The development of safe and effective radioprotective agents with minimal side effects, particularly for high-dose exposure, remains a global priority. E0703, a novel steroidal compound structurally derived from estradiol, has shown promising radioprotective efficacy with limited estrogenic activity in prior pharmacodynamic studies. In this study, E0703 was found to significantly increase the abundance of Akkermansia muciniphila (AKK) in the intestines of both irradiated and non-irradiated mice. Co-administration of E0703 and AKK markedly improved the 7-day survival rate of mice exposed to a lethal 8.5 Gy dose of radiation. E0703 induced beneficial transcriptional changes in AKK, with enrichment in metabolic pathways such as amino acid biosynthesis, aminoacyl-tRNA biosynthesis, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis. These alterations supported the production of glucosamine 6-phosphate (GlcN-6-P) by AKK, which contributed to intestinal tissue regeneration following irradiation. Single-cell transcriptomic analysis revealed that E0703 significantly increased the proportion of intestinal stem cells and goblet cells by Day 5 post irradiation. Mechanistically, E0703 modulated the oxidative phosphorylation pathway in these cell types, including regulation of Muc2 production. E0703 also enhanced AKK abundance in irradiated mice, particularly in the presence of mucin, thereby elevating the availability of GlcN-6-Pu2014a critical substrate for intestinal organoid repair. These findings indicate that E0703 exerts direct effects on goblet cells and AKK, promoting hostu2013microbe interactions that facilitate intestinal regeneration and improve survival following radiation exposure.

Abstract The development of safe and effective radioprotective agents with minimal side effects, particularly for high‐dose exposure, remains a global priority. E0703, a novel steroidal compound structurally derived from estradiol, has shown promising radioprotective efficacy with limited estrogenic activity in prior pharmacodynamic studies. In this study, E0703 was found to significantly increase the abundance of Akkermansia muciniphila (AKK) in the intestines of both irradiated and non‐irradiated mice. Co‐administration of E0703 and AKK markedly improved the 7‐day survival rate of mice exposed to a lethal 8.5 Gy dose of radiation. E0703 induced beneficial transcriptional changes in AKK, with enrichment in metabolic pathways such as amino acid biosynthesis, aminoacyl‐tRNA biosynthesis, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis. These alterations supported the production of glucosamine 6‐phosphate (GlcN‐6‐P) by AKK, which contributed to intestinal tissue regeneration following irradiation. Single‐cell transcriptomic analysis revealed that E0703 significantly increased the proportion of intestinal stem cells and goblet cells by Day 5 post irradiation. Mechanistically, E0703 modulated the oxidative phosphorylation pathway in these cell types, including regulation of Muc2 production. E0703 also enhanced AKK abundance in irradiated mice, particularly in the presence of mucin, thereby elevating the availability of GlcN‐6‐P—a critical substrate for intestinal organoid repair. These findings indicate that E0703 exerts direct effects on goblet cells and AKK, promoting host–microbe interactions that facilitate intestinal regeneration and improve survival following radiation exposure.

The development of compounds triggering intestinal stem cells (ISCs) proliferation represents a promising strategy to alleviate irradiation (IR)-induced gastrointestinal syndrome. Here, cannabidiol (CBD)-a nonpsychotomimetic phytocannabinoid derived from the Cannabis sativa plant-was found to dramatically improve body weight loss of mice and stimulate Lgr5+ ISCs proliferation upon a lethal dose of IR. Using absolute quantitative lipidomics, we found that the dysregulation of fatty acids in crypts induced by IR was rescued by CBD, which was indispensable for ISCs regeneration. Integrative analysis of transcriptome and lipidomics unveiled the critical role of PPARα in regulating fatty acid β-oxidation (FAO) by transcriptionally upregulating Slc27a2 and Acox1. Further experiments showed that CBD could trigger the enrichment of Stat2 on the promoter region of Pparα, ultimately facilitating the FAO program and subsequent ISCs proliferation following IR exposure. In addition,THOC3 was identified as a direct target of CBD, which stabilized the THOC3 protein and substantially alleviated the IR-induced blockade of Stat2 mRNA nuclear export. This study reveals a connection between CBD-driven ISCs proliferation and the FAO program during IR damage, providing a promising avenue for IR-induced gastrointestinal syndrome treatment. The binding of CBD to THOC3 maintains its radiation stability, which then supports the nuclear export of Stat2 mRNA for the subsequent transactivation of Pparα. The upregulation of PPARα will ultimately stimulate the FAO program, thereby facilitating ISCs regeneration during IR exposure.

