is no significant difference between the two groups of patients in terms of gender,age,smoking history,history of hypertension,diabetes and stroke before and after treatment,and they are comparable;2.There was no significant difference in the NIHSS scores and TCM syndrome scores between the two groups before treatment.After treatment,both indicators showed a downward trend within the group.In the inter-group comparison,the Blood-heat Recipe group had a more significant decrease in the two indicators than the control group;3.The species richness and diversity of the intestinal flora of patients treated with Jingyure Recipe were significantly improved;at the phylum level,the abundance of Bacteroidetes,Proteobacteria,and unclassified Bacteroidetes was significantly reduced,and the abundance of Firmicutes and Verrucomicrobia was significantly reduced.The abundance of bacterial phyla was increased;the abundance of Bacteroides,unclassified Bacteroidetes and Prevotella was decreased at the genus level,and the abundance of Akkermansia and Fischina was increased;at the species level,muciniphila Akkermansia is enriched in Blood-heat Recipe group and may be an important bacterial species regulated by Blood-heat Recipe;4.There was no significant difference in the levels of LPS,IL-1β,IL-6 and TNF-α between the groups before treatment.After treatment,there were significant differences within and between the groups.The comparison between the groups showed that the four indicators of the Blood-heat Recipe group were lower than those of the control group much more significantly.5.There were no significant differences in Zonulin,TLR4,and NF-κB between the groups before treatment.After treatment,there were statistical differences within and between groups.In the inter-group comparison,the Blood-heat Recipe group had a more significant decrease in these three indicators than the control group.Experimental StudyObjective:Through the detection of intestinal factors and related signaling pathways,the possible mechanism of Blood-heat Recipe in treating neuroinflammatory reaction in ischemic stroke was explored.Methods:1.Experiment on constructing ischemic stroke mouse model:Adult male C57BL/6 mice were selected as the experimental subjects.After MCAO modeling,they were divided into sham operation group,model group,low-dose Blood-heat Recipe group,high-dose Blood-heat Recipe group and control group.Different intervention plans were given to observe the changes in neurological function scores and cerebral infarct volume of mice in each group.16S rRNA technology was used to detect gut microbiota,and Elisa was used to detect intestinal permeability and mechanical barriers,endotoxins,and brain tissue inflammatory factors.Western blotting and RT-qPCR detect intestinal TLR4/NF-κB signaling pathway;2.Experiment on construction of pseudo-germ-free(SGF)mouse mode:Quadruple broad-spectrum antibiotics(vancomycin 100 mg/kg,neomycin sulfate 200mg/kg,metronidazole 200mg/kg,and ampicillin 200mg/kg)were used to construct a pseudo-sterile(SGF)mouse model.After MCAO modeling they were divided into sham operation group,model group,high-dose Blood-heat Recipe group and control group.They were given different intervention plans respectively.The changes in neurological function scores and cerebral infarct volume of mice in each group were observed.Intestinal microbiota was detected by 16S rRNA technology.Elisa Detect intestinal permeability and mechanical barrier,endotoxin,and brain tissue inflammatory factors.Western blotting and RT-qPCR detect intestinal TLR4/NF-κB signaling pathway.3.Experiment on construction of Fecal Microbiota Transplantation(FMT)Mouse mode:After successfully constructing pseudo-sterile mice,mice in the high-dose Blood-heat Recipe group in Chapter 1 Animal Experiment were intragastrically transplanted with 200uL/mouse of fecal bacterial suspension.After MCAO modeling,they were divided into sham operation group,model group,and high-dose group.The Blood-heat Recipe group and the control group were given different intervention plans respectively,and the changes in neurological function scores and cerebral infarction volume of mice in each group were observed.Intestinal microbiota was detected by 16S rRNA technology,and intestinal permeability,mechanical barrier,and intestinal microbiota were detected by Elisa.Toxins,brain tissue inflammatory factors,Western blotting and RT-qPCR to detect intestinal TLR4/NF-κB signaling pathway;4.Experiment on construct a high-concentration LPS intervention mouse mode:A mouse model of ischemic stroke stimulated by high-concentration LPS was constructed by intraperitoneal injection of 5ug/mouse of LPS.After MCAO modeling,the mice were divided into a sham operation group,a model group,a high-concentration LPS group and a normal saline group,and each received different treatment interventions.Changes in neurological function scores and cerebral infarct volume of mice in each group were observed,16S rRNA technology was used to detect intestinal microbiota,Elisa was used to detect intestinal permeability and mechanical barrier,endotoxemia,and brain tissue inflammatory factors,Western blotting and RT-qPCR detection of intestinal TLR4/NF-κB signaling pathway.Results:1.