远端肢体缺血后处理通过线粒体自噬减轻大鼠局灶型脑缺血再灌注损伤的研究

doi:10.3877/cma.j.issn.1674-6880.2020.04.001

目的

探讨远端肢体缺血后处理通过线粒体自噬减轻大鼠局灶型脑缺血再灌注损伤的作用。

方法

将105只成年雄性Sprague-Dawley大鼠分为假手术组、缺血再灌注组（A组）、缺血再灌注+远端缺血后处理组（B组）、缺血再灌注+远端缺血后处理+等渗NaCl溶液组（C组）和缺血再灌注+远端缺血后处理+线粒体分裂抑制剂（Mdivi-1）组（D组），每组各21只大鼠。假手术组仅暴露和游离右侧颈动脉，A、B、C、D组采用大脑中动脉阻断法制备大鼠局灶性脑缺血再灌注损伤模型。B、C、D组大鼠于再灌注开始夹闭双侧股动脉实行3个循环的10 min缺血/10 min再灌注，C、D组大鼠在缺血前5 min分别腹腔注射等容量的等渗NaCl溶液和3 mg/kg的Mdivi-1。比较各组大鼠神经功能缺陷量表（NDS）评分、脑梗塞体积百分比、脑缺血半暗区细胞凋亡率、微管相关蛋白1轻链3（LC3）-Ⅱ/Ⅰ比值、超氧化物歧化酶（SOD）、丙二醛及15-F_2t-Isoprostane的表达水平。

结果

假手术组大鼠未发生神经功能缺损和脑梗塞。A、B、C、D组大鼠NDS评分[（2.8±0.6）、（1.6±0.4）、（1.6±0.5）、（2.5±0.5）分]和脑梗塞体积百分比[（48±3）%、（28±4）%、（28±4）%、（41±3）%]比较，差异均有统计学意义（F = 39.237、53.278，P均< 0.001），且B、C组大鼠NDS评分和脑梗塞体积百分比均较A、D组显著降低（P均< 0.05）。假手术组、A组、B组、C组及D组大鼠脑缺血半暗区细胞凋亡率[（2.3±0.8）%、（54.6±5.2）%、（29.3±3.1）%、（29.8±3.3）%、（51.2±4.5）%]、LC3-Ⅱ/Ⅰ比值[（0.13±0.03）、（0.32±0.05）、（0.53±0.06）、（0.48±0.08）、（0.35±0.06）]、SOD [（168±19）、（92±13）、（162±21）、（165±23）、（94±15）U/mg]、丙二醛[（4.22±0.28）、（8.41±0.42）、（5.14±0.27）、（5.26±0.31）、（7.93±0.44）nmol/mg]及15-F_2t-Isoprostane [（179±86）、（389±105）、（208±89）、（215±85）、（364±103）mg/g]的表达水平比较，差异均有统计学意义（F = 54.658、32.358、59.677、46.195、193.962，P均< 0.001）。进一步两两比较发现，A、B、C、D组大鼠脑缺血半暗区细胞凋亡率、LC3-Ⅱ/Ⅰ比值、丙二醛及15-F_2t-Isoprostane表达水平均较假手术组显著升高，A、D组大鼠SOD表达水平均较假手术组显著降低（P均< 0.05）；与A、D组比较，B、C组大鼠LC3-Ⅱ/Ⅰ比值和SOD表达水平均显著升高，脑缺血半暗区细胞凋亡率、丙二醛及15-F_2t-Isoprostane表达水平均显著降低（P均< 0.05）。

结论

远端肢体缺血后处理可能通过增加线粒体自噬水平抑制氧化应激反应，从而减轻大鼠局灶性脑缺血再灌注损伤。

关键词: 再灌注损伤, 脑, 缺血后处理, 线粒体自噬

Objective

To investigate the role of remote limb ischemic postconditioning in mitigation of focal cerebral ischemia-reperfusion injury in rats via mitochondrial autophagy.

Methods

Totally 105 adult male Sprague-Dawley rats were divided into a sham operation group, a ischemia-reperfusion group (A group), a ischemia-reperfusion + remote ischemic postconditioning group (B group), a ischemia-reperfusion + remote ischemic postconditioning + isotonic NaCl solution group (C group) and a ischemia-reperfusion + remote ischemic postconditioning + mitochondrial division inhibitor-1 (Mdivi-1) group (D group), 21 rats in each group. The right carotid artery was exposed and freed in the sham operation group, while a model of focal cerebral ischemia-reperfusion injury was prepared by middle cerebral artery blockade in the A, B, C and D groups. Then 10 min of ischemia/10 min of reperfusion were performed for three cycles in the B, C and D groups, and an equal volume of isotonic NaCl solution and 3 mg/kg of Mdivi-1 were injected intraperitoneally in the C and D groups 5 min before ischemia respectively. The neurological deficit score (NDS), percentage of cerebral infarction volume, and apoptosis rate, microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ/Ⅰ ratio, superoxide dismutase (SOD), malondialdehyde and 15-F_2t-Isoprostane of cerebral ischemic semidark cells were compared.

