System Xc-/GPX4/Nrf2介导的铁死亡对脓毒症大鼠肠道机械屏障的影响

Influence of System Xc-/GPX4/Nrf2-mediated ferroptosis on intestinal mechanical barrier in septic rats

目的 探讨System Xc-/GPX4/Nrf2通路介导的铁死亡对脓毒症大鼠肠道机械屏障损伤程度及炎症状态的影响。方法 24只SPF级雄性大鼠随机分为假手术组、脓毒症组、铁死亡组、治疗组各6只。脓毒症组、铁死亡组、治疗组采用盲肠结扎穿孔法制备脓毒症大鼠模型,假手术组开腹游离盲肠后还纳关腹。造模即刻,治疗组皮下注射去铁胺20 mg/kg,铁死亡组腹腔注射铁死亡激活剂Erastin 20 mg/kg,假手术组和脓毒症组腹腔注射等量生理盐水,注射后4组均皮下注射37℃生理盐水50 mg/kg液体复苏。造模24 h大鼠麻醉后开腹,取十二指肠(空肠),机械法组织匀浆后收集细胞悬液,应用荧光探针法检测小肠平滑肌细胞活性氧(ROS);透射电镜下观察小肠上皮组织中线粒体形态。然后腹主动脉采血离心取血清,采用比色法检测Fe^(3+)、D-乳酸脱氢酶(D-LDH)水平,采用硫代巴比妥酸缩合法检测丙二醛(MDA)水平,采用ELISA法检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6水平。采血后处死大鼠,取小肠组织,采用HE染色观察组织病理学改变,采用免疫组织化学ABC法检测GPX4、Nrf2、Claudin-1阳性面积百分比,采用Western blot法检测GPX4、Nrf2、Claudin-1蛋白相对表达量。结果 (1)治疗组[(56 528±182)×10~5/mL]、脓毒症组[(67 964±844)×10~5/mL]、铁死亡组[(71 958±1 306)×10~5/mL]小肠平滑肌细胞ROS荧光强度均高于假手术组[(52 551±438)×10~5/mL](P<0.05),脓毒症组、铁死亡组高于治疗组(P<0.05),铁死亡组高于脓毒症组(P<0.05)。(2)假手术组小肠上皮组织线粒体形态完整、嵴丰富整齐、双膜结构存在且体积较大;脓毒症组线粒体体积变小,膜信号变强,线粒体嵴松散并发生溶解现象;治疗组线粒体体积较脓毒症组略有恢复,双膜结构部分存在,线粒体嵴少量溶解;铁死亡组线粒体体积较脓毒症组进一步缩小,膜密度增高,线粒体嵴发生溶解及消失。(3)脓毒症组、治疗组、铁死亡组血清Fe^(3+)含量低于假手术组(P<0.05),MDA、D-LDH水平均高于假手术组(P<0.05);脓毒症组、铁死亡组血清Fe^(3+)含量低于治疗组,MDA、D-LDH水平高于治疗组(P<0.05);铁死亡组血清Fe^(3+)含量低于脓毒症组,MDA、D-LDH水平高于脓毒症组(P<0.05)。(4)脓毒症组、治疗组、铁死亡组血清IL-1β、TNF-α、IL-6水平均高于假手术组(P<0.05),脓毒症组、铁死亡组高于治疗组(P<0.05),铁死亡组高于脓毒症组(P<0.05)。(5)假手术组小肠绒毛丰富规整、未见基底层分离及毛细血管充血;脓毒症组绒毛顶端出现溶解、破碎,炎症细胞浸润明显,毛细血管充血;治疗组绒毛顶端部分破裂情况略有好转,但仍有毛细血管充血、炎症细胞浸润;铁死亡组小肠绒毛已出现崩解、脱落,炎症组织浸润明显,部分延伸到绒毛两侧,黏膜与黏膜下层出现分离。(6)脓毒症组、治疗组、铁死亡组Nrf2阳性面积百分比大于假手术组(P<0.05),GPX4、Claudin-1阳性面积百分比小于假手术组(P<0.05);脓毒症组、铁死亡组Nrf2阳性面积百分比大于治疗组,GPX4、Claudin-1阳性面积百分比小于治疗组(P<0.05);铁死亡组Nrf2阳性面积百分比大于脓毒症组,GPX4、Claudin-1阳性面积百分比小于脓毒症组(P<0.05)。(7)脓毒症组、铁死亡组Nrf2蛋白相对表达量高于治疗组,GPX4、Claudin-1蛋白相对表达量均低于治疗组(P<0.05);铁死亡组Nrf2蛋白相对表达量高于脓毒症组,GPX4、Claudin-1蛋白相对表达量均低于脓毒症组(P<0.05)。结论 脓毒症大鼠模型肠道损伤中存在铁死亡现象;抑制System Xc-/GPX4/Nrf2通路介导的铁死亡,可改善脓毒症大鼠肠道机械屏障的损伤程度及炎症状态。

