OBJECTIVE:This study aimed to investigate the effects of low-dose radiation on the abdominal aorta of mice and vascular endothelial cells. METHODS:Wild-type and tumor-bearing mice were exposed to 15 sessions of low-dose irradiation, resulting in cumulative radiation doses of 187.5, 375, and 750 mGy. The effect on the cardiovascular system was assessed. Immunohistochemistry analyzed protein expressions of PAPP-A, CD62, P65, and COX-2 in the abdominal aorta. Microarray technology, Gene Ontology analysis, and pathway enrichment analysis evaluated gene expression changes in endothelial cells exposed to 375 mGy X-ray. Cell viability was assessed using the Cell Counting Kit 8 assay. Immunofluorescence staining measured γ-H2AX levels, and real-time polymerase chain reaction quantified mRNA levels of interleukin-6 (IL-6), ICAM-1, and Cx43. RESULTS:Hematoxylin and eosin staining revealed thickening of the inner membranes and irregular arrangement of smooth muscle cells in the media membrane at 375 and 750 mGy. Inflammation was observed in the inner membranes at 750 mGy, with a clear inflammatory response in the hearts of tumor-bearing mice. Immunohistochemistry indicated increased levels of PAPP-A, P65, and COX-2 post-irradiation. Microarray analysis showed 425 up-regulated and 235 down-regulated genes, associated with processes like endothelial cell-cell adhesion, IL-6, and NF-κB signaling. Cell Counting Kit 8 assay results indicated inhibited viability at 750 mGy in EA.hy926 cells. Immunofluorescence staining demonstrated a dose-dependent increase in γ-H2AX foci. Reverse transcription quantitative PCR results showed increased expression of IL6, ICAM-1, and Cx43 in EA.hy926 cells post 750 mGy X-ray exposure. CONCLUSION:Repeated low-dose ionizing radiation exposures triggered the development of pro-atherosclerotic phenotypes in mice and damage to vascular endothelial cells.

Purpose: UVB exposure accelerates skin aging and age-associated pigmentation, but their relationship remains unclear. UVB induces premature senescence and melanin production within melanocytes, accompanied by the up-regulation of p53 and cellular tyrosinase (TYR). As a tumor suppressor gene, p53 can keep the genome intact by modulating cell apoptosis and growth arrest during DNA injury. P53 also relates to age-associated pigmentation, directly or indirectly regulating pigment-related gene expression. Melatonin effectively regulates tyrosinase activity and resists aging. This study focused on the regulation of p53 on TYR to understand the association between premature senescence and senescence-associated pigmentation and determine the mechanism behind melatonin affecting UVB stimulated melanin production.Methods: Primary melanocytes were extracted and identified from the male foreskin. The primary melanocytes were transduced using lentivirus pLKD-CMV-EGFP-2A-Puro-U6-TYR to knock down TYR expression. The melanin content was determined using the NaOH method, the oxidation of 3,4-Dihydroxy-L-phenylalanine (L-DOPA) into dopachrome to determine TYR activity, and Western blotting was used to detect the level of TYR protein. After being pretreated with Nutlin-3 or PFT-α to up or down-regulate p53 levels or melatonin for 12 h, primary melanocytes were under UVB irradiation at 80 mJ/cm2. The senescence-associated beta-galactosidase (SA-β-gal) kit analyzed premature senescence. At the same time, the level of p53, p-p53, and TYR protein were detected using the automated capillary electrophoresis western analysis in melanocytes at 72 h after UVB irradiation. Wild-type and TYR (–/–) or TYR (+/–) knockout C57BL/6J mice were used to verify the regulatory role of TYR on melanin synthesis in vivo . Moreover, the effect of melatonin on skin erythema and pigmentation induced by UVB irradiation was illustrated in vivo.Results: Primary melanocytes showed a deep black color after L-DOPA staining, higher TYR protein, and mRNA expression. Tyrosinase activity and melanin levels induced by UVB irradiation were significantly alleviated after being infected with pLKD-CMV-EGFP-2A-Puro-U6-TYR (P <0.05). Premature senescence, tyrosinase activity, and melanin levels were increased under UVB irradiation induction. Furthermore, there was a dramatic increase following the Nutlin-3 treatment while significantly inhibited after being treated with PFT-α (P <0.05) in the primary melanocytes. Melatonin inhibited UVB-induced premature senescence, associated with decreased p53 level and phosphorylation on serine-15 position, decreased UVB-induced tyrosinase activity and melanin levels, and reduced TYR expression. TYR (–/–) knockout mice were recognized through white hair colors, whiskers, paws, and pigment loss in the eyes. The tyrosinase activity and melanin levels in the whiskers follicles of TYR (–/–) knockout mice were also significantly decreased (P <0.05) relative to the wild-type (WT) counterparts. Skin erythema and melanin pigmentation induced by UVB irradiation were reduced in the dorsal and ear skin of C57BL/6J mice topically pretreated with 2.5% melatonin.Conclusion: Melanin synthesis induced by UVB irradiation is dependent on TYR in primary melanocytes and C57BL/6J mice. Moreover, p53 links the UVB irradiation-induced premature senescence and senescence-associated pigmentation in the primary melanocytes, and directly regulates TYR in primary mel

