Modification of m6 A in human embryonic lung fibroblasts induced by neodymium oxide exposure and its mechanism / 环境与职业医学

Background Occupational and environmental particulate matter may cause fibrosis, accompanied by RNA m6A modification changes. Neodymium oxide (Nd2O3) can cause mouse lung fibrosis, which contains a large number of fibroblasts. Objective To investigate m6A modification of tumor necrosis factor receptor-associated protein 6/nuclear factor-κB (TRAF6/NF-κB) signaling pathway in fibrosis of human embryonic lung fibroblasts induced by Nd2O3, and identify the key m6A modification sites of TRAF6. Methods Designed concentrations of Nd2O3 (0, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg∙L−1) were infected with HELF cells for 24 and 48 h, and cell viability was detected to determine exposure time and dose. Measurements included indicators of fibrosis [hydroxyproline (HYP) and transforming growth factor-β1 (TGF-β1)], m6A methylation level, methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), reading proteins (YTHDC2 and YTHDF2), fibrosis-associated genes (collagen-І, vimentin, and α-SMA), and proteins related to signaling pathway (TRAF6, NFKB1, P65, and P-P65). The enrichment of m6A in TRAF6 mRNA was measured by methylated RNA immunoprecipitation-quantitative real-time PCR (MeRIP-qPCR). Results The results of cell viability indicated that 6.25, 12.5, 25 mg∙L−1 Nd2O3 and 48 h exposure time were used for subsequent experiments. After 48 h exposure, compared with the control group, the HYP level in the 25 mg∙L−1 Nd2O3 group was increased, and the levels of TGF-β1 in the 6.25, 12.5, and 25 mg∙L−1 Nd2O3 groups were increased (P<0.05); the overall m6A methylation levels of HELF cells in the 12.5 and 25 mg∙L−1 Nd2O3 groups were increased (P<0.05). At mRNA level, compared with the control group, the mRNA expression levels of methyltransferases METTL3 and METTL14 (6.25, 12.5, and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the mRNA expression level of reading protein YTHDF2 (6.25, 12.5, and 25 mg∙L−1 Nd2O3) was increased (P<0.05), while the mRNA expression level of YTHDC2 (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the mRNA expression levels of demethylases FTO (12.5 and 25 mg∙L−1 Nd2O3) and ALKBH5 (25 mg∙L−1 Nd2O3) were decreased (P<0.05); the mRNA expression levels of fibrosis-related genes vimentin, α-SMA, and collagen-Ⅰ (6.25, 12.5, and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the mRNA expression levels of pathway-related genes TRAF6 (25 mg∙L−1 Nd2O3) and NFKB1 (12.5 and 25 mg∙L−1 Nd2O3) were increased (P<0.05). At protein level, compared with the control group, the expression levels of methyltransferases METTL3 (25 mg∙L−1 Nd2O3) and METTL14 (12.5 and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the expression level of reading protein YTHDF2 (12.5 and 25 mg∙L−1 Nd2O3) was increased, while the expression level of YTHDC2 (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the expression level of demethylase FTO (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the expression level of fibrosis-associated protein vimentin was increased at 25 mg∙L−1 Nd2O3, and the expression levels of α-SMA and collagen-Ⅰ were increased at 12.5 and 25 mg∙L−1 Nd2O3 (P<0.05); the expression levels of TRAF6 and P-P65 were increased at 25 mg∙L−1 Nd2O3 (P<0.05). The MeRIP-qPCR results showed that compared with the control group, the concentrations of m6A in all Nd2O3 groups were significantly increased (P<0.05). Conclusions Upon exposure of HELF cells to Nd2O3, the alterations in fibrosis-related indexes increase the expression of some m6A methylases and decrease the expression of demethylases, thereby increasing the m6A methylase level, and may promote the progression of fibrosis by activating the TRAF6/NF-κB signaling pathway.

The role of Keap1/Nrf2/HO-1 signal pathway in liver injury induced by rare earth neodymium oxide in mice / 中华劳动卫生职业病杂志

Objective: To investigate the role of Keap1/Nrf2/HO-1 signaling pathway in liver injury induced by neodymium oxide (Nd(2)O(3)) in mice. Methods: In March 2021, forty-eight SPF grade healthy male C57BL/6J mice were randomly divided into control group (0.9% NaCl), low dose group (62.5 mg/ml Nd(2)O(3)), medium dose group (125.0 mg/ml Nd(2)O(3)), and high dose group (250.0 mg/ml Nd(2)O(3)), each group consisted of 12 animals. The infected groups were treated with Nd(2)O(3) suspension by non-exposed tracheal drip and were killed 35 days after dust exposure. The liver weight of each group was weighed and the organ coefficient was calculated. The content of Nd(3+) in liver tissue was detected by inductively coupled plasma mass spectrometry (ICP-MS). HE staining and immunofluorescence was used to observe the changes of inflammation and nuclear entry. The mRNA expression levels of Keap1, Nrf2 and HO-1 in mice liver tissue were detected by qRT-PCR. Western blotting was used to detect the protein expression levels of Keap1 and HO-1. The contents of catalase (CAT), glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) were detected by colorimetric method. The contents of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. The data was expressed in Mean±SD. Two-independent sample t-test was used for inter-group comparison, and one-way analysis of variance was used for multi-group comparison. Results: Compared with the control group, the liver organ coefficient of mice in medium and high dose groups were increased, and the Nd(3+) accumulation in liver of mice in all dose groups were significantly increased (P<0.05). Pathology showed that the structure of liver lobules in the high dose group was slightly disordered, the liver cells showed balloon-like lesions, the arrangement of liver cell cords was disordered, and the inflammatory exudation was obvious. Compared with the control group, the levels of IL-1β and IL-6 in liver tissue of mice in all dose groups were increased, and the levels of TNF-α in liver tissue of mice in high dose group were increased (P<0.05). Compared with the control group, the mRNA and protein expression levels of Keap1 in high dose group were significantly decreased, while the mRNA expression level of Nrf2, the mRNA and protein expression levels of HO-1 were significantly increased (P<0.05), and Nrf2 was successfully activated into the nucleus. Compared with the control group, the activities of CAT, GSH-Px and T-SOD in high dose group were significantly decreased (P<0.05) . Conclusion: A large amount of Nd(2)O(3) accumulates in the liver of male mice, which may lead to oxidative stress and inflammatory response through activation of Keap1/Nrf2/HO-1 signal pathway. It is suggested that Keap1/Nrf2/HO-1 signal pathway may be one of the mechanisms of Nd(2)O(3) expose-induced liver injury in mice.

