Baicalin attenuates acute skin damage induced by ultraviolet B via inhibiting pyroptosis.

As the outermost layer of the human body, the skin suffers from various external factors especially light damage, among which ultraviolet B (UVB) irradiation is common and possesses a relatively high biological damage capacity. Pyroptosis is a newly discovered type of programmed cell death, which can induce cell rupture and induce local inflammatory response. However, the molecular mechanisms of pyroptosis in photodamaged skin is poorly understood. Baicalin, a flavonoid extracted from the desiccated root of Scutellaria baicalensis Georgi (Huang Qin). Despite its antioxidant abilities, whether baicalin protects skin by attenuating UVB-induced pyroptosis remains unclear, which was the aim of this study. The UVB-induced acute skin damage model was established by using human immortalized keratinocytes (HaCaT cells) and Kunming (KM) strain mice. The protective dose selection for baicalin is 50 µM in vitro and 100 mg/kg in vivo. In in vitro study, UVB irradiation significantly decreased cell viability, increased cell death and oxidative stress in HaCaT cells, while pretreatment with baicalin improved these phenomena. Furthermore, the baicalin pretreatment notably suppressed nuclear factor kappa B (NF-κB) translocation, the NLRP3 inflammasome activation and gasdermin D (GSDMD) maturation, thus effectively attenuating UVB-induced pyroptosis. In in vivo study, the baicalin pretreatment mitigated epidermal hyperplasia, collagen fiber fragmentation, oxidative stress and pyroptosis in UVB-irradiated mouse skin. In a nutshell, this study suggests that baicalin could be a potential protective agent to attenuate acute skin damage induced by UVB irradiation through decreasing oxidative stress and suppressing NF-κB/NLRP3/GSDMD-involved pyroptosis.

Lycium barbarum polysaccharide inhibits blue-light-induced skin oxidative damage with the involvement of mitophagy.

Although blue light can damage the skin to a certain extent, the pathogenesis of its damage remains still unclear. The available evidence suggests that oxidative stress may be the main cause of its damage. Lycium barbarum polysaccharide (LBP) has antioxidative effects in a variety of cells. In this paper, we investigated the protective role of LBP and its mechanism of action related to mitophagy in blue-light-damaged skin cells. The findings indicated that in HaCaT cells and mouse skin, LBP pretreatment was effective in reducing blue-light-induced apoptosis and ameliorating the elevated level of cellular autophagy/mitophagy caused by excessive blue light exposure. The markers reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were used to assess oxidative stress. LBP could effectively inhibit blue-light-induced oxidative stress. It was also found that blue light exposure caused mitochondrial dysfunction in HaCaT cells, including increased intracellular calcium ion levels and decreased mitochondrial membrane potential. LBP pretreatment significantly relieved mitochondrial dysfunction in HaCaT cells. These findings imply that LBP pretreatment protects skin cells from damage induced by blue light irradiation and that mitophagy may be a significant factor in skin photodamage.

Hydrogen sulfide protects retina from blue light-induced photodamage and degeneration via inhibiting ROS-mediated ER stress-CHOP apoptosis signal.

Background: Hydrogen sulfide (H2S) is a small reducing gas molecule with various biological functions such as anti-oxidative, anti-apoptotic and anti-inflammatory activities. In this study, we investigated the therapeutic effects of exogenous H2S in the experimental models of retinal photodamage in vivo and in vitro.Methods: Rats with open eyelids were pretreated with H2S (80~120 µmol/kg) for 10 days and then continuously exposed to blue light (435~445nm, 11.2W/m2) for 8 h to establish in vivo experimental model. ARPE-19 cells were pretreated with H2S and then exposed to blue light to establish in vitro experimental model.Results: In vivo experiments, H2S significantly ameliorated blue light-induced retinal oxidative stress, apoptosis and degeneration. Moreover, H2S inhibited the activation of blue light-induced endoplasmic reticulum (ER) stress CHOP apoptotic signaling. In vitro experiments, H2S improved blue light-induced oxidative stress and oxidative damage. H2S inhibited ROS-mediated activation of ER stress CHOP apoptotic signaling. H2S alleviated blue light-induced apoptosis and increases cell viability. The ER stress inhibitor 4-PBA alleviated blue light-induced apoptosis and increases cell viability.Conclusion: Taken together, these results indicate that H2S can inhibit ROS-mediated ER stress-CHOP apoptosis signal, thereby alleviating blue light-triggered retinal apoptosis and degeneration.

