艾司洛尔通过阻断NLRP3/Caspase-1/GSDMD通路抑制早期脓毒症大鼠心脏炎症反应及焦亡

Esmolol inhibits cardiac inflammation and pyroptosis in early sepsis rats by blocking NLRP3/Caspase-1/GSDMD pathway

目的研究艾司洛尔(Esmolol,ES)是否可通过抑制焦亡的经典通路进而发挥心脏保护作用。方法 72只大鼠随机分为假手术(sham operation,Sham)组、盲肠结扎穿孔(cecal ligation and puncture,CLP)组和ES组,每组24只。每组随机抽取8只大鼠观察生存情况,剩余16只大鼠根据处理时间分为6 h和24 h两个亚组,每组8只。Sham组仅给予开腹,翻动盲肠处理;CLP组和ES组采用CLP法建立脓毒症大鼠模型。Sham组、CLP组持续经大鼠左颈内静脉泵入生理盐水6 h;ES组持续泵入艾司洛尔稀释液6 h。分别于相应时间点处死大鼠,采用酶联免疫吸附法(ELISA)测定血清IL-1β、IL-18水平,采用免疫印迹法(Western blot)检测心肌组织NLRP3、Caspase-1及GSDMD蛋白表达水平;光学显微镜下观察心肌病理改变;荧光显微镜下观察心肌细胞焦亡情况。结果 (1)ELISA结果显示,与各时间点的Sham组比较,ES组大鼠血清IL-1β及IL-18水平均明显升高,24 h组最高;而与CLP组比较,上述因子水平在6 h与24 h均明显下降(P <0.05)。(2)Western blot结果显示,术后各时间点,大鼠心肌组织中焦亡经典通路相关蛋白[NOD样受体蛋白3(nucleotide-binding domain(NOD)-like receptor protein 3,NLRP3)、天冬氨酸蛋白水解酶-1 (cysteinyl aspartate-specific proteases-1,Caspase-1)、消皮素D(Gasdermin D,GSDMD)]在CLP组均显示出高表达状态,较Sham组明显升高(P <0.05),24 h组表达量最高,而ES组各时间点上述蛋白表达水平均较CLP组有所减少,且差异统计学意义(P <0.05)。(3)病理结果显示,Sham组大鼠心肌组织可见轻微的病理损伤,CLP组心肌细胞损伤最重,ES组大鼠的心肌细胞破坏较CLP组减轻程度多,各组24 h损伤程度均较6 h重。(4)24 h Tunel与DAPI染色显示,与Sham组可见的少量阳性细胞比较,CLP组可见大量被染色的阳性细胞,且差异有统计学意义(P <0.05);而与CLP组比较,ES组心肌细胞阳性染色数量明显减少(P <0.05)。(5) 7 d生存分析显示,CLP组大鼠死亡率最高,ES组大鼠生存天数较CLP组明显延长,差异有统计学意义(P <0.01)。结论 ES能减轻心肌损伤,提高脓毒症大鼠生存率,其机制可能与抑制焦亡经典通路中NLRP3/Caspase-1/GSDMD蛋白表达及减少炎症因子释放有关。

Objective To study whether Esmolol can protect the heart by inhibiting the classical pathway of pyroptosis. Methods 72 rats were randomly divided into sham operation group( Sham group),cecal ligation and puncture( CLP) group and Esmolol( ES) group. Each group had 24 rats. The three groups were further divided into two subgroups,8 rats was used to observe the survival days,and16 rats was randomly divided into 6 h and 24 h groups according to the treatment time( n = 8). Sham group was only given laparotomy and turning cecum,CLP group and ES group were treated with CLP to establish sepsis models. In Sham group and CLP group,normal saline was continuously pumped through left internal jugular vein for 6 h;Esmolol dilution was continuously pumped into ES group for 6 h. The rats were killed at the corresponding time points. The levels of serum IL-1β and IL-18 were detected by ELISA. The expression levels of NLRP3,Caspase-1 and GSDMD protein in myocardial tissue were detected by Western blot. The pathological changes of myocardium were observed under optical microscope,and the pyroptosis of myocardial cells was observed under fluorescence microscope. Results(1)The results of ELISA showed that compared with Sham group at each time point,the levels of IL-1β and IL-18 in the serum of ES group were significantly increased,especially at 24 h;compared with CLP group,the levels of above factors were significantly decreased at 6 h and 24 h( P < 0. 05).(2)Western blot showed that the expression of classical pathway related proteins of pyroptosis [nucleotide-binding domain( NOD)-like receptor protein 3( NLRP3),Cysteinyl aspartate-specific proteases-1( Caspase-1) and Gasdermin D( GSDMD) ] in CLP group was significantly higher than that in Sham operation group( P < 0. 05),and the expression at 24 h after operation was the highest. The expression of above proteins in ES group was lower than that in CLP group at each time point,and the difference was statistically significant( P < 0. 05).(3)The pathological results showed that the myocardial tissue of Sham group had slight pathological damage,and the myocardial cell damage of CLP group was the most serious. The myocardial cell damage of ES group was significantly lighter than that of CLP group,and the damage degree of each group at 24 h was heavier than that at 6 h.(4)At 24 h group,Tunel and DAPI staining showed that compared with a small number of positive cells in Sham group,a large number of positive cells were observed in CLP group,and the difference was statistically significant( P < 0. 05).Compared with CLP group,the number of positive staining cells in ES group was significantly reduced( P < 0. 05).(5)The 7-day survival analysis showed that the mortality of CLP group was the highest,and the survival days of ES group were significantly longer than that of CLP group( P < 0. 01). Conclusions Esmolol can alleviate myocardial injury and improve the survival rate of septic rats. The mechanism may be related to the inhibition of NLRP3/Caspase-1/GSDMD protein expression and the reduction of inflammatory factor release.