柴胡加龙骨牡蛎汤对心肌梗死合并焦虑大鼠海马区NLRP3/GSDMD炎性信号通路的影响

Effect of Chaihu plus Longgu Muli Decoction on NLRP3/GSDMD inflammatory signal pathway in hippocampus of rats with myocardial infarction and anxiety

目的探讨心肌梗死合并焦虑大鼠海马区NLRP3/GSDMD炎性信号通路变化及柴胡加龙骨牡蛎汤对大鼠海马区炎性损伤的作用效应。方法将60只SD大鼠随机分为假手术组9只、心梗并焦虑组17只、心梗组17只、心梗并焦虑+中药组17只。心梗组采用结扎冠状动脉左前降支方法进行造模;假手术组穿线不结扎;心梗并焦虑组和心梗并焦虑+中药组先采用空瓶刺激方法进行焦虑造模(周期21 d),于造模第15天采用结扎冠状动脉左前降支方法进行心肌梗死造模。心肌梗死造模后第2天,心梗合并焦虑+中药组给予柴胡加龙骨牡蛎汤10 mL/kg灌胃,其余组灌胃等量蒸馏水,均1次/d,连续7 d。灌胃结束后进行心脏超声检查,采用高架十字迷宫实验、旷场实验评价各组大鼠行为学,大脑海马区HE、尼氏染色观察脑神经元病理改变,免疫组化检测大鼠海马区NLRP3、Caspase-1、GSDMD表达情况,Western blot法检测大鼠海马组织中NLRP3、Caspase-1、GSDMD、IL-1β、IL-18蛋白表达水平。结果与心梗并焦虑组比较,心梗并焦虑+中药组左室射血分数(LVEF)、左室短轴缩短率(LVFS)均明显升高(P均<0.05),左心室舒张末期内径(LVIDd)和左心室收缩末期内径(LVIDs)均明显缩短(P均<0.05),大鼠进入开放臂次数所占比和开放臂停留时间所占比、大鼠水平运动得分和垂直运动得分均明显升高(P均<0.05)。心梗并焦虑组和心梗组大鼠海马区尼氏阳性面积均明显低于假手术组(P均<0.05),心梗并焦虑+中药组明显高于心梗并焦虑组和心梗组(P均<0.05)。心梗并焦虑组和心梗组大鼠海马区NLRP3、Caspase-1、GSDMD表达IOD值和海马组织中NLRP3、Caspase-1、GSDMD、IL-1β、IL-18蛋白表达水平均明显高于假手术组(P均<0.05),且心梗并焦虑组GSDMD表达IOD值和NLRP3、Caspase-1、IL-18蛋白表达水平均明显高于心梗组(P均<0.05),而心梗并焦虑+中药组NLRP3、Caspase-1、GSDMD表达IOD值和NLRP3、Caspase-1、GSDMD、IL-1β、IL-18蛋白表达水平均明显低于心梗并焦虑组(P均<0.05)。结论柴胡加龙骨牡蛎汤可能通过调控海马区NLRP3/GSDMD炎性信号通路,抑制心肌梗死合并焦虑大鼠海马区的炎性反应,缓解大鼠焦虑状态。

Objective It is to investigate the changes of NLRP3/GSDMD inflammatory signal pathway in hippocampus of rats with myocardial infarction combined with anxiety and the effect of Chaihu plus Longgu Muli Decoction on inflammatory injury in hippocampus of rats.Methods Sixty SD rats were randomly divided into sham operation group(n=9),myocardial infarction and anxiety group(n=17),myocardial infarction group(n=17),and myocardial infarction and anxiety+Chinese medicine group(n=17).The left anterior descending coronary artery was ligated for modeling in the myocardial infarction group;the threading without ligation was performed in the sham operation group;the anxiety model was established firstly by empty bottle stimulation method(cycle 21 d),and the myocardial infarction model was modeled by ligating the left anterior descending coronary artery on the 15th day of modeling in the myocardial infarction and anxiety group and the myocardial infarction and anxiety+traditional Chinese medicine group.On the second day after myocardial infarction modeling,the myocardial infarction with anxiety+traditional Chinese medicine group was given Chaihu plus Longgu Muli Decoction 10 mL/kg by gavage,and the other groups were given the same amount of distilled water,all once a day for 7 consecutive days.After the gavage,the echocardiography was performed.The behavior of the rats in each group was evaluated by the elevated cross maze experiment and the open field experiment.The brain hippocampus HE and Nissl staining were used to observe the pathological changes of brain neurons.The expression of NLRP3,Caspase-1,GSDMD in the rat hippocampus were detected by immunohistochemical method,the protein expression levels of NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 in rat hippocampus were detected by Western blot method.Results Compared with the myocardial infarction and anxiety group,the left ventricular ejection fraction(LVEF)and the left ventricular short axis shortening rate(LVFS)in the myocardial infarction with anxiety+traditional Chinese medicine group were significantly increased(P<0.05),and the left ventricular end-diastole inner diameter(LVIDd)and the left ventricular end-systolic diameter(LVIDs)were significantly shortened(both P<0.05),the proportion of the number of times the rats entered the open arm and the proportion of the staying time in the open arm,the horizontal movement score and the vertical movement score of the rats were significantly increased(P<0.05).The Niss-positive area in the hippocampus of rats in the myocardial infarction and anxiety group and the myocardial infarction group was significantly lower than that of the sham operation group(P<0.05),and the myocardial infarction and anxiety+Chinese medicine group was significantly higher than the myocardial infarction and anxiety group and the myocardial infarction group(P<0.05).The IOD value of expression of NLRP3,Caspase-1,GSDMD,and the expression levels of Caspase-1,GSDMD and NLRP3,Caspase-1,IL-18 protein in in the hippocampus of rats in the myocardial infarction and anxiety group and the myocardial infarction group were significantly higher than those in the sham operation group(all P<0.05),the IOD value of GSDMD expression and the protein expression levels of NLRP3,Caspase-1 and IL-18 in the myocardial infarction and anxiety group were significantly higher than those in the myocardial infraction group(all P<0.05),the IOD value of expression of NLRP3,Caspase-1,GSDMD,and the expression levels of Caspase-1,GSDMD and NLRP3,Caspase-1,IL-18 protein in the hippocampus of rats in the myocardial infarction and anxiety+Chinese medicine group were significantly lower than those in the myocardial infarction and anxiety group.Conclusion Chaihu plus Longgu Muli Decoction could inhibit the inflammatory response in the hippocampus and relieve the anxiety of rats with myocardial infarction and anxiety maybe by regulating the hippocampal NLRP3/GSDMD inflammatory signal pathway.