Toxic effects of nano-sized neodymium oxide on central nervous system in mice / 中国职业医学

OBJECTIVE: To investigate the toxic effects of nano-sized neodymium oxide( nano-Nd_2O_3) on the central nervous system in mice. METHODS: Specific pathogen free female ICR mice were randomly divided into control group,lowdose group and high-dose group,with 12 rats in each group. The mice in low-dose group and high-dose group were treated with nano-Nd_2O_3 by nasal drip method at 80 and 160 mg/( kg·d) body weight for 30 days,while the mice in the control group were given 0. 9% sodium chloride solution. The water maze experiment and jump platform experiment were used to evaluate learning and memory ability. Hippocampus was examined using Hematoxylin-Eosin( HE) staining and glial fibrillary acidic protein( GFAP) immunohistochemical staining. The level of malondialdehyde( MDA) and the activity of total superoxide dismutase( T-SOD) in brain tissue were detected by microplate reader. RESULTS: The escape latency increased and the step down latency decreased in the low-dose group and high-dose group compared with the control group(P < 0. 05). No obvious pathological changes were observed by HE staining in brain hippocampus. Immunohistochemistry staining showed that the expression of GFAP protein in the hippocampal astrocytes of the low-and high-dose groups was higher than that in the control group,especially in the high-dose group,when compared with the control group. The MDA level increased and the T-SOD activity decreased in the low-and high-dose groups compared with the control group( P <0. 05). CONCLUSION: nano-Nd2 O3 can reduce the learning and memory ability of mice and increased GFAP expression in hippocampal astrocytes. The mechanism may be related to oxidative stress.

Damaging effect of nano-neodymium oxide on PC12 cells / 中国职业医学

OBJECTIVE: To study the damaging effect of nano-neodymium oxide( Nano-Nd_2O_3) on rat adrenal pheochromocytoma cell PC12. METHODS: PC12 cells in the logarithmic growth phase were divided into control group and 7 drug treatment groups. The control group was not treated with Nano-Nd_2O_3. The drug treatment groups were treated with 7 different dosing with the final mass concentrations of 3. 13,6. 25,12. 50,25. 00,50. 00,75. 00,100. 00 mg/L of NanoNd_2O_3 for 24 hours,then the follow-up selected experimental concentration were 3. 13,6. 25,12. 50,25. 00,50. 00 and75. 00 mg/L. The CCK-8 essay was used to detect the cell viability. The levels of glutathione( GSH) and superoxide dismutase( SOD) in the cells were measured by enzyme-linked immunosorbent assay. The expression of B cell lymphoma-2( Bcl-2),Bcl-2 associated X protein( Bax) and Caspase-3 protein were detected by Western blot. RESULTS: The survival rate,GSH level and Bcl-2/Bax ratio of PC12 cells decreased with the increase of the Nano-Nd_2O_3 dose,and showed doseresponse relationship( P < 0. 01). The SOD activity of PC12 cells in Nano-Nd_2O_3 group of 12. 50,25. 00,50. 00 and75. 00 mg/L were lower than that of control group( P < 0. 05). The ratio of Caspase-3/β-actin in PC12 cells was higher than that in control group( P < 0. 05). In the range of 0. 00-25. 00 mg/L of Nano-Nd_2O_3,with the increase of NanoNd_2O_3 dose,the ratio of Caspase-3/β-actin in PC12 cells increased gradually,and showed a dose-response relationship( P < 0. 01). CONCLUSION: Nano-Nd_2O_3 can induce oxidative stress in PC12 cells,leading to the decrease of Bcl-2/Bax ratio,activate Caspase-3 expression,and than inhibits cell proliferation in a dose-dependent manner.

Determination of Impurities in High Pure Neodymium Oxide with Inductively Coupled Plasma Mass Spectrometry / 分析化学

A method for the determination of impurities in high pure Nd_2O_3 employing inductively coupled plasma mass spectrometry(ICP-MS) was established, and emphasise was put on the elimination of matrix induced NdO~+ and NdOH~+ spectral interferences using cell collision technology(CCT). Using 10% O_2-10% Ar-80% He as the reaction/collision gas and with proper adjustment of the instrumental parameters, ~(159)Tb,~(165)Ho,~(163)Dy could be quantified at ~(175)TbO,~(179)DyO and ~(181)HoO respectively, while the matrix induced NdO~+ and NdOH~+ spectral interferences could be reduced by 25-70 folds. In addition, different elements were tested as internal standards for matrix effect correction under the CCT mode, and it was found that Re was best. Other impurities including La, Ce, Sm, Eu, Gd, Er, Tm, Yb and Lu were detected under conventional mode, except that Pr was detected under high instrumental resolution mode to eliminate peak tailing interference from Nd. In combination with mathematic correction, the established method can be applied for the determination of impurities in 99.999% Nd_2O_3 products with satisfactory RSDs and recoveries.