Yang Yongli, Li Zeshan, Ye Hongbing, Liu Haizhen
Department of Critical Care Medicine, Hubei NO.3 People's Hospital of Jianghan University, Wuhan 430033, Hubei, China.
Department of Internal Medicine, Jiayu Hospital of Traditional Chinese Medicine (Jiayu Branch of Hubei NO.3 People's Hospital of Jianghan University), Jiayu 437200, Hubei, China.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2022 Jan;34(1):54-58. doi: 10.3760/cma.j.cn121430-20210706-01012.
To investigate the role and mechanism of microRNA-499 (miR-499) regulating α-myosin heavy chain (α-MHC) and β-myosin heavy chain (β-MHC) gene axis in septic myocardial dysfunction (SMD) and its significance.
Sixty healthy adult male Sprague-Dawley (SD) rats were divided into phosphate buffered saline (PBS) control group (PBS group), lipopolysaccharide (LPS) induced SMD model group (LPS group), miR-499 agonist pretreatment group (agomir+LPS group), and miR-499 inhibitor pretreatment group (antagomir+LPS group) by random number table, with 15 rats in each group. SMD rat model was reproduced by intraperitoneal injection of LPS 10 mg/kg. The PBS group was intraperitoneally injected with the same amount of PBS. The two pretreatment groups were injected with agomir 30 mg/kg or antagomir 80 mg/kg through the caudal vein for 3 days, once a day. PBS group and LPS group were not pretreated. Echocardiography was detected 5 hours after LPS injection, and relevant indexes were recorded. The expression of miR-499 in plasma and myocardial tissue was detected by real-time quantitative polymerase chain reaction (qPCR). Western blotting was used to detect the protein expressions of α-MHC and β-MHC in myocardial tissue. Plasma N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of heart failure, was measured by electrochemiluminescence.
Compared with the PBS group, the rats in LPS group were depressed. Additionally, LPS down-regulated the level of miR-499 in plasma and myocardial tissue, decreased α-MHC expression in myocardial tissue and up-regulated the expression of β-MHC. Echocardiography showed that left ventricular ejection fraction (LVEF), left ventricle fractional shortening (LVFS), cardiac output (CO), stroke volume (SV) and heart rate (HR) decreased by 49.1%, 59.2%, 48.8%, 39.4% and 15.9%, respectively, and the level of plasma NT-proBNP increased significantly in LPS group, indicating that LPS could induce cardiac dysfunction in rats. Compared with the LPS group, after pretreatment with agomir to overexpress the miR-499, LVEF and LVFS were significantly increased [LVEF: 0.662±0.020 vs. 0.323±0.024, LVFS: (36.16±1.43)% vs. (20.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was improved in agomir+LPS group. At the same time, pretreatment with agomir significantly down-regulated the β-MHC protein expression (β-MHC/GAPDH: 0.74±0.04 vs. 2.97±0.34, P < 0.01), significantly up-regulated α-MHC protein expression (α-MHC/GAPDH: 1.59±0.05 vs. 0.74±0.14, P < 0.01), and significantly decreased the plasma NT-proBNP level (ng/L: 114.49±6.85 vs. 334.13±4.36, P < 0.01). After pretreatment with antagomir to inhibit the expression of miR-499, echocardiography showed that LVEF and LVFS were significantly lower than those in the LPS group [LVEF: 0.297±0.021 vs. 0.323±0.024, LVFS: (19.38±1.52)% vs. (21.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was significantly inhibited. At the same time, pretreatment with antagomir significantly down-regulated α-MHC protein expression in myocardial tissue (α-MHC/GAPDH: 0.63±0.03 vs. 0.74±0.14, P < 0.01), significantly up-regulated β-MHC protein expression (β-MHC/GAPDH: 3.03±0.47 vs. 2.97±0.34, P < 0.01), and significantly increased the level of plasma NT-proBNP (ng/L: 373.91±4.23 vs. 334.13±4.36, P < 0.05).
miR-499 could regulate the expression of α-MHC and β-MHC which improved cardiac dysfunction caused by sepsis. Targeted regulation of miR-499 expression may be an effective way to treat SMD.
探讨微小RNA-499（miR-499）调控α-肌球蛋白重链（α-MHC）和β-肌球蛋白重链（β-MHC）基因轴在脓毒症性心肌功能障碍（SMD）中的作用、机制及其意义。
将60只健康成年雄性Sprague-Dawley（SD）大鼠通过随机数字表法分为磷酸盐缓冲液（PBS）对照组（PBS组）、脂多糖（LPS）诱导的SMD模型组（LPS组）、miR-499激动剂预处理组（agomir+LPS组）和miR-499抑制剂预处理组（antagomir+LPS组），每组15只。通过腹腔注射10 mg/kg LPS复制SMD大鼠模型。PBS组腹腔注射等量PBS。两个预处理组通过尾静脉注射30 mg/kg agomir或80 mg/kg antagomir，连续3天，每天1次。PBS组和LPS组不进行预处理。LPS注射后5小时进行超声心动图检测，并记录相关指标。采用实时定量聚合酶链反应（qPCR）检测血浆和心肌组织中miR-499的表达。采用蛋白质印迹法检测心肌组织中α-MHC和β-MHC的蛋白表达。采用电化学发光法检测血浆N末端脑钠肽前体（NT-proBNP），作为心力衰竭的标志物。
与PBS组相比，LPS组大鼠精神萎靡。此外，LPS下调了血浆和心肌组织中miR-499的水平，降低了心肌组织中α-MHC的表达，上调了β-MHC的表达。超声心动图显示，LPS组左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）、心输出量（CO）、每搏输出量（SV）和心率（HR）分别下降了49.1%、59.2%、48.8%、39.4%和15.9%，血浆NT-proBNP水平显著升高，表明LPS可诱导大鼠心脏功能障碍。与LPS组相比，经agomir预处理使miR-499过表达后，LVEF和LVFS显著升高[LVEF：0.662±0.020比0.323±0.024，LVFS：（36.16±1.43）%比（20.20±1.32）%，P均<0.01]，提示agomir+LPS组大鼠心脏功能得到改善。同时，agomir预处理显著下调β-MHC蛋白表达（β-MHC/GAPDH：0.74±0.04比2.97±0.34，P<0.01），显著上调α-MHC蛋白表达（α-MHC/GAPDH：1.59±0.05比0.74±0.14，P<0.01），并显著降低血浆NT-proBNP水平（ng/L：114.49±6.85比334.13±4.36，P<0.01）。经antagomir预处理抑制miR-499表达后，超声心动图显示LVEF和LVFS显著低于LPS组[LVEF：0.297±0.021比0.323±0.024，LVFS：（19.38±1.52）%比（20.20±1.32）%，P均<0.01]，提示大鼠心脏功能受到显著抑制。同时，antagomir预处理显著下调心肌组织中α-MHC蛋白表达（α-MHC/GAPDH：0.63±0.03比0.74±0.14，P<0.01），显著上调β-MHC蛋白表达（β-MHC/GAPDH：3.03±0.47比2.97±0.34，P<0.01），并显著升高血浆NT-proBNP水平（ng/L：373.91±4.23比334.13±4.
