The leader in the zinc family-Polyprezinc
- Categories:Stomach healthy
- Time of issue:2020-12-02
The leader in the zinc family-Polyprezinc
- Categories:Stomach healthy
- Time of issue:2020-12-02
Zinc can reduce the incidence, severity and duration of diarrhea in children in developing countries. Many studies have found that zinc can treat Shigellosis by regulating immune function. Zinc is essential for T-cell maturation, because zinc is an important cofactor for the synthesis of thymosin. Thymosin is a thymus hormone that is critical to T cell synthesis and function. Therefore, zinc is likely to be an effective auxiliary therapeutic agent for the treatment of microbial infections.
Polaprezinc (Polaprezinc, PZ) is a chelate composed of zinc ions and L-carnosine, and is widely used clinically to treat gastric ulcers. Studies have shown that the combination of polyprezinc and antibacterial agents can significantly improve Helicobacter pylori (Hp) infection. In addition, supplementing with zinc elements such as polyprezinc can inhibit autoimmune diseases by inhibiting T-cell activation. At present, polyprezinc has been further used to treat liver fibrosis, acetaminophen-induced hepatitis and endotoxin shock.
The above-mentioned effect of polyprezinc is related to the effect of zinc contained. Therefore, you may ask whether the effect of polyprezinc is different from that of pure zinc salt (such as ZnCl2)? In this study using fluorescent probes and rat thymocytes for cell counting, we compared polyprezinc Cellular effect of zinc and ZnCl2 to determine whether polyprezinc has a better zinc-related effect than ZnCl2.
1 Materials and methods
1.1 Animal and cell preparation
The study was approved by the Animal Experiment Committee of Tokushima University (No. 05279). To prepare the cell suspension: at 3-4°C, cut the thymus isolated from the ether anesthetized rat into a thickness of 1-2 mm with a blade. The sections were shaken gently in frozen Tyrode's solution (in mM: NaCl 150, KCl 5, CaCl2 2, MgCl2 1, glucose 5, HEPES 5; adjusted pH to 7.3-7.4 with NaOH) to dissociate thymocytes. After that, the Tyrode's solution containing cells was passed through a mesh (10 μm in diameter) to prepare a cell suspension. Before the experiment, the cell suspension was incubated for 1 h in a 36–37°C water bath. Although the Tyrode solution does not contain ZnCl2, the cell suspension usually contains 200–230 nM of zinc, which comes from cell preparations. Rat thymocytes can prepare single cells without enzyme treatment and can maintain the integrity of cell membranes. In addition, the modification effect of zinc on apoptosis and necrosis has been extensively studied in mouse thymocytes.
1.2 Fluorescence measurement of cell and membrane parameters
Use a flow cytometer equipped with an argon laser and fluorescent probes to measure cell and membrane parameters, and analyze fluorescence with JASCO software. In order to evaluate the lethality of the cells, propidium iodide was added to the cell suspension at a final concentration of 5 μM. Propidium iodide stains dead cells, and the lethality of cells is evaluated by measuring the fluorescence of the cells. The fluorescence intensity of 2 min after the addition of propidium iodide was detected by flow cytometry. The excitation wavelength is 488nm, and the emission wavelength is 600±20nm. Use FluoZin-3-AM as an indicator of intracellular Zn²+. Before the fluorescence measurement, the cells were incubated with 500nM FluoZin-3-AM for 60min to evaluate the change of Zn²+ concentration in rat thymocytes. FluoZin-3 fluorescence was measured in cells not stained with 5 μM propidium iodide. The excitation wavelength of FluoZin-3 is 488nm, and the emission wavelength is 530±20nm. 5-CMF-DA is used to monitor the changes of non-protein sulfhydryl groups in cells. Before performing fluorescence measurement, cells were incubated with 1 μM 5-CMF-DA for 30 min. 5-CMF fluorescence was measured in cells not stained with 5 μM propidium iodide. The excitation wavelength of 5-CMF is 488nm, and the emission wavelength is 530±15nm. Dihydroethidine was used to detect intracellular superoxide anions. Before the fluorescence measurement, the cells were incubated in 10 μM dihydroethidine for 60 min. The excitation wavelength is 488nm, and the emission wavelength is 600±15nm. 1.3 Oxidative stress Add H2O2 (2μL) to the cell suspension (2mL). The cell density is about 5×105 cells/mL to detect the effects of polyprezinc and ZnCl2. The cells were incubated with polyprezinc or ZnCl2 and H2O2 at 36-37°C. A preliminary study examined the effect of H2O2 on thymocytes. When the exposure time is 60min or longer, 2 mM H2O2 is lethal to thymocytes. This is based on prolonged exposure of thymocytes to 2 mM H2O2, which increases the number of cells stained with propidium iodide (possibly dead cells and/or cell membrane damage). After incubating the cells with 2 mM H2O2 for 90–120 min, the cell lethality increased slightly (10–20%), which is suitable for testing the enhancement of H2O2-induced cytotoxicity. H2O2 is not lethal at 100μM, but it increases the fluorescence intensity of FluoZin-3.
