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Prevention of acute injury caused by radiotherapy-Polyprezinc particles

Prevention of acute injury caused by radiotherapy-Polyprezinc particles

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2020-12-01
  • Views:0

(Summary description)

Prevention of acute injury caused by radiotherapy-Polyprezinc particles

(Summary description)

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2020-12-01
  • Views:0
Information

  Preface

  Exposure to ionizing radiation in the intestine will cause rapid apoptosis of stem cells. Stem cells are responsible for regeneration in the intestinal wall. If the stem cells are damaged, it will cause crypt disorder. At an appropriate high dose, crypt injury will be accompanied by functional changes, such as malabsorption, which is clinically manifested as an acute intestinal reaction. Apoptosis is an active cell death process, including the concentration of nuclear chromatin, the densification of cytoplasmic organelles, and the fragmentation of membrane vesicles and cells into apoptotic bodies. The identification and counting of apoptotic bodies in hematoxylin-eosin (H&E) stained sections has been widely used to study intestinal cell apoptosis caused by ionizing radiation and other genotoxic stimuli. The p53 tumor suppressor gene is one of the main cytokines that determines growth arrest and cell death after ionizing radiation. The increase of p53 protein level in irradiated cells and the up-regulation of p21WAF-1/CIP1 (cyclin-dependent kinase inhibitor protein) are the key factors for G1 arrest. Radiation-induced apoptosis of mouse intestinal cells is regulated by members of the Bcl-2 family including Bax.

  Polaprezinc (Polaprezinc, PZ) is a chelate composed of zinc ions and L-carnosine, and is often used clinically to treat gastric ulcers. Studies have shown that polyprezinc can effectively prevent gastric mucosal damage without affecting gastric acid secretion and endogenous prostaglandins. This protective effect may be related to its anti-oxidation, membrane stabilization and promotion of mucus secretion. This study aims to evaluate whether polyprezinc can prevent X-ray radiation-induced apoptosis of jejunal gland cells in rats, and to explore its mechanism of radiation-induced jejunal gland cell apoptosis. H&E staining confirmed the existence of cell apoptosis, TUNEL method identified cell apoptosis, and immunohistochemical method was used to study the key effector caspase-3 in the apoptosis pathway of jejunal crypt cells. At the same time, observe the effect of polyprezinc on the expression of p53, p21WAF-1/CIP1 and Bax after irradiation.

  1 Materials and methods

  1.1 Animals: 60 male Wistar rats (230-310g) aged 7-8 weeks. Divided into 2-3 groups, each group of 2-3 animals, reared in a 24 ℃ environment, 7a.m.-9p.m. lights on, free eating and drinking water. One hour before irradiation, rats were pretreated with PZ (100 mg/kg) dissolved in 2.5 mL of sodium carboxymethyl cellulose (CMC), and control rats were pretreated with 2.5 mL of 2% CMC solution.

  1.2 Irradiation

  7a.m.-9p.m. for radiation. Rats received whole body X-ray radiation, using Toshiba EXS-300 X-ray, 200KV, 15mA device, 0.5mm aluminum filter, and the dose rate was 0.864 Gy/min.

  1.3 Evaluation of histology and apoptosis

  Rats in each group were sacrificed 1h, 2h, 4h, 6h after 2Gy radiation. The jejunum and colon are removed. The tissue specimens were fixed overnight in 4% neutral formalin buffer and embedded in paraffin blocks for H&E staining. The TUNEL method was used to identify cell apoptosis. This method used deoxynucleotide terminal transferase to label the 3'-OH end and detect the DNA fragments related to apoptosis. Anti-active caspase-3 polyclonal antibody staining was used to detect active caspase-3, which specifically recognizes amino acids 163-175 of caspase-3, but does not detect the precursor form. Immunohistochemical staining of active caspase-3 was performed with DAKO LSAB-2 kit.

