Mechanism of extracellular vesicles derived from intestinal flora regulating atrial structural remodeling in rats with atrial fibrillation

XIAO Peng; XIE Feng; HU Xinyu; CHEN Shiyu; LIAO Xiaobo; LEI Yu; LI Yunong; NING Lin

doi:10.3969/j.issn.1674-4055.2025.11.015

Abstract：Objective To analyze the action mechanism of extracellular vesicles(EVs)derived from intestinal flora regulating atrial structural remodeling in rats with atrial fibrillation.Methods A total of 48 rats were randomly divided into control group,model group,EVs group and probiotic group(Bifidobacterium lactis triple-activated bacterial tablets),12 rats in each group.Except for control group,tail vein injection of adrenocortical hormone(Ach)-CaCl2 mixed solution was performed in the other groups to replicate atrial fibrillation models.The pathological changes of myocardial tissues were observed by HE staining and Masson staining.The cardiac function indexes,collagenⅠ(COL-Ⅰ)and collagenⅠ(COL-Ⅰ)in myocardial tissues,tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-px),and malondialdehyde(MDA)were compared,and Western blot was used to detect the changes of BCL-2 related X protein(Bax)/B lymphoma-2 gene(Bcl-2),NOD-like receptor thermal protein domain associated protein 3(NLRP3),cysteine protease-1(Caspase-1),and gasdermin D(GSDMD)in rat cardiomyocytes in each group.Results There were statistically significant differences in LVEF,LVFS,LVEDD,LVESD,collagen area percentage,COL-I positive expression rate,COL-Ⅲ positive expression rate,TNF-α,IL-1β,IL-6,SOD,GSH-Px,MDA,Bax/Bcl-2,NLRP3,Caspase-1,GSDMD protein levels among the control group,model group,experimental group and probiotic group(F=76.234,66.584,77.864,63.472,83.124,77.894,80.443,116.102,107.896,96.482,82.035,75.258,79.453,62.488,55.324,49.154,50.643,P<0.05).Compared with the control group,the LVEF and LVFS levels in the model group were decreased,and the LVEDD and LVESD levels were increased(t=15.666,14.242,16.872,13.015,P<0.05).Compared with the model group,the levels of LVEF and LVFS in the experimental group were increased,and the levels of LVEDD and LVESD were decreased(t=7.631,7.633,9.995,6.585,P<0.05).There was no significant difference in LVEF,LVFS,LVEDD and LVESD levels between the experimental group and the probiotic group(t=0.480,P=0.636;t=0.385,P=0.704;t=1.791,P=0.087;t=1.376,P=0.183).The HE staining results showed that the myocardial cells of rats in the control group were complete and evenly distributed,with no obvious abnormalities;the myocardial cells of rats in the model group were hypertrophy and irregularly arranged,myocardial fibers were broken,and a large number of inflammatory cells were infiltrated;the myocardial cells in the experimental group and the probiotic group were significantly alleviated.The collagen area percentage of myocardial tissue in the model group was higher than that in the control group(t=26.212,P<0.05),and the collagen area percentage of the experimental group was lower than that in the model group(t=20.509,P<0.05).There was no statistical difference in the percentage of collagen area in myocardial tissue between the experimental group and the probiotic group(t=0.794,P=0.436).The positive expression rates of COL-Ⅰ and COL-Ⅰ in the model group were higher than those in the cotrol group(t=21.882,20.899,P<0.05),and the positive expression rates of COL-Ⅰ and COL-Ⅰ in the experimental group were lower than those in the model group(t=17.139,16.370,P<0.05).There was no significant difference between the positive expression rates of COL-Ⅰ and COL-Ⅰ between the experimental group and the model group(t=0.471,P=0.642;t=0.335,P=0.741).The levels of TNF-α,IL-1β,and IL-6 in the model group were higher than those in the control group(t=24.363,28.145,21.733,P<0.05),and the levels of TNF-α,IL-1β,and IL-6 in the experimental group were lower than those in the model group(t=7.582,17.976,17.374,P<0.05).There was no significant difference in the levels of TNF-α,IL-1β,and IL-6 between the experimental group and the probiotic group(t=0.056,P=0.956;t=0.361,P=0.721;t=0.317,P=0.754).The SOD and GSH-Px levels in the model group were lower than those in the control group,and the MDA levels were higher than those in the control group(t=12.561,12.425,9.267,P<0.05).The levels of SOD and GSH-Px in the experimental group were higher than those in the model group,and the levels of MDA were lower than those in the model group(t=5.533,7.696,5.509,P<0.05).There was no significant difference in the levels of SOD,GSH-Px and MDA between the experimental group and the probiotic group(t=0.261,P=0.797;t=0.906,P=0.375;t=1.138,P=0.268).The protein levels of Bax/Bcl-2,NLRP3,Caspase-1 and GSDMD in the model group were higher than those in the control group(t=7.926,21.309,22.562,24.100,P<0.05),and the protein levels of Bax/Bcl-2,NLRP3,Caspase-1 and GSDMD in the experimental group were lower than those in the model group(t=6.010,17.488,19.620,18.641,P<0.05).There was no significant difference in the protein levels of Bax/Bcl-2,NLRP3,Caspase-1,and GSDMD between the experimental group and the probiotic group(t=0.653,P=0.520;t=1.623,P=0.119;t=1.386,P=0.180;t=1.386,P=0.180).Conclusion Intestinal flora EVs can effectively improve pathological injury of myocardial tissues and myocardial function,inhibit myocardial fibrosis and oxidative stress response,reduce inflammatory response and attenuate myocardial pyroptosis in rats with atrial fibrillation.The mechanism may be related to the regulation of NLRP3/Caspase-1/GSDMD signaling pathways.