| Objective To explore the influence of intestinal flora on ferroptosis and its upstream mechanism,and to provide potential therapeutic targets for clinic.Methods(1)C57BL/6 mice aged 6 to 8 weeks and weighing about 25 g were screened and randomly grouped.Firstly,animal models were established according to the progressive relationship of experimental purposes:Group 1: Control group and Radiotherapy group;Group 2: Control group,Vehicle group,ferroptosis agonist RSL-3 group,ferroptosis inhibitor Liproxstatin-1 group and RSL-3+Liproxstatin-1 group.The mice in ferroptosis agonist RSL-3 group were intraperitoneally injected with 10 mg/kg RSL-3.The mice in the ferroptosis inhibitor Liproxstatin-1 group were intraperitoneally injected with 2.5 mg/kg Liproxstatatin-1 2days before radiotherapy.The mice in RSL-3+Liproxstatin-1 group were intraperitoneally injected with RSL-3 10 mg/kg and Liproxstatin-1 2.5 mg/kg daily 2 days before radiotherapy;Group 3: Mice were divided into control group,Vehicle group,anti-bacterial group and anti-fungal group.The anti-bacterial group was fed metronidazole 1 mg/ml,vancomycin 0.5mg/ml,streptomycin 0.5 mg/ml,ampicillin 0.5 mg/ml for 2 weeks,and the anti-fungal group was given fluconazole 2 mg/ml intraperitoneal injection for 2 weeks.Group 4: The mice were divided into control group,Vehicle group and Troglitazone group.Troglitazone group was given intraperitoneal injection of Troglitazone 12 hours before radiotherapy.Group 5: Mice were divided into control group,Vehicle group and Troglitazone group.The mice in Troglitazone group were continuously observed for 14 days after radiotherapy,and mice in Troglitazone group were given Troglitazone intraperitoneal injection 12 hours before radiotherapy.(2)The mice were anesthetized with ether and fixed on the foam board,and the whole body was exposed to radiation.Irradiation with Varian 23 EX linear accelerator device,the irradiation dose is 14 Gy and the dose rate is 400 Mu/min.(3)RNA sequencing was performed on liver,lung and intestinal tissues of mice,and the differentially expressed genes were obtained by analyzing the sequencing results.At the same time,total RNA was extracted from tissues for quantitative PCR(RT-qPCR)to further verify the RNA sequencing analysis results.(4)Mouse tissues were stained by Hematoxylin & Eosin staining(HE),then observed under a dehydrating sealed microscope and quantified evidence of intestinal mucosal damage(0 = none,1 = mild,2 = moderate,3 = high).The histological severity of radiation intestinal injury was evaluated by the degree of epithelial structure maintenance,recess injury,vascular dilatation,and inflammatory cell infiltration in lamina.(5)The positive staining was quantified objectively by immunohistochemistry,and the differences of each group were observed and compared under the microscope.(6)The mice fasted for 4 hours and then injected FITC-glucan(4 k Da,600mg/kg)into the esophagus by gavage.Fluorescence values of each group were detected by fluorescent enzyme label.(7)MDA content in intestinal tissue of mice was determined by using Lipid Peroxidation(MDA)kit(ab118970)to monitor lipid peroxidation.(8)GSH/GSSG radio in intestinal tissue of mice was determined by using GSH/GSSG Radio Detection Assay kit(ab138881)to determinate cell redox state.Results(1)(Group 1)HE staining results showed that compared with the control group,the radiotherapy group mice suffered serious tissue damage,the lung showed alveolar septum thickening and a large number of inflammatory cell infiltration,the liver showed hepatocyte necrosis and balloonlike degeneration,and the intestine showed intestinal recess disorders and mucosal edema.To systematically investigate the underlying mechanisms of IR-induced acute tissue injury,RNA sequencing of liver,lung,and intestinal tissues was performed to compare transcriptome changes between control and irradiated mice.The results showed that transcription profiles of liver,lung and intestine were significantly different.Compared with lung and liver tissues,the expression of genes that promote ferroptosis was significantly increased in intestinal tissues,while the expression of genes that inhibit ferroptosis was significantly decreased.Further RT-qPCR studies confirmed the results.Compared with lung and liver tissues after radiotherapy,the ratio of reduced glutathione/oxidized glutathione(GSH/GSSG)in intestinal tissues was significantly decreased,and malondialdehyde(MDA)was significantly increased.(2)(Group 2)HE staining results showed that compared with Vehicle group,intestinal tissue in RSL-3 group was significantly damaged.In contrast,intestinal tissue damage was significantly reduced in the Liproxstatin-1 group.The tissue damage score of RSL-3+ Liproxstatin-1 group was lower than that of RSL-3 group and higher than that of Liproxstatin-1 group.Ki67 staining analyzed these cell vitality in accordance with the results of the organization.GSH/GSSG and MDA were determined at the same time.The results showed that the ratio of GSH/GSSG in RSL-3 group was significantly decreased and MDA was significantly increased compared with that in Vehicle group,while that in Liproxstatin-1 group was the opposite,and RSL-3+Liproxtatin-1 group was in between.In addition,intestinal permeability was analyzed in these groups.Intestinal permeability was significantly higher in mice treated with RSL-3 compared to the Vehicle group.However,in mice treated with RSL-3+ Liproxstatin-1,intestinal permeability was lower than in the RSL-3 group,and Liproxstatin-1 group had the lowest intestinal permeability.(3)(Group 3)The expression of genes related to ferroptosis was significantly changed in the intestinal tissues of anti-bacterial and anti-fungal groups.The expression of GPX4,which promotes ferroptosis,was increased,while the expression of ACSL4,which inhibits ferroptosis,was decreased.HE staining showed that intestinal tissue damage was significantly reduced in the anti-bacterial and anti-fungal groups compared with the Vehicle group.Ki67 immunohistochemical staining showed a significant increase in Ki67+ cells in the antifungal and antibacterial groups compared with the Vehicle group.In addition,the GSH/GSSH ratio in the treatment group was significantly higher than that in the Vehicle group,and MDA was significantly lower than that in the Vehicle group.(4)(Group 4)HE staining showed that compared with Vehicle group,Liproxstatin-1 group had less tissue damage.Immunohistochemistry showed that Liproxstatin-1 treatment resulted in increased staining of Ki67.The GSH/GSSH ratio in Liproxstatin-1 group increased significantly,while MDA decreased significantly.(5)(Group 5)We found that the intestinal damage caused by radiotherapy was most obvious on day 3,and the tissue damage gradually recovered on day 7 and day 14.Similar results were obtained when MDA content was detected.Liproxstatin-1 significantly reduced tissue damage and lipid peroxidation on days 3 and 7,and intestinal tissue was almost normal on day 14.Conclusions Radiotherapy can affect the transcriptome of liver,lung and intestine in mice and induce ferroptosis.Intestinal bacteria and fungi can promote ferroptosis by inducing the expression of ACSL4,thus aggravating the radiation damage of intestine.ACSL4 may be a potential target for the treatment of radiation enteritis. |
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