摘要
目的探究瑞马唑仑通过间隙蛋白43(connexin-43,CX43)调控铁死亡减轻小鼠肠缺血再灌注(ischemia-reperfusion,I/R)损伤。方法利用分子对接技术预测瑞马唑仑与CX43蛋白的结合亲和力。采用SPF级健康成年雄性C57BL/6J小鼠72只,6~8周龄,体质量20~25 g。实验一,采用随机数字表法将30只小鼠分为(n=6):假手术组(S组)、I/R组(I/R1组)、I/R+瑞马唑仑10 mg/kg组(RM10组)、I/R+瑞马唑仑20 mg/kg组(RM20组)、I/R+瑞马唑仑40 mg/kg组(RM40组);实验二,采用随机数字表法将30只小鼠分为(n=6):I/R组(I/R2组)、Erastin组(E组)、I/R+瑞马唑仑40 mg/kg组(RM40-2组)、I/R+Fer-1组(Fer-1组)、Erastin+瑞马唑仑40 mg/kg组(ERM组);实验三,采用随机数字表法将12只小鼠分为(n=6):I/R+RM+oe-NC组(oe-NC组)和I/R+RM+oe-CX43组(oe-CX43组)。Fer-1组于再灌注前1 h腹腔注射Fer-15 mg/kg,E组于造模前1 d腹腔注射Erastin 10 mg/kg,各剂量瑞马唑仑组、oe-NC组及oe-CX43组于造模前30 min尾静脉注射瑞马唑仑,oe-NC组和oe-CX43组分别于给瑞马唑仑前48 h尾静脉注射空载载体病毒及过表达CX43载体病毒。采用夹闭肠系膜上动脉45 min再灌注30 min的方法构建小鼠肠I/R损伤模型。光镜下观察小肠组织病理学变化并行Chiu’s评分评估肠黏膜损伤程度;比色法检测Fe^(2+)、总铁、丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)、超氧化物歧化酶(superoxide dismutase,SOD)的含量;DHE探针测定活性氧(reactive oxygen species,ROS)含量;RT-qPCR检测铁死亡相关基因表达;Western blot法检测CX43、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)的表达水平。结果分子对接结果表明,瑞马唑仑与CX43蛋白结合能为-6.699 kcal/mol。与S组相比,I/R1组Chiu’s评分升高,CX43表达上调(P<0.05),Fe^(2+)、总铁、ROS、MDA含量升高(P<0.05),GPX4和SLC7A11表达下调(P<0.05),肠道组织学损伤程度加重。与I/R组相比,RM40组Chiu’s评分降低;RM40组和Fer-1组CX43表达均显著下调(P<0.05),Fe^(2+)、总铁、ROS和MDA含量降低(P<0.05),GPX4和SLC7A11表达上调(P<0.05),肠道组织学损伤程度减轻。与E组相比,ERM组CX43表达下调(P<0.05),Fe^(2+)、总铁、ROS、MDA含量降低(P<0.05),GPX4和SLC7A11表达上调(P<0.05),肠道组织学损伤程度减轻。与oe-NC组相比,oe-CX43组CX43表达上调(P<0.05),Fe^(2+)、总铁、ROS、MDA含量升高(P<0.05),GPX4和SLC7A11表达下调(P<0.05),肠道组织学损伤程度加重。结论瑞马唑仑通过下调CX43表达抑制铁死亡减轻肠I/R损伤。
Objective To investigate whether remimazolam attenuates intestinal ischemia-reperfusion(I/R)injury in mice by regulating ferroptosis through connexin-43(CX43).Methods Molecular docking was applied to predict the binding affinity of remimazolam to CX43.A total of 72 SPF-grade adult male C57BL/6J mice(6~8 weeks old,weighing 20~25 g)were subjected.Thirty of them were randomly divided into sham operation group(Sham group),I/R group 1,and I/R+10,20 and 40 mg/kg remimazolam groups(RM10,RM20 and RM40 groups),with 6 mice in each group.Another 30 mice were randomly assigned into 5 groups(n=6),I/R group 2,erastin group(E group),I/R+40 mg/kg remimazolam group 2(RM40 group 2),I/R+Fer-1 group(Fer-1 group),and erastin+40 mg/kg remimazolam group(ERM group).The left 12 mice were randomly and equally grouped into I/R+RM+oe-NC group(oe-NC group)and I/R+RM+oe-CX43 group(oe-CX43 group).The Fer-1 group was given an intraperitoneal injection of 5 mg/kg Fer-1 in 1 h prior to reperfusion,the E group was given 10 mg/kg erastin intraperitoneally 1 d before modeling,and all the remimazolam groups,the oe-NC group and the oe-CX43 group were injected intravenously with corresponding doses of remimazolam 30 min pre-modeling,while the oe-NC and oe-CX43 groups were injected with empty vector virus and overexpression of CX43 vector virus,respectively,48 h before the administration of remimazolam.A mouse intestinal I/R injury model was constructed by clamping the superior mesenteric artery for 45 min and reperfusion for 30 min.The small intestine tissues were harvested and observed for pathological changes,and the intestinal mucosal damage was assessed with Chiu’s score.The contents of Fe^(2+),total iron,malondialdehyde(MDA),glutathione(GSH),and superoxide dismutase(SOD)were detected by colorimetric assay;the production of reactive oxygen species(ROS)was determined by DHE probe;the expression of ferroptosis-related genes was determined by RT-qPCR;and the expression levels of CX43,GPX4,and SLC7A11 were detected by Western blotting.Results Molecular docking indicated that remimazolam had a binding energy of-6.699 kcal/mol with CX43 protein,suggesting good binding affinity between them.Compared with the Sham group,the I/R group 1 showed increases in Chiu’s scores and CX43 expression(P<0.05),along with pathological damage to intestinal tissues,and elevated contents of Fe^(2+),total iron,ROS and MDA(P<0.05),and down-regulated GPX4 and SLC7A11(P<0.05).Compared with the I/R group 1,Chiu’s score was reduced in the RM40 group,and CX43 was significantly down-regulated(P<0.05),contents of Fe^(2+),total iron,ROS,and MDA were decreased(P<0.05),and expression levels of GPX4 and SLC7A11 were enhanced(P<0.05),and severity of intestinal histological damage was attenuated in both the RM40 and Fer-1 groups.Compared with the E group,the ERM group had the decreases in CX43 expression level(P<0.05),Fe^(2+),total iron,ROS,and MDA contents(P<0.05),and increases in GPX4 and SLC7A11 expression levels(P<0.05),with the improvement in intestinal tissue.Compared with the oe-NC group,overexpression of CX43 resulted in the increased CX43 expression,elevated contents of Fe^(2+),total iron,ROS and MDA(P<0.05)and decreased expression of GPX4 and SLC7A11(P<0.05),leading to the exacerbated injury in intestinal tissue.Conclusion Remimazolam attenuates intestinal I/R injury by inhibiting ferroptosis through down-regulating CX43 expression.
作者
孔令国
冷玉芳
马小杰
慕佳璐
KONG Lingguo;LENG Yufang;MA Xiaojie;MU Jialu(First Clinical Medical College of Lanzhou University,Lanzhou,Gansu;Department of Anaesthesiology,Gansu Provincial Maternity and Child Health Hospital,Lanzhou,Gansu;Department of Anesthesiology,First Hospital of Lanzhou University,Lanzhou,Gansu,China)
出处
《陆军军医大学学报》
北大核心
2025年第15期1771-1781,共11页
Journal of Army Medical University
基金
国家自然科学基金地区科学基金项目(82260381,82460382)。