Lycium Barbarum polysaccharide protects HaCaT cells from PM2.5-induced apoptosis via inhibiting oxidative stress, ER stress and autophagy.

Objectives: Lycium barbarum polysaccharide (LBP) is a natural polysaccharide extracted from Lycium barbarum that has anti-inflammatory, anti-apoptotic and anti-aging effects, and plays a role in the prevention and treatment of various diseases. In this study, we investigated the therapeutic effect of LBP on particulate matter 2.5 (PM2.5)-induced skin damage.Methods: Cell viability was analyzed by MTT and LDH assays. Apoptosis was analyzed by Annexin V-FITC/PI staining. Oxidative stress/damage were assessed by intracellular ROS levels, MDA content and SOD activity. The intracellular protein expression was analyzed by Western blot. Mitochondrial damage was assayed by mitochondrial membrane potential with JC-1 probe. LC3-GFP adenovirus was transfected into HaCaT cells to analyze intracellular autophagosome levels.Results: In PM2.5-treated HaCaT cells, LBP pretreatment reduced PM2.5-induced cytotoxicity, ameliorated cell morphology and reduced cell apoptosis. LBP also inhibited the expression levels of GRP78 and CHOP, reduced the conversion of LC3I to LC3II, inhibited Bax protein and activated Bcl-2 protein. Furthermore, LBP inhibited PM2.5-induced mitochondrial autophagy (mitophagy) and mitochondrial damage. PM2.5-induced autophagy was regulated by endoplasmic reticulum (ER) stress.Conclusion: LBP protects skin cells from PM2.5-induced cytotoxicity by regulating the oxidative stress-ER stress-autophagy-apoptosis signaling axis, revealing that LBP has a great potential for the skin protection.

Macrophage-Mediated Bystander Effects after Different Irradiations through a p53-dependent Pathway.

The goal of this work was to elucidate the mechanisms of bystander effects outside the localized irradiation field and their potential hematological toxicity. In this study, an in vitro multicellular co-culture system was used to investigate the intercellular commutation and related signaling pathways between either irradiated A549 cells or Beas-2B cells and bystander lymphoblast TK6 cells with or without macrophage U937 cells as an intermediator. Results showed that the proliferation ability of bystander TK6 cells was inhibited after co-culture with A549 cells irradiated with γ rays rather than carbon ions. When macrophages were contained in the co-culture system, the cell viability damage to the bystander TK6 cells were further enhanced. However, the proliferation inhibition of bystander TK6 cells after co-culture with irradiated Beas-2B cells was observed only when intermediator macrophages existed in the cell co-culture system. More serious cell injury was detected after carbon-ion irradiation compared with γ-ray irradiation. The p53-relevant apoptosis pathway was activated in both irradiated A549 and Beas-2B cells, each to a different extent. When the p53 pathway of irradiated cells was inhibited by PFT-α, PFTµ or p53 siRNA, the bystander damage to TK6 cells were clearly alleviated. In conclusion, the bystander lymphoblast damage was induced in different cells using different LET radiations. An amplified bystander response was modulated by the intermediator macrophage. The underlying molecular mechanisms of these bystander effects were dependent on the activation of p53 and its relevant apoptosis pathway in the irradiated cells. These results suggest that the bystander and macrophage-mediated bystander effects contribute to the common acute side effect of lymphocytopenia after local irradiation.

G(2)-M phase-correlative bystander effects are co-mediated by DNA-PKcs and ATM after carbon ion irradiation.

