Objective To evaluate the role of PI 3 K/Akt/Nrf 2 pathway in electroacupuncture (EA)-induced reduction of acute lung injury induced by endotoxic shock in rabbits. Methods Eighty healthy male New Zealand white rabbits were randomly divided into 8 groups (n=10): normal control group (group C), endotoxic shock-induced ALI group (group L), wortmannin+endotoxic shock-induced ALI group (group WL), wortmannin group (group W), dimethyl sulfoxide (DMSO) group (group D), EA at acupoints+endotoxic shock-induced ALI (group EL), EA at non-acupoints+endotoxic shock-induced ALI (group SEL) and EA at acupoints+wortmannin+endotoxic shock-induced ALI group (group ELW). Bilateral electroacupuncture stimulation of Zusanli and Feishu acupoints for 30 min was performed once daily for 4 d before establishment of the model and during the process of establishment of the model in groups EL and ELW. In group SEL, EA was performed at the points 0.5 cm lateral to the above acupoints with the same parameters. The animals were anesthetized with iv 20% urethane (5 mL/kg), tracheostomized and kept spontaneous breathing. LPS (5 mg/kg) was given via the auricular vein to establish the model of endotoxic shock-induced ALI in groups L, EL, SEL, ELW and WL, while the equal volume of normal saline was given in groups C, D and W. Before 30 min of the model establishment, wortmannin (0.6 mg/kg) was iv injected in groups WL and ELW, the equal volume of wortmannin was given in group W, while the equal volume of DMSO was given in group D as control. The rabbits were sacrificed by bloodletting at 6 h after LPS or normal saline injection. Blood samples were collected and the lung tissue was taken for microscopic examination of pathologic changes, determination of lung W/D ratio, serum levels of TNF-α and IL-10 concentration, activities of SOD and contents of MDA, detection of p-Akt protein, HO-1 protein, Nrf2 nuclear and total protein, as well as HO-1 mRNA and Nrf2 mRNA expression. Results The scores of lung injury in groups L, WL, EL, SEL and ELW were 6.8±0.9, 8.3±1.1, 4.5±0.6, 6.5±0.7 and 5.9±0.9, respectively. The concentration of TNF-α in these groups was (3.14±0.23) ng/mL, (4.62±0.32) ng/mL, (2.03±0.18) ng/mL, (3.18±0.25) ng/mL and (3.96±0.28) ng/mL, respectively. The concents of MDA of lung tissue in these groups were (4.34±0.39) nmol/mg, (6.29±0.54) nmol/mg, (3.09±0.26) nmol/mg, (4.31±0.35) nmol/mg and (5.74±0.47) nmol/mg,respectively. These indexes were higher than those in group C (P<0.05). The concentration of IL-10 in these groups was (5.37±0.47) pg/mL, (3.14±0.25) pg/mL, (6.98±0.54) pg/mL, (5.34±0.43) pg/mL and (5.19±0.39) pg/mL,respectively. The concents of SOD in these groups were (67.6±3.8) U/mg, (42.3±2.6) U/mg, (87.5±5.6) U/mg, (62.3±4.1) U/mg and (57.4±3.5) U/mg,respectively. These indicators were lower than those of group C (P<0.05). The concentrations of p-Akt protein in the lung tissue in these groups were 1.58±0.29, 1.23±0.21, 1.93±0.32, 1.52±0.28 and 1.46±0.24,respectively. The HO-1 protein expression was 0.67±0.09, 0.48±0.07, 1.05±0.12, 0.69±0.08 and 0.74±0.09. The Nrf2 protein expression was 0.84±0.11, 0.61±0.09, 1.37±0.18, 0.89±0.12 and 0.94±0.13. And the total protein of Nrf2 expression was 2.14±0.43, 1.82±0.32, 3.25±0.58, 2.18±0.45 and 2.46±0.49. The expression of these indicators was higher than that of group C (P<0.05). Compared with group L, the lung W/D ratio, lung injury scores, serum TNF-α concentration and MDA contents were significantly decreased(P>0.05), while the serum IL-10 levels and activities of SOD were increased, the expression of p-Akt protein, HO-1 protein, Nrf2 nuclear and total protein was up-regulated in group EL, and the lung W/D ratio, lung injury scores, serum TNF-α concentration and MDA contents were significantly increased (P<0.05), while the serum IL-10 levels and activities of SOD were decreased, the expression of p-Akt protein, HO-1 protein, Nrf2 nuclear and total protein was down-regulated in groups WL and ELW (P<0.05). Compared with group EL, the lung W/D ratio, lung injury scores, serum TNF-α concentration and MDA contents were significantly increased (P<0.05), while the serum IL-10 levels and activities of SOD were decreased, the expression of p-Akt protein, HO-1 protein, Nrf2 nuclear and total protein was down-regulated in group ELW (P<0.05). Compared with group ELW, the lung W/D ratio, lung injury scores, serum TNF-α concentration and MDA contents were significantly increased (P<0.05), while the serum IL-10 levels and activity of SOD were decreased, the expression of p-Akt protein, HO-1 protein, Nrf2 nuclear and total protein was down-regulated in group WL (P<0.05). Conclusion PI3K/Akt/Nrf2 pathway mediated reduction of endotoxic shock-induced ALI by electroacupuncture of Zusanli and Feishu acupoints in rabbits.%目的:评价磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/核因子E2相关因子2(Nrf2)信号通路在电针刺减轻兔内毒素休克诱发急性肺损伤中的作用.方法:健康清洁级雄性新西兰大白兔80只,随机分成8组(n=10):对照组(C组)、内毒素休克诱发急性肺损伤模型组(L组)、渥曼青霉素+模型组(WL组)、渥曼青霉素组(W组)、二甲基亚砜组(D组)、模型+电针刺组(EL)、模型+电针刺激非穴位组(SEL)、模型+电针刺+渥曼青霉素组(ELW).EL组、ELW组于模型制备前1~4 d及制备过程中电针刺激兔双侧足三里和肺俞穴,SEL组用同样方法电针刺激足三里和肺俞穴旁开0.5 cm处.L组、WL组、EL组、SEL组和ELW组经耳缘静脉注射脂多糖(LPS)5 mg/kg,C组、W组和D组给予等容量生理盐水.WL组、W组和ELW组在模型制备前30 min静注渥曼青霉素0.6 mg/kg,D组静注0.08 mL/kg DMSO, C组、L组静注生理盐水0.08 mL/kg.注射LPS或生理盐水后6 h,采集动脉血,检测TNF-α和IL-10浓度,处死动物后取肺组织,进行病理学损伤评分,测定SOD活性及MDA含量及检测p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白的表达.结果:L组、WL组、EL组、SEL组和ELW组肺损伤评分分别为(6.8±0.9)、(8.3±1.1)、(4.5±0.6)、(6.5±0.7)、(5.9±0.9),血清TNF-α浓度分别为(3.14±0.23) ng/mL、(4.62±0.32) ng/mL、(2.03±0.18) ng/mL、(3.18±0.25) ng/mL、(3.96±0.28) ng/mL,以及肺组织MDA含量分别为(4.34±0.39) nmol/mg、(6.29±0.54) nmol/mg、(3.09±0.26) nmol/mg、(4.31±0.35) nmol/mg、(5.74±0.47) nmol/mg,均较C组升高,IL-10浓度分别为(5.37±0.47) pg/mL、(3.14±0.25) pg/mL、(6.98±0.54) pg/mL、(5.34±0.43) pg/mL、(5.19±0.39) pg/mL,以及SOD含量分别为(67.6±3.8) U/mg、(42.3±2.6) U/mg、(87.5±5.6) U/mg、(62.3±4.1) U/mg、(57.4±3.5) U/mg,较C组降低,肺组织p-Akt蛋白分别为(1.58±0.29)、(1.23±0.21)、(1.93±0.32)、(1.52±0.28)、(1.46±0.24),HO-1蛋白分别为(0.67±0.09)、(0.48±0.07)、(1.05±0.12)、(0.69±0.08)、(0.74±0.09),Nrf2核蛋白分别为(0.84±0.11)、(0.61±0.09)、(1.37±0.18)、(0.89±0.12)、(0.94±0.13),以及总蛋白分别为(2.14±0.43)、(1.82±0.32)、(3.25±0.58)、(2.18±0.45)、(2.46±0.49),表达水平较C组上调(P<0.05),W组、D组上述各指标差异无统计学意义(P>0.05);与L组比较,EL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量降低,而IL-10浓度及SOD活性升高,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平上调,WL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P<0.05),而SEL组上述各指标差异无统计学意(P>0.05);与EL组比较,ELW组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P<0.05);与ELW组比较,WL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P<0.05).结论:PI3K/Akt/Nrf2信号通路介导电针刺激足三里和肺俞穴减轻兔内毒素休克诱发急性肺损伤的作用,机制可能与上调HO-1表达有关.
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