Objective:To investigate the protective effect and underlying mechanism of Eleutheroside E(EE),derived from Acanthopanax senticosus,against γ-ray radiation-induced splenic injury. Methods:The Cell Counting Kit-8 assay was used to determine the toxicity of the drug.Hematoxylin and eosin,terminal deoxynucleotidyl transferase dUTP nick end-labeling,and live/dead cell double staining assays were used to evaluate the protective effects of EE on the spleen.Immunofluorescence,immunohistochemical,and enzyme-linked immunosorbent assays(ELISA)were used to investigate the underlying mechanisms of EE.Cross-talk between the absent in melanoma 2(AI M2)inflammasome and the protein kinase A(PKA)signaling pathway was clarified using selective inhibitors. Results:EE reduced IR-induced splenic injury-the increased apoptosis rates and the levels of γ-H2AX and inflammatory factors in splenic cells were significantly alleviated.ELISA showed that EE reduced the level of interferon-γ(IFN-γ),interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α)in irradiated tissues.The radioprotective effects of EE were mediated by inhibition of the AIM2 inflammasome and stimulation of PKA signaling,in which EE inhibited the increased levels of AIM2,apoptosis-associated speck-like protein containing a CARD(ASC),and cleaved caspase-1 proteins upon ionizing radiation damage but rescued the decreased protein levels in PKA signaling.Suppression of AIM2 signaling was dependent on the activation of PKA signaling by EE treatment. Conclusions:EE exerts a significant radioprotective effect on the spleen in vitro and in vivo.Activation of the PKA signaling pathway leads to AIM2 inflammasome inhibition,thereby attenuating radiation-induced DNA damage.This was demonstrated to be the mechanism involved in the radioprotective effects of EE.Thus,EE can be used as a potential radioprotective drug in clinical practice.

Bone marrow serves as the residence of hematopoietic stem cells and is recognized as one of the most radiosensitive tissues. Exposure to acute radiation leads to severe damage to bone marrow hematopoiesis which can be fatal, while few clinically applicable medication or specific therapeutic targets have been discovered. In this study, we found that the administration of cannabidiol significantly enhanced individual survival and restored the reconstitution capacity of bone marrow hematopoietic stem cells within 14 days after irradiation. Single-cell RNA sequencing analysis demonstrated that the expression levels of genes associated with stemness along with Wnt and BMP signaling pathways were restored by the cannabidiol treatment through the upregulation of Atf2, a transcription factor possessing multifunctional properties. Atf2 upregulation induced by cannabidiol treatment potentially upregulated the expression of Lrp6 to improve the stemness of hematopoietic stem cells. Further functional experiments validated the crucial role of Atf2 in regulating multilineage differentiation potential of bone marrow hematopoietic stem and progenitor cells. Overall, our findings provide evidence for a promising radioprotective function of cannabidiol and Atf2 as a candidate therapeutic target for acute radiation-induced hematopoietic injury, thereby paving the way for future research in the field.

Radiotherapy for malignant tumor treatment and irradiation (IR)-related diagnosis damage lymphocytes, which inevitably suppresses immunity and leads to unwanted clinical outcomes. However, a few agents have been approved by the Food and Drug Administration (FDA) to alleviate IR-induced injury. Here, the radioprotective effect and underlying mechanism of a new steroidal compound optimized from estradiol (E0703) were investigated. Mice were exposed to γ-ray IR to establish an in vivo model of radiation injury, and human peripheral blood B lymphocytes (AHH-1) were employed to investigate injury in lymphocytes. Protein level changes in cell and tissue samples were detected by western blot and immunofluorescence. DNA damage was assessed by the comet assay and γH2AX staining. RNA sequencing was used to screen the critical genes mediating the radioprotective effect of E0703. To determine the direct target of E0703, cellular thermal shift (CETSA), drug affinity responsive target stability (DARTS), molecular docking, and surface plasmon resonance (SPR) assays were adopted. GLI3 transactivation by estrogen receptor β (ERβ) was determined by the chromatin immunoprecipitation (ChIP) assay, while protein interactions were detected by coimmunoprecipitation (Co-IP). IP products were subjected to label-free proteomics assay to screen GLI3 conjugates. E0703 significantly improved survival and tissue injury in mice exposed to IR damage. In lymphocytes, IR-induced DNA damage was ameliorated with E0703 in an ataxia-telangiectasia mutated protein (ATM)-checkpoint kinase 2 (CHK2)-dependent manner. ERβ but not ERα was a direct target of E0703, wherein ERβ enhancement on the promoter region of GLI3 triggered by E0703 could sustain its protein expression. The interaction between GLI3 and eIF4G1 favored by E0703 was critical for the formation of the eIF4F translation–initiation complex. eIF4F assembly was indispensable for the stimulation of ATM-CHK2 signaling involved in DNA damage repair. E0703 alleviated IR-induced DNA damage in lymphocytes by selectively targeting ERβ. The formation of the eIF4F complex in a GLI3-dependent manner was critical for ATM-CHK2 activation triggered by E0703. Our study provides an alternative countermeasure to alleviate IR-induced lymphopenia in individuals undergoing radiotherapy or IR-related diagnosis.