Objective To investigate the influence of ferroptosis mediated by System Xc-/GPX4/Nrf2 pathway on intestinal mechanical barrier injury and inflammatory status in septic rats.Methods Twenty-four SPF male rats were randomly divided into sham operation group,sepsis group,ferroptosis group and treatment group,with 6 rats in each group.Sepsis group,ferroptosis group and treatment group were prepared septic rat models by cecal ligation and perforation,and sham operation group was opened the abdomen which was closed after the cecum was freed.Immediately after modeling,treatment group was subcutaneously injected with deferoxamine 20 mg/kg,ferroptosis group was intraperitoneally injected with ferroptosis activator Erastin 20 mg/kg,and sham operation group and sepsis group were injected with the same amount of normal saline.The rats in four groups were subcutaneously injected with 37℃ normal saline 50 mg/kg for fluid resuscitation.When the rats got anesthesia 24 h after modeling,the abdominal cavity was opened,and the duodenum(jejunum) was taken.The cell suspension was collected after mechnical tissue homogenization.The reactive oxygen species(ROS) in small intestinal smooth muscle cells was detected by fluorescence probe method,and the mitochondrial morphology in small intestinal epithelial tissue was observed under transmission electron microscope.The blood was taken from the abdominal aorta and the serum was separated by centrifugation.The Fe~(3+) and D-lactate dehydrogenase(D-LDH) levels were detected by colorimetry,the level of malondialdehyde(MDA) was detected by thiobarbituric acid condensation method(TAB method),and the levels of serum tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6 were detected by ELISA.The rats were sacrificed after blood collection from the abdominal aorta,and the small intestinal tissues were taken.The histopathological changes were observed by HE staining.The percentages of positive areas of GPX4,Nrf2 and Claudin-1 were detected by immunohistochemical ABC method.The relative expressions of GPX4,Nrf2 and Claudin-1 proteins were detected by Western blot.Results(1) The ROS fluorescence intensity of small intestinal smooth muscle cells was higher in treatment group [(56 528±182) × 10~5/mL],sepsis group [(67 964±844) × 10~5/mL] and ferroptosis group[(71 958±1 306) × 10~5/mL] than that in sham operation group [(52 551±438) × 10~5/mL](P<0.05),was higher in sepsis group and ferroptosis group than that in treatment group(P<0.05),and was higher in ferroptosis group than that in sepsis group(P<0.05).(2) In sham operation group,the mitochondrial morphology of the small intestinal epithelial tissue was complete,the cristae were rich and neat,the double membrane structure existed and the volume was large.In sepsis group,the mitochondrial volume became smaller,the membrane signal became stronger,and the mitochondrial cristae became loose and dissolved.In treatment group,the mitochondrial volume was slightly restored compared with sepsis group,the double membrane structure was partially present,and the mitochondrial cristae were slightly dissolved.In ferroptosis group,the mitochondrial volume was further reduced compared with sepsis group,the membrane density increased,and the mitochondrial cristae dissolved and disappeared.(3) The serum Fe~(3+) content was lower in sepsis group,treatment group and ferroptosis group than that in sham operation group(P<0.05),was lower in sepsis group and ferroptosis group than that in treatment group(P<0.05),and was lower in ferroptosis group than that in sepsis group(P<0.05).The levels of MDA and D-LDH were higher in sepsis group,treatment group and ferroptosis group than those in sham operation group(P<0.05),were higher in sepsis group and ferroptosis group than those in treatment group(P<0.05),and were higher in ferroptosis group than those in sepsis group(P<0.05).(4) The serum levels of IL-1β,TNF-α and IL-6 were higher in sepsis group,treatment group and ferroptosis group than those in sham operation group(P<0.05),were higher in sepsis group and ferroptosis group than those in treatment group(P<0.05),and were higher in ferroptosis group than those in sepsis group(P<0.05).(5) In sham operation group,the small intestinal villi were rich and regular,and there was no basal layer separation and capillary congestion.In sepsis group,the top of the villi was dissolved and broken,inflammatory cell infiltration was obvious,and capillary congestion was observed.In treatment group,the rupture of the top part of the villi was slightly improved,but there was still capillary congestion and inflammatory cell infiltration.In ferroptosis group,the small intestinal villi had disintegrated and fallen off,the inflammatory tissue infiltration was obvious and some extended to both sides of the villi,and the mucosa and submucosa were separated.(6) The percentage of Nrf2 positive area was larger in sepsis group,treatment group and ferroptosis group than that in sham operation group(P<0.05),was larger in sepsis group and ferroptosis than that in treatment group(P<0.05),and was larger in ferroptosis group than that in sepsis group(P<0.05).The percentages of GPX4 and Claudin-1 positive areas were smaller in sepsis group,treatment group and ferroptosis group than those in sham operation group(P<0.05),were smaller in sepsis group and ferroptosis group than those in treatment group(P<0.05),and were smaller in ferroptosis group than those in sepsis group(P<0.05).(7) The relative expression of Nrf2 protein was higher in sepsis group and ferroptosis group than that in treatment group,and was higher in ferroptosis group than that in sepsis group(P<0.05).The relative expressions of GPX4 and Claudin-1 proteins were lower in sepsis group and ferroptosis group than those in treatment group(P<0.05),and were lower in ferroptosis group than those in sepsis group(P<0.05).Conclusions Ferroptosis is found in the septic rats with intestinal injury.To inhibit ferroptosis mediated by System Xc-/GPX4/Nrf2 pathway can decrease the degree of intestinal mechanical barrier injury and inflammatory state in septic rats.