ABSTRACT:Quantification of gamma-H2AX foci can estimate exposure to ionizing radiation. Most nuclear and radiation accidents are partial-body irradiation, and the doses estimated using the total-body irradiation dose estimation formula are often lower than the actual dose. To evaluate the dose-response relation of gamma-H2AX foci in human peripheral blood lymphocytes after partial-body irradiation and establish a simple and high throughput model to estimate partial-body irradiation dose, we collected human peripheral blood and irradiated with 0-, 0.5-, 1-, 2-, 3-, 4-, 5-, 6-, and 8-Gy gamma rays to simulate total-body irradiation in vitro. Gamma-H2AX foci were quantitated by flow cytometry at 1 h after irradiation, and a dose-response curve was established for total-body irradiation dose estimation. Then, a partial-body irradiation dose-response calibration curve was established by adding calibration coefficients based on the Dolphin method. To reflect the data distribution of all doses more realistically, the partial-body irradiation dose-response calibration curve was divided into two sections. In addition, partial-body irradiation was simulated in vitro, and the PBI data were substituted into curves to verify the accuracy of the two partial-body irradiation calibration curves. Results showed that the dose estimation variations were all less than 30% except the 25% partial-body irradiation group at 1 Gy, and the partial-body irradiation calibration dose-response curves were YF 1 = - 3.444 x 2 + 18.532 x + 3.109, R 2 = 0.92 (YF ≤ 27.95); YF 2 = - 2.704 x 2 + 37.97 x - 56.45, R 2 = 0.86 (YF > 27.95). Results also suggested that the partial-body irradiation dose-response calibration curve based on the gamma-H2AX foci quantification in human peripheral blood lymphocytes is a simple and high throughput model to assess partial-body irradiation dose.

The intestinal compensatory proliferative potential is a key influencing factor for susceptibility to radiation-induced intestinal injury. Studies indicated that the carnitine palmitoyltransferase 1 (CPT1) mediated fatty acid β-oxidation (FAO) plays a crucial role in promoting the survival and proliferation of tumor cells. Here, we aimed to explore the effect of 60Co gamma rays on CPT1 mediated FAO in the radiation-induced intestinal injury models, and investigate the role of CPT1 mediated FAO in the survival and proliferation of intestinal cells after irradiation. We detected the changed of FAO in the plasma and small intestine of Sprague Dawley (SD) rats at 24 h after 60Co gamma irradiation (0, 5 and 10 Gy), using target metabolomics, qRT-PCR, immunohistochemistry (IHC), western blot (WB) and related enzymatic activity kits. We then analyzed the FAO changes in radiation-induced intestinal injury models regardless of ex vivo (mice enteroids), or in vitro (normal human intestinal epithelial cell lines, HIEC-6). HIEC-6 cells were transduced with lentivirus vector GV392 and treated with puromycin for obtaining CPT1 stable knockout cell lines, named CPT1 KO. CPT1 enzymatic activities of HIEC-6 cells and mice enteroids were also inhibited by pharmaceutical inhibitor ST1326 and Etomoxir (ETO), to study the function of CPT1 in the survival and proliferation of HIEC-6 cells after 60Co gamma irradiation. We found that CPT1 mediated FAO was altered in the small intestine of the SD rats after irradiation, especially, the expression level and enzymatic activity of CPT1 were significantly increased. Similarly, the expression levels of CPT1 were also remarkably enhanced in mice enteroids and HIEC-6 cells after irradiation. CPT1 inhibition decreased the proliferation of the HIEC-6 cells and mice enteroids after irradiation partially by reducing the extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways activation, CPT1 inhibition also reduced the proliferation of mice enteroids after irradiation partially by down-regulating the Wnt/β-catenin signaling activity. In conclusion, our study indicated that CPT1 plays a crucial role in promoting intestinal epithelial cell proliferation after irradiation.

电离辐射会对人体造成损伤,根据受照剂量、时间等因素的不同可诱发多种生物效应.目前对于低剂量辐射产生的健康效应仍有争议,筛选对低剂量敏感的辐射响应生物标志物,对于完善低剂量辐射生物效应机制、拓宽低剂量辐射在临床中的应用均具有重要理论意义.综述探讨各核糖核酸(RNA)在低剂量辐射反应中的变化及其对辐射敏感性的调节作用,同时评估各RNA作为低剂量辐射响应标志物的潜力.

