Heat-killed Salmonella Typhimurium protects mice against carbon ion radiation

Abstract

Objective: Patients receiving carbon-ion radiation therapy and astronauts exploring outer space are inevitably exposed to heavy ion radiation. The aim of this study was to develop radioprotectors to minimize the injuries induced by carbon ion radiation.

Methods: Heat-killed Salmonella Typhimurium (HKST) was administered to mice by gavage prior to irradiation with a ¹²C⁶⁺ heavy ion accelerator. Hematoxylin and eosin staining and immunofluorescence TdT-mediated dUTP Nick-End Labeling staining were used to assess the radioprotective effect of HKST on organ damage and levels of apoptosis, respectively, in mice. To investigate the mechanism underlying the radioprotective effect of HKST, levels of the pro-apoptotic proteins BAX and caspase 3 as well as interferon-regulatory factor (IRF) 3/7 in the femur, testis and intestine were assessed using immunofluorescence.

Results: Injuries induced by carbon ion radiation were significantly eased by pretreatment with HKST. Both apoptosis and high expression levels of pro-apoptotic proteins induced by heavy ion radiation were inhibited by HKST pretreatment. The radioprotective effect of HKST was associated with stimulation of Toll-like receptor signaling mediated by enhanced IRF3 and IRF7 signaling.

Conclusion: HKST was an effective radioprotector alleviating damage to multiple organs caused by heavy ion radiation.

Keywords: Carbon ion radiation; IRF3/7; Toll-like receptors; apoptosis; heat-killed Salmonella Typhimurium; radioprotector.

Figure 1.

Tissue damage induced by carbon ion radiation was alleviated by HKST pretreatment. Mice receiving ¹²C⁶⁺ heavy ion radiation were pretreated with PBS or HKST. Twenty-four hours post irradiation, mice were sacrificed and the protective effects of HKST on femur (a), intestine (b) and testis (c) were assessed by HE staining. Abbreviations: HKST, heat-killed Salmonella Typhimurium; IR, ionizing radiation.

Figure 2.

Post-carbon ion irradiation apoptosis was inhibited by HKST pretreatment. Intestine (a), testis (b) and femur (c) were isolated 24 hours post-irradiation and levels of apoptosis was measured IFTUNEL staining. Abbreviations: Con, control; DAPI, 4′,6-diamidino-2-phenylindole; HKST, heat-killed Salmonella Typhimurium; IR, ionizing radiation; ns, not significant.

Figure 3.

Influence of HKST treatment on the expression level of BAX protein post-carbon ion irradiation. Femur (a), intestine (b) and testis (c) were cut into 3-μm-thick sections, then blocked with bovine serum albumin. Tissues were stained with an anti-BAX antibody followed by a secondary antibody. Levels of BAX protein in tissues were detected by fluorescent microscopy and analyzed using ImageJ software. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; HKST, heat-killed Salmonella Typhimurium; IR, ionizing radiation; ns, not significant.

Figure 4.

Upregulation of caspase 3 protein in different tissues post-carbon ion irradiation was inhibited by HKST pretreatment. Tissues (a: femur, b: intestine, and c: testis) were stained with anti-caspase 3 antibody followed by a secondary antibody. Levels of caspase 3 protein in tissues were assessed by fluorescent microscopy and analyzed using ImageJ software. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; HKST, heat-killed Salmonella Typhimurium; IR, ionizing radiation.

Figure 5.

The IRF3/7 pathways were stimulated by HKST treatment. Tissues (a: femur, b: intestine, and c: testis) were cut into 3-μm-thick sections and stained with anti-IRF3 or -IRF7 antibodies followed by a secondary antibody. Levels of IRF3 or IRF7 protein in tissues were assessed by fluorescent microscopy and analyzed with ImageJ software. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; HKST, heat-killed Salmonella Typhimurium; IR, ionizing radiation; ns, not significant.