Lysosomes represent a promising target for cancer therapy and reducing drug resistance. However, the short treatment time and low efficiency of lysosomal targeting have limited the application in lysosome-targeting anticancer drugs. In this study, we proposed an adhesive-bandage approach and synthesized a new lysosomal targeting drug, namely long-term lysosome-targeting anticancer drug (LLAD). It contains a SLC38A9-targeting covalently bound moiety and an alkaline component both to prolong the inhibition of SLC38A9 in lysosomes and alkalinize lysosomes. Upon short term and low-dose treatment of HeLa cells, at passage 0, with LLAD, it rapidly alkalinized lysosomes and also can be detected in lysosomes even at passage 15. LLAD induced apoptosis in HeLa cells through long-term lysosomal damage, and showed better long-term anticancer effect than cisplatin in vivo. Overall, our study paves the way for developing long-term lysosomal targeting drugs to treat cancer and overcome the drug resistance of cancer cells, and also provides a candidate drug, LLAD, for treating cancer.

Objective:To investigate the changes in the expression levels of long non-coding ribonucleic acids (lncRNAs) in human lymphocytes induced by low-dose ionizing radiation (LDIR) and the potential of lncRNAs as radiation biomarkers.Methods:Human immortalized lymphocytes (AHH-1) were irradiated with 0, 0.05, and 0.1 Gy of γ-rays at 24 h to extract RNAs for whole transcriptome sequencing. The sequencing was performed based on the 0, 0.05, and 0.1 Gy groups. The differentially expressed lncRNAs induced by LDIR were identified. The molecular functions, biological processes, and signaling pathway enrichment of differentially expressed genes were analyzed through the Gene Ontology (GO) analysis. Candidate lncRNAs were preliminarily validated using the qRT-PCR method. AHH-1 cells were irradiated with 0, 0.02, 0.05, 0.075, 0.1, and 0.2 Gy to extract the total RNAs at 4, 24, 48, 72, 96, and 120 h. The dose-response relationship of candidate lncRNAs was detected and analyzed. Peripheral blood sampled from eight healthy persons was irradiated with 0, 0.02, 0.05, 0.075, 0.1, and 0.2 Gy in vitro, followed by culturing for 24 h and 48 h to further verify the changes in the expression levels of radiation-responsive lncRNAs at the cellular level. Results:A total of 44 lncRNAs that were significantly up- or down-regulated after 0.05 and 0.1 Gy irradiation were initially identified through transcriptome sequencing. Among them, lncRNAs with over two-fold differential expression included SNHG1, SNHG15, NEAT1, and PRC1-AS1. At the cellular level, compared to 0 Gy, the relative expression level of PRC1-AS1 after 4 h to 48 h of γ-ray irradiation, was significantly elevated at 0.05, 0.075, and 0.1 Gy( t= -3.11 to 1.23, P < 0.05). In contrast, the relative expression level of NEAT1 was significantly up-regulated in a dose range of 0.02 to 0.1 Gy ( t=-2.47 to 2.10, P < 0.05). At the level of human peripheral blood, the relative expression levels of PRC1-AS1 and NEAT1 were significantly increased at 24 h after 0 to 0.2 Gy irradiation ( t=-3.79 to -1.96, P < 0.05). Conclusion:The PRC1-AS1 and NEAT1 with significant changes in expression levels serve as potential LDIR biomarkers.

