Chemical materials
NPs-TPP-NIT was prepared in our laboratory refer to previous study [25, 26]. Amifostine (WR2721) was purchased from MedChemExpress LLC. (Shanghai, China). All chemicals were of commercial analytical reagent grade.
Cell lines
Human normal hepatocytes cells (L-02) and human liver cancer cells (HepG2) were provided by the Department of Chemistry, the Air Force Medical University (AFMU) of the People’s Liberation Army of China (No. 169, Changle West Road, Xi’an, Shaanxi 710032, PR China). The cells were cultured in RPMI-1640 and Dul-becco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 50 µg/mL streptomycin, and 10,000 units/mL penicillin. The cultivation conditions were 37 ℃, 5% CO2 and saturated humidity.
Animals model
The male BALB/c mice (weight 18-22 g) and the nude mice (weight 20 ± 1.5 g) were both provided by the Experimental Animal Center of the AFMU. Animal experiments were approved by the Animal Care and Use Committee of the AFMU (Certificate No. KY20194116) following the Guidelines for the Care and Use of Laboratory Animals in China. In the experimental process, we tried to avoid animal pain and minimize the number of animals used. All mice were maintained on a 12-hour light/12-hour dark cycle at 25 °C. Tumor-bearing mice were subcutaneously injected HepG2 cells into lateral abdomen skin of nude mice at a concentration of 1 - 2 × 107 cells/mL of phosphate saline. Observe and measure the formation of tumors every 1 or 2 days. Approximately 7 - 10 days after injection of HepG2 cells, the tumor grew to a size of approximately 500 mm3 (10 × 10 × 5mm3), the mice were subjected to the in vivo experiments.
Survival status, spleen index and hematology assay
The male BALB/c mice were received i.v. injections of PBS, WR2721, or NPs-TPP-NIT. Thirty minutes after administration, all mice except for those in the normal group received a single dose of 6 Gy X-ray TBI, with an irradiation duration of 254 s and a dose rate of 1.414 Gy·min− 1 using biological irradiator (Rad Source irradiators RX-2000 XE, Atlanta, USA). We monitored the body weights (BW) daily, as well as their overall health status, including fur condition, behavior, food intake, and bowel movements. On different time point after irradiation, we measured the BW of the mice, after which mice were sacrificed by cervical dislocation and their spleens excised. We weighed the spleens of mice in each group to calculate the spleen index according to the following formula: (spleen weight/BW) × 1000. This experiment was performed in triplicate. On the 1st, 3rd, 7th, and 14th days after radiation, the blood samples were collected and determined using automatic animal blood cell analyzer (Mindray, BC-2800VET, Shenzhen, China).
Histopathology and TUNEL staining
The animal grouping and radiation modes were the same as before. We collected the spleens of mice from different groups on day 3 and 7 after irradiation exposure. Then, spleens were fixed with 10% (v/v) buffered formalin for 24 h, dehydrated in ethanol, and embedded in paraffin. Next, we sliced the spleens into 5-µm-thick sections, stained them with hematoxylin and eosin (H&E), and observed them under an Olympus BX41 microscope (Olympus, Tokyo, Japan). Spleen slices of the different group mice on day 7 were incubated with a terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) reaction mixture in a dark humidified chamber at 37 °C for 1 h for subsequent TUNEL staining experiments. Then, we washed the slices with PBS, counterstained them with 4′,6-diamidino-2-phenylindole (DAPI; C10012, Beyotime, Haimen, China) for 15 min, and then covered them with glycerol. An Olympus BX51 fluorescence microscope (Olympus Corporation, Japan) was used to capture and view images.
Mitochondrial ultrastructure of the spleens
The spleen tissue transmission electron microscopy (TEM) samples of mice in different groups on days 7 after radiation exposure was observed under the TEM (HT-7800, Hitachi Co., Japan), taking an ultrastructural image. The specific magnification was shown in the scale on the image. We measured and recorded the pixel area of mitochondria using Adobe Photoshop 22.0 (Adobe, Inc., San José, CA, USA).
Mitochondrial membrane potential assay
The mitochondrial membrane potential (∆Ψm) was determined using a mitochondrial membrane potential assay kit (JC-1). When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria, forming J-aggregates that can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 is a monomer that can produce green fluorescence. In 6-well plates, L-02 cells at a concentration of 1 × 105 cells per well were allowed to attach for 24 h. Before being exposed to 6 Gy X-ray irradiation (1.414 Gy·min− 1), the cells were treated with 0.15 µM NPs-TPP-NIT solution for 1 h. At 0.5 h after X-ray irradiation, the cells were washed with PBS three times and then incubated in DMEM culture for 24 h. Then, according to the kit method, JC-1 staining working solution was used (C2003S, Beyotime Biotechnology, Shanghai, China) to measure mitochondrial membrane potential. When detecting JC-1 monomer, the excitation light was set to 514 nm and the emission light to 529 nm; when detecting JC-1 polymer, the excitation light was set to 585 nm and the emission light was set to 590 nm.
