The Role Of Nrf2 In Ferroptosis Of T Lymphocytes In Sepsis

Objective : The objective of this research is to detect the changes of the important indicators of ferroptosis in T cells as the severity of sepsis varies,in order to determine the relationship between sepsis and ferroptosis,and to study the mechanism of Nrf2 in ferroptosis of T lymphocytes by establishing a mouse model of cecal ligation and perforation(CLP).Methods : A total of 51 C57 mice were divided into 4 groups: survival analysis group,sham operation group,mild sepsis group,severe sepsis group.Survival analysis group was further divided into 3 subgroups: sham operation group,mild sepsis group,severe sepsis group.And at different time points,the manifestation and survival rate of the mice were observed.In the sham operation group,the cecum was returned to the abdominal cavity immediately after the laparotomy.In the mild sepsis group and the severe sepsis group,the cecum and some of the vessels were ligated at 1/3 and 2/3 from the distal end of the cecum,and then the 27 G needle was inserted for 3 times.Then the cecum was returned and the wound was sutured.The blood,peritoneal lavage fluid,spleen tissue,bronchoalveolar lavage fluid and lung tissue were obtained.The number of T cells and the expression levels of iron,transferrin and ferritin in peritoneal lavage fluid and serum were detected.The expression of mRNA of caspase 3,caspase 4,caspase 8,caspase 9,caspase 12,Bcl-1,Bax,CHOP in mononuclear cells of each group was detected by RT-PCR.The contents of ROS and GSH in T cells were detected,and Nrf2 expression in T cells was detected by RT-PCR and Western Blot.Flow cytometry was used to determine the percentage of T cell apoptosis and lung permeability index(LPI)was calculated.Results :(1)the survival rate of mice in each subgroup was analyzed by SPSS software.The results showed that the mortality rate of mice increased significantly with the aggravation of sepsis.(2)The lung permeability index(LPI)was measured and the results showed that LPI in mild sepsis had no significant change compared with the sham operation group,while LPI in severe sepsis was higher than that in sham operation group and mild sepsis group.(3)The expression level of Nrf2 in lung tissue was measured:The expression of Nrf2 protein and its phosphorylation in lung tissue were detected by Western Blot.The results showed that the expression of Nrf2 in mild sepsis group was slightly reduced,and the expression of Nrf2 in the severe sepsis group was significantly higher than that in the sham operation group.Even though the expression of phosphorylated Nrf2 was significantly different among the mice,the expression of phosphorylated Nrf2 in sepsis group was still higher than that in sham operated group.The expression of Nrf2 in mRNA was detected by RT-PCR.The results showed that the expression of Nrf2 in mRNA was significantly increased in septic mice,and that in severe sepsis mice was significantly higher than that in mild sepsis mice.(4)The amount of iron,transferrin and ferritin in the peritoneal lavage fluid of each group were detected.The results showed that all three of them were increased.In mild sepsis group,iron,ferritin and transferrin all increased slightly but not significantly.In severe sepsis group,iron and transferrin have increased significantly,while ferritin increased slightly but not significantly.(5)The number of T cells in the spleen was detected.The results showed that the positive rates of CD3,CD4 and CD8 T cells in the spleen of septic mice were significantly lower than those in sham operation group.Compared with sham operation group,the positive rate of CD4 T cells in mild sepsis group was not statistically significantly increased,while the positive rate of CD4 T cells was increased significantly in severe sepsis group.(6)The amount of iron,transferrin,ferritin in the serum were detected.Results showed that after the mice model of sepsis was established,the amount of iron decreased,and with the aggravation of sepsis,it was decreased more significantly.In serum,ferritin and transferrin in mild sepsis group increased slightly but not significantly,while in severe sepsis group they were significantly increased.(7)To verify whether T cell death was ferroptosis:The expression of caspase 3,caspase 4,caspase 8,caspase 9,mRNA 12,caspase,Bcl-1,Bax,CHOP in mononuclear cells of each group was detected by RT-PCR.The results showed that the expression of each gene in mild sepsis group was slightly increased or had no significant change.In the severe sepsis group,the expression of each gene was significantly higher than that of sham operation group and mild sepsis group,except for caspase 12,which was also increased,but the differences between each sample was significant.The amont of ROS in T cells was increased in CLP mice,and the amont of ROS in T cells of severe sepsis mice was higher than that in mild sepsis mice.The amont of GSH was significantly reduced,and GSH in severe sepsis group was slightly lower than that in mild sepsis group,but not significantly.The expression of Nrf2 was increased,and the expression of Nrf2 in severe sepsis group was higher than that in mild sepsis group.Compared with the sham operation group,the apoptosis rate of T cells in sepsis group was significantly higher than that in sham operation group,and the proportion of T cell apoptosis was increased with the aggravation of sepsis.Conclusion: After establishing the model of cecal ligation and perforation(CLP),glutathione(GSH)synthesis is inhibited,and the oxidative stress system(ROS)is activated,which leads to ferroptosis in T cells and acute lung injury in sepsis mice.With this research,it has been proved that Nrf-2 plays an important role in ferroptosis of T lymphocytes in mice with severe sepsis.