Bailong Li

Hydrogen-rich saline attenuates radiation-induced male germ cell loss in mice through reducing hydroxyl radicals

Our recent studies suggest that H2 (hydrogen) has a potential as a novel radioprotector without known toxic side effects. The present study was designed to examine the underlying radioprotective mechanism of H2 and its protective role on irradiated germ cells. Produced by the Fenton reaction and radiolysis of H2O, hydroxyl radicals (•OH) were identified as the free radical species that were reduced by H2. We used a H2 microelectrode to dynamically detect H2 concentration in vivo, and found H2 significantly reduced in situ fluorescence intensity of hydroxyphenyl fluorescein; however, as we treated the mice with H2 after irradiation, the decrease is not significant. We found that pre-treatment of H2 to IR (ionizing radiation) significantly suppressed the reaction of •OH and the cellular macromolecules which caused lipid peroxidation, protein carbonyl and oxidatively damaged DNA. The radioprotective effect of H2 on male germ cells was supported by ameliorated apoptotic findings examined by morphological changes and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) in testicular tissue, and by preserved viability of stem spermatogonia examined for testicular histological parameters, daily sperm production and sperm quality; we used WR-2721 [S-2-(3-aminopropylamino)ethyl phosphorothioic acid] as a reference compound. Our results represent the first in vivo evidence in support of a radioprotective role of H2 by neutralizing •OH in irradiated tissue with no side effects.

Inhibition of TBK1 attenuates radiation-induced epithelial-mesenchymal transition of A549 human lung cancer cells via activation of GSK-3β and repression of ZEB1.

Radiotherapy is an effective treatment method for lung cancer, particularly when the disease is at an advanced stage. However, previous researchers have observed that the majority of patients with conventional radiation therapy develop distant metastases and succumb to the disease. Thus, identifying and understanding novel pathways for the development of new therapeutic targets is a major goal in research on pulmonary neoplasms. Recent studies suggest that epithelial–mesenchymal transition (EMT) is the most important contributor to cancer metastasis. Induction of this complex process requires endogenously produced microRNAs; specifically, downregulation of the miRNA-200c causes an induction of EMT. We recently identified the tank-binding kinase-1 (TBK1) as a downstream effector of the miR-200c-driven pathway, but the biological function of TBK1 in EMT remains unknown. In this study, we tested whether TBK1 has a role in radiation-induced EMT and identified associated potential mechanisms. Human alveolar type II epithelial carcinoma A549 cells were irradiated with 60Co γ-rays. Western blotting revealed a time- and dose-dependent decrease in E-cadherin with a concomitant increase in vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. TBK1 siRNA significantly inhibited radiation-induced suppression of the epithelial marker E-cadherin and upregulation of the mesenchymal marker vimentin. The invasion and migratory potential of lung cancer cells upon radiation treatment was also reduced by TBK1 knockdown. Furthermore, radiation-induced EMT attenuated by TBK1 depletion was partially dependent on transcriptional factor ZEB1 expression. Finally, we found glycogen synthase kinase-3β (GSK-3β) is involved in regulation of radiation-induced EMT by TBK1. Thus, our findings reveal that TBK1 signaling regulates radiation-induced EMT by controlling GSK-3β phosphorylation and ZEB1 expression. TBK1 may therefore constitute a useful target for treatment of radiotherapy-induced metastasis diseases.

Hydrogen-rich saline protects immunocytes from radiation-induced apoptosis.

Summary Background Radiation often causes depletion of immunocytes in tissues and blood, which results in immunosuppression. Molecular hydrogen (H2) has been shown in recent studies to have potential as a safe and effective radioprotective agent through scavenging free radicals. This study was designed to test the hypothesis that H2 could protect immunocytes from ionizing radiation (IR). Material/Methods H2 was dissolved in physiological saline or medium using an apparatus produced by our department. A 2-[6-(4′-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoate (HPF) probe was used to detect intracellular hydroxyl radicals (•OH). Cell apoptosis was evaluated by annexin V-FITC and Propidium iodide (PI) staining as well as the caspase 3 activity. Finally, we examined the hematological changes using an automatic Sysmex XE 2100 hematology analyzer. Results We demonstrated H2-rich medium pretreatment reduced •OH level in AHH-1 cells. We also showed H2 reduced radiation-induced apoptosis in thymocytes and splenocytes in living mice. Radiation-induced caspase 3 activation was also attenuated by H2 treatment. Finally, we found that H2 rescued the radiation-caused depletion of white blood cells (WBC) and platelets (PLT). Conclusions This study suggests that H2 protected the immune system and alleviated the hematological injury induced by IR.

