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

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

  • 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 Two

(Summary description)

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

 

 Helicobacter pylori (Hp) infection is the cause of gastrointestinal mucosal lesions such as gastroduodenal ulcer and chronic active gastritis. Studies have found that in Hp-induced chronic active gastritis, mucosal infiltrating neutrophils (PMN) is a characteristic finding. Hp eradication can significantly reduce PMN infiltration, indicating that there may be a correlation between Hp infection and PMN invasion Sex.

  Relevant data show that Hp or its water extract (HPE) can promote the production of interleukin (IL)-8 by gastric mucosal epithelial cells. IL-8 is considered to be a chemotactic or activator of PMN and promotes the interaction of PMN and vascular endothelial cells. Adhesion, this high adhesion is related to the expression of adhesion molecules (CD11a/CD18 and CD11b/CD18) on the PMN cell membrane, and HPE can induce its expression. Based on these findings, Hp that infects the gastric mucosa promotes the production of IL-8 by epithelial cells and induces the expression of adhesion molecules on PMN to cause PMN to migrate and infiltrate.

  Polyprezinc is a chelate compound composed of zinc and L-carnosine. It is a unique anti-ulcer drug that can adhere to and penetrate the gastric mucosa. Studies have shown that polyprezinc protects experimental gastric mucosal damage in animal models by inhibiting lipid peroxidation and neutrophil accumulation. So what is the effect of polyprezinc on the gastric inflammatory response induced by Hp? This article explores the effect of polyprezinc on the interaction of IL-8 and PMN endothelial cell adhesion in human gastric cancer cells (MKN 45 cells) .

  Materials and Methods

  1

  drugs

  Polyprezinc, L-carnosine and zinc sulfate. Dissolve polyprezinc in the same amount of hydrochloric acid and dilute to the specified concentration with Hanks' balanced salt solution (HBSS); the same applies to L-carnosine and zinc sulfate.

  2

  Hp Water Extract (HPE)

  HPE is prepared from the standard strain NCTC 11637.

  3

  Human PMN preparation

  Use Ficoll-Paque to collect heparinized peripheral blood from healthy adults for dextran precipitation and density gradient centrifugation to separate PMN and store it in HBSS. The PMN population produced by this process has 95-98% viability (except Trypan Blue) and 98% purity (acetic acid-crystal violet staining).

  4

  Endothelial cells

  Human umbilical vein endothelial cells (HUVEC) were harvested from the umbilical cord by collagenase treatment. The cells were inoculated in 199 medium containing 10% heat-inactivated fetal bovine serum, thymine, glutamine, heparin sodium, antibiotics (penicillin, streptomycin and amphotericin B) and endothelial cell growth factor. The cell culture was cultured in a humidified environment of 37°C and 5% CO₂ and amplified by trypsin. The primary to third-generation HUVEC were inoculated into a 96-well tissue culture plate coated with 0.1% gelatin and 25 μg/mL fibronectin for use.

  5

  Cell viability

  In order to study the effects of polyprezinc, L-carnosine and zinc sulfate on the viability of PMN and MKN 45 cells, cells were incubated with each compound (final concentration 10-5M) for 30 min and 6 h, respectively. The cell viability was determined by trypan blue dye exclusion method.

  6

  MKN 45 cells produce IL-8

  Human gastric cancer cell line MKN 45 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics in a humid environment at 37°C and 5% CO₂. MKN 45 cells were seeded in 48 culture plates with 2×105 cells and cultured under the above conditions. About 72 hours after inoculation, when the cells were fused, HPE (final concentration 2%) was added, incubated at 37°C for 6 hours, and the IL-8 released from the culture supernatant was quantitatively detected with an enzyme-linked immunosorbent kit.

  7

  PMN adhesion measurement

  The adhesion of PMN to endothelial cells grown in 96-well culture plates was measured by enzyme immunoassay. Add PMN suspension (2×105PMN) and HPE (final concentration 2%) to each well. After culturing in a CO² incubator at 37°C for 30 minutes, the cells were washed three times to remove non-adhesive PMN, and the adhesive PMN was fixed in PBS with 1% paraformaldehyde for 15 minutes and washed. The anti-CD11a monoclonal antibody was added to the wells and incubated for 30 minutes. After washing, the microwells were incubated with biotinylated anti-mouse immunoglobulin in PBS containing carrier protein and 15 mM sodium azide for 15 minutes. Wash the wells, add peroxidase-conjugated streptavidin for 15 minutes, and wash the wells again. Subsequently, 200μL of 0.4 mg/mL o-phenylenediamine dihydrochloride and 0.012% H²O² were added to the citrate buffer (pH5). The reaction was terminated by adding 50 μL of 1.5 M sulfuric acid. The absorbance was measured at 492 nm using a microplate reader to quantify the amount of antibody bound to the adhered PMN.

