D3T Targeting GPX4 Mediates Ferroptosis And Participates Sepsis-induced Intestinal Barrier Injury In Mice

Objective:The research was designed to investigate the effect of pretreatment with3H-1,2-dithiole-3-thione(D3T)on intestinal barrier injury in septic mice and explore the possible mechanism of ferroptosis in the regulation of intestinal barrier injury induced by sepsis,which may provide new strategy for clinical diagnosis and treatment of sepsis.Methods: Sepsis model was induced by cecal ligation and puncture(CLP)in mice.The expression of GPX4 was up-regulated by using 3H-1,2-dithiole-3-thione(D3T).Thirty-two male C57BL/6J mice were randomly divided into The mice were randomly divided into different groups with 8 mice in each group,including sham operation group(Sham),sham operation treatment group(Sham+ D3T),sepsis group(CLP)and sepsis treatment group(CLP+ D3T).30 minutes before the experiment,the mice in D3 T group were injected with D3 T suspension(dissolved in dimethyl sulfoxide(DMSO),50mg/kg)by intraperitoneal injection,while the mice in the sham operation group and sepsis group were injected with DMSO + PBS at the same dose by intraperitoneal injection.After 20 hours,we observe the survival condition in each group and the detect the contents of serum TNF-α and IL-6 to evaluate the level of inflammation in mice of each group.Partial small intestine was collected to evaluate the intestinal injury by using hematoxylin and eosin(HE)staining.The remaining small intestine was stored for detecting the expression of intestinal tight junction proteins ZO-1,Occludin and ferroptosis related proteins GPX-4,ACSL-4 and SLC7A11/x CT by using Western blotting.In the 72-hour survival observation experiment,64 male C57BL/6J mice were divided into the same groups and treated as follows above,with 16 mice in each group.Only the death of mice in each group was counted and no other treatment was given.Results:1.Survival condition of mice in each group: The mice in CLP and CLP+ D3 T groups occured septic symptoms such as listlessness,lethargy,curling up,piloerection,hypokinesia,antifeedant or hypophagia,and secretion in the eye corners at 12 hours after the operation.Furthermore the above symptoms showed a progressive worsening trend.However,there were no obvious septic symptoms in the Sham group and Sham + D3 T groups.In the 72 hour survival observation test,no mice died in Sham and Sham + D3 T groups.In CLP group,12 mice died,the survival rate was 33% (P<0.05),while there were 5 mice died in CLP+ D3 T group and the survival rate was 68% (P<0.05).2.Histopathological changes in small intestine of mice in each group: At 20 hours after CLP,there was no abnormal histology changes in small intestine of Sham and Sham+ D3 T groups,where showed normal arrangement of intestinal epithelial cells,no edema and inflammatory cell infiltration,and complete and abundant villi under microscope(200X).While in CLP group,there were some histology changes in intestine,including the disappearation of small intestinal epithelial tight junction,the local infiltration of inflammatory cells,the edema and breaks of small intestinal villi.Furthermore,the small intestinal Chiu ’s score significantly increased in CLP group(P<0.01).Compared with CLP group,the mice in CLP+ D3 T group showed more intact intestinal epitheliums,more distinguished villi structure,slight villi edema and less broken villi.Besides,the Chiu ’s score was significantly improved when compared to CLP group(P<0.0001).3.Changes of serum inflammatory factors in mice of each group: The levels of TNF-α and IL-6 in the CLP group were significantly higher than those in the Sham group(P<0.01).The levels of TNF α and IL 6 in CLP+ D3 T group were lower than those in CLP group(P<0.05).4.Changes of intestinal tight junction protein expression in small intestine of mice in each group:Compared with Sham group,the expression of Occludin and ZO-1 in CLP group were downregulated (P<0.01).Compared with CLP group,the expression of Occludin and ZO-1 in CLP +D3 T group were significantly upregulated(P<0.01).5.Changes in expression of ferroptosis-related proteins in small intestine of mice in each group:In CLP group,GPX4 and SLC7A11/xCT protein levels were upregulated(P<0.01),while ACSL4 protein level was significantly downregulated(P<0.01).The expression of GPX4 and SLC7A11/x CT in CLP+ D 3T group was higher than that in CLP group(P<0.01),and the expression of ACSL4 was lower than that in CLP group(P<0.01).Conclusions:D3T can protect the intestinal barrier by reducing the release of inflammatory factors to ameliorate the inflammatory response and alleviating the damage of intestinal barrier induced by sepsis,which may owe to the inhibition of GPX4-mediated ferroptosis.