Objective:To investigate the changes in the expression levels of long non-coding ribonucleic acids (lncRNAs) in human lymphocytes induced by low-dose ionizing radiation (LDIR) and the potential of lncRNAs as radiation biomarkers.Methods:Human immortalized lymphocytes (AHH-1) were irradiated with 0, 0.05, and 0.1 Gy of γ-rays at 24 h to extract RNAs for whole transcriptome sequencing. The sequencing was performed based on the 0, 0.05, and 0.1 Gy groups. The differentially expressed lncRNAs induced by LDIR were identified. The molecular functions, biological processes, and signaling pathway enrichment of differentially expressed genes were analyzed through the Gene Ontology (GO) analysis. Candidate lncRNAs were preliminarily validated using the qRT-PCR method. AHH-1 cells were irradiated with 0, 0.02, 0.05, 0.075, 0.1, and 0.2 Gy to extract the total RNAs at 4, 24, 48, 72, 96, and 120 h. The dose-response relationship of candidate lncRNAs was detected and analyzed. Peripheral blood sampled from eight healthy persons was irradiated with 0, 0.02, 0.05, 0.075, 0.1, and 0.2 Gy in vitro, followed by culturing for 24 h and 48 h to further verify the changes in the expression levels of radiation-responsive lncRNAs at the cellular level. Results:A total of 44 lncRNAs that were significantly up- or down-regulated after 0.05 and 0.1 Gy irradiation were initially identified through transcriptome sequencing. Among them, lncRNAs with over two-fold differential expression included SNHG1, SNHG15, NEAT1, and PRC1-AS1. At the cellular level, compared to 0 Gy, the relative expression level of PRC1-AS1 after 4 h to 48 h of γ-ray irradiation, was significantly elevated at 0.05, 0.075, and 0.1 Gy( t= -3.11 to 1.23, P < 0.05). In contrast, the relative expression level of NEAT1 was significantly up-regulated in a dose range of 0.02 to 0.1 Gy ( t=-2.47 to 2.10, P < 0.05). At the level of human peripheral blood, the relative expression levels of PRC1-AS1 and NEAT1 were significantly increased at 24 h after 0 to 0.2 Gy irradiation ( t=-3.79 to -1.96, P < 0.05). Conclusion:The PRC1-AS1 and NEAT1 with significant changes in expression levels serve as potential LDIR biomarkers.

Based on an overreview of Chinese radiological health standards, the Chinese radiological health standards currently in effect for biodosimetry were analyzed with respect to their current status, application and existing problems. Furthermore the improvement measures and development trends were put forward.

Objective:To study the influence of circular RNA hsa_circZDHHC21_004 on the proliferation of human small intestinal epithelial cells HIEC-6 after 60Co γ-rays exposure. Methods:HIEC-6 cells were exposed to 60Co γ-rays at 0, 5, 10, and 15 Gy with a dose rate of 1 Gy/min. The expression level of hsa_circZDHHC21_004 in the irradiated HIEC-6 cell was detected. Hsa_circZDHHC21_004 was knocked-down to investigate the influences of hsa_circZDHHC21_004 on the proliferation of irradiated HIEC-6 cells by CCK-8 assay and colony formation assay. Results:The expression level of hsa_circZDHHC21_004 in HIEC-6 cells was upregulated by (1.00±0.24), (1.34±0.28), (1.85±0.31), and (2.80±0.64) times of control after 0, 5, 10, and 15 Gy irradiation, respectively and there were significant difference between 10 or 15 Gy group and 0 Gy group ( F=10.86, P=0.008). Knockdown of hsa_circZDHHC21_004 significantly increased the proliferation rate of HIEC-6 cells at 24, 48, and 72 h after 10 Gy irradiation compared with non-irradiated control ( t=-6.25, -5.83, -7.75, P < 0.001). Under 2 and 5 Gy irradiation, the clone formation rates of the hsa_circZDHHC21_004 knockdown cells were significantly higher than those of the control ( t=-7.45, -8.83, P<0.01). Conclusions:Hsa_circZDHHC21_004 is increased after irradiation and influenced the proliferation of irradiated HIEC-6 cells.