  Quantify the incidence of apoptosis (apoptosis index) in the jejunum and colon by counting the number of apoptotic cells in each crypt through HE stained sections under an optical microscope of ×400 times.

  1.4 Western blot

  The jejunum tissues of rats in the control group and PZ group were taken 1h, 2h, 4h and 6h after 2Gy radiation, and frozen immediately. Then the tissue was suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate and 0.05% sodium lauryl sulfate, pH 7.4), broken into pieces on ice, and performed 3 times Freeze-thaw cycle. Centrifuge to remove insoluble cell debris, collect the supernatant, and determine the protein concentration with a protein analysis reagent. The protein sample (30μg) was subjected to SDS polyacrylamide gel electrophoresis and transferred to Hybond-ECL nitrocellulose membrane. Cell membranes were incubated with mouse monoclonal anti-p53 (Pab 421) antibody, rabbit anti-p21WAF-1/CIP1 antibody, Bax or rabbit polyclonal anti-actin antibody. Horseradish peroxidase combined with anti-mouse IgG antibody or horseradish peroxidase combined with anti-rabbit IgG (Amersham). Chemiluminescence (ECL+Amersham) is used to analyze protein levels. The blot was processed with ultra-thin film ECL (Amersham). The density of each protein band was measured with NIH-image1.61 software. The actin level was used as the standard to determine the protein level after irradiation.

  1.5 Statistical analysis

  Data are expressed as mean±SEM. Use Student’s test to test whether the differences between groups are statistically significant. P<0.05 is statistically significant.

  2 Results and analysis

  2.1 Polyprezinc reduces cell apoptosis index

  As shown in Figure 1, the histological sections of the jejunal crypts (Figure 1A, B), TUNEL (Figure 1C, D) and active caspase-3 antibody (Figure 1E, F) of the jejunal crypts of rats in the control group and PZ group stained with H&E . Two hours after 2Gy radiation, H&E staining and TUNEL staining showed a large number of apoptotic cells at the base of the jejunal crypts of the control group (Figure 1A, C). In contrast, the number of apoptotic cells in the jejunal crypts of rats in the PZ group decreased sharply (Figure 1B, D). The active caspase-3 immune response is mainly confined to the apoptotic bodies at the bottom of the intestinal crypts, which are the areas where apoptotic bodies most frequently appear in H&E stained sections (Figure 1E). Compared with control rats, the active caspase-3 immunoreactivity of PZ group rats was also significantly reduced (Figure 1F).

  Figure 1 The jejunum tissue sections of the control group (A, C, E) and the PZ group (B, D, F) 2h after 2Gy irradiation, stained with H&E (A, B), TUNEL method (C, D) ) Shows cells containing DNA fragments and active caspase-3 (E, F) (×400).

  As shown in Figure 2A, the time course of apoptosis index detected by H&E staining, TUNEL staining and active caspase-3 expression in the jejunum and colon of rats in the control and PZ groups 6 h after 2 Gy irradiation is compared. As shown in Figure 2A, the apoptotic index of jejunum cells in the control group showed an upward trend at 2h after irradiation, and gradually decreased at 4h and 6h. The apoptosis of jejunal gland cells in the PZ group after 2h, 4h and 6h radiation decreased to 13% (p<0.001), 44% (p<0.05) and 41% (p<0.01) of the control group, respectively. There was no significant difference in the level of apoptosis between the two groups at 0h.

  As shown in Figure 2B, the number of TUNEL-positive cells in the control group increased at 2h and 4h after irradiation, and decreased slightly after 6h. The number of TUNEL positive cells in the control group after irradiation was not higher than the apoptosis index. When rats in the PZ group were irradiated for 2h, 4h and 6h, the number of TUNEL positive cells decreased to 17% (p<0.001), 49% (p<0.05) and 47% (p<0.05) of the control group, respectively.

  As shown in Figure 2C, the number of caspase-3 positive cells in the control group reached a peak 2h after irradiation, and dropped sharply after 4h. In the PZ group, the number of caspase-3 positive cells decreased to 17% (p<0.001) and 33% (p<0.001) of the control group at 2h and 6h irradiation.