Results

No neurological deficit and cerebral infarction occurred in the sham operation group. The NDS [(2.8 ± 0.6), (1.6 ± 0.4), (1.6 ± 0.5), (2.5 ± 0.5) scores] and percentage of cerebral infarction volume [(48 ± 3)%, (28 ± 4)%, (28 ± 4)%, (41 ± 3)%] were statistically significantly different in the A, B, C and D groups (F = 39.237, 53.278; both P < 0.001). Moreover, they were significantly lower in the B and C groups than in the A and D groups (all P < 0.05). The apoptosis rate [(2.3 ± 0.8)%, (54.6 ± 5.2)%, (29.3 ± 3.1)%, (29.8 ± 3.3)%, (51.2 ± 4.5)%], LC3-Ⅱ/Ⅰ ratio [(0.13 ± 0.03), (0.32 ± 0.05), (0.53 ± 0.06), (0.48 ± 0.08), (0.35 ± 0.06)], SOD [(168 ± 19), (92 ± 13), (162 ± 21), (165 ± 23), (94 ± 15) U/mg], malondialdehyde [(4.22 ± 0.28), (8.41 ± 0.42), (5.14 ± 0.27), (5.26 ± 0.31), (7.93 ± 0.44) nmol/mg] and 15-F_2t-Isoprostane [(179 ± 86), (389 ± 105), (208 ± 89), (215 ± 85), (364 ± 103) mg/g] of cerebral ischemic semidark cells were statistically significantly different in the sham operation, A, B, C and D groups (F = 54.658, 32.358, 59.677, 46.195, 193.962; all P < 0.001). Further pairwise comparison showed that the apoptosis rate, LC3-Ⅱ/Ⅰ ratio, malondialdehyde and 15-F_2t-Isoprostane in the A, B, C and D groups were significantly higher than those in the sham operation group, while the SOD expression level in the A and D groups was significantly lower than that in the sham operation group (all P < 0.05). Compared with the A and D groups, the LC3-Ⅱ/Ⅰ ratio and SOD expression level of rats in the B and C groups significantly increased, and the apoptosis rate, malondialdehyde and 15-F_2t-Isoprostane significantly decreased (all P < 0.05).

Conclusion

Remote limb ischemia postconditioning may reduce focal cerebral ischemia-reperfusion injury in rats by increasing the mitochondrial autophagy level and inhibiting the oxidative stress response.

Key words: Reperfusion injury, Brain, Ischemic postconditioning, Mitophagy

表1 5组大鼠脑缺血半暗区LC3-Ⅱ/Ⅰ比值、SOD、丙二醛及15-F_2t-Isoprostane表达水平比较（ ± s）

组别	例数	LC3-Ⅱ/Ⅰ	SOD（U/mg）	丙二醛（nmol/mg）	15-F_2t-Isoprostane（mg/g）
假手术组	5	0.13 ± 0.03	168 ± 19	4.22 ± 0.28	179 ± 86
A组	5	0.32 ± 0.05^a	92 ± 13^a	8.41 ± 0.42^a	389 ± 105^a
B组	5	0.53 ± 0.06^ab	162 ± 21^b	5.14 ± 0.27^ab	208 ± 89^ab
C组	5	0.48 ± 0.08^ab	165 ± 23^b	5.26 ± 0.31^ab	215 ± 85^ab
D组	5	0.35 ± 0.06^acd	94 ± 15^acd	7.93 ± 0.44^acd	364 ± 103^acd
F值		32.358	59.677	46.195	193.962
P值		< 0.001	< 0.001	< 0.001	< 0.001

表1 5组大鼠脑缺血半暗区LC3-Ⅱ/Ⅰ比值、SOD、丙二醛及15-F_2t-Isoprostane表达水平比较（ ± s）

组别	例数	LC3-Ⅱ/Ⅰ	SOD（U/mg）	丙二醛（nmol/mg）	15-F_2t-Isoprostane（mg/g）
假手术组	5	0.13 ± 0.03	168 ± 19	4.22 ± 0.28	179 ± 86
A组	5	0.32 ± 0.05^a	92 ± 13^a	8.41 ± 0.42^a	389 ± 105^a
B组	5	0.53 ± 0.06^ab	162 ± 21^b	5.14 ± 0.27^ab	208 ± 89^ab
C组	5	0.48 ± 0.08^ab	165 ± 23^b	5.26 ± 0.31^ab	215 ± 85^ab
D组	5	0.35 ± 0.06^acd	94 ± 15^acd	7.93 ± 0.44^acd	364 ± 103^acd
F值		32.358	59.677	46.195	193.962
P值		< 0.001	< 0.001	< 0.001	< 0.001

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Study of remote limb ischemic postconditioning in mitigation of focal cerebral ischemia-reperfusion injury in rats via mitochondrial autophagy

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Study of remote limb ischemic postconditioning in mitigation of focal cerebral ischemia-reperfusion injury in rats via mitochondrial autophagy