安氟醚减轻心脏手术中小鼠的疼痛作用研究

目的研究安氟醚对心肌缺血小鼠心肌细胞保护作用的潜在机制。方法(1)用随机数字表法将成模后小鼠分为2组:对照组和安氟醚组,每组20只。小鼠经腹腔注射异丙肾上腺素3 mg·kg^-1制备心肌缺血模型。在心脏搭桥术前,对照组小鼠用异戊巴比妥(150 mg·kg^-1)麻醉,搭桥完成后腹腔注射等体积生理盐水;安氟醚组用安氟醚(1.0 mg·kg^-1)麻醉,术后4 h腹腔注射(0.2 mg·kg^-1)安氟醚处理;每天1次,连续3 d。用视觉模拟评分(VAS)评估小鼠术后疼痛程度。(2)处死2组小鼠后获取心肌细胞,将细胞分为2组:安氟醚组和对照组;另外,将心肌细胞分为4组:对照组(等体积PBS)、安氟醚组(安氟醚1 mg·mL^-1)、安氟醚+磷酸肌醇3激酶(PIK3)抑制剂组(安氟醚+PI3K抑制剂,均1 mg·mL^-1)和PI3KI抑制剂组(PI3K抑制剂1 mg·mL^-1)。通过细胞计数试剂盒法检测细胞活力(光密度值),MTT法测定细胞增殖(光密度值),缺口末端标记法和流式细胞仪分别检测2组和4组的细胞凋亡情况,蛋白质印迹法检测免疫球蛋白结合蛋白(BIP)、CCAAT增强子结合蛋白(CCAAT enhancer binding protein,CHOP)和PI3KI/蛋白激酶B(Akt)通路相关蛋白表达水平。结果(1)安氟醚组和对照组的VAS分别为(6.53±1.37)和(13.21±2.35)分,组间比较差异有统计学意义(P<0.05)。(2)安氟醚组和对照组的BIP蛋白相对表达水平分别为0.23±0.03和1.04±0.12;这2组的CHOP蛋白表达分别为0.47±0.01和0.98±0.09;这2组的caspase-3蛋白表达分别为0.34±0.01和1.00±0.12;这2组的caspase-8蛋白表达分别为0.27±0.01和1.03±0.08;这2组的p-PI3K蛋白表达分别为1.53±0.16和0.98±0.08;这2组的p-Akt蛋白表达分别为1.69±0.19和0.99±0.11;这2组的心肌细胞存活率分别为(0.87±0.12)%和(0.62±0.03)%;这2组的细胞凋亡率分别为(0.33±0.01)%和(1.08±0.09)%,安氟醚组与对照组比较,上述指标差异均有统计学意义(均P<0.05)。安氟醚+PI3K抑制剂组和对照组的细胞存活率分别为(0.91±0.05)%和(0.98±0.06)%;这2组心肌细胞凋亡率分别为(1.12±0.12)%和(1.02±0.09)%;安氟醚+PI3K抑制剂组与对照组比较,上述指标的差异均无统计学意义(均P>0.05)。结论安氟醚有助于减轻心脏手术中小鼠的疼痛并通过PI3K/Akt信号通路抑制心肌细胞的凋亡。

Effects of enflurane on the reduction of pain in mice during cardiac surgery

Objective To study the potential mechanism of the protective effect of enflurane on myocardial cells in myocardial ischemic mice.Methods(1)Myocardial ischemia mice model was established,and the mice were randomly divided into two groups according to random number table:enflurane group and control group,20 mice in each group.Myocardial ischemia model was established by intraperitoneal injection of isoproterenol 3 mg·kg^-1 in mice.Before cardiac bypass surgery,mice in control group were anesthetized with isopentyl barbiturate(150 mg·kg^-1)and intraperitoneally injected with saline of equal volume;mice in the enflurane group were anesthetized with enflurane(1.0 mg·kg^-1)and intraperitoneal injectionwas given enflurane(0.2 mg·kg^-1)treatment 4 h after surgery;inject once a day for 3 consecutive days.Visual analogue scale(VAS)was performed after cardiac bypass surgery to assess postoperative pain in mice.(2)Mice in the enflurane group and the control group were sacrificed and myocardial cells were harvested.The cells were divided into two groups:enflurane group and control group;In addition,cardiomyocytes were divided into 4 groups:control group(with PBS of equal volume),enflurane group(1 mg·m L^-1 enflurane),enflurane+phosphoinositol 3 kinase(PIK3)inhibitor group(enflurane+PI3 K inhibitor,both 1 mg·m L^-1)and PI3 K inhibitor group(1 mg·m L^-1 PI3 K inhibitor).Cell activity(OD value)was measured by cell counting kit and cell proliferation(OD value)was measured by MTT method.TdT-mediated d UTP nick-end labeling and flow cytometer were used to detect apoptosis of the two groups and four groups,respectively.Western blot was used to detect the expression levels of immunoglobulin binding protein(BIP),CCAAT enhancer binding protein(CHOP)and PI3 KI/protein kinase B(Akt)pathways.Results(1)VAS scores of the enflurane group and the control group were(6.53±1.37)and(13.21±2.35)point,respectively.The difference between the two groups was significant(P<0.05).(2)The relative expression levels of BIP protein in enflurane group and control group were 0.23±0.03 and 1.04±0.12,respectively;CHOP protein in the two groups were 0.47±0.01 and 0.98±0.09,respectively;Caspase-3 protein in the two groups were 0.34±0.01 and1.00±0.12,respectively;Caspase-8 protein in the two groups were 0.27±0.01 and 1.03±0.08,respectively;p-PI3 K protein in the two groups were 1.53±0.16 and 0.98±0.08,respectively;p-AKT protein in the two groups were 1.69±0.19 and 0.99±0.10,respectively;the survival rate of myocardial cells in the two groups were(0.87±0.12)%and(0.62±0.03)%,respectively;the apoptosis rate were(1.12±0.12)%and(1.02±0.09)%,respectively.Comparison between enflurane+PI3 K inhibitor group and control group,the differences of above indicators showed statistically significant(all P<0.05).After p-PI3 K treated,the cell survival rate in enflurane+PI3 K inhibitor group and control group were(0.91±0.05)%and(0.98±0.06)%,respectively;the apoptosis rate in the two groups were(1.12±0.12)%and(1.02±0.09)%,respectively.Comparison between enflurane group and control group,the differences of above indicators showed no statistically significant difference(all P>0.05).Conclusion Enflurane could reduce pain in mice during cardiac surgery and inhibit myocardial cell apoptosis through PI3 K/Akt signaling pathway.