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How much do you know about polyprezine except for the secluded game? Series one

How much do you know about polyprezine except for the secluded game? Series one

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

(Summary description)

How much do you know about polyprezine except for the secluded game? Series one

(Summary description)

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

 

 Epithelial cells play an important role in gastric mucosal cytokine network. In Helicobacter pylori (Hp) infection, gastric epithelial cells secrete large amounts of interleukin (IL)-8, which can aggregate and activate neutrophils in response Locally produced pro-inflammatory cytokines attach to the cell surface in response to Hp. The expression of IL-8 mRNA is regulated by the transcription factor NF-κB, NF-IL-6 and AP-1. Studies have shown that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1β, and Hp attached to the cell surface can activate NF-κB in gastric epithelial cells. This epithelial chemokine response is the main factor leading to gastric inflammation caused by Hp infection. Because persistent inflammation is associated with mucosal damage and possible gastric cancer, drugs that can down-regulate NF-κB activation and IL-8 expression have great therapeutic significance.

  Carnosine can promote the growth of granulation tissue and accelerate the healing of ulcers. Zinc can protect the stomach from damage and has an anti-ulcer effect. Combining the beneficial effects of zinc and carnosine, a new type of drug commonly used in clinical anti-ulcers-polyprezinc (a chelate composed of zinc ions and L-carnosine) is formed. Studies have confirmed that Polyprezinc can protect stomach damage and accelerate the healing of gastric ulcers. So does it have a stimulating effect on mucus secretion? In this study, we explored whether polyprezinc affects the activation of NF-κB and the expression of IL-8 in NKN28 gastric epithelial cells.

  1 Experimental method

  1.1 Cell culture

  MKN28 cells (gastric cancer cell line) are used as gastric epithelial cell model. Culture in a medium containing 10% fetal bovine serum, and eliminate fetal bovine serum 24 hours before the start of the experiment.

  1.2 Quantification of IL-8

  KN28 cells were cultured in a 24-well dish (2×105 cells/well), and the cells were incubated with TNF-α and IL-1β for 16 hours with and without polyprezinc, and the supernatant was collected. The concentration of IL-8 in the supernatant (0.5 mL) was determined using a human IL-8 specific enzyme-linked immunosorbent assay kit.

  1.3 Northern Blot

  After incubating with the test reagent, scrape off the MKN28 cells growing in a 10 cm culture dish, and extract the total RNA with Trizol reagent.

  1.4 EMSA

  Prepare nuclear extract from 5 x 106 cells.

  1.5 Western Blot

  Treat the MKN28 cells cultured in a 10cm petri dish with reagents, and directly collect them in 150μL SDS-sample containing 62.5mM Tris-HCl (pH 6.8), 2% SDS, 25% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue Buffer. LumiGLO chemiluminescence reagent was used to observe its specific bands.

  1.6 Statistics

  The data are expressed as an average of 6 standard deviations, and the unpaired data is statistically analyzed by Student't test, and p<0.05 is statistically significant.

  2 Results

  2.1 The effect of polyprezinc on the secretion of IL-8 induced by pro-inflammatory cytokines

  In Figure 2A, 10ng/mL TNF-α was used alone to increase the concentration of polyprezinc (10-1000μM). Incubation with 10 ng/mL TNF-α can significantly increase the IL-8 secreted by MKN28 cells (a 14-fold increase). However, compared with the IL-8 level measured in the presence of only 10ng/mL TNF-α, Polyprezinc caused a dose-dependent decrease in IL-8 secretion in MKN28 cells. In the presence of polyprezinc as low as 10 μM, IL-8 levels were significantly reduced. In this experiment, 10 μM, 100 μM, 300 μM, and 1000 μM polyprezinc inhibited the IL-8 secretion induced by 10ng/mL TNF-α by 57%, 60%, 96%, and 96%, respectively. Dose-dependently inhibited IL-8 secretion induced by TNF-α (10ng/mL).