Hydrogen-rich saline protects spermatogenesis and hematopoiesis in irradiated BALB/c mice

Summary Background Recent studies show that molecular hydrogen (dihydrogen, H2) has potential as an effective and safe radioprotective agent through reducing oxidative stress. The aim of this study was to investigate whether H2 is able to protect spermatogenesis and hematopoiesis from radiation-induced injuries. Material/Methods H2 was dissolved in physiological saline using an apparatus produced by our department. 60Co-gamma rays in the irradiation centre were used for irradiation. Spermatid head counts and histological analysis were used to evaluate spermatogenesis. Endogenous hematopoietic spleen colony formation (endoCFUs), bone marrow nucleated cells (BMNC) and peripheral blood (PB) leukocytes were used to evaluate hemopoiesis. Results This study demonstrates that treating mice with H2 before ionizing radiation (IR) can increase the spermatid head count and protect seminiferous epithelium from IR. This study also demonstrates that H2 could significantly increase the number of endoCFUs, BMNC and PB leukocyte. Conclusions This study suggests that hydrogen-rich saline could partially protect spermatogenesis and hematopoiesis in irradiated mice.

A possible prevention strategy of radiation pneumonitis: combine radiotherapy with aerosol inhalation of hydrogen-rich solution.

Summary Radiotherapy is an important modality of cancer treatment. Radiation pneumonitis is a major obstacle to increasing the radiation dose in radiotherapy, and it is important to prevent this radiation-induced complication. Recent studies show that hydrogen has a potential as an effective and safe radioprotective agent by selectively reducing hydroxyl and peroxynitrite radicals. Since most of the ionizing radiation-induced cellular damage is caused by hydroxyl radicals, we hypothesize that a treatment combining radiotherapy with aerosol inhalation of a hydrogen-rich solution may be an effective and novel prevention strategy for radiation pneumonitis (hydrogen is explosive, while a hydrogen-rich solution such as physiological saline saturated with molecular hydrogen is safer).

Radiation-driven lipid accumulation and dendritic cell dysfunction in cancer

Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. Here we report our findings on the dysfunction of DCs in radiation-induced thymic lymphomas, and the up-regulation of the expression of the lipoprotein lipase (LPL) and the fatty acid binding protein (FABP4), and the level of triacylglycerol (TAG) in serum after total body irradiation, which contribute to DCs lipid accumulation. DCs with high lipid content showed low expression of co-stimulatory molecules and DCs-related cytokines, and were not able to effectively stimulate allogeneic T cells. Normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the function of DCs. A high-fat diet promoted radiation-induced thymic lymphoma growth. In all, our study shows that dysfunction of DCs in radiation-induced thymic lymphomas was due to lipid accumulation and may represent a new mechanism in radiation-induced carcinogenesis.

GSK-3β Inhibition Attenuates LPS-Induced Death but Aggravates Radiation-Induced Death via Down-Regulation of IL-6

Background: Exposure of high dose ionizing radiation is lethal. Signal pathways involved in radiation biology reaction still remain illdefined. Lipopolysaccharides (LPS), the ligands of Toll-like receptor 4(TLR4), could elicit strong immune responses. Glycogen synthase kinase-3β(GSK-3β) promotes the production of inflammatory molecules and cell migration. Inhibition of GSK-3β provides protection against inflammation in animal models. The aim of the study was to investigate role of GSK-3β in LPS shock and ionizing radiation. Methods: WT or IL-6-/-mice or cells were pretreated with SB216763, a GSK-3β inhibitor, and survival of the mice was determined. Cell viability was assayed by Cell Counting Kit. Apoptosis was assayed by Annexin V-PI double staining. Serum concentrations of IL-6 and TNF-α were determined by ELISA. Results: SB216763 attenuated LPS induced mice or cell death but aggravated radiation induced mice or cell death. SB216763 reduced IL-6, but not TNF-α levels in vivo. IL-6-/- mice were more resistant to LPS-induced death but less resistant to radiation-induced death than wild type mice. Conclusions: Inhibition of GSK-3β conferred resistance to LPS shock but fostered death induced by ionizing radiation. Inhibition of GSK-3β was effective by reducing IL-6.