  Figure 2D shows that the apoptotic index of colon cells in the two groups of rats increased first and then decreased. There was no significant difference between the control group and the PZ group.

  Figure 2 In the control group (□) and PZ group (●) 0-6h after 2Gy irradiation, the jejunal crypt cell apoptosis index (A), TUNEL (B) and active caspase-3 (C) positive cells, colon Crypt cell apoptosis index (D). The data is expressed as the mean±SEM value of 3-5 rats. *Compared with the control group, p<0.05, **p<0.01 and ***p<0.001. 2.2 Western blot analysis of p53, p21WAF-1/CIP1 and Bax is to explore the pathway of PZ protection radiation-induced apoptosis, Western blot was used to analyze the expression of p53, p21WAF-1/CIP1 and Bax after 2 Gy irradiation. As shown in Figure 3, the accumulation of p53 at 1h, 2h, 4h, and 6h after irradiation in the control group increased by 1.2, 2.8, 2.9 and 2.6 times, respectively, compared with the non-irradiated control group. The accumulation of p53 in PZ group rats increased by 1.2, 1.5, and 1.4 times at 1h, 2h and 4h after irradiation, and decreased to 0.9 times after 6h. At 6h, there was a significant difference between the control group and the PZ group (p<0.05) (Figure 3A).

  After irradiation, the p21WAF-1/CIP1 of rats in the control group increased by 1.3, 2.4, 3.0, and 3.4 times at 1h, 2h, 4h, and 6h, respectively, compared with the non-irradiated control group, while the rats in the PZ group were increased by 1h, 2h, and 4h, respectively. The radiation group increased 1.4, 1.8, and 2.3 times, and dropped to 1.3 times after 6 hours. At 6h, there was a significant difference between the control group and PZ group rats (p<0.05) (Figure 3B).

  The Bax expression of rats in the control group increased by 1.5, 2.5, 1.9, and 3.7 times respectively at 1h, 2h, 4h, and 6h after irradiation, while the PZ group rats increased by 1.5 and 1.3 times at 1h and 4h, respectively. 2h and 6h dropped to 0.9 and 1.0 times respectively. There were significant differences between the control group and the PZ group at 2h (p<0.05), 4h (p<0.01) and 6h (p<0.05) (Figure 3C).

  Figure 3 Western blot analysis and kinetic study of p53 (A), p21WAF-1/CIP1 (B) and Bax (C) expression in the jejunum of rats in the control group and PZ group. Density analysis was used to determine the contents of p53, p21WAF-1/CIP1 and Bax. In the control group (□) and the PZ group (●), the protein level was expressed as the ratio of unirradiated rats (0h). The data is expressed as the mean±SEM value of 3-4 independent experiments in 2 rats. *Compared with the control group, p<0.05 and **p<0.01.

  Discuss

  In this study, the apoptotic index of the H&E stained sections of the control group gradually increased within 2h and gradually decreased after 6h, while the activation of caspase-3 reached a peak at 2h, and then dropped sharply at 4h, and at 4h and 6h No further increase. At 2 h and 6 h after irradiation, it was observed that the caspase-3 positive cell activity of rats in the PZ group was significantly inhibited (Figure 2C). These results indicate that polyprezinc can protect radiation-induced apoptosis and inhibit the activation of caspase-3. After 6h of X-ray radiation, compared with control rats, polyprezinc can significantly reduce the accumulation of p53 and the expression of p21WAF-1/CIP1 and Bax (Figure 3). During radiotherapy for malignant tumors, taking Relaisheng (polyprezinc particles) can prevent acute radiation damage and effectively protect the intestinal cell apoptosis induced by radiation. This protective effect may be related to the mechanism of membrane stability and promotion of mucus secretion. Prezinc is of great significance in radiotherapy.

  

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