丙泊酚对肠缺血再灌注损伤大鼠心肌损伤的保护作用及相关信号通路研究

doi:10.3877/cma.j.issn.2095-1221.2021.05.007

目的

探究丙泊酚对肠缺血再灌注损伤大鼠心肌损伤的保护及其作用机制。

方法

构建肠缺血再灌注损伤大鼠模型，使用随机数字表法分为模型组、假手术组（假手术组和模型组腹腔注射脂肪乳）、低、中、高剂量丙泊酚组（在手术前30 min分别腹腔注射5、10、20 mg/ kg的丙泊酚）。再灌注2 h后，苏木素-伊红（HE）染色观察大鼠小肠组织和心肌组织病理学改变；TUNEL法检测大鼠心肌细胞凋亡情况；酶联免疫吸附法（ELISA）检测大鼠血清丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）以及白细胞介素1β（IL- 1β）、IL-6、肿瘤坏死因子α （TNF-α）的水平；蛋白免疫印迹法（Western blot）检测大鼠心肌组织沉默信息调节因子2相关酶1 （Sirt1）、乙酰化叉头状转录因子1 （Ac-Foxo1）蛋白的表达。多组间比较采用单因素方差分析，进一步两组间比较采用SNK-q检验。

结果

与假手术组相比，模型组小肠组织出现严重水肿且部分坏死，小肠黏膜结构受损，绒毛明显脱落，心肌细胞肿胀，间质出现大量炎性细胞浸润，心肌细胞凋亡率[（4.05±0.37）﹪比（34.24±4.28）﹪]、Ac- Foxo1表达量（0.80±0.14比2.21±0.25）、MDA含量[（4.09±0.81） nmol/ mg比（12.64±2.10） nmol/ mg]、TNF-α [（19.26±3.48）pg/mg比（85.37±10.41）pg/mg]、IL- 1β[（9.53±1.14） pg/mg比（57.82±7.36）pg/ mg]和IL- 6[（8.49±2.03） pg/ mg比（50.76±6.28） pg/ mg]水平均升高，Sirt1表达量（1.15±0.17比0.42±0.05）、GSH-Px [（33.65±5.64） U/ mg比（8.42±1.87） U/ mg]和SOD活性[（45.96±7.83） U/ mg比（13.65±4.34） U/ mg]均降低，差异有统计学意义（P均< 0.05）；与模型组相比，中、高剂量丙泊酚组大鼠心肌细胞凋亡率[（34.24±4.28）﹪比（19.44±3.53）﹪，（13.61±2.29）﹪]、Ac- Foxo1表达量（2.21±0.25比1.50±0.19，1.16±0.17）、MDA含量[（12.64±2.10）nmol/ mg比（9.24±1.02） nmol/mg，（7.16± 0.87） nmol/mg]以及TNF-α[（85.37±10.41）pg/mg比（58.82±5.43） pg/mg，（42.15±7.38）pg/mg]、IL-1β[（57.82±7.36） pg/mg比（36.58±6.12）pg/mg，（26.75±5.34）pg/mg]、IL-6 [（50.76±6.28） pg/ mg比（35.26±4.99）pg/mg，（23.51±4.32）pg/mg]水平均降低，Sirt1表达量（0.42±0.05比0.65±0.07，0.86±0.09）、GSH-Px [（8.42±1.87）U/mg比（20.45±2.86）U/mg，（26.26±3.03）U/mg]和SOD活性[（13.65±4.34）U/mg比（24.62±4.19） U/ mg，（32.95±4.13）U/mg]均升高，且呈一定的剂量依赖性，差异均有统计学意义（P均< 0.05）。

结论

丙泊酚可提高肠缺血再灌注损伤大鼠心肌细胞抗氧化能力，降低炎症反应，从而对心肌损伤起到保护作用，其作用机制可能与激活Sirt1/Foxo1通路有关。

关键词: 丙泊酚, 缺血再灌注损伤, 心肌损伤, 沉默信息调节因子2相关酶1, 叉头状转录因子1

Objective

To explore the protective effect of propofol on myocardial injury in rats with intestinal ischemia-reperfusion injury and mechanism thereof.

