Abstract：
BACKGROUND: Limb remote ischemic postconditioning (LRIP) and paeoniflorin (PF) both can ameliorate cerebral ischemia reperfusion (I/R) injury. At present, whether LRIP combined with PF can achieve better therapeutic effect is unknown.
OBJECTIVE: This study explored the alleviating effect and mechanism of LRIP in combination with PF on cerebral I/R injury in rats.
METHODS: Middle cerebral artery occlusion (MCAO) surgery was performed on rats except Sham group. Then PF (2.5 mg/kg, 5 mg/kg, 10 mg/kg) was administrated by intraperitoneal injection 10 min before the start of reperfusion. LRIP was operated on the left femoral artery at 0 h of reperfusion. Behavioral testing was used to assess neurological impairment, while TTC staining was used to examine infarct volume. Protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox in neutrophils from rat peripheral blood were tested by Western blot. Rat bone marrow neutrophils were extracted and incubated for 24 h with serum from rats after LRIP combined with PF. p38 MAPK inhibitor group was administrated SB203580 while the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor group was administrated Apocynin. Neutrophils were stimulated by fMLP (10 μM). Reactive oxygen species (ROS) production and protein expression of MyD88, TRAF6, p38 MAPK, and p47phox (ser 304 and ser 345) were detected.
RESULTS: LRIP combined with PF (5 mg/kg) reduced cerebral infarct volume, ameliorated neurological deficit score (NDS), decreased fMLP-stimulated ROS release and downregulated the protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox (ser 304 and ser 345) in neutrophils.
CONCLUSIONS: The protective effect of LRIP combined with PF on cerebral I/R injury was better than either alone. Taken together, we provided solid evidence to demonstrate that the combination of LRIP and PF had potential to alleviate cerebral I/R injury, which was regulated by MyD88-TRAF6-p38 MAPK pathway and neutrophil NADPH oxidase pathway.
OBJECTIVE: This study explored the alleviating effect and mechanism of LRIP in combination with PF on cerebral I/R injury in rats.
METHODS: Middle cerebral artery occlusion (MCAO) surgery was performed on rats except Sham group. Then PF (2.5 mg/kg, 5 mg/kg, 10 mg/kg) was administrated by intraperitoneal injection 10 min before the start of reperfusion. LRIP was operated on the left femoral artery at 0 h of reperfusion. Behavioral testing was used to assess neurological impairment, while TTC staining was used to examine infarct volume. Protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox in neutrophils from rat peripheral blood were tested by Western blot. Rat bone marrow neutrophils were extracted and incubated for 24 h with serum from rats after LRIP combined with PF. p38 MAPK inhibitor group was administrated SB203580 while the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor group was administrated Apocynin. Neutrophils were stimulated by fMLP (10 μM). Reactive oxygen species (ROS) production and protein expression of MyD88, TRAF6, p38 MAPK, and p47phox (ser 304 and ser 345) were detected.
RESULTS: LRIP combined with PF (5 mg/kg) reduced cerebral infarct volume, ameliorated neurological deficit score (NDS), decreased fMLP-stimulated ROS release and downregulated the protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox (ser 304 and ser 345) in neutrophils.
CONCLUSIONS: The protective effect of LRIP combined with PF on cerebral I/R injury was better than either alone. Taken together, we provided solid evidence to demonstrate that the combination of LRIP and PF had potential to alleviate cerebral I/R injury, which was regulated by MyD88-TRAF6-p38 MAPK pathway and neutrophil NADPH oxidase pathway.
摘要：
背景：肢体远隔缺血后处理（LRIP）和芍药苷（PF）均可改善脑缺血再灌注（I/R）损伤。目前,LRIP联合PF能否取得更好的治疗效果未知。
目的：本研究探讨LRIP联合PF对大鼠脑缺血再灌注损伤的缓解作用及机制。
方法：除Sham组外，对大鼠进行大脑中动脉阻塞（MCAO）手术。然后PF（2.5mg/kg，5mg/kg，10mg/kg)于再灌注开始前10min腹腔打针给药。再灌注0h左股动脉行LRIP手术。行为测试用于评估神经功能缺损，而TTC染色用于检查梗死体积。Westernblot检测大鼠外周血中性粒细胞中MyD88，TRAF6，p38-MAPK的蛋白表达和p47phox的磷酸化。LRIP联合PF后，提取大鼠骨髓中性粒细胞，与大鼠血清孵育24h。p38MAPK抑制剂组给予SB203580，烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂组给予Apocynin。用fMLP(10μM)刺激嗜中性粒细胞。活性氧（ROS）产生及MyD88、TRAF6、p38MAPK蛋白表达，和p47phox（ser304和ser345）被检测到。
结果：LRIP联合PF（5mg/kg）可减少脑梗死体积，改善神经功能缺损评分（NDS），减少fMLP刺激的ROS释放并下调中性粒细胞中MyD88，TRAF6，p38-MAPK的蛋白表达和p47phox（ser304和ser345）的磷酸化。
结论：LRIP联合PF对脑I/R损伤的保护作用优于单独使用。一起来看,我们提供了确凿的证据证明LRIP和PF的组合具有减轻脑I/R损伤的潜力，受MyD88-TRAF6-p38MAPK通路和中性粒细胞NADPH氧化酶通路的调控。
目的：本研究探讨LRIP联合PF对大鼠脑缺血再灌注损伤的缓解作用及机制。
方法：除Sham组外，对大鼠进行大脑中动脉阻塞（MCAO）手术。然后PF（2.5mg/kg，5mg/kg，10mg/kg)于再灌注开始前10min腹腔打针给药。再灌注0h左股动脉行LRIP手术。行为测试用于评估神经功能缺损，而TTC染色用于检查梗死体积。Westernblot检测大鼠外周血中性粒细胞中MyD88，TRAF6，p38-MAPK的蛋白表达和p47phox的磷酸化。LRIP联合PF后，提取大鼠骨髓中性粒细胞，与大鼠血清孵育24h。p38MAPK抑制剂组给予SB203580，烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂组给予Apocynin。用fMLP(10μM)刺激嗜中性粒细胞。活性氧（ROS）产生及MyD88、TRAF6、p38MAPK蛋白表达，和p47phox（ser304和ser345）被检测到。
结果：LRIP联合PF（5mg/kg）可减少脑梗死体积，改善神经功能缺损评分（NDS），减少fMLP刺激的ROS释放并下调中性粒细胞中MyD88，TRAF6，p38-MAPK的蛋白表达和p47phox（ser304和ser345）的磷酸化。
结论：LRIP联合PF对脑I/R损伤的保护作用优于单独使用。一起来看,我们提供了确凿的证据证明LRIP和PF的组合具有减轻脑I/R损伤的潜力，受MyD88-TRAF6-p38MAPK通路和中性粒细胞NADPH氧化酶通路的调控。