  Figure 2A Polyprezinc inhibits IL-8 secretion induced by TNF-a (10ng/ml) in a dose-dependent manner Control, medium alone; T10: TNF-a (10 ng/ml); T10/P10: TNF-a (10 ng/ml) + 10 μM polaprezinc; T10/P100: TNF-a (10 ng/ml) + 100 μM polaprezinc; T10/P300: TNF-a (10 ng/ml) + 300 μM polaprezinc; T10/P1000 : TNF-a (10 ng/ml) + 1000 μM polaprezinc (n= 4-6). *p<.01 versus TNF-a.

  Figure 2B shows that TNF-α dose-dependently increased the IL-8 secretion of MKN28 cells (100ng/mL TNF-α, 22-fold increase), and 300 μM polyprezinc completely inhibited the stimulating effect of TNF-α.

  Figure 2B The effect of 300 μM polyprezinc on IL-8 secretion induced by TNF-a at different concentrations (0.1-100ng/ml) Control, medium alone control; T100, 100 ng/ml TNF-a; T10, 10 ng/ ml TNF-a; T1, 1 ng/ml TNF-a; T0.1, 0.1 ng/ml TNF-a (n= 4-6). *p<.01 versus polaprezinc(-).

  2.2 The effect of polyprezinc on IL-1β-induced IL-8 secretion

  The experiment is the same as 2.1. Figure 3A shows that IL-1β can induce MKN28 cells to secrete IL-8 more than TNF-α, and 10 ng/mL IL-1β can increase IL-8 secretion by about 25 times. Although 10 μM polyprezinc did not inhibit the IL-8 secretion induced by 10ng/mL IL-1β, 100 μM polyprezinc inhibited about 56%, and 300 μM polyprezinc almost completely inhibited the secretion of IL-8 .

  Figure 3A Polyprezinc has a dose-dependent inhibitory effect on IL-1β (10 ng/ml)-induced IL-8 secretion Control, medium alone; B10, IL-1β (10ng/ml); B10/P10, IL -1β (10ng/ml) +10 μM polaprezinc; B10/P100, IL-1β (10ng/ml) + 100 μM polaprezinc; B10/P300, IL-1β (10ng/ml) + 300 μM polaprezinc; B10/P1000, IL-1β (10ng/ml) + 1000 μM polaprezinc (n= 4-6). *p <.01 versus IL-1β.

  Figure 3B shows the IL-8 secretion induced by increasing the concentration of IL-1β (0.1-100ng/mL) with and without polyprezinc (300μM). Polyprezinc at this concentration has a significant inhibitory effect on IL-8 secretion induced by IL-1β at various concentrations, and the inhibitory rate is> 90%. At the same time, experiments proved that the effect of polyprezinc on IL-8 secretion was not caused by cytotoxicity.

  Figure 3B The effect of 300 μM polyprezinc on IL-8 secretion induced by IL-1β at different concentrations (0.1-100 ng/ml) Control, medium alone; B100, 100 ng/ml IL-1β; B10, 10 ng/ ml IL-1β; B1, 1 ng/ml IL-1β; B0.1, 0.1 ng/ml IL-1β (n = 4-6). p <.01 versus polaprezinc (-).

  2.3 The effect of polyprezinc on the expression of IL-8 mRNA induced by pro-inflammatory cytokines

  Northern blot was used to study whether polyprezinc down-regulated the expression of IL-8 mRNA induced by TNF-α and IL-1β in MKN28 cells. Figure 4A shows that TNF-α (10ng/mL) significantly up-regulates IL-8 mRNA expression, while Polyprezinc inhibits IL-8 mRNA expression in a dose-dependent manner. Optical density analysis (Figure 4B) showed that compared with only incubation with 10ng/mL TNF-α, in the presence of 300 μM polyprezinc, the ratio of IL-8 mRNA: glyceraldehyde-3-phosphate dehydrogenase mRNA Was suppressed by 90%.