Objective: Patients with colon adenocarcinoma (COAD) who undergo radiation therapy develop radiation enteritis (RE). The predictive value of RE in COAD is yet to be established. Berberine, an active compound derived from the traditional Chinese medicinal plant, Coptis chinensis, has notable anti-inflammatory properties and offers protection to the intestinal mucosa. This study aimed to evaluate the possible therapeutic effect and mechanism of berberine as a treatment for COAD complicated with RE (COAD&RE). Methods: Relevant genetic features of diverse COAD&RE populations were analyzed using bioinformatics and the Cox proportional hazards regression model. The therapeutic targets of berberine were predicted using network pharmacology and molecular docking. In vivo and in vitro experiments were conducted to validate the core genes identified using molecular docking. Results: RE has a certain impact on the prognosis of COAD and berberine may play an important role in the treatment of COAD&RE. In addition, we identified five core therapeutic targets of berberine by network pharmacology and molecular docking: CCND1, MYC, AR, LEP, and CYP19A1. In vivo experiments showed that berberine increased short-term survival rate, body weight, and intestinal epithelial cell recovery in mice after radiation. In an in vitro study, berberine promoted the proliferation of human intestinal epithelial cells and enhanced the radiosensitivity of HT29 cells after radiation, and the relative mRNA expression levels of CCND1 and MYC closely correlated with these effects. Conclusions: This study predicted the potential therapeutic effects of berberine on COAD&RE and verified the relevant mechanisms, which may provide insights and suggestions for the clinical treatment of COAD&RE.

Radiation enteritis remains a major challenge for radiotherapy against abdominal and pelvic malignancies. Nevertheless, there is no approved effective therapy to alleviate irradiation (IR)-induced gastrointestinal (GI) toxicity. In the current study, Cannabidiol (CBD) was found to mitigate intestinal injury by GPX4-mediated ferroptosis resistance upon IR exposure. RNA-sequencing was employed to investigate the underlying mechanism involved in the radio-protective effect of CBD, wherein runt-related transcription factor 3 (RUNX3) and its target genes were changed significantly. Further experiment showed that the transactivation of GPX4 triggered by the direct binding of RUNX3 to its promoter region, or by stimulating the transcriptional activity of NF-κB via RUNX3-mediated LILRB3 upregulation was critical for the anti-ferroptotic effect of CBD upon IR injury. Specially, CBD was demonstrated to be a molecular glue skeleton facilitating the heterodimerization of RUNX3 with its transcriptional chaperone core-biding factor β (CBFβ) thereby promoting their nuclear localization and the subsequent transactivation of GPX4 and LILRB3. In short, our study provides an alternative strategy to counteract IR-induced enteritis during the radiotherapy on abdominal/pelvic neoplasms.

Low-dose radiation has been extensively employed in clinical practice, including tumor immunotherapy, chronic inflammation treatment and nidus screening. However, the damage on the spleen caused by low-dose radiation significantly increases the risk of late infection-related mortality, and there is currently no corresponding protective strategy. In the present study, a novel compound preparation named CB001 mainly constituted of Acanthopanax senticosus (AS) and Oldenlandia diffusa (OD) was developed to alleviate splenic injury caused by fractionated low-dose exposures. As our results show that, white pulp atrophy and the excessive apoptosis in spleen tissue induced by radiation exposure were significantly ameliorated by CB001. Mechanistically, BAX-caspase-3 signaling and nucleotide-binding domain and leucine-rich-repeat-containing family pyrin 3 (NLRP3) inflammasome signaling were demonstrated to be involved in the radio-protective activity of CB001 with the selective activators. Furthermore, the crosstalk between apoptosis signaling and NLRP3 inflammasome signaling in mediating the radio-protective activity of CB001 was clarified, in which the pro-apoptotic protein BAX but not the anti-apoptotic protein Bcl2 was found to be downstream of NLRP3. Our study demonstrated that the use of a novel drug product CB001 can potentially facilitate the alleviation of radiation-induced splenic injury for patients receiving medical imaging diagnosis or fractionated radiation therapy.