Objective:To observe the effect of methylprednisolone (MP) combined with cyclophosphamide (CTX) on inflammation and immune cell activity in bleomycin (BLM)-induced pulmonary fibrosis rat model.Methods:Forty healthy 6 to 8-week-old SD rats were randomly divided into blank control, BLM model, BLM+MP, and BLM+MP+CTX groups, with 10 rats in each group. The rat model of pulmonary fibrosis was prepared by intratracheal infusion of BLM (5 mg/kg, only once). From the 7th day of modeling, MP (3 mg/kg) was injected in rats in the BLM+MP group and MP (3 mg/kg)+CTX (8 mg/kg) was injected via tail vein in rats in the BLM+MP+CTX group, once daily for 21 days. The degree of lung inflammation and fibrosis in rats was detected using HE and Masson staining methods. The numbers of granu-locytes and neutrophils in bronchoalveolar lavage fluid (BALF) and blood T cell subsets in rats were detected using flow cytometry.Results:On the 7th day of modeling, the external morphology, HE and Masson staining results of rat lung tissue showed that BLM-induced pulmonary fibrosis model was successfully prepared. On the 28th day of modeling, the lung tissue structure of the BLM group was disordered with obvious collagen deposition, the number of granulocytes and neutrophils in BALF increased significantly, the proportion of blood T cells, CD4 + T cells, and regulatory T cells (Tregs) decreased, the proportion of CD8 + T cells, and the CD4 +/CD8 + T cells ratio decreased significantly (all P<0.05). Compared with the BLM group, the degree of pulmonary fibrosis in the BLM+MP+CTX group was improved significantly, the number of granulocytes and neutrophils in BALF decreased significantly, the proportion of blood T cells, CD4 + T cells and Tregs cells increased significantly, the proportion of CD8 + T cells decreased, and the ratio of CD4 +/CD8 + T cells increased significantly (all P<0.05). The improvement effect in rats of BLM+MP+CTX group was better than that of BLM+MP group, and the difference was statistically significant ( P<0.05). Conclusion:MP combined with CTX can reduce the degree of inflammatory reaction in rats with pulmonary fibrosis and improve T cell immune activity.

Objective:To observe the effect of methylprednisolone (MP) combined with cyclophosphamide (CTX) on inflammation and immune cell activity in bleomycin (BLM)-induced pulmonary fibrosis rat model.Methods:Forty healthy 6 to 8-week-old SD rats were randomly divided into blank control, BLM model, BLM+MP, and BLM+MP+CTX groups, with 10 rats in each group. The rat model of pulmonary fibrosis was prepared by intratracheal infusion of BLM (5 mg/kg, only once). From the 7th day of modeling, MP (3 mg/kg) was injected in rats in the BLM+MP group and MP (3 mg/kg)+CTX (8 mg/kg) was injected via tail vein in rats in the BLM+MP+CTX group, once daily for 21 days. The degree of lung inflammation and fibrosis in rats was detected using HE and Masson staining methods. The numbers of granu-locytes and neutrophils in bronchoalveolar lavage fluid (BALF) and blood T cell subsets in rats were detected using flow cytometry.Results:On the 7th day of modeling, the external morphology, HE and Masson staining results of rat lung tissue showed that BLM-induced pulmonary fibrosis model was successfully prepared. On the 28th day of modeling, the lung tissue structure of the BLM group was disordered with obvious collagen deposition, the number of granulocytes and neutrophils in BALF increased significantly, the proportion of blood T cells, CD4 + T cells, and regulatory T cells (Tregs) decreased, the proportion of CD8 + T cells, and the CD4 +/CD8 + T cells ratio decreased significantly (all P<0.05). Compared with the BLM group, the degree of pulmonary fibrosis in the BLM+MP+CTX group was improved significantly, the number of granulocytes and neutrophils in BALF decreased significantly, the proportion of blood T cells, CD4 + T cells and Tregs cells increased significantly, the proportion of CD8 + T cells decreased, and the ratio of CD4 +/CD8 + T cells increased significantly (all P<0.05). The improvement effect in rats of BLM+MP+CTX group was better than that of BLM+MP group, and the difference was statistically significant ( P<0.05). Conclusion:MP combined with CTX can reduce the degree of inflammatory reaction in rats with pulmonary fibrosis and improve T cell immune activity.

