Maresin1通过Sirt1抑制Caspase11/GSDMD通路减轻小肠缺血再灌注损伤的研究

Maresin1 alleviates small intestinal ischemia-reperfusion injury by inhibiting the Caspase11/GSDMD pathway via Sirt1

摘要:

目的 探讨巨噬素1（Maresin1，Mar1）在小鼠肠缺血再灌注（ischemia-reperfution，IR）中的作用及其可能机制。方法 通过夹闭肠系膜上动脉（superior mesenteric artery，SMA）建立小肠IR模型。第一部分①组12只小鼠随机分为Control组、IR组和IR+Mar1组。第二部分②组20只小鼠随机分为Control组、IR组、IR+Mar1组、IR+EX527组、IR+Mar1+EX527组。Mar1于术前30 min采用5 μg/kg腹腔注射。EX527于术前1 d 10 mg/kg经腹腔注射。Control组仅分离SMA而不夹闭。其余模型组均用无损伤血管夹夹闭SMA根部，45 min后松开血管夹形成小肠IR模型。各组小鼠均于再灌注后4 h采集静脉血和回肠标本。检测①组小鼠肠组织超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）含量；检测血清4 kd荧光素异硫氰酸酯-葡聚糖（FITC-Dextran 4000，FD-4）含量；免疫荧光染色检测肠Occludin蛋白表达，HE染色观察肠组织病理形态。Western blot检测①、②组肠组织Sirt1、P-NF-κB p65（P-p65）、Caspase11、GSDMD-N蛋白表达。结果 ①组中与Control组相比，IR组肠组织MDA水平，血清FD-4含量，病理学损伤程度均升高（P<0.01）；SOD、GSH水平，Sirt1蛋白表达下降；P-p65、Caspase11、GSDMD-N蛋白表达增加。与IR组比较，IR+Mar1组肠组织MDA水平及血清FD-4含量，病理组织学损伤程度均下降（P<0.05）；SOD、GSH水平，Sirt1蛋白表达增加；P-p65、Caspase-11、GSDMD-N蛋白表达减少。②组中与IR+Mar1组比较，IR+Mar1+EX527组Sirt1蛋白表达下降；P-p65、Caspase11、GSDMD-N蛋白表达增加。而IR+EX527组与IR+Mar1+EX527组蛋白表达差异无统计学意义。结论 Mar1预处理通过Sirt1抑制Caspase11/GSDMD通路减少肠组织缺血再灌注损伤。

Abstract:

Objective To investigate the role and possible mechanisms of Maresin1（Mar1） in intestinal ischemia-reperfusion（IR） in mice.Methods Clamping of the superior mesenteric artery（SMA） was performed to establish a model of small intestinal IR. In the first part of the experiment，12 mice were randomly divided into Control group，IR group，and IR+Mar1 group，and in the second part，20 mice were randomly divided into Control group，IR group，IR+Mar1 group，IR+EX527 group，and IR+Mar1+EX527 group. Mar1 5 μg/kg was injected intraperitoneally at 30 min before surgery，and EX527 10 mg/kg was injected intraperitoneally at 1 day before surgery. In the control group，the SMA was isolated without clamping，and in the other model groups，the root of the SMA was clamped with a damage-free vascular clip，which was released after 45 min to establish a model of small intestinal IR. Venous blood and ileal specimens were collected at 4 hours after reperfusion in all groups. For the first part of the experiment，the levels of superoxide dismutase（SOD），malondialdehyde（MDA），and glutathione（GSH） in the intestinal tissue of each group were measured，as well as the serum level of FITC-Dextran 4000（FD-4）； immunofluorescent staining was used to measure the protein expression level of intestinal Occludin；HE staining was used to observe the pathological morphology of intestinal tissue. For the first and second parts of the experiment，western blot was used to measure the protein expression levels of Sirt1，P-NF-κB p65（P-p65），Caspase11，and GSDMD-N in intestinal tissue.Results In the first part of the experiment，compared with the Control group，the IR group had significant increases in the level of MDA in intestinal tissue，the content of FD-4 in serum，and the degree of pathological damage（P<0.01），significant reductions in the protein expression levels of SOD，GSH，and Sirt1，and significant increases in the protein expression levels of P-p65，Caspase11，and GSDMD-N； compared with the IR group，the IR+Mar1 group had significant reductions in the level of MDA in intestinal tissue，the content of FD-4 in serum，and the degree of pathological damage（P<0.05），significant increases in the protein expression levels of SOD，GSH，and Sirt1，and significant reductions in the protein expression levels of P-p65，Caspase11，and GSDMD-N. In the second part of the experiment，compared with the IR+Mar1 group，the IR+Mar1+EX527 group had a significant reduction in the protein expression level of Sirt1 and significant increases in the protein expression levels of P-p65，Caspase11，and GSDMD-N，while there was no significant difference in the expression of proteins between the IR+EX527 group and the IR+Mar1+EX527 group.Conclusion Mar1 pretreatment can alleviate small intestinal IR injury by inhibiting the Caspase11/GSDMD pathway via Sirt1.