| Objective:In this paper,the clinical efficacy and safety of Zhongfeng Xingnao Liquid in the treatment of acute cerebral infarction(ACI)were evaluated,and the effects of Zhongfeng Xingnao Liquid on intestinal-brain Treg/Th17 immune homeostasis,intestinal flora diversity,and short-chain fatty acid metabolism in animal models of middle cerebral artery occlusion(MCAO)were investigated based on the microbial-intestinal-brain axis system.This study aims to further explore the potential pharmacological mechanisms of Zhongfeng Xingnao Liquid and provide clinical and experimental data to improve the level of evidence for the prevention and treatment of ischaemic stroke in Traditional Chinese Medicine.Methods:Part Ⅰ:Clinical efficacy of Zhongfeng Xingnao Liquid in the treatment of acute cerebral infarction.In the clinical part of the study,a central zone group randomization method was used to divide the eligible subjects included in the four clinical centers into a trial group and a control group on a 1:1 basis according to a random number table generated by SAS software.Patients in the control group were given basic treatment and patients in the trial group were given basic treatment combined with Zhongfeng Xingnao Liquid.The incidence of death or severe functional impairment(mRS>4),NIHSS score,Barthel Index,time to first lavage,and constipation major symptom score were observed in both groups after 30 days of treatment,and survivors were followed up for 90 days to evaluate the efficacy and safety of Zhongfeng Xingnao Liquid for ACI.Part Ⅱ:Effect of Zhongfeng Xingnao Liquid on the immune homeostasis of intestinebrain Treg/Th17 in mice with ACI.In this part,96 male SPF-grade C57BL/6J mice were selected from 6-8 weeks of age to prepare a mouse middle cerebral artery infarction model(MCAO).A laser scatter imaging system was used to detect blood flow in the brain during the modeling process,and the models were screened using the Longa score and stained with TTC to verify the ischemia.MCAO mice,sham-operated mice,and mice without any manipulation were randomly divided into ZFXN,Mod,Sham,and NC groups,and each group was stratified according to 2-time points at 3 and 7 days post-operatively(4 groups,2 observation points,12 mice per group).The recovery of neurological function was assessed using the Longa score for each group of mice at different time points.Brain and intestinal histopathological changes and inflammatory infiltration were observed by hematoxylin-eosin(HE)staining.The mRNA expression of IL-10,IL-17A,FOXP3,and RORyt in brain and intestinal tissues was measured by qRTPCR.The expression of IL-10,IL-17A,and leukocyte aggregation factors CXCL1 and CXCL2 in brain and intestinal tissues were measured by ELISA and Western-Blot.Part III:Effect of Zhongfeng Xingnao Liquid on gut microbiota and Microbial metabolites(SCFAs)in mice with ACI.The animal modeling,model evaluation,and grouping methods in this part are the same as in Part 2.The diversity and abundance of intestinal microbiota in each group were analyzed by 16s rRNA sequencing on day 7 after modeling,and genes that were significantly differentially regulated in the ZFXN group were screened for functional prediction.The metabolism of short-chain fatty acids(including acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid,and isovaleric acid)in the intestine of each group of mice was measured by liquid chromatography tandem mass spectrometry(LC-MS)on day 3 and day 7 after molding.Results:Part Ⅰ(1)From 1 July 2021 to 30 November 2022,a total of 134 subjects were recruited across the 4 study centers,127 of whom eventually completed the program.(2)At 30 days:there were no statistically significant differences in overall mortality and severe disability rates between the two groups(P>0.05).The improvement in NIHSS scores was more pronounced in the trial group compared to the control group scores,but the difference was not statistically significant(P>0.05).The total BI index score was not statistically significant in the trial group compared to the control group(P>0.05),but in the BI index interval distribution,more patients in the trial group had a BI≥75 than in the control group(P<0.05).The time to first stool after admission was significantly shorter in the trial group than in the control group(P<0.05),and all patients in the trial group had lower scores of major constipation symptoms during treatment than in the control group(P<0.05).(3)90-day follow-up:The overall mortality rate in the trial group was lower than that in the control group,but the difference was not statistically significant(P>0.05).Patients in the trial group had better improvement in total NIHSS scores than the control group(P<0.05).The number of patients with mRS≤2 was higher in the trial group than in the control group(P<0.05).There was no significant difference in the number of patients with BI≥75 in the trial group compared to the control group(P>0.05),but there were more patients in the BI≥95 intervals in the trial group than in the control group(P<0.05).(4)Safety evaluation:There was no significant difference in the incidence of adverse events during treatment between the two groups(P>0.05),and there were no significant abnormalities in the safety indicators between the two groups during drug administration(P>0.05).Part Ⅱ(1)Neurological function scores:There was no significant difference between the Longa scores of the ZFXN and Mod groups at 3 days after modeling(P>0.05).The Longa score was lower in the ZFXN group than in the Mod group at 7 days after modeling(P<0.05).(2)Pathological sections of brain and small intestine:At 7 days,the Mod group showed severe histopathological changes with significant inflammatory infiltration and tissue necrosis in the brain and small intestine compared with the Sham group;the ZFXN group showed significantly less inflammatory damage and immune infiltration in both the brain and small intestine compared with the Mod group.