(1)High-dose Blood-heat Recipe can significantly reduce the neurological function scores of mice and reduce cerebral infarction volume;(2)High-dose Blood-heat Recipe can significantly reduce the abundance of Bacteroidetes and up-regulate Firmicutes in the intestinal tract of mice at the phylum level.The abundance of phyla at the genus level,the proportion of Bacteroidetes was significantly reduced,the proportions of Akkermansia and Blautia in the intestinal microbiota of mice in the H-YRF group were significantly increased,and the intestinal flora of mice in the HYRF group was significantly increased.The diversity of intestinal microbiota;(3)Compared with the L-YRF group and the Nim group,the H-YRF group showed lower FD4,LPS and Zonulin concentrations,as well as lower brain tissue IL-1β,TNF-α and IL-6 levels,and the expression of TLR4 and NF-κB mRNA and protein in small intestinal tissue decreased;2.(1)There is no significant difference in the neurological function scores between each group of pseudo-germ-free mice.The effect of high-dose Blood-heat Recipe on reducing cerebral infarction volume in the pseudo-germ-free mouse model is weakened;(2)SGF-Compared with the SGF-M group,the abundance of Proteobacteria in group H was down-regulated,and the abundance of Firmicutes was up-regulated,and the intestinal flora distribution and proportion were similar to those in group SGF-S;at the genus level,SGF-H group performed better than SGF-M group.Lower abundance of Bacteroidetes and higher abundance of Blautia and Lachnospira NK4A136 were found;the diversity index of intestinal flora of high-dose Blood-heat prescription in pseudo-germ-free mice had no difference Significant difference;(3)High-dose Blood-heat Recipe can regulate blood FD4,LPS and Zonulin levels,brain tissue IL-1β,IL-6 and TNF-αconcentrations,and small intestinal tissue TLR4,NF-κB mRNA and protein expression are weakened.3.(1)There is no significant difference in neurological function scores and cerebral infarct volume between the various groups of mice after FMT.The cerebral infarct volume of the FMTH group is close to that of the FMT-S group;(2)Firmicutes in mice in the FMT-H group at the phylum level Phylum Bacteroidetes and Bacteroidetes are absolutely dominant,and other bacteria are basically depleted;at the genus level,the abundance of Bacteroidetes and Lactobacillus in the FMT-H group is down-regulated,and MuribacuLaceae,Mycobacterium and Lachnospira are unclassified NK4A136 The abundance is increased;the intestinal microbiota changed by highdose Blood-heat Recipe has the characteristics of richness and diversity;(3)The intestinal flora changed by high-dose Blood-heat Recipe can effectively reduce blood FD4,LPS and Zonulin levels,as well as brain Tissue IL-1β,IL-6 and TNF-α contents,and down-regulate the mRNA and protein expression of TLR4 and NF-κB in small intestinal tissue.4.(1)High-dose Blood-heat Recipe can significantly reduce the neurological function scores and cerebral infarction volume of mice treated with high-concentration LPS;(2)High-dose Bloodheat Recipe can regulate the intestinal microbiota of mice treated with high-concentration LPS at the phylum level.It is close to the Mcao group,Bacteroidetes is down-regulated at the genus level,and other bacterial genera and proportions are close to the Sham group;high-dose Blood-heat Recipe can increase the intestinal microbiota diversity index of mice treated with highconcentration LPS;(3)High Dosage of Blood-heat Recipe can effectively reduce FD4,LPS and Zonulin in the peripheral blood of mice treated with high concentrations of LPS,down-regulate the levels of IL-6,TNF-α,and IL-1β in brain tissue,and inhibit TLR4 and NF-κB mRNA and protein in small intestinal tissue expression.In conclusions:1.Blood-heat Recipe can significantly reduce NIHSS scores and TCM syndrome scores,inhibit early neuroinflammatory reactions in patients with ischemic stroke,and improve neurological dysfunction.2.Blood-heat Recipe can regulate richness and diversity of the structure of intestinal microbiota in patients with ischemic stroke,reducing the proportion of Gram-negative bacteria and increasing the proportion of Gram-positive bacteria;Akkermansia muciniphila may be the specific bacterial group regulated by Blood-heat Recipe.3.The effect of Blood-heat Recipe of the neuroprotective effect may be related to repairing intestinal barrier damage,reducing endotoxin and inhibiting neuroinflammatory pathways.4.Blood-heat Recipe may improve the prognosis of ischemic stroke by regulating the composition of gut microbiota.