摘要
目的分析肠道菌群来源细胞外囊泡调控心房颤动大鼠心房结构重塑的作用机制。方法选取48只健康雄性成年SD大鼠,随机分为对照组、模型组、实验组与益生菌组,每组12只。除对照组外,其余各组大鼠均于尾静脉注射肾上腺皮质激素(Ach)-CaCl2混合液以建立心房颤动模型。采用超声心电图监测各组大鼠心功能指标的变化,采用HE染色和Masson染色观察各组大鼠心肌组织的病理变化,采用免疫组织化学法检测各组大鼠心肌组织中Ⅰ型胶原蛋白(COL-Ⅰ)、COL-Ⅲ水平的变化,采用酶联免疫吸附法检测各组大鼠心肌组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平的变化,采用蛋白免疫印迹法检测各组大鼠心肌细胞Bcl-2相关X蛋白(Bax)/B淋巴细胞瘤-2基因(Bcl-2)、NOD样受体热蛋白结构域3(NLRP3)、半胱氨酸蛋白酶-1(Caspase-1)、消皮素D(GSDMD)的变化。结果在对照组、模型组、实验组和益生菌组中,大鼠的左心室射血分数(LVEF)、左心室缩短分数(LVFS)、左室舒张末内径(LVEDD)、左室收缩末内径(LVESD)、胶原面积百分数、COL-Ⅰ阳性表达率、COL-Ⅲ阳性表达率、TNF-α、IL-1β、IL-6、SOD、GSH-Px、MDA、Bax/Bcl-2、NLRP3、Caspase-1、GSDMD蛋白水平比较,差异有统计学意义(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均<0.05)。与对照组比较,模型组大鼠的LVEF、LVFS水平降低,LVEDD和LVESD水平升高(t=15.666、14.242、16.872、13.015,P<0.05)。与模型组比较,实验组大鼠的LVEF、LVFS水平升高,LVEDD和LVESD水平降低(t=7.631、7.633、9.995、6.585,P均<0.05)。实验组和益生菌组大鼠的LVEF、LVFS、LVEDD和LVESD水平比较,差异无统计学意义(t=0.480,P=0.636;t=0.385,P=0.704;t=1.791,P=0.087;t=1.376,P=0.183)。HE染色结果显示,对照组大鼠的心肌细胞结构完整,分布均匀,未见明显异常;模型组大鼠心肌细胞肥大,排列不规则,心肌纤维断裂,大量炎性细胞浸润;实验组、益生菌组心肌细胞损伤均明显缓解。模型组大鼠的心肌组织胶原面积百分数高于对照组(t=26.212,P<0.05),实验组大鼠的胶原面积百分数低于模型组(t=20.509,P<0.05)。实验组和益生菌组大鼠的心肌组织胶原面积百分数比较,差异无统计学意义(t=0.794,P=0.436)。模型组大鼠的COL-Ⅰ、COL-Ⅲ阳性表达率高于对照组(t=21.882、20.899,P<0.05),实验组大鼠的COL-Ⅰ、COL-Ⅲ阳性表达率低于模型组(t=17.139、16.370,P均<0.05)。实验组和模型组大鼠的COL-Ⅰ阳性表达率和COL-Ⅲ阳性表达率比较,差异无统计学意义(t=0.471,P=0.642;t=0.335,P=0.741)。模型组大鼠的TNF-α、IL-1β、IL-6水平高于对照组(t=24.363、28.145、21.733,P<0.05),实验组大鼠的TNF-α、IL-1β、IL-6水平低于模型组(t=7.582、17.976、17.374,P<0.05)。实验组和益生菌大鼠的TNF-α、IL-1β、IL-6水平比较,差异无统计学意义(t=0.056,P=0.956;t=0.361,P=0.721;t=0.317,P=0.754)。模型组大鼠的SOD、GSH-Px水平低于对照组,MDA水平高于对照组(t=12.561、12.425、9.267,P<0.05)。实验组大鼠的SOD、GSH-Px水平高于模型组,MDA水平低于模型组(t=5.533、7.696、5.509,P<0.05)。实验组和益生菌组大鼠的SOD、GSHPx和MDA水平比较,差异无统计学意义(t=0.261,P=0.797;t=0.906,P=0.375;t=1.138,P=0.268)。模型组大鼠的Bax/Bcl-2、NLRP3、Caspase-1、GSDMD蛋白水平高于对照组(t=7.926、21.309、22.562、24.100,P<0.05),实验组大鼠的Bax/Bcl-2、NLRP3、Caspase-1、GSDMD蛋白水平低于模型组(t=6.010、17.488、19.620、18.641,P<0.05)。实验组和益生菌组大鼠的Bax/Bcl-2、NLRP3、Caspase-1、GSDMD蛋白水平比较,差异无统计学意义(t=0.653,P=0.520;t=1.623,P=0.119;t=1.386,P=0.180;t=1.386,P=0.180)。结论肠道菌群来源细胞外囊泡可有效改善心房颤动大鼠心肌组织病理损伤,提高心肌功能,抑制心肌纤维化与氧化应激反应,降低炎症反应,改善心肌焦亡,其机制可能与NLRP3/Caspase-1/GSDMD信号通路有关。
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-Ⅰand COL-Ⅰin 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-Ⅰand COL-Ⅰin 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-Ⅰand COL-Ⅰbetween 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.
作者
肖鹏
谢锋
胡鑫渝
陈施羽
廖小波
雷雨
李雨浓
宁琳
XIAO Peng;XIE Feng;HU Xinyu;CHEN Shiyu;LIAO Xiaobo;LEI Yu;LI Yunong;NING Lin(Department of Cardiovascular Medicine,Chongqing University Fuling Hospital,Chongqing 408000,China)
出处
《中国循证心血管医学杂志》
2025年第11期1358-1363,1368,共7页
Chinese Journal of Evidence-Based Cardiovascular Medicine
基金
重庆市涪陵区科研项目(FLKJ,2024AAN3073)。
关键词
心房颤动
心房结构重塑
细胞外囊泡
肠道菌群
心肌纤维化
炎症因子
atrial fibrillation
atrial structural remodeling
extracellular vesicle
intestinal flora
myocardial fibrosis
inflammatory factors