Objective: Radiotherapy is used to treat patients with tumors; however, radiation (IR)-induced testicular injury, which has no effective treatment approved in clinical practice, significantly influences their prognosis and quality of life. The protective effects and underlying mechanisms of action of isofraxidin (IF) against IR-induced testicular injury were investigated. Methods: A mouse testis injury model was established using 5 Gy irradiation. Hematoxylin and eosin (H&E) staining, immunofluorescence staining, and enzyme-linked immunosorbent assay were used to measure DNA damage, apoptosis, inflammatory reactions, and oxidative stress in the testes of mice after irradiation. The effectiveness of IF irradiation on testicular injury was evaluated, and the mechanisms of the related oxidative stress and inflammatory response pathways were discussed. Results: IF can improve IR-induced testicular injury by inhibiting the increased levels of DNA damage, apoptosis rate, oxidative stress, and inflammatory factors. The radioprotective effects of IF on testicular injury are mediated by the stimulation of nuclear factor E2-related factor 2 (Nrf2)/heme oxidase-1 (HO-1) or suppression of NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signaling pathways. In addition, crosstalk between the Nrf2/HO-1 and NLRP3 inflammasome signaling pathways was elucidated, in which the inhibition of the NLRP3 inflammasome was mediated by the activation of Nrf2 signaling with IF upon IR exposure. Conclusion: IF can be a potent radioprotective agent to mitigate testicular damage, and may provide a new therapeutic option to alleviate the side effects of radiotherapy in male patients with tumors.

Background : The efficacy of Liangxue Guyuan Yishen Decoction (LGYD), a traditional Chinese medicine, has been scientifically proven in the treatment of radiation-induced intestinal injury (RIII) and preservation of intestinal integrity and function following high-dose radiation exposure. However, further investigation is required to comprehensively elucidate the precise mechanisms underlying the therapeutic effects of LGYD in order to provide potential pharmaceutical options for radiation protection. Purpose This study aims to elucidate the potential mechanism through which LGYD exerts its therapeutic effects on RIII by modulating the gut microbiota (GM). Methods 16s rRNA analysis was employed to assess the impact of varying doses of whole body irradiation (WBI) on GM in order to establish an appropriate model for this study. The effects of LGYD on GM and SCFA were evaluated using 16s rRNA and Quantification of SCFA. UHPLC-QE-MS was utilized to identify the active components in LGYD as well as LGYD drug containing serum (LGYD-DS). Subsequently, immunofluorescence and immunohistochemical staining were conducted to validate the influence of LGYD and/or characteristic microbiota on RIII recovery in vivo. The effects of LGYD-DS, characteristic flora, and SCFA on intestinal stem cell (ISC) were assessed by measuring organoid surface area in intestinal organoid model. Results The species composition and abundance of GM were significantly influenced by whole-body irradiation with a dose of 8.5 Gy, which was used as in vivo model. LGYD significantly improves the survival rate and promotes recovery from RIII. Additionally, LGYD exhibited a notable increase in the abundance of Akkermansia muciniphila (AKK) and levels of SCFA, particularly isobutyric acid. LGYD-DS consisted of seven main components derived from herbs of LGYD. In vivo experiments indicated that both LGYD and AKK substantially enhanced the survival rate after radiation and facilitated the recovery process for intestinal structure and function. In the organoid model, treatment with LGYD-DS, AKK supernatant or isobutyric acid significantly increased organoid surface area. Conclusions LGYD has the potential to enhance RIII by promoting the restoration of intestinal stem cell, which is closely associated with the upregulation of AKK abundance and production of SCFA, particularly isobutyric acid.