Accumulated evidence has shown that radiation-induced bystander effect (RIBE) may have significant implications to the efficiency of radiotherapy. Although cellular radiosensitivity relies on cell cycle status, it is largely unknown how about the relationship between RIBE and cell cycle distribution, much less the underlying mechanism. In the present study, the lung cancer A549 cells were synchronized into different cell cycle phases of G1, S and G2/M and irradiated with high linear energy transfer (LET) carbon ions. By treating nonirradiated cells with the conditioned medium from these irradiated cells, it was found that the G2-M phase cells had the largest contribution to RIBE. Meanwhile, the activity of DNA-PKcs but not ATM was increased in the synchronized G2-M phase cells in spite of both of them were activated in the asynchronous cells after carbon ion irradiation. When the G2-M phased cells were transferred with DNA-PKcs siRNA and ATM siRNA individually or treated with an inhibitor of either DNA-PKcs or ATM before carbon ion irradiation, the RIBE was effectively diminished. These results provide new evidence linking cell cycle to bystander responses and demonstrate that DNA-PKcs and ATM are two associated factors in co-regulating G2-M phase-related bystander effects.

The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation.

The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in the γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation, while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury.

Role of the MAPK pathway in the observed bystander effect in lymphocytes co-cultured with macrophages irradiated with γ-rays or carbon ions.

AIMS: The radiation-induced bystander effect (RIBE) has potential implications in cancer risks from space particle radiation; however, the mechanisms underlying RIBE are unclear. The role of the MAPK pathway in the RIBEs of different linear energy transfer (LET) was investigated. MAIN METHODS: Human macrophage U937 cells were irradiated with γ-rays or carbon ions and then co-cultured with nonirradiated HMy2.CIR (HMy) lymphocytes for different periods. The activation of MAPK proteins and the generation of intracellular nitric oxide (NO) and reactive oxygen species (ROS) in the irradiated U937 cells were measured. Micronuclei (MN) formation in the HMy cells was applied to evaluate the bystander damage. Some U937 cells were pretreated with different MAPK inhibitors before irradiation. KEY FINDINGS: Additional MN formation was induced in the HMy cells after co-culturing with irradiated U937 cells, and the yield of this bystander MN formation was dependent on the co-culture period with γ-ray irradiation but remained high after 1h of co-culture with carbon irradiation. Further investigations disclosed that the time response of the RIBEs had a relationship with LET, where ERK played a different role from JNK and p38 in regulating RIBEs by regulating the generation of the bystander signaling factors NO and ROS. SIGNIFICANCE: The finding that the RIBE of high-LET radiation could persist for a much longer period than that of γ-rays implies that particle radiation during space flight could have a high risk of long-term harmful effects. An appropriate intervention targeting the MAPK pathway may have significant implications in reducing this risk.

Differential effects of p53 on bystander phenotypes induced by gamma ray and high LET heavy ion radiation.

High LET particle irradiation has several potential advantages over γ-rays such as p53-independent response. The purpose of this work is to disclose the effect of p53 on the bystander effect induced by different LET irradiations and underlying mechanism. Lymphocyte cells of TK6 (wild type p53) and HMy2.CIR (mutated p53) were exposed to either low or high LET irradiation, then their mitochondrial dysfunction and ROS generation were detected. The micronuclei (MN) induction in HL-7702 hepatocytes co-cultured with irradiated lymphocytes was also measured. It was found that the mitochondrial dysfunction, p66(Shc) activation, and intracellular ROS were enhanced in TK6 but not in HMy2.CIR cells after γ-ray irradiation, but all of them were increased in both cell lines after carbon and iron irradiation. Consistently, the bystander effect of MN formation in HL-7702 cells was only triggered by γ-irradiated TK6 cells but not by γ-irradiated HMy2.CIR cells. But this bystander effect was induced by both lymphocyte cell lines after heavy ion irradiation. PFT-µ, an inhibitor of p53, only partly inhibited ROS generation and bystander effect induced by 30 keV/µm carbon-irradiated TK6 cells but failed to suppress the bystander effect induced by the TK6 cells irradiated with either 70 keV/µm carbon or 180 keV/µm iron. The mitochondrial inhibitors of rotenone and oligomycin eliminated heavy ion induced ROS generation in TK6 and HMy2.CIR cells and hence diminished the bystander effect on HL-7702 cells. These results clearly demonstrate that the bystander effect is p53-dependent for low LET irradiation, but it is p53-independent for high LET irradiation which may be because of p53-independent ROS generation due to mitochondrial dysfunction.