Purpose: Ionizing radiation (IR) can induce autophagy and premature senescence; however, the link between them has not been clarified. Our research has shown that X-ray irradiation induces premature senescence in lung adenocarcinoma cells, and its occurrence partially depends on the signal transducer and activator of transcription 3 (STAT3). STAT3 can bind to the promoter region of Beclini and regulate its expression. Therefore, it is speculated that there may be a close link between premature senescence and autophagy induced by ionizing radiation in lung adenocarcinoma cells. p62 plays a regulatory role in both autophagy and premature senescence, and it is also an irreplaceable molecule that causes the senescence -associated secretory phenotype (SASP) and a substrate for selective autophagy. This study focused on STAT3, Beclin1 and p62 to clarify the regulatory relationship between 1R-induced autophagy and premature senescence. Materials and methods: After exposure to 4 Gy X-rays, a beta-galactosidase staining kit was used to detect the positive rate of premature senescence. STAT3 was overexpressed by pcDNA3.0-STAT3 transfection, and was inhibited by AG490 and rapamycin. Lung adenocarcinoma cells were transduced with the adenovirus vector GV119-Beclin1 to knockdown the expression of Beclin1, or treated with ATM and ATR inhibitors to inhibit premature senescence. Western blotting was used to examine alterations in the radiation response proteins STAT3 and p-STAT3, senescence-related proteins p62 and GATA4, autophagy-related proteins Beclin1, and LC3-II/LC3-1. The mRNA expression levels of SASP factors, including IL-6 and IL-8, were examined by real-time polymerase chain reaction. Results: The activity of SA-beta-gal increased significantly (p < .05), and the expression of p62 decreased significantly at 72 h after 4 Gy X-ray irradiation, accompanied by the increased expression of STAT3, p-STAT3, Beclin1, and the LC3-II/LC3-I ratio. Up- or down-regulation of STAT3 expression was followed by an increase or decrease in Beclin1 expression. After treatment with ATM and ATR inhibitors, there were no significant changes in Beclin1 expression or LC3-II/LC3-I ratio in A549 cells after 4 Gy X-ray irradiation. The p62 expression, the percentage of the SA-beta-gal-positive staining cells, and the expression of IL-6 and IL-8 mRNA in cells transduced with GV119-Beclin1 were also decreased significantly after 4Gy X-ray irradiation compared with that of the 0 Gy group. Conclusion: Radiation induces premature senescence and autophagy in lung adenocarcinoma cells. Autophagy regulates X-ray radiation-induced premature senescence through the STAT3-Beclin1-p62 pathway in lung adenocarcinoma cells.

Objective To evaluate the clinical efficacy and safety of baricitinib, a Janus kinase (JAK) inhibitor, in treating patient with progressing vitiligo, and to further explore the regulation of baricitinib on melanocytes (MCs) in vitro. Methods Four patients with progressing vitiligo were treated with oral baricitinib for a total of 12 weeks. MCs were cultured in vitro and irradiated by high-dose ultraviolet B (UVB, 150mJ/cm 2 ) to make an MC damaged model (MC-Ds). Baricitinib was added at a final concentration of 25 μM. Dopamine staining and NaOH method were used to measure the tyrosinase activity and melanin level, respectively, real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the mRNA levels of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1). Results Significant re-pigmentation was observed in the week 12 without obvious side effects. Depigmentation occurred in 2 patients at the 3-month follow-up. Laboratory research found that higher doses of UVB irradiation (150mJ/cm 2 ) could decrease melanin content of MCs, baricitinib (25 μM) could significantly promote tyrosinase activity, melanin content, and TYR, TRP-1 gene expression of MC-Ds. Conclusion Our preliminary study showed that baricitinib was effective and safe in treating progressing vitiligo. Baricitinib could promote tyrosinase activity, melanin content and TYR, TRP1 gene expression of MC-Ds in vitro.

