Study On The Mechanism Of The Effect Of Microbiota Derived Cognitive Impairment Via Ferroptosis In CIRI Mic

Objective: To explore the cognitive impairment of antibiotics induced microbiota disorder in cerebral ischemia-reperfusion mice,and to explore the mechanism of microbial metabolite trimethylamine oxide in iron death of neurons.Methods: Firstly,48 8-week-old male C57/BL-6 mice were randomly divided into control group(CON),antibiotic treatment group(ABX),cerebral ischemia-reperfusion group(CIR)and antibiotic + cerebral ischemia-reperfusion group(ACIR).Four weeks after the administration of mixed antibiotics with drinking water,the feces of mice were collected for intestinal microbiological analysis and TMAO detection,and then bilateral carotid artery clamping was used to establish the model.The neurological deficit was evaluated by Longa score one day after operation,and the cognitive behavior function of mice was detected by water maze and new object recognition test three days after operation.Then,48 8-week-old male C57/BL-6mice were randomly divided into control group(CON),cerebral ischemia-reperfusion group(CIR),TMAO intraperitoneal injection + cerebral ischemia-reperfusion group(TMAO)and celecoxib intraperitoneal injection +TMAO+ cerebral ischemia-reperfusion group(CELE).TMAO was given one week before operation,and celecoxib was given 3 days before operation,once a day for 1 week respectively.The cognitive behavior was detected on the third day after operation.immediately after the test,the mice were killed,the blood of ophthalmic artery was taken,the brain tissue was collected and the hippocampal samples were separated.IL-1 β,IL-6 and TNF-α in hippocampal tissue were detected by ELISA,MDA and GSH level in hippocampal tissue was detected by assay kit,and PTGS2 and GPX4 protein expression was detected by Western Blot.Results: Longa score showed that cerebral ischemia-reperfusion(CIR)could cause neurological deficit in mice(compared with CON group,P<0.05),and the use of antibiotics would aggravate this injury(compared with CON group,P<0.01 and CIR group,P<0.05).The behavioral results further confirmed that CIR caused cognitive impairment in mice,and the use of antibiotics further aggravated the cognitive impairment.Compared with CON group,CIR group and ACIR group showed increased latency,decreased times of crossing platform and limited stay time in target area,and decreased object recognition index in water maze and new object recognition experiments(P<0.01).Compared with the CIR group and the ACIR group,the mice in the ACIR group showed longer latency,less times of crossing the platform,limited residence time in the target area,and a further decrease in object recognition index.The results of microbial diversity analysis showed that the use of antibiotics reshaped the microbial composition,structure and species richness of mouse intestines,and significantly decreased the α-diversity and β-diversity of intestinal microorganisms in mice.Lachnospiraceae,Bacteroidaceae and Tannerellaceae were significantly increased in ABX group,while the abundance of Lactobacillaceae decreased significantly.The results of ELISA showed that compared with CON group,the plasma TMAO level of ABX group and ACIR group increased after operation,and the plasma TMAO level of ACIR group further increased compared with ABX group,and the difference was statistically significant.Further studies showed that compared with CIR group,the latency of TMAO group in water maze and new object recognition test increased,the times of crossing the platform and the limited stay time of target area decreased,and the object recognition index decreased after CIR.This cognitive impairment was reversed in CELE group treated with PTGS2 inhibitor celecoxib.ELISA detection of inflammatory factors in hippocampus showed that TNF-α,IL-1 β and IL-6 in CIR group,TMAO and CELE mice were significantly higher than those in CON group.Compared with CIR group,TNF-α,IL-1 β and IL-6 in TMAO group were further increased.Compared with TMAO group,the expression of TNF-α,IL-1 β and IL-6 in CELE group was lower than that in CIR group.Compared with CIR group,the expression of TNF-α in hippocampus of CELE group was not significantly different,but the levels of IL-1 β and IL-6 decreased significantly.These results suggest that CIR upregulates hippocampal inflammatory cytokines and TMAO aggravates inflammation,but it can be partially reversed by PTGS2 inhibitor celecoxib.The detection of key indexes of iron death showed that compared with CON group,the level of GSH in hippocampus of CIR group and TMAO group decreased and the level of MDA increased.There was no significant difference in the level of GSH and MDA in hippocampus of mice in CELE group.Compared with CIR group,the level of GSH in hippocampus of TMAO group decreased and the level of MDA increased.Finally,WB results showed that compared with CON group,the expression of PTGS2 protein in hippocampus of CIR group,TMAO group and CELE group was up-regulated and GPX4 protein expression was down-regulated.Compared with CIR group,the expression of PTGS2 was up-regulated and the expression of GPX4 was down-regulated in TMAO group.There was no significant difference in the expression of PTGS2 and GPX4 in CELE group.Compared with TMAO group,the expression of PTGS2 was down-regulated and the expression of GPX4 was up-regulated in CELE group.These results suggest that CIR can cause iron death in hippocampal neurons of mice,and TMAO aggravates hippocampal iron death and can be partially reversed by celecoxib,an inhibitor of PTGS2.Conclusion: Intestinal flora imbalance is induced by antibiotics.Intestinal metabolite TMAO promotes neuronal iron death through PTGS2 inflammatory pathway and aggravates cognitive impairment after CIRI.Celecoxib,an inhibitor of PTGS2,can alleviate cognitive impairment caused by TMAO.