Amphotericin B (AmB) is a widely used antifungal agent especially for the therapy of systemic fungal infections. However, the severe side effects of AmB often leads to the premature termination of the treatment. So it is imperative to find the drugs that can both reduce the dosage and enhance the antifungal efficacy of AmB. Here we demonstrated that Nicotinamide (NAM), a cheap and safe vitamin, could enhance the antifungal activities of AmB. We demonstrated the synergistic interaction of NAM and AmB against Candida albicans as well as other Candida spp. and Cryptococcus neoformans. Moreover, NAM could enhance of the activity of AmB against biofilm. This enhancement was also observed in disseminated candidiasis in vivo. Our further study revealed that AmB could induce oxidative damage through the modification of histone acetylation. AmB could inhibit the expression of HST3, an H3K56 deacetylase in C. albicans. The immunoblotting test revealed excessive H3K56ac in AmB-treated fungal cells. Consistantly, the hst3Δ mutant displayed high sensitivity to AmB, while addition of NAM, an H3K56 deacetylation inhibitor, resulted in an even severe inhibition in the growth of this strain. These results indicated that AmB could execute antifungal activity via boosting H3K56ac which was mediated by HST3, and the mechanism for the synergistic interaction of NAM and AmB was based on exacerbating this process, which led to even excessive H3K56ac and oxidative damage. This finding provided theoretical basis for better understanding the antifungal mechanisms of AmB and clinical application of this drug.

Gastrointestinal (GI) toxicity caused by ionizing radiation (IR) is a dose limiting factor in radiotherapy and a great threat for individual nuclear-related military missions. However, there are currently no available strategies to effectively prevent the damage on the intestine induced by IR. In the present study, the protective activity of Heat Killed Salmonella typhimurium (HKST) on intestine against IR was investigated. Through mouse intestinal organoids and whole body irradiation of mice, we found that the pretreatment with HKST significantly preserved the structure of small intestine upon IR exposure and promoted the proliferation of intestinal cells post-IR. Further study revealed that the radioprotective effects of HKST were involved in DNA damage response (DDR) signaling. Moreover, the stimulation of DDR signaling by HKST upon radiation damage was mediated by Wnt signaling, in which the inhibition of Wnt signaling diminished the radioprotective effects of HKST. To sum up, our study suggested HKST as a potential radioprotectant used for prevention of IR-induced GI toxicity.

Intestinal stem cells (ISCs) are essential for the regeneration of intestinal cells upon radiation or chemical agent damage. As for radiation-induced damage, the expression of AIM2, YAP, TLR3, PUMA or BVES can aggravate ISCs depletion, while the stimulation of TLR5, HGF/MET signaling, Ass1 gene, Slit/Robo signaling facilitate the radio-resistance of ISCs. Upon chemical agent treatment, the activation of TRAIL or p53/PUMA pathway exacerbate injury on ISCs, while the increased levels of IL-22, β-arrestin1 can ease the damage. The transformation between reserve ISCs (rISCs) maintaining quiescent states and active ISCs (aISCs) that are highly proliferative has obtained much attention in recent years, in which ISCs expressing high levels of Hopx, Bmi1, mTert, Krt19 or Lrig1 are resistant to radiation injury, and SOX9, MSI2, clusterin, URI are essential for rISCs maintenance. The differentiated cells like Paneth cells and enteroendocrine cells can also obtain stemness driven by radiation injury mediated by Wnt or Notch signaling. Besides, Mex3a-expressed ISCs can survive and then proliferate into intestinal epithelial cells upon chemical agent damage. In addition, the modulation of symbiotic microbes harboring gastrointestinal (GI) tract is also a promising strategy to protect ISCs against radiation damage. Overall, the strategies targeting mechanisms modulating ISCs activities are conducive to alleviating GI injury of patients receiving chemoradiotherapy or victims of nuclear or chemical accident.

Radiation-induced lung injury (RILI) is a common serious complication and dose-limiting factor caused by radiotherapy for lung cancer.This study was to investigate radioprotective effects of grape seed proanthocyanidins (GSP) on normal lung as well as radiosensitizing effects on lung cancer.In vitro, we demonstrated radioprotective effects of GSP on normal alveolar epithelial cells (MLE-12 and BEAS/2B) and radiosensitizing effects on lung cancer cells (LLC and A549).In vivo, we confirmed these two-way effects in tumor-bearing mice.The results showed that GSP inhibited tumor growth, and played a synergistic killing effect with radiotherapy on lung cancer.Meanwhile, GSP reduced radiation damage to normal lung tissues.The two-way effects related to the differential regulation of the MAPK signaling pathway by GSP on normal lung and lung cancer.Moreover, GSP regulated secretion of cytokines IL-6 and IFN-γ and expression of p53 and Ki67 on normal lung and lung cancer.Our findings suggest that GSP is expected to be an ideal radioprotective drug for lung cancer patients who are treated with radiotherapy.

