| 秦佳月,朱志鹏,周红梅,路建,吴城.七氟醚后处理对缺血再灌注大鼠心肌内质网应激反应的影响及可能机制[J].浙江中西医结合杂志,2022,32(11): |
| 七氟醚后处理对缺血再灌注大鼠心肌内质网应激反应的影响及可能机制 |
| Effect of sevoflurane postconditioning on endoplasmic reticulum stress response of myocardium in rats with ischemia-reperfusion and the possible mechanism Qin Jiayue,Zhu Zhipeng, Zhou Hongmei, Liu Jing, Wu Cheng |
| 投稿时间：2022-04-07 修订日期：2022-10-19 |
| DOI： |
| 中文关键词: 七氟醚 内质网应激 缺血再灌注 H9C2细胞 细胞低氧 |
| 英文关键词: |
| 基金项目:浙江省医药卫生科技计划(2019ZD053) |
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| 中文摘要: |
| 摘要 目的 明确七氟醚后处理对缺血再灌注大鼠心肌损伤的保护作用，初步阐明其对心肌内质网应激反应的影响及可能靶点。方法 取SPF级雄性SD大鼠25只，分为对照组（sham）和缺血再灌注组（I/R）。I/R组分别于缺血30min之后进行再灌注2h、4h、6h和8h，制造在体心脏左冠状动脉前降支结扎缺血再灌注动物模型，获取心肌损伤和内质网应激反应最理想模型参数。另取大鼠30只（n=5），随机分为sham组、I/R组、七氟醚后处理组（sevoP，分为2.5%、3%亚组）、4-苯基丁酸（4-PBA）复合七氟醚组（4-PBA +2.5%、3% sevoP组）。sevoP组动物在行微创左冠状动脉前降支结扎后即刻，吸入相应浓度的七氟醚15min后洗脱，松开结扎线恢复血流至额定时间，观测心肌HE形态，血清的乳酸脱氢酶LDH（U/L）、肌钙蛋白cTnI，以及组织的GRP78、CHOP mRNA和蛋白表达，sham组只放线不结扎。利用体外H9C2心肌细胞缺氧/复氧损伤模型（3H/3R），除正常对照组和H/R组外，sevoP组复氧前以2.5%七氟醚处理缺氧箱内H9C2细胞30min，另两组H9C2细胞分别以小干扰肌浆网Ca2+-ATP酶（siSERCA）和阴性对照（ncSERCA）处理48h后进行sevoP同样的操作，检测上清中的上述指标，以及细胞ERS蛋白指标。结果 与sham组相比，I/R组的大鼠心肌缺血0.5h再
灌注2h后血清LDH（3.44 vs5.62）、cTnI（2.72 vs5.64）浓度的急剧升高（P<0.05），GRP78蛋白表达直到缺血0.5h再灌注6h后才增高明显（0.38 vs 2.36）。与对照组鼠相比，I/R组大鼠肺组织GRP78、CHOP的mRNA和蛋白显著高表达，而七氟醚后处理能够有效抑制其高表达，4-PBA能够进一步减弱七氟醚的抑制作用。H9C2细胞的H/R损伤和ERS可以引起GRP78和CHOP的4倍高表达和细胞上清中LDH浓度上升（417 vs 834），以2.5%七氟醚后处理降低过度的升高的GRP78和CHOP（GRP78, 2.46 vs 1.52;CHOP, 2.1 vs 1.32,P<0.05），siSERCA+sevoP+H/R组基因敲低能够减弱七氟醚的保护作用。结论 七氟醚后处理能够抑制心肌的缺血再灌注损伤和过度的ERS，与心肌细胞Ca2+-ATP酶活性有关。 |
| 英文摘要: |
| Effect and the possible mechanism of sevoflurane postconditioning on endoplasmic reticulum stress response of myocardium in rats with ischemia-reperfusion in rats and the possible mechanism Qin Jiayue,Zhu Zhipeng, Zhou Hongmei, Liu Jing, Wu Cheng. Department of anesthesiology, the second affiliated hospital of Jiaxing university, Jiaxing 314000, China
ABSTRACT Objective To clarify the protective effect of sevoflurane postconditioning on ischemic re-perfusion injury of myocardium in rats, the influence on endoplasmic reticulum stress response and even the possible target of endoplasmic reticulum stress responsethis process. Methods Fifty-five SPF SD rats were used in this study. Of which, During the first part, tTwenty-five rats (n=5), male, were randomized divided into control group (sham) and ischemic re-perfusion group (I/R). The I/R group was reflooded at 2h, 4h, 6h and 8h after 30min' ischemic, respectively, to create an animal model of ligation ischemic re-perfusion in front of the left coronary artery of the body heart, and also to obtain the ideal model parameters for myocardial injury and endoplasmic stress response. For the second part, 30 rats were selected (n=5), and randomly divided into sham group, I/R group, sevoflurane postconditioning group (sevoP, divided