(3)Expression of mRNA for Treg and Th17 transcriptional activator(Foxp3,RORyt):In brain tissue,there was no significant difference in the expression of FOXP3 mRNA between Mod,Sham,and ZFXN groups at 3 and 7 days(P>0.05).(At 7 days,the expression of RORyt mRNA was significantly higher in the Mod group compared to the Sham group(P<0.05),while the expression of RORyt mRNA decreased to near normal level in the ZFXN group compared to the Mod group(P<0.05).In small intestine tissues,Foxp3 mRNA expression tended to decrease in the Mod and Sham groups at 3 days(P>0.05),while Foxp3 mRNA expression was significantly upregulated in the ZFXN group compared to the Mod group(P<0.05);Foxp3 mRNA expression was slightly increased in the Mod and Sham groups at 7 days(P>0.05).The expression of Foxp3 mRNA was upregulated in the ZFXN group compared to the Mod group(P>0.05).RORyt mRNA expression was significantly increased in the Mod group compared to the Sham group at both 3 and 7 days(P<0.05),while RORyt mRNA expression was significantly decreased in the ZFXN group compared to the Mod group(P<0.05).(4)Expression of pro-inflammatory/inflammation-related factors:In brain tissue,at 3 days,IL-10 protein levels were significantly higher in the Mod group than in the Sham group(P<0.05)and slightly lower in the ZFXN group compared to the Mod group(P>0.05).At 7 days,IL-10 protein levels were higher in the Mod group than in the Sham group(P>0.05)and slightly lower in the ZFXN group compared to the Mod group(P>0.05).PAt 3 days,IL-17A protein levels were higher in the Mod group compared to the Sham group(P<0.05)and slightly lower in the ZFXN group compared to the Mod group(P>0.05).CXCL1 and CXCL2 expression followed the same trend as IL-17A and was significantly higher in the Mod group compared to the Sham group at 3 and 7 days(P<0.05).CXCL1 and CXCL2 protein levels were down-regulated in the ZFXN group compared to the Mod group,with significant differences in CXCL2 down-regulation in the ZFXN group at 3 days(P<0.05)and CXCL1 down-regulation in the ZFXN group at 7 days(P<0.05).In the small intestinal tissue,IL-10 protein expression was significantly reduced in the Mod group compared to the Sham group at 3 days(P<0.05)and increased in the ZFXN group compared to the Mod group(P>0.05).At 7 days,there was a tendency for IL-10 protein expression to decrease in the Mod group compared to the Sham group(P>0.05),and IL-10 protein expression was significantly upregulated in the ZFXN group compared to the Mod group(P<0.05).At 3 days,there was no significant difference in IL-17A protein expression between the Mod,Sham,and ZFXN groups(P>0.05).At 7 days,IL-17A protein expression was significantly increased in the Mod group compared to the Sham group(P<0.05),while IL-17A protein expression was significantly decreased in the ZFXN group compared to the Mod group(P<0.05).Part Ⅲ(1)16s rRNA gene sequencing:In terms of species distribution,there were some differences in the intestinal flora of all groups of mice,but in general the abundance of the main dominant flora in the gut was altered in the Mod group compared to the Sham group.At the phylum level,the abundance of Firmicutes was significantly higher in the Mod group.At the Class level,Bacilli abundance was significantly higher.At the Order level,Lactobacillales abundance was elevated.At the family level,the abundance of Lactobacillaceae is elevated.At the genus level,Ligilactobacillus abundance was significantly higher,while Lactobacillus abundance decreased.In contrast,the ZXF group moderated the abnormal flora of the Mod group,making it essentially the same as the Sham group.Alpha diversity:the Mod group showed a decrease in diversity compared to the Sham group,while the ZFXN group showed an increase in diversity compared to the Mod group.Beta diversity:the ZFXN group differed less from the Sham group in terms of intestinal flora compared to the Mod group.Functional prediction of genes for intestinal flora differences between ZFXN and Mod mice by KEGG.In Level2,upregulated differential genes in the ZFXN group were mainly associated with senescence,cofactor,and vitamin metabolism,terpenoid and polyketide metabolism and energy metabolism,while downregulated differential genes were mainly associated with biosynthesis of other secondary metabolites and carbohydrate metabolism.In Level 3,the upregulated differential genes in the ZFXN group were mainly associated with the pentose phosphate pathway and the metabolism of nicotinic acid and nicotinamide,while the downregulated differential genes were mainly associated with the biosynthesis of arginine and the metabolism of cysteine and methionine.(2)LC-MS detection:the Mod group showed a decreasing trend for several short-chain fatty acids compared to the Sham group,with the 3-day group showing a more pronounced decrease than the 7-day group;the ZFXN group showed an upward trend for acetic acid,butyric acid and propionic acid,and a downward trend for isobutyric acid,valeric acid and isovaleric acid compared to the Mod group(P>0.05).Conclusion:(1)Zhongfeng Xingnao Liquid can effectively restore the neurological function and consciousness of ACI patients,improve post-stroke intestinal dysfunction,reduce the degree of long-term disability,and improve the quality of life of patients,and it has few side effects and is safe for long-term use.(2)Zhongfeng Xingnao Liquid can promote neurological recovery in MCAO mice,reduce inflammatory damage in intestinal and brain tissues,and restore Treg/Th17 immune balance in the intestine.(3)Zhongfeng Xingnao Liquid can,to a certain extent,correct the disorder of intestinal microbiota in MCAO mice,maintain the abundance of intestinal microorganisms,restore intestinal microbial diversity,and regulate short-chain fatty acid metabolism.(4)Zhongfeng Xingnao Liquid can effectively improve the prognosis of ACI and reduce the inflammatory damage of ischemic brain tissue.The mechanism of action may be related to the regulation of intestinal microbiota and short-chain fatty acid metabolism and improvement of intestinal Treg/Th17 immune balance. |
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