Measurement of antioxidant-enzyme activity and level of inflammatory cytokines
On the seventh day after irradiation exposure, we prepared a certain amount of splenic tissue as 10% tissue homogenate, which was centrifuged at 3,500 rpm at 4 ℃ for 10 min. We measured SOD (A001-3-2), GSH peroxidase (GSH-Px; A005-1-1), and catalase (CAT; A007-1-1) activity as well as lactate dehydrogenase (LDH; A020-1-2) and MDA content (A003-4-1) in splenic tissue supernatant according to the reagent kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Enzymatic activity was expressed as U/mg or U/µg protein and MDA and LDH content as nmol/mg protein. We determined the level of the inflammatory cytokines IL-1β (H002-1-1), TNF-α (H052-1-2), IL-18 (H015-1-1), and IL-4 (H005-1-1) in serum using commercially available enzyme-linked immunosorbent assay kits (R&D systems, MN) per manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Immunofluorescent staining
ROS generation in splenic tissues were measured using a dihydroethylenediamine (DHE) fluorescent probe (D7008, Sigma Germany, Munich, Germany) [27]. In brief, frozen spleen sections were incubated with DHE (50 µM) for 1 h at room temperature in the dark. We observed specimen images at excitation wavelengths of 525 nm and emission wavelengths of 610 nm under an Olympus DP80 fluorescence microscope (Olympus Corporation, Japan). In 6-well plates, L-02 cells at a concentration of 1 × 106 cells per well were allowed to attach for 24 h. Before being exposed to 6 Gy X-ray irradiation (1.414 Gy·min− 1), the cells were treated with 0.15 µM NPs-TPP-NIT solution for 1 h. At 0.5 h after X-ray irradiation, the cells were washed with PBS three times and then incubated in RPMI-1640 culture for 24 h. Subsequently, the cells were fixed and blocked, followed by incubation at 4 ℃ with anti-p-NF-κB (1:1,000; GB113882; Servivebio, China) overnight at 4 ℃. On the next day, the cells were washed with PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:300; GB21303; Servivebio, China) for one hour at room temperature in the dark, then fixed with DAPI solution, and photographed via a fluorescence microscope (Nikon Eclipse C1, Japan).
Western blot analysis
On the seventh day after irradiation exposure, splenic tissues were dissected from mice in different groups and prepared for tissue homogenization. We obtained the supernatant by centrifuging the homogenate at 12,000 g for 10 min at 4 °C and took 10 µL supernatant to determine total protein concentration. Then, 10% separation glue and 5% concentration glue were prepared using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kit (W003-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). We transferred the membrane to a polyvinylidene difluoride (PVDF) membrane (Millipore Sigma, Burlington, MA, USA) and blocked it with 5% nonfat dairy milk for 2 h in TBST. Then, antibodies (Abs) against Bax (1: 1,000, GB12690, Servicebio, China), Bcl-2 (1: 1,000, GB124830, Servicebio, China), cleaved cysteine–aspartic acid–specific protease/proteinase (cleaved-caspase-3, 1:1,000, GB11767C, Servicebio, China, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB, 1: 2,000, GB11997, Servicebio, China), inhibitor of NF-κB kinase α + β (IKKα + β, 1: 2,000, 2682, CST, America), phosphorylated IKKα + β (p-IKKα + β, 1: 2,000, 2697, CST, America), nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α (IκBα, 1: 2,000, GB111509, Servicebio, China), p-IκBα (1: 2,000, AF2002, Affinity, America), IL-1β (1: 2,000, GB11113, Servicebio, China), TNF-α (1: 2,000, GB11188, servicebio, China), IL-18 (1: 2,000, 10663-1-AP, Proteintech Group, Inc, China), IL-4 (1: 2,000, ab62351, ABCAM, America), proliferating cell nuclear antigen (PCNA, 1: 2,000, GB11010, Servicebio, China), and β-actin (1: 2,000, GB11001, Servicebio, China) were added to the membrane for overnight incubation at 4℃. Next, we washed the membrane with TBST three times for 10 min each time and then incubated it with horseradish peroxide (HRP)-labeled goat anti-rabbit immunoglobulin1 G (IgG; 1:5,000; ZB-2301; ZSGB-BIO Technology Co. Ltd, Beijing, China) or HRP-labeled goat anti-mouse IgG (1:2,000; TA-09; ZSGB-BIO) at RT for 1 h. An electrochemiluminescent (ECL) Western blot detection reagent was prepared to detect antigen Abs. After scanning the strip, we performed grayscale analysis to calculate the relative expression level of the protein.