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 12 C 6+ 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.

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 12 C 6+ 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 , heat-killed , Typhimurium , radioprotector , apoptosis , IRF3/7 , Toll-like receptors

With high incidence and mortality, non-small cell lung cancer (NSCLC) represent 85-90% in all lung cancer patients. In addition to surgery and chemotherapy, radiotherapy is an indispensable approach for cancer treatment. However, cellular resistance to ionizing radiation often results in failure in treatment. In this study, we aimed to investigate the role of Sirt3 in radiotherapy on NSCLC. Briefly, survival as well as apoptosis assay were used to determine the cellular radiosensitivity, and an in situ lung cancer model to test the radiosensitivity in vivo. Firstly, our data show that Sirt3 is upregulated in NSCLC cell lines, as well as tissues compared with normal tissues. Then we generated Sirt3 knockdown cells and overexpression cells and found that Sirt3 knockdown increased radiosensitivity, while Sirt3 overexpression caused resistance. Sirt3 knockdown also aggravated the G2/M cell cycle arrest caused by irradiation. Furthermore, Sirt3 was found to be critical for the activation of ATM-Chk2 pathway upon irradiation. Finally, our in vivo model showed that targeting Sirt3 significantly sensitized lung cancer to radiotherapy. In conclusion, our findings identified a significant role of Sirt3 in radioresistanct of NSCLC, which provides novel mechanism as well as target for radiotherapy.

Radiation protection on male testis is an important task for ionizing radiation-related workers or people who receive radiotherapy for tumours near the testicle. In recent years, Toll-like receptors (TLRs), especially TLR4, have been widely studied as a radiation protection target. In this study, we detected that a low-toxicity TLR4 agonist monophosphoryl lipid A (MPLA) produced obvious radiation protection effects on mice testis. We found that MPLA effectively alleviated testis structure damage and cell apoptosis induced by ionizing radiation (IR). However, as the expression abundance differs a lot in distinct cells and tissues, MPLA seemed not to directly activate TLR4 singling pathway in mice testis. Here, we demonstrated a brand new mechanism for MPLA producing radiation protection effects on testis. We observed a significant activation of TLR4 pathway in macrophages after MPLA stimulation and identified significant changes in macrophage-derived exosomes protein expression. We proved that after MPLA treatment, macrophage-derived exosomes played an important role in testis radiation protection, and specially, G-CSF and MIP-2 in exosomes are the core molecules in this protection effect.

As a common serious complication of thoracic radiotherapy, radiation-induced pulmonary fibrosis (RIPF) severely limits radiation therapy approaches. Epithelial-mesenchymal transition (EMT) is a direct contributor to the fibroblast pool during fibrogenesis, and prevention of EMT is considered an effective strategy to inhibit tissue fibrosis. Our previous study revealed that TANK-binding kinase 1 (TBK1) regulates EMT in lung cancer cells. In the present study, we aimed to investigate the therapeutic potential of targeting TBK1 to prevent RIPF and EMT progression. We found radiationinduced EMT and pulmonary fibrosis in normal alveolar epithelial cells and lung tissues. TBK1 knockdown or inhibition significantly reversed EMT in vivo and in vitro and attenuated pulmonary fibrosis and collagen deposition. Moreover, we observed that TBK1 was elevated in a time- and dose-dependent manner by radiation. Meanwhile, radiation also induced time- and dose-dependent activation of AKT and ERK, each of whose inhibitors suppressed radiation-induced EMT. Intriguingly, silencing of TBK1 with shRNA also blocked the radiation-induced activation of AKT and ERK signaling. The ERK inhibitor did not obviously affect the expression of TBK1 or phosphorylated AKT, while AKT inhibition suppressed activation of ERK without changing the expression of TBK1. Finally, we found that a TBK1 inhibitor inhibited inflammatory cytokine expression in a RIPF model and Amlexanox protected normal cells and mice from ionizing radiation. In conclusion, our results indicate that the TBK1-AKT-ERK signaling pathway regulates radiation-induced EMT in normal alveolar epithelial cells, suggesting that TBK1 is a potential target for pulmonary fibrosis prevention during cancer radiotherapy.