Influence of System Xc-/GPX4/Nrf2-mediated ferroptosis on intestinal mechanical barrier in septic rats
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 Fe3-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)× 105/mL],sepsis group[(67 964±844)× 105/mL]and ferroptosis group[(71 958±1 306)× 105/mL]than that in sham operation group[(52 551±438)× 105/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 Fe3+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.
NETL
NSTL
万方数据
维普