The value is expressed as the average value of 4 experiments ± standard deviation. Statistical analysis adopts Tukey multivariate analysis. P<0.05 has a significant difference.
2 Results and analysis
2.1 The effect of polyprezinc and ZnCl2 on the intracellular Zn²+ concentration
When the concentration of polyprezinc was 0.3μM, the fluorescence intensity of FluoZin-3 increased slightly, and further increasing the concentration of polyprezinc (up to 10μM) showed a concentration-dependently induced increase in fluorescence intensity. 90 minutes after starting the medication, the ability of polyprezinc to enhance the fluorescence of FluoZin-3 is similar to that of ZnCl2.
2.2 The effect of polyprezinc and ZnCl2 on the content of non-protein sulfhydryl groups in cells
Polyprezinc is incubated in the concentration range of 0.3μM-10μM to increase the fluorescence intensity of 5-CMF in a concentration-dependent manner. When the concentration of polyprezinc is 1 μM or more, the strength increases significantly. This shows that polyprezinc at a concentration of 1 μM or higher can increase the content of non-protein sulfhydryl groups in cells. Similar to the FluoZin-3 fluorescence result, the ability of Polyprezinc to enhance the fluorescence of 5-CMF is similar to that of ZnCl2 90 minutes after starting the medication.
2.3 The effect of polyprezinc and ZnCl2 on cell viability under the action of H2O2
Micromolar zinc is cytotoxic to H2O2-induced oxidative stress cells. In order to observe whether this situation exists in polyprezinc, the effect of simultaneous incubation of polyprezinc with H2O2 on cells was studied. Incubation with 10μM polyprezinc or ZnCl2 for 90min has no effect on cell lethality. However, when incubated with 2mM H2O2 at the same time, the cell lethality increased. 10μM polyprezinc significantly enhanced the cytotoxicity of 2mM H2O2, but the increase in cell mortality caused by the combination of H2O2 and polyprezinc was significantly less than that of H2O2 and zinc Combined. It shows that the enhancement effect of polyprezinc on the cytotoxicity induced by H2O2 is weaker than that induced by ZnCl2. Compared with ZnCl2, polyprezinc reduces the peroxidative damage caused by H2O2.
2.4 The effect of polyprezinc and ZnCl2 on oxidative stress induced by H2O2
In order to study whether there is a difference in the cellular effects of polyprezinc and ZnCl2 under oxidative stress, under the conditions of H2O2 induced oxidative stress, we investigated their effects on the intracellular Zn²+ concentration and intracellular superoxide anion content. As shown in Figure 4, 2mM H2O2 significantly enhanced the fluorescence intensity of FluoZin-3. Simultaneous application of 2mM H2O2 and 10μM polyprezinc also enhanced the fluorescence intensity of FluoZin-3, which was comparable to the fluorescence enhancement of 2mM H2O2 and 10μM ZnCl2. Therefore, under oxidative stress conditions, polyprezinc and ZnCl2 increase the intracellular Zn²+ concentration to almost the same extent. The same is true when 100μM H2O2 is combined with 3μM polyprezinc or 3μM ZnCl2.
The effect of simultaneous incubation of PZ and ZnCl2 with H2O2 on cell fluorescence. Zinc pyrithione increases the intracellular superoxide anion content by increasing the intracellular Zn²+ concentration. As mentioned above, under the oxidative stress induced by H2O2, polyprezinc and ZnCl2 greatly enhance FluoZin-3 fluorescence. Regarding the effect of polyprezinc and ZnCl2 on the content of superoxide anion in cells under oxidative stress, when H2O2 significantly increased the acetylene fluorescence intensity, the combination of H2O2 with polyprezinc and ZnCl2 did not further increase the intensity. Therefore, polyprezinc and ZnCl2 may not further increase the content of superoxide anion in cells under oxidative stress conditions.
Although there is no significant difference in the cellular effects of polyprezinc and ZnCl2, both zinc preparations can significantly increase the fluorescence intensity of FluoZin-3 and 5-CMF, and can detect intracellular Zn²+ and non-protein sulfhydryl groups. However, polyprezinc and ZnCl2 are used under the conditions of oxidative stress induced by H2O2, both of which can increase cell lethality by inducing excessive Zn²+ into the cells. However, compared with ZnCl2, it was found that polyprezinc can significantly reduce the peroxide damage caused by H2O2, and the cytotoxicity of polyprezinc is much lower than that of ZnCl2. Boda Weiye Ruilaisheng (Polyprezinc Granules) is a chelate composed of zinc ions and L-carnosine, and its effectiveness and safety are significantly better than other zinc preparations in the family.
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