Simulated microgravity increases heavy ion radiation-induced apoptosis in human B lymphoblasts.

AIMS: Microgravity and radiation, common in space, are the main factors influencing astronauts' health in space flight, but their combined effects on immune cells are extremely limited. Therefore, the effect of simulated microgravity on heavy ion radiation-induced apoptosis, and reactive oxygen species (ROS)-sensitive apoptosis signaling were investigated in human B lymphoblast HMy2.CIR cells. MAIN METHODS: Simulated microgravity was achieved using a Rotating Wall Vessel Bioreactor at 37°C for 30 min. Heavy carbon-ion irradiation was carried out at 300 MeV/u, with a linear energy transfer (LET) value of 30 keV/µm and a dose rate of 1Gy/min. Cell survival was evaluated using the Trypan blue exclusion assay. Apoptosis was indicated by Annexin V/propidium iodide staining. ROS production was assessed by cytometry with a fluorescent probe dichlorofluorescein. Malondialdehyde was detected using a kit. Extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase phosphatase-1 (MKP-1) and caspase-3 activation were measured by immunoblotting. KEY FINDINGS: Simulated microgravity decreased heavy ion radiation-induced cell survival and increased apoptosis in HMy2.CIR cells. It also amplified heavy ion radiation-elicited intracellular ROS generation, which induced ROS-sensitive ERK/MKP-1/caspase-3 activation in HMy2.CIR cells. The above phenomena could be reversed by the antioxidants N-acetyl cysteine (NAC) and quercetin. SIGNIFICANCE: These results illustrated that simulated microgravity increased heavy ion radiation-induced cell apoptosis, mediated by a ROS-sensitive signal pathway in human B lymphoblasts. Further, the antioxidants NAC and quercetin, especially NAC, might be good candidate drugs for protecting astronauts' and space travelers' health and safety.

The local skin dose conversion coefficients of electrons, protons and alpha particles calculated using the Geant4 code.

The skin tissue-equivalent slab reported in the International Commission on Radiological Protection (ICRP) Publication 116 to calculate the localised skin dose conversion coefficients (LSDCCs) was adopted into the Monte Carlo transport code Geant4. The Geant4 code was then utilised for computation of LSDCCs due to a circular parallel beam of monoenergetic electrons, protons and alpha particles <10 MeV. The computed LSDCCs for both electrons and alpha particles are found to be in good agreement with the results using the MCNPX code of ICRP 116 data. The present work thus validates the LSDCC values for both electrons and alpha particles using the Geant4 code.

Investigation of fragment doses produced by heavy ions in tissue-like material.

In this paper, the distribution of fragments produced by 55 MeV/n 4°Ar¹7⁺ ions in tissue-equivalent material is reported as a function angles relative to the incident particle direction. The relative fragment doses at different angles and the total dose due to the primary beam have been estimated. The results show that the fragments produced by primary beam were concentrated at very small angles, so the fraction of particle fluence diverging from the primary beam was very small. Even though the diverging fluence is a small fraction of the total, it should still be taken into account because biological systems are very sensitive to low doses of heavy ion irradiation.

Comparison doses of secondary neutron with the heavy ions in a 75-Mev/n heavy ion beam.

The angular distributions for neutrons of energy >6 MeV that are induced by 75 MeV/n 12C6+ and 16O8+ ions were measured with the activation method of Al threshold detectors at the radiobiological terminal of HIRFL. The data were obtained by a high-purity Ge(HpGe) detector. The results show that the neutron angular distributions produced by heavy ion beams are strongly peaked in the forward direction and decreased exponentially with angles in experimental area. The experimental conditions for these measurements were similar to those for biological experiments, so the results should be representative of neutrons produced by heavy ions during the biological experiments and tumour therapy. Comparing with the neutron doses produced by the heavy ion beam, the heavy ion dose is the main factor in biological effects and tumour therapy response, so the contribution of neutron dose can be neglected.