Methods

Rat models of intestinal ischemia-reperfusion injury were constructed and divided into model group, sham operation group (sham operation group and model group by intraperitoneal injection of fat emulsion) , low, medium and high dose propofol group (5, 10, and 20 mg/kg propofol were injected intraperitoneally 30 minutes before surgery) . After 2 hours of reperfusion, hematoxylin-eosin (HE) staining was used to observe the pathological changes of the small intestinal tissue and cardiac muscle tissue in the rats; TUNEL assay was used to detect rat cardiomyocyte apoptosis; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum malondialdehyde (MDA) , glutathione peroxidase (GSH-Px) , superoxide dismutase (SOD) and interleukin 1β (IL-1β) , IL-6, and tumor necrosis factor α (TNF-α) in the rats; Western blot was used to detect the expressions of silent information regulation factor 2 related enzyme 1 (Sirt1) and acetylated forkhead transcription factor 1 (Ac- Foxo1) proteins in rat myocardial tissues. One-way analysis of variance was used for the comparison between multiple groups, and SNK-q test was used for further comparison between two groups.

Results

Compared with the sham operation group, the small intestinal tissue of the model group was severely edematous and partially necrotic, the structure of the small intestinal mucosa was damaged, the villi were obviously shedding, myocardial cells were swollen, and a large number of inflammatory cell infiltration appeared in the interstitium. The myocardial cells apoptosis rate [ (4.05±0.37) ﹪vs (34.24±4.28) ﹪], Ac-Foxo1 expression (0.80±0.14 vs 2.21±0.25) , MDA content [ (4.09±0.81) nmol/mg vs (12.64±2.10) nmol/ mg], and the levels of TNF-α [ (19.26±3.48) pg/ mg vs (85.37±10.41) pg/ mg], IL-1β [ (9.53±1.14) pg/ mg vs (57.82±7.36) pg/ mg], and IL-6 [ (8.49±2.03) pg/mg vs (50.76±6.28) pg/mg] were increased significantly (P < 0.05) ; Sirt1 expression (1.15±0.17 vs 0.42±0.05) , and the activity of GSH-Px[ (33.65±5.64) U/mg vs (8.42±1.87) U/ mg] and SOD [ (45.96±7.83) U/mg vs (13.65±4.34) U/ mg]were decreased, with the difference statistically significant (all P < 0.05) . Compared with the model group, the apoptotic rate of cardiomyocytes in the middle and high dose propofol group[ (34.24±4.28) ﹪ vs (19.44±3.53) ﹪, (13.61±2.29) ﹪], Ac- Foxo1 expression (2.21±0.25 vs 1.50±0.19, 1.16±0.17) , MDA content [ (12.64±2.10) nmol/ mg vs (9.24±1.02) nmol/mg, (7.16±0.87) nmol/ mg] and the levels of TNF-α [ (85.37± 10.41) pg/ mg vs (58.82±5.43) pg/ mg, (42.15±7.38) pg/ mg], IL-1β [ (57.82±7.36) pg/ mg vs 36.58±6.12) pg/ mg, (26.75±5.34) pg/ mg], and IL-6 [ (50.76±6.28) pg/ mg vs (35.26±4.99) pg/ mg, (23.51±4.32) pg/ mg] were reduced; Sirt1 expression (0.42±0.05 vs 0.65±0.07, 0.86±0.09) , and the activity of GSH-Px[ (8.42±1.87) U/ mg vs (20.45±2.86) U/ mg, (26.26±3.03) U/mg] and SOD [ (13.65±4.34) U/mg vs (24.62±4.19) U/ mg, (32.95±4.13) U/ mg]were all increased in a certain dose-dependent manner, and the difference was statistically significant (all P < 0.05) .

Conclusion

Propofol can increase the antioxidant capacity of myocardial cells in rats with intestinal ischemia-reperfusion injury, reduce inflammation, and thus protect the myocardium from injury. Its mechanism may be related to the activation of the Sirt1/Foxo1 pathway.

Key words: Propofol, Ischemia-reperfusion injury, Myocardial injury, Silent information regulation factor 2 related enzyme 1, Forkhead transcription factor 1

图1 光学显微镜下观察实验大鼠小肠组织（HE染色，×100）

图2 光学显微镜下观察大鼠心肌组织（HE染色，×200）

图3 光学显微镜下观察心肌组织（TUNEL染色，×200）

图4 丙泊酚对心肌组织Sirt1/Foxo1通路蛋白表达的影响

表1 丙泊酚对心肌组织Sirt1/Foxo1通路蛋白表达的影响（ ± s）

分组	动物数	Sirt1/β-actin	Ac-Foxo1/β-actin
假手术	6	1.15±0.17	0.80±0.14
模型	6	0.42±0.05^a	2.21±0.25^a
低剂量丙泊酚	6	0.49±0.04^a	1.93±0.22^a
中剂量丙泊酚	6	0.65±0.07^abc	1.50±0.19^abc
高剂量丙泊酚	6	0.86±0.09^abcd	1.16±0.17^abcd
F值		57.411	45.590
P值		< 0.001	< 0.001