Mass-spectrometric analysis and protein identification
Using SDT lysis buffer to treat the splenic tissue samples from the normal, X-ray, and X-ray + NPs-TPP-NIT groups for protein extraction. Protein samples were boiled for 3 min and then sonicated and boiled again for 2 min. Then, we collected the supernatant via centrifugation at 16,000 g for 20 min. The supernatant was quantified using a BCA Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). We digested the proteins (100 µg) for each sample according to the method described in the literature [28]. In brief, reduced cysteine was blocked by adding DTT, the detergent, and iodoacetamide (IAA) in universal assay (UA) buffer. We added trypsin (Promega Corp., Fitchburg, WI, USA) at a ratio of 50:1 to the protein suspension for digestion overnight at 37 °C. Then, the suspension was centrifuged for 15 min at 16,000 g to collect the peptides, which was desalted and then subjected to liquid chromatography-mass spectrometry (LC-MS) using the C18 cartridge. The peptide concentration was measured at an optical density of 280 nm (OD280) on a Nanodrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) using MS database retrieval software MSFragger version 3.4 (https://msfragger.arsci.com/upgrader/). We used the UniProt protein database: UniProt Mus (Mouse) [10090]-88534-20230417.fasta (https://www.uniprot.org/taxonomy/10090), a total of 88,534 protein sequences. Microsoft Excel (Microsoft Corp., Redmond, WA, USA), Perseus software (Max-Planck-Institute of Biochemistry, Planegg, Germany) [29], and R statistical software (R Foundation for Statistical Computing, Vienna, Austria) were used to analyze bioinformatics data. Based on the open-source statistical language R25, this method uses Euclidean distance as the distance metric and the complete method as the aggregation method.
The analysis of annotated sequences relied on UniProtKB/Swiss-Prot (Boutet, 2016), Gene Ontology (GO) [30], and the Kyoto Encyclopedia of Genes and Gene Ontology (KEGG) for information extraction [31, 32]. GO is a controlled vocabulary of terms that represent biology in a structured way; the database uses three distinct ontologies that represent different biological aspects to describe genes and gene products: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). We performed Fisher’s exact test for GO and KEGG enrichment analyses, as well as false discovery rate (FDR) correction for multiple testing.
The influence of NPs-TPP-NIT on the radiotherapy treatment in vitro
After one hour of treatment with WR2721 or NPs-TPP-NIT, the HepG2 cells were exposed to a dose of 6 Gy X-ray irradiation (1.414 Gy·min− 1) except for control group. Then, the cell viability was determined using the CCK-8 method. Five replicates were used for each concentration. Cell apoptosis were detected by assay kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). Before being exposed to 6 Gy X-ray irradiation, HepG2 and L-02 cells were both treated with 0.15 µM NPs-TPP-NIT solution for 1 h. At 0.5 h after X-ray irradiation, the cells were washed with PBS three times and then incubated in RPMI-1640 and DMEM culture for 24 h. Then, the apoptosis rate of cells was determined using the annexin V-fluorescein isothiocyanate (annexin V-FITC) apoptosis detection assay kit. The Annexin-V was labeled with fluorescein isothiocyanate (FITC) to detect apoptotic cells, and PI was used to detect necrotic cells, the total cell death was calculated by the sum of apoptotic and necrotic cells. The experiments were performed at least three times. Cell cycle was measured by Flow cytometry (Beckman, USA). HepG2 and L-02 cells were pretreated with NPs-TPP-NIT and irradiated with 6 Gy X-ray the same as before. Then the cells were digested with trypsin and collected in centrifuge tubes. They were centrifuged at 1,000 rpm for 5 min, and the culture medium was removed. The cell pellet was washed once with 2 mL of PBS buffer and fixed with ice pre cooled 70% ethanol, overnight at 4 °C, centrifuged at 1,000 rpm for 5 min. 100 µL PBS was added to resuspend the cells. Then the cells were digested with RNase (50 µg/mL) (Servicebio, Wuhan, China) at 37 °C for 30 min, centrifuged at 1,200 rpm for 5 min, and PBS buffer was removed; cells were resuspended in 1 mL of PI staining solution (100 µg/mL) and stained avoiding light at 4 °C for 30 min. Flow cytometry was used with an excitation wavelength of 488 nm and an emission wavelength of 585 ± 21 nm for determination of the cell cycle. The cell cycle was analyzed using Modfit software to determine the cell cycle distribution.
The effect of NPs-TPP-NIT on the radiotherapy treatment
The tumor bearing mice treated with 5 mg/kg NPs-TPP-NIT and X-ray irradiation. Tumor bearing mice were irradiated with a dose rate of 1.414 Gy\({\cdot}\)min-1 on the tumor site, and the mice were sacrificed on the 7th day after irradiation. Then, the blood samples were collected for hematology and blood biochemistry examinations. The hematology assay was conducted using automatic animal blood cell analyzer (Mindray, BC-2800VET, Shenzhen, China). The blood biochemistry examinations was conducted using Automatic biochemical analyzer (Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China), including Alanine aminotransferase (ALT), aspertate aminotransferase (AST), creatinine (CREA), Urea and blood Urea Nitrogen (BUN). The tumor tissues were collected and kept in formalin (10%), and stained them with hematoxylin and eosin (H&E), and conducted to TUNEL staining and ki67 immunohistochemical staining.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Prism, Inc., La Jolla, California, USA). The statistical differences between groups were assessed using Student’s t-test for two-group comparisons and two-way analysis of variance (ANOVA) for multiple comparisons. Mean standard deviation (mean ± S.D.) was used to represent the experimental results. The experimental results were repeated more than three times. In all cases, P < 0.05 or less indicated a significant difference; P values greater than 0.05 were considered not significant (ns).
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