Hydrogen protects rats from dermatitis caused by local radiation

Background: Radiation therapy produced unwanted side effect on normal tissues, such as radiodermatitis. Hydrogen was previously shown capable of radiation protective in both animals and cell cultures. The effect of hydrogen was now to be investigated on radiation-induced cutaneous. Objective: Development of dermatitis is a frequent side effect of radiotherapy of patients with head-and-neck cancer. Here we analyzed the radioprotective efficacy of hydrogen under conditions of local, single-dose or fractionated radiation treatment, and its possible molecular mechanisms. Methods: Rats received either single-dose or fractioned irradiation of the head-and-neck area with or without subcutaneous injection of hydrogen solution before irradiation. In vitro, the effect of hydrogen medium on radiation-induced cell viability, apoptosis, and biochemical assays was measured. Result: Hydrogen significantly reduced the severity of dermatitis, accelerated tissue recovery, and reduced the extent of radiation-induced weight loss in rats after a single dose of 15 or 20 Gy but not 25 Gy of radiation. Hydrogen was also protective from cumulative doses of 30 Gy delivered in three fractions, respectively. Hydrogen also protect HaCaT cells from radiation-induced injury, it could significantly inhibit ionizing injury. Conclusion: These results suggest that hydrogen has a positive effect on acute radiodermatitis.

CpG-Oligodeoxynucleotide Treatment Protects against Ionizing Radiation-Induced Intestine Injury

Background the bone marrow and the intestine are the major sites of ionizing radiation (IR)-induced injury. Our previous study demonstrated that CpG-oligodeoxynucleotide (ODN) treatment mitigated IR-induced bone marrow injury, but its effect on the intestine is not known. In this study, we sought to determine if CpG-ODN have protective effect on IR-induced intestine injury, and if so, to determine the mechanism of its effect. Methods and Findings Mice were treated with CpG-ODN after IR. The body weight and survival were daily monitored for 30 days consecutively after exposure. The number of surviving intestinal crypt was assessed by the microcolony survival assay. The number and the distribution of proliferating cell in crypt were evaluated by TUNEL assay and BrdU assay. The expression of Bcl-2, Bax and caspase-3 in crypt were analyzed by Immunohistochemistry assay. The findings showed that the treatment for irradiated mice with CpG-ODN diminished body weight loss, improved 30 days survival, enhanced intestinal crypts survival and maintained proliferating cell population and regeneration in crypt. The reason might involve that CpG-ODN up-regulated the expression of Bcl-2 protein and down-regulated the expression of Bax protein and caspase-3 protein. Conclusion CpG-ODN was effective in protection of IR-induced intestine injury by enhancing intestinal crypts survival and maintaining proliferating cell population and regeneration in crypt. The mechanism might be that CpG-ODN inhibits proliferating cell apoptosis through regulating the expression of apoptosis-related protein, such as Bax, Bcl-2 and caspase-3.

Molecular hydrogen protects human lymphocyte AHH-1 cells against 12C6+ heavy ion radiation.

Abstract Purpose: To investigate the potential protective role of molecular hydrogen (H2) against 12C6+ heavy ion radiation, which is a major hazard for space travel and has been also widely used in heavy ion radiotherapy. Materials and methods: H2 was dissolved in Roswell Park Memorial Institute (RPMI) 1640 medium under high pressure (0.4 Mpa) to a saturated level by using an apparatus produced by our department. A 2-[6-(4′-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoate (HPF) probe and a 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFH-DA) fluorescent dye were used to measure the intracellular reactive oxygen species (ROS) level. Cell apoptosis were determined by double-staining with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) as well as a Hoechst 33342 staining method alternatively. Subsequently, cell cycle analysis was performed using a PI staining method and the expression of apoptotic protein was examined by Western blot. Results: In this study, we demonstrated H2 reduced ROS level in Human lymphocyte AHH-1 cells as well as in the radiolysis of water. Our data also showed H2 attenuated 12C6+ radiation- induced cell apoptosis and also alleviated radiation-induced G2/M cell cycle arrest. Heavy ion radiation-induced Caspase 3 activation was also inhibited by H2 treatment. Conclusion: In conclusion, these data showed that H2 attenuated 12C6+ radiation-induced cell apoptosis through reducing the ROS level and modulating apoptotic molecules, thus indicating the potential of H2 as a safe and effective radioprotectant.