表1 丙泊酚对心肌组织Sirt1/Foxo1通路蛋白表达的影响（ ± s）

分组	动物数	Sirt1/β-actin	Ac-Foxo1/β-actin
假手术	6	1.15±0.17	0.80±0.14
模型	6	0.42±0.05^a	2.21±0.25^a
低剂量丙泊酚	6	0.49±0.04^a	1.93±0.22^a
中剂量丙泊酚	6	0.65±0.07^abc	1.50±0.19^abc
高剂量丙泊酚	6	0.86±0.09^abcd	1.16±0.17^abcd
F值		57.411	45.590
P值		< 0.001	< 0.001

表2 丙泊酚对心肌组织MDA、GSH-Px、SOD水平的影响（ ± s）

分组	动物数	MDA （nmol/mg）	GSH-Px （U/mg）	SOD （U/mg）
假手术	6	4.09±0.81	33.65±5.64	45.96±7.83
模型	6	12.64±2.10^a	8.42±1.87^a	13.65±4.34^a
低剂量丙泊酚	6	11.35±0.86^a	13.37±2.95^a	16.21±3.76^a
中剂量丙泊酚	6	9.24±1.02^abc	20.45±2.86^abc	24.62±4.19^abc
高剂量丙泊酚	6	7.16±0.87^abcd	26.26±3.03^abcd	32.95±4.13^abcd
F值		45.643	49.231	40.421
P值		< 0.001	< 0.001	< 0.001

表2 丙泊酚对心肌组织MDA、GSH-Px、SOD水平的影响（ ± s）

分组	动物数	MDA （nmol/mg）	GSH-Px （U/mg）	SOD （U/mg）
假手术	6	4.09±0.81	33.65±5.64	45.96±7.83
模型	6	12.64±2.10^a	8.42±1.87^a	13.65±4.34^a
低剂量丙泊酚	6	11.35±0.86^a	13.37±2.95^a	16.21±3.76^a
中剂量丙泊酚	6	9.24±1.02^abc	20.45±2.86^abc	24.62±4.19^abc
高剂量丙泊酚	6	7.16±0.87^abcd	26.26±3.03^abcd	32.95±4.13^abcd
F值		45.643	49.231	40.421
P值		< 0.001	< 0.001	< 0.001

表3 丙泊酚对心肌组织TNF-α、IL-1β、IL-6水平的影响（pg/mg， ± s）

分组	动物数	TNF-α	IL-1β	IL-6
假手术	6	19.26±3.48	9.53±1.14	8.49±2.03
模型	6	85.37±10.41^a	57.82±7.36^a	50.76±6.28^a
低剂量丙泊酚	6	74.69±6.35^a	49.61±7.97^a	44.58±5.72^a
中剂量丙泊酚	6	58.82±5.43^abc	36.58±6.12^abc	35.26±4.99^abc
高剂量丙泊酚	6	42.15±7.38^abcd	26.75±5.34^abcd	23.51±4.32^abcd
F值		84.622	58.711	71.612
P值		< 0.001	< 0.001	< 0.001

表3 丙泊酚对心肌组织TNF-α、IL-1β、IL-6水平的影响（pg/mg， ± s）

分组	动物数	TNF-α	IL-1β	IL-6
假手术	6	19.26±3.48	9.53±1.14	8.49±2.03
模型	6	85.37±10.41^a	57.82±7.36^a	50.76±6.28^a
低剂量丙泊酚	6	74.69±6.35^a	49.61±7.97^a	44.58±5.72^a
中剂量丙泊酚	6	58.82±5.43^abc	36.58±6.12^abc	35.26±4.99^abc
高剂量丙泊酚	6	42.15±7.38^abcd	26.75±5.34^abcd	23.51±4.32^abcd
F值		84.622	58.711	71.612
P值		< 0.001	< 0.001	< 0.001

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Protective effect of propofol on myocardial injury and related signal pathways in rats with intestinal ischemia-reperfusion injury

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Protective effect of propofol on myocardial injury and related signal pathways in rats with intestinal ischemia-reperfusion injury