  8

  Statistical Analysis

  The results are expressed as mean ± standard deviation. Data comparison uses analysis of variance, and then Scheffe’s test is performed according to the situation. P<0.05 is statistically significant.

  results and analysis

  1

  Cell viability

  After co-cultivating PMN and MKN 45 cells with HBSS (control group), polyprezinc, zinc sulfate and L-carnosine, the cell survival rate was higher than 97%, indicating that the compound would not affect cell viability (Table 1).

  2

  MKN45 cells produce IL-8

  Figure 1 shows the effect of polyprezinc on the IL-8 production of MKN 45 after exposure to HPE. The results show that culturing MKN 45 cells with HPE can significantly increase IL-8 in the culture supernatant. 1×10-6 M-1×10-5 M) and zinc sulfate (1×10-6 M-1×10-5 M) have a significant inhibitory effect on the production of IL-8 by MKN 45 cells, but L-carnosine can It has no obvious inhibitory effect.

  Figure 1 The effect of polyprezinc, zinc sulfate and L-carnosine on the production of IL-8 by MKN 45 cells exposed to HPE. Each value is the mean ± SE of three repeated experiments. *Compared with the normal group without HPE, P<0.05; #Compared with the HPE group alone, P<0.05.

  3

  Expression of adhesion molecules on PMN

  Immunofluorescence flow cytometry was used to observe the effect of polyprezinc and its components on the expression of PMNCD11b and CD18 after HPE stimulation. As shown in Figures 2 and 3, after exposure to HPE, the expression of CD11b and CD18 on PMN increased significantly. Polyprezinc (1×10-7M-1×10-5M) has a concentration-dependent inhibitory effect on the expression of CD11b and CD18. At the same concentration, L-carnosine has no inhibitory effect, while zinc sulfate has an inhibitory effect similar to polyprezinc.

  Figure 2 The effect of polyprezinc, zinc sulfate, and L-carnosine on the expression of CD11b in PMN treated by HPE. The surface expression of CD11b in flow cytometry is the average fluorescence intensity. Each value is the average of four experiments ± SE. *With Comparison of normal group without HPE, P<0.05; #Compared with HPE group alone, P<0.05.

  Figure 3 The effect of polyprezinc, zinc sulfate, and L-carnosine on the expression of CD18 in PMN treated by HPE. The surface expression of CD18 in flow cytometry is the average fluorescence intensity. Each value is the average of four experiments ± SE. *With Comparison of normal group without HPE, P<0.05; #Compared with HPE group alone, P<0.05.

  4

  PMN adhesion to HUVEC

  Figure 4 shows the effect of polyprezinc and its components on the adhesion of PMN caused by HPE to HUVEC. HPE enhanced the adhesion interaction between PMN and endothelial cells, while polyprezinc and zinc sulfate reduced the adhesion of HPE-stimulated PMN to HUVEC in a dose-dependent manner, but L-carnosine had no effect on the adhesion of PMN.

  Figure 4 The effect of polyprezinc, zinc sulfate, and L-carnosine on HPE-induced adhesion of PMN to endothelial cells. In enzyme immunoassay, the adhesion ratio of polymorphonuclear leukocytes is expressed in optical density (OD). Each value It is the mean ± SE of three repeated experiments. *Compared with no HPE group, P<0.01; #Compared with HPE alone group, P<0.05.

  in conclusion

  In this study, polyprezinc has an inhibitory effect on HPE-induced CD11b/CD18 expression and PMN adhesion, and is concentration-dependent. Polyprezinc down-regulates the expression of PMN adhesion molecules, thereby reducing the infiltration of PMN caused by Helicobacter pylori-derived chemotaxis. CD11b/CD18 glycoprotein is present in resting neutrophil granules. After activation, it is transferred to the cell surface through particle fusion. Neutrophil activation can also trigger the activation of CD11b/CD18 on the cell surface. Polyprezinc has a direct effect on any signal transduction or translocation and conformation pathway of the CD11b/CD18 complex.

  Polyprezinc can inhibit the production of IL-8 in human gastric cancer cells MKN 45 and inhibit the expression of adhesion molecules on PMN induced by HPE. It can be seen that polyprezinc may prevent PMN-mediated inflammation of Helicobacter pylori infection It is effective for gastric mucosal diseases. Therefore, polyprezinc has great potential in the treatment of Helicobacter pylori-related gastric mucosal diseases.

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