Objective:To explore the characteristics of lipid metabolism in rat plasma after total body irradiation(TBI) in order to provide scientific evidence of radiation biomarkers.Methods:For the non-targeted lipidomics study, 50 SD rats were divided into 6 groups and irradiated with 0, 1, 2, 3, 5 or 8 Gy 60Co γ-rays, respectively. For the targeted lipidomics study, 25 rats were divided into 5 groups and irradiated with 0, 0.5, 2.5, 4 or 6 Gy. Venous blood samples were collected and plasma were separated 4 h after TBI. Radiation-sensitive lipids were screened and their concentrations were determined. Receiver operating characteristic curve (ROC) and dose-response were analyzed. Results:A total of 15 radiation differential lipids were screened out based on non-targeted lipidomics study and 7 of them were identified as radiosensitive lipids by targeted lipidomics analysis. The ROC of radiosensitive lipids distinguished area under curve (AUC) of samples in 0 Gy group and > 0 Gy group, < 2 Gy group and ≥ 2 Gy group were all > 0.75. The AUC values were increased to 0.96 and 0.94 after the panel of radiation sensitive lipids ROC analysis. The concentrations of LysoPC(18: 2), LysoPC(22: 0), PC(18: 0/18: 2), PE(18: 2/16: 0) and PE(18: 2/18: 0) decreased with irradiation dose within 0-6 Gy.Conclusions:A total of 7 plasma radiosensitive lipids in rat plasma were identified 4 h after TBI, and the panel of them could be used for specific dose classification. Five of the lipids had good dose-response relationship.

Objective:To screen radiosensitive lipid metabolites in rat small intestine and analyze their metabolic pathways, in order to provide scientific basis for radiation enteropathy biomarkers.Methods:The total body irradiation of 60Co γ rays was performed to rats with different doses of 0, 1, 2, 3, 5 and 8 Gy. The changes of lipids in small intestine were studied by targeted lipidomics method based on liquid chromatography coupled mass spectrometry (LC-MS). Results:Fifteen lipids in small intestine were screened as radiosensitive metabolites at 3 d after irradiation, including 4 up-regulated lipids and 11 down-regulated lipids( t=-6.395, 5.998, 5.836, -5.503, -5.449, -5.422, 4.841, 4.802, 4.621, 4.457, 4.426, 4.373, 4.110, 3.945, 3.902, P< 0.05 and FDR < 0.05). The metabolic pathways of sphingolipid, glycerophosphoplipid were significantly enriched. Four phosphatidyl serines (PS)increased while 1 phosphatidic acid(PA), 2 sphingomyelins(SM) and 4 fatty acids(FA)decreased in a good dose-response manner( R2> 0.80, P< 0.05), which were more potential radiation enteropathy biomarkers. Conclusions:Lipid metabolites in rat small intestine were significantly changed after the rat was total body irradiated with 60Co γ-rays.Eleven lipids with good dose-response relationship were more potential to be radiation enteropathy biomarkers.

Objective:To explore the feasibility of the optimized cytokinesis-block (CB) assay on radiation-induced nucleoplasmic bridge (NPB), and to provide a scientific basis for the application of NPB in biological dose estimation.Methods:Human peripheral blood in vitro was irradiated with 2 Gy 60Co γ-rays at a dose rate of 1 Gy/min (0 Gy control group). According to the culture time after irradiation, blood samples were divided into group 48, 56, 68 and 72 h. Cytochalasin-B (Cyt-B) with a concentration of 6 μg/ml was added into the samples at 28 h and harvested at 48, 56, 68 and 72 h after irradiation, respectively. On the other hand, the blood samples were treated with different concentration of Cyt-B i. e., 0.6, 1, 2, 6 and 10 μg/ml at the beginning of culture (0 h) and harvested at 68 h after irradiation. The proportion of mononucleated, binucleated and multinucleated cells, radiation-induced NPB and micronucleus (MN) frequencies were analyzed. Results:The nuclear division index (NDI) and proportion of binucleated cells at 2 Gy and 0 Gy had tendency of increasing with cell culture time. NPB frequencies (0.023 0-0.033 0/cell) and MN frequencies had no significantly difference ( P> 0.05). With the increase of Cyt-B concentration, NDI and the proportion of binucleated cells in group 2 Gy and 0 Gy also increased, but NPB frequencies (0.023 0-0.047 0/cell) had no significant difference ( P> 0.05). MN frequencies of group 10 μg/ml were significantly lower than that of group 6 μg/ml ( U=2.74, P< 0.01). Conclusions:Cell culture time and Cyt-B concentration had no significant influence on radiation-induced NPB frequencies, suggesting that NPB could be obtained by appropriately reducing cell culture time and Cyt-B could be added into blood samples at the beginning of culture. But this protocol reduced the number of cells for further analysis, and thus its feasibility for dose estimation still need to be studied.