into 2.5% and 3% subgroup), 4-phenybutyric acid (4-PBA)-sevoflurane complex group (4-PBA group plus 2.5% and 3% sevoP group). SevoP group Aanimals in sevoP group in the line of minimally invasive left coronary artery before the reduction of branch ligation immediately, after inhaling inhaled the corresponding concentration of sevoflurane for 15min after undergoing the minimally invasive ligation of the left anterior descending coronary, and eluted. While loosening the ligation line to restore blood flow for the rated time, the myocardial HE, serum lactic acid dehydrogenase LDH (U/L), troponin cTnI, as well as tissue GRP78, CHOP mRNA and protein expression were examined, except the sham group which only putlack of linesligation. Using the in vitro Hypoxia reoxygenation model (3H/3R), H9C2 cells in the hypoxia tank were treated with 2.5% sevoflurane before reoxygenation for 30min in the sevoP group except the cells in the normal control group and the H/R group. The other two groups of H9C2 cells carry out the same operation of as sevoP after incubation with small interference Ca2+-ATP enzyme (siSERCA) and negative control (ncSERCA) for 48h. Detection of the above indicators in the supernatant and cell lysates were conducted. Results Compared with the sham group, the LDH and cTnI concentrations of in serum increased sharply after 0.5h’ ischemia and 2h's re-perfusion in rats in the I/R group respectively，i.e., 3.44±0.54 vs 5.62±0.68, 2.72±0.61 vs 5.64±1.01(all P<0.05), and GRP78 protein expression increased significantly until 0.5h of ischemic and 6h’re-perfusion of 6h, i.e.,0.38±0.18 vs 2.36±0.49(P<0.05). Compared to the sham group, GRP78 and CHOP mRNA and protein expression level in I/R group dramatically increased, i.e., （1.02±0.27 vs 22.22±8.22，1.01±0.22 vs 14.84±2.49 for mRNA expression，0.59±0.11 vs 2.06±0.36，0.44±0.12 vs 1.97±0.42 for protein expression; all P <0.05）sevoflurane postconditioning can effectively inhibit the significant increase of above indicators in I/R group, which was further reversed by 4-PBA. In H/R cellular experiment, H/R resulted extensive cellular injury such as 4-fold GRP78 and CHOP expression, as well as the LDH high expression (417.6±57.07 vs 834.6±93.7; P<0.05). The Sevoflurane postconditioning inhibits the high GRP78 and CHOP expression of GRP78 and CHOP for H9C2 cells (2.46±0.50 vs 1.52±0.38，2.1±0.43 vs 1.32±0.23; all P <0.05). Moreover, the GRP78 and CHOP expression in siSERCA+sevoP+H/R group went down to normal by the knockdown of SERCA. Conclusion Sevoflurane postconditioning inhibits ischemic re-perfusion damage and excessive ERS in rat myocardium, which is associated with the activity of the Ca2+-ATP enzyme.
KEY WORDS Sevoflurane ；Endoplasmic reticulum stress；Ischemia perfusion；H9C2 cells；cell hypoxia |
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