  Figure 4A Northern blot analysis shows the effect of polyprezinc on the expression of IL-8 mRNA induced by TNF-a (10ng/ml) in MKN28 cells Lane 1, medium alone; Lane 2, TNF-a (10ng/ml); Lane 3, TNF-a (10ng/ml) + 10μM polaprezinc; Lane 4, TNF-a (10ng/ml) + 100μM polaprezinc; Lane 5, TNF-a (10ng/ml) + 300μM polaprezinc; Lane 6, TNF-a (10ng/ml) + 1000μM polaprezinc. Figure 4B Optical density analysis

  2.4 The effect of polyprezinc on IL-1β-induced IL-8 mRNA expression

  "IL-1β can significantly up-regulate the expression of IL-8 mRNA, and polyprezinc can also inhibit the expression of IL-8 mRNA in a dose-dependent manner (Figure 5A). Optical density analysis (Figure 5B) showed that 300μM polyprezinc almost completely inhibited the expression of IL-8 mRNA. Therefore, these results indicate that the inhibitory effect of polyprezinc on IL-8 secretion induced by TNF-α and IL-1β is related to the inhibition of IL-8 mRNA expression.

  Figure 5A Northern blot results show the effect of polyprezinc on IL-1β (10ng/ml)-induced IL-8 mRNA expression in MKN28 cells Lane 1, medium alone; Lane 2, IL-1β (10ng/ml); Lane 3 , IL-1β (10ng/ml) + 10μM polaprezinc; Lane 4, IL-1β (10ng/ml) + 100μM polaprezinc; Lane 5, IL-1β (10ng/ml) + 300 μM polaprezinc; Lane 6, IL-1β (10ng/ml) + 1000 μM polaprezinc. Figure 5B Density analysis

  2.5 The effect of polyprezinc on the activation of IL-8 and NF-κB induced by pro-inflammatory cytokines

  The expression of IL-8 is mainly regulated by the transcription factor NF-κB. The effect of polyprezinc on the activation of NF-κB induced by pro-inflammatory cytokines was studied through EMSA experiments. Incubation of MKN28 cells with TNF-α (10ng/mL) for 90 min can significantly up-regulate the specific DNA binding activity of NF-κB (Figure 6).

  Lane 1: Nuclear extract (-); Lane 2, medium alone; Lane 3: TNF-α (10ng/ml); Lane 4, TNF-α (10ng/ml) + anti-p50 antibody; Lane 5, TNF-α (10ng/ml) + anti-p52 antibody; lane 6, TNF-α (10ng/ml) + anti-p65 antibody; lane 7, TNF-α (10ng/ml) + anti-c-Rel antibody; lane 8, TNF-α (10ng / ml)+100-fold amount of cold competitor oligo; Lane 9, TNF-a(10ng / ml)+a 100-fold amount of cold noncompetitor oligo.

  2.6 The effect of different concentrations of polyprezinc (10-1000 μM) on the activation of NF-κB induced by TNF-α (10 ng/mL)

  Figure 7 shows that polyprezinc has a dose-dependent inhibitory effect on TNF-α-induced NF-κB activation, while 300 μM polyprezinc completely inhibits TNF-α-induced NF-κB activation. The activation of NF-κB induced by IL-1β (10 ng/mL) was also inhibited by polyprezinc. Because the intracellular signal transduction pathways of TNF-α and IL-1β are different, these results indicate that the role of polyprezinc in the process of NF-κB activation is at a stage where the two cytokine signal transduction pathways converge.

  Lane 1, nuclear extract (-); Lane 2, medium alone; Lane 3, TNF-a (10ng/ml); Lane 4, TNF-a (10ng/ml) + 10μM polaprezinc; Lane 5, TNF-a( 10ng / ml) + 100μM polaprezinc; Lane 6, TNF-a (10ng / ml) + 300μM polaprezinc; Lane 7, TNF-a, (10 ng / ml) + 1000μM polaprezinc; Lane 8, TNF-a (10ng/ml ) + 100-fold amount of cold competitor oligo; Lane 9, TNF-a (10ng/ml) + a 100-fold amount of cold noncompetitor olig

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