Objective To explore the effect and mechanism of ultraviolet B (UVB) on melanin synthesis and premature senescence in human immortalized keratinocytes (HaCaT) cells. Methods HaCaT cells were irradiated with 0, 20, 50, 80, 100, 150, and 200 mJ/cm 2 of UVB. NaOH method was used for melanin content assay, cellular tyrosinase (TYR) activity was determined by 3,4-Dihydroxy-L-phenylalanine (L-DOPA) oxidation to dopachrome, premature senescence was analyzed by senescence-associated beta-galactosidase (SA-β-gal) staining kit, and the levels of p21, p16, p62, and GATA4 proteins were detected by Western blotting. Premature senescence was inhibited by the inhibitors of ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3–related (ATR), and the p53 signaling pathway was activated by Nutlin-3. The mRNA levels of senescence-associated secretory phenotype (SASP) factors including tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor A (VEGF-A), and interleukin-8 (IL-8) were measured by real-time quantitative polymerase chain reaction in HaCaT cells after 80 mJ/cm 2 of UVB irradiation. Results The melanin level increased significantly with the elevation of irradiation dose ( F = 28.19, 43.82, 143.60, P < .05), reaching the peak at the dose of 80 mJ/cm 2 . The tyrosinase activity increased significantly ( F = 84.50, P < .05), the percentage of premature senescence increased ( F = 16.31, P < .05), the levels of p62 decreased, and the level of GATA4 increased obviously with the increase of UVB dose after irradiation. The UVB-induced promotion of GATA4 level was significantly inhibited by being treated with ATM or ATR inhibitor. However, this did not occur in the Nutlin-3-treated group. The mRNA and protein expression of TNF-α increased significantly at 72 h at 80 mJ/cm 2 of UVB irradiation. Conclusions Melanin contents increased first and decreased afterward with the increasing of UVB irradiation. The decrease of p62-mediated selective autophagy was accompanied by the accumulation of GATA4 after different doses of UVB irradiation. Activation of this p62/GATA4 pathway depends on the ATM and ATR but is independent of p53, and the SASP factor was activated in HaCaT cells at 80 mJ/cm 2 of UVB irradiation.

Melatonin is an effective antioxidant hormone produced mainly by the pineal gland. Premature senescence refers to senescence induced by declined cell proliferation and physiological functions when cells are stimulated by non-telomeric signals. The anti-aging effect of melatonin is exerted by upregulating the expression of silent information regulator 1/Sirtuin 1 (SIRT1), which reduces oxidative stress damage, decreases p53 activation, and inhibits the NF-κB pathway. Radiation can induce premature senescence through direct or indirect oxidative stress damage. The mechanism by which melatonin regulates radiation-induced premature senescence includes the inhibition of the p53-mediated senescence pathway through elimination of reactive oxygen species or p53 deacetylation induced by upregulation of SIRT1 expression. Melatonin exhibits different regulatory effects for different cells and types of radiation.

Objective:To explore the mechanism and regulatory effects of melatonin on UVB-induced melanin synthesis in human immortalized keratinocytes (HaCaT), so as to provide a theoretical basis for the skin protection of melatonin.Methods:HaCaT cells were pretreated with 10 -5 mol/L melatonin and then irradiated with 80 mJ/cm 2UVB. The melanin content was detected by NaOH assay, the proportion of premature senescence cells was detected by β-galactosidase staining kit, and the protein expression levels of both p53 and tyrosinase (TYR) were detected by Western blot at 72 h after UVB exposure. After 12 h pretreatment of ATM/ATR inhibitor, p53 inhibitor and melatonin, the proportion of premature senescence and the change of melanin content in HaCaT cells were detected at 72 h after 80 mJ/cm 2 UVB irradiation. Results:Melatonin inhibited UVB-induced increases of melanin content ( t=56.65, 13.39, P<0.05) and TYR expression ( t=16.46, P<0.05) in HaCaT cells. Melatonin alleviated UVB-induced premature senescence ( t=7.139, P<0.05) and inhibited UVB-induced increase of p53 expression ( t=19.08, P<0.05) in HaCaT cells. In addition, ATM/ATR inhibitor, p53 inhibitor and melatonin all inhibited UVB-induced increase of melanin content in HaCaT cells. Conclusions:Melatonin inhibits TYR-mediated melanin synthesis by regulating p53-related premature senescence in HaCaT cells after UVB irradiation.

目的:探讨信号转导和转录激活因子3(STAT3)和小凹蛋白1(Cav-1)在X射线诱导A549细胞早衰过程中的调控关系,完善X射线诱导A549细胞早衰的分子机制.方法:利用蛋白免疫印迹(Western blot)法检测不同肿瘤细胞A875、BGC-823、A549细胞和正常细胞株Hacat细胞中STAT3和Cav-1蛋白表达水平,并检测X射线照射和STAT3过表达及抑制剂处理后A549细胞中p53蛋白、Cav-1蛋白表达水平.结果:肿瘤细胞A875、BGC-823、A549细胞及正常细胞Hacat细胞中STAT3、Cav-1蛋白呈现阳性表达;X射线照射A549细胞后p53蛋白表达增加,STAT3过表达及活化可引起p53蛋白表达水平升高,同时抑制Cav-1蛋白的表达.结论:X射线诱导的A549细胞p53蛋白早衰通路部分依赖于STAT3激活,STAT3负向调控Cav-1蛋白表达.