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Polyprene zinc particles | up-regulate heat shock protein to protect intestinal injury

Polyprene zinc particles | up-regulate heat shock protein to protect intestinal injury

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2020-12-02
  • Views:0

(Summary description)

Polyprene zinc particles | up-regulate heat shock protein to protect intestinal injury

(Summary description)

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2020-12-02
  • Views:0
Information

  Preface

  Polaprezinc (Polaprezinc, PZ) is a chelate composed of zinc ions and L-carnosine, and is widely used clinically to treat gastric ulcers. It has antioxidant effects and anti-Helicobacter pylori (Hp) activity. Studies have shown that polyprezinc can effectively prevent gastric mucosal damage and have a therapeutic effect on ulcerative colitis. At the same time, polyprezinc can up-regulate the expression of heat shock protein (HSP) in the stomach and colon.

  HSP is a highly conserved and widespread protein. When various physiological stress conditions such as infection and ischemia occur, HSP is up-regulated to play a protective role. The HSP family is artificially regarded as a key anti-inflammatory molecule to prevent physiological and external Stress plays a key role. Over-expressed HSP is thought to regulate intracellular mediators to closely participate in apoptosis signals to prevent cell apoptosis. In the small intestine, HSP can be upregulated through chemical substances or non-lethal heat stress to protect small intestinal epithelial cells and colon tissues. Especially HSP27 and HSP72 can protect cells and tissues from chemical, infectious and ischemic damage.

  "Previous studies have shown that polyprezinc up-regulates the expression of HSP27 and HSP72 in the colon of mice, but there is no research on the expression of HSP in intestinal epithelial cells cultured in vitro by polyprezinc. Therefore, this study aimed to explore the effect of polyprezinc on cell damage induced by oxidative stress. In addition, it was also investigated whether the protective effect of polyprezinc depends on the function of HSPs in human colon cells.

  1 Materials and methods

  1.1 Cell culture and PZ pretreatment

  CaCo2 cells (between passage 8 and 14) were cultured in DMEM, and 100 mL/l fetal bovine serum was added. The cells were cultured at 37°C with 50 mL/l CO2 and 90% humidity. Dilute polyprezinc with DMEM, add it to the cells at the final concentration (10-100μmol/L), and culture for 6h.

  1.2 Cell viability determination

  The MTT method was used to evaluate cell viability. The cells were grown at a concentration of about 104 cells/well. The cells were incubated at 37°C, and the cell viability was measured by the methoxazine produced in the substrate. The absorbance at 490nm (A) was measured with a spectrometer at 0 and 90 minutes after adding the celltiter96 aqueous solution and cell proliferation reagent. Calculate the difference between 0 and 90 min after incubation to assess cell viability. Untreated cells served as a negative control. Compare with the result of the negative control group. All experiments were repeated more than 3 times to confirm repeatability.

  1.3 Western blot analysis

  Use 1g/L trypsin to collect the cells and homogenize with a multi-tube homogenizer. Dissolve the same amount of homogenate in 20μL Tris HCL containing 10 g/L 2-mercaptoethanol, 20 g/L sodium lauryl sulfate, 200 mL/L glycerol and 0.4 g/L bromophenol blue, 50 mmol /L(pH 6.8). The sample was heated at 100°C for 5 min, and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoresis was transferred to a nitrocellulose membrane. The cell membrane was blocked with 10ml/L skimmed milk powder in PBS, and HSP27 and 72 antibody probes were used to react with goat anti-rabbit IgG antibody combined with horseradish peroxidase (HRP). The protein concentration was quantified with a trace BCA protein analysis kit.

  1.4 Quercetin treatment

  Quercetin has an inhibitory effect on HSP synthesis. In order to explore the effect of quercetin on cells, the cells were pretreated with 200μmol/L quercetin for 2h before polyprezinc pretreatment, and the expression of HSP27 and HSP72 was observed. 1.5 Statistical analysis The data is expressed as the mean ± standard deviation, and the analysis of variance (ANOVA) is used for statistical analysis, and then the Turkish comparison test is performed. P<0.05 is statistically significant.

  2 Results and analysis

  2.1 Polyprezinc protects CaCo2 cells from H2O2 damage

  After pre-treatment with polyprezinc, no morphological changes and growth inhibition of CaCo2 cells were observed. The MTT colorimetric method was used to determine the effect of ammonia (NH2Cl) on the activity of CaCo2 cells. The results showed that when the final concentration of H2O2 was 20mol/L (6.0%±4.4%), the cell difference at 490nm was significantly reduced. In contrast, 10μmol/L, 30μmol/L and 100μmol/L polyprezinc (35.0%±7.7%, 58.3%±14.6%, and 64.2%±8.2%, respectively) significantly improved the survival rate of cells ( p<0.01).

  2.2 Polyprezinc significantly increases colonic HSP expression

  "In untreated CaCo2 cells, HSP27 and HSP72 are constitutively expressed. After 6 hours of pretreatment with 10, 30 and 100 μmol/L polyprezinc, the expression of HSP25 and HSP72 is significantly up-regulated.

  2.3 The up-regulation effect of quercetin on HSP27 is not as obvious as that of polyprezinc

  In order to clarify the effect of HSP on polyprezinc-induced cell damage, the effect of quercetin, a HSP synthesis inhibitor, on polyprezinc and H2O2 damaged CaCo2 cells was studied. Compared with the cells exposed to H2O2, the cell viability of 200μmol/L quercetin pretreated with 200μmol/L quercetin greatly reduced the cell viability of CaCo2 cells treated with 30μmol/L polyprezinc. (P <0.01; respectively 12.0%±0.3% points and 53.6%±1.9%), while the cell viability of CaCo2 cells treated with quercetin and not exposed to H2O2 differed little from that of untreated cells (respectively They are 94.3%±0.1% and 98.8%±0.1%). As shown in Figure 2, in HSP expression, 200μmol/L quercetin completely inhibited the upregulation of polyprezinc on HSP27 and HSP72. But quercetin did not change the baseline levels of HSP27 and HSP72 that were not pre-treated with polyprezinc.

  Discuss

  Research shows that polyprezinc can effectively prevent CaCo2 cells from oxidative damage caused by H2O2 and improve cell survival. At the same time, polyprezinc significantly increases the expression of HSP27 and HSP72 in the colon, which plays an important role in protecting cells from stress. In addition, quercetin is an inhibitor of HSP synthesis, which weakens the protective effect of polyprezinc against H2O2 damage and the upregulation of HSP27 and HSP72 in CaCo2 cells. Reliance (Polyprezinc Granules) protects human colon cancer cells from oxidative damage induced by H2O2. This protective mechanism is closely related to the up-regulation of cytoprotective heat shock protein and has potential therapeutic significance for intestinal damage (such as colitis).

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