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Does Zuprezinc have additional benefits for patients with lung injury?

Does Zuprezinc have additional benefits for patients with lung injury?

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

(Summary description)

Does Zuprezinc have additional benefits for patients with lung injury?

(Summary description)

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

 

 Polyprezinc

  Polyprezinc is a chelate of zinc and L-carnosine, and is often used in clinical anti-ulcer drugs. A study published in Metallomics in 2019 confirmed that polyprezinc has a protective effect on cadmium-induced lung epithelial cell damage. Cadmium is a toxic metal contained in food, water and the atmosphere. Cadmium exposure can cause human respiratory diseases. Various problems caused by cadmium are caused by oxidative stress-dependent cell damage. Low-dose environmental cadmium exposure can cause lung inflammation in mice by inducing the oxidation of mitochondrial proteins (including isocitrate dehydrogenase, malate dehydrogenase and ATP synthase). After acute exposure to cadmium, inflammation is most likely related to thiobarbital acid reactive substances (TBARS) in lung tissue. In addition, cadmium induces oxidative stress by generating reactive oxygen species, thereby inducing apoptotic cell death. Metallothionein is a metal-binding protein rich in cysteine ​​in cells, which has a detoxification effect on cadmium and other heavy metals in various organs. In addition, the expression of metallothionein is induced by metals with low biological toxicity, such as zinc. Therefore, in this study, we examined whether polyprezinc, a chelating compound composed of carnosine and zinc, can effectively inhibit cadmium-induced apoptosis of lung epithelial cells; at the same time, the mechanism was further studied.

  experimental method

  Using A549 cells (human lung epithelial cell line) to study the effect of polyprezinc on cadmium-induced apoptosis of lung epithelial cells.

  01

  Cell culture:

  Dulbecco's modified Eagle medium (DMEM) was added with 10% fetal bovine serum (FBS), and A549 (human lung epithelial cells) was cultured. After trypsinization, the cells were resuspended in 1% FBS DMEM, distributed to petri dishes, and cultured in an incubator (5% CO₂) at 37°C.

  02

  The number of viable cells and the determination of cytotoxicity, and the detection of apoptosis:

  Using a 96-well culture plate, add A549 cells to a concentration of 0.5 × 10⁴ cells per well, and add 200 μL of culture medium. After incubating for 24 hours, the cells are treated with polyprezinc (0-40 μM), zinc acetate ( 0-40 μM) or carnosine (0-40 μM) pretreated for 24 h. Then, the number of live cells is quantitatively detected, and the number of apoptosis is measured.

  03

  Reactive oxygen species (ROS) level measurement:

  A549 cells were pre-cultured in a black 96-well microplate (0.5×10⁴ cells). The cells were then pretreated with polyprezinc (0-40 μM) for 24 h, and then treated with DCFHDA (100 μM) and polyprezinc (0-40 μM), and CdCl₂ was added to the medium. After 1 hour of exposure, the ROS level was measured using a microplate reader.

  04

  RT-PCR:

  Use the RNeasy kit to extract total RNA from A549 cells (2.7×10⁴ cells per well in 0.5 mL of medium) grown in 24-well culture plates. The specificity was further confirmed by electrophoretic analysis of the reaction products and including a control group without template or reverse transcriptase. To normalize the amount of total RNA present in each reaction, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA was used as an internal standard. Use Primer-BLAST to design primers.

  05

  ELISA:

  A549 cells are pre-cultured in a 6-well culture plate (1.4×10⁵ cells per well). After 24 hours of incubation, the cells were pretreated with polyprezinc (0–40 μM), zinc acetate (0–40 μM) or carnosine (0–40 μM) for 24 h, and then collected in 200 mL PBS. The samples were frozen and thawed twice, incubated at 100±1°C for 10 minutes, and finally centrifuged at 13000×g for 10 minutes. The supernatant was diluted 10-fold and used for ELISA.

  06

  Western blotting:

  A549 cells are pre-cultured in a 6-well culture plate (1.4 × 10 cells per well). After 24 hours of incubation, the cells were treated with polyprezinc (0-40 μM) and collected in 200 mL RIPA buffer. The samples were frozen and thawed, and finally centrifuged at 13000 × g for 15 minutes. For recombinant MT-1, the protein supernatant was incubated with N,N,N,N’-tetrakis-(2-pyridylmethyl)ethylenediamine (100 μM) overnight, and the protein concentration was measured with Bradford reagent.

  Experiment result

  01

  Induce lung epithelial cell apoptosis through CdCl₂

  CellTiter-Glos 2.0 and Cell Counting kit-8 were used to measure ATP levels and mitochondrial activity in A549 cells to determine cell viability; as shown in Figure 1A, CdCl₂ (0-80 μM) dose-dependently reduced the intracellular levels of A549 cells ATP level. CdCl₂ (0-80 μM) also reduced the mitochondrial activity of A549 cells in a dose-dependent manner (Figure 1B). At the same time, the release of LDH in A549 cells was measured to monitor cytotoxicity, and CdCl₂ (0-80 μM) increased LDH release in a dose-dependent manner.

  Figure 1 A549 cells were incubated with the specified concentration of CdCl₂ for 24 hours. Use CellTiter-Glos 2.0 (A) or WST analysis kit (B) to determine the number of viable cells. The LDH activity assay kit (C) was used to determine the release of LDH into the cell culture medium.

  02

  The effect of polyprezinc on the apoptosis of lung epithelial cells induced by CdCl₂

  As shown in 2A, treating A549 cells with CdCl₂ (40 μM) can reduce intracellular ATP levels, and polyprezinc significantly restores the intracellular ATP levels reduced by CdCl₂ in A549 cells in a dose-dependent manner; at the same time, under these conditions, poly Prezinc did not affect the viability of A549 cells (Figure 2B). Polyprezinc treatment can also restore the mitochondrial activity reduced by CdCl₂ (Figure 2C). In addition, polyprezinc treatment can significantly reduce the cytotoxicity caused by CdCl₂-induced LDH release. (Figure 2D). The above results indicate that polyprezinc can inhibit the apoptosis of lung epithelial cells induced by CdCl₂.

  Figure 2 A549 cells were pre-incubated with polyprezinc at the specified concentration for 24 hours, and then pre-incubated with polyprezinc at the specified concentration for 24 h without CdCl₂ (40μM) or in the presence of CdCl₂ (40μM) ) Or 5 h(E). CellTiter-Glos 2.0 (A and B) or WST analysis kit (C) was used to determine the number of viable cells. LDH activity assay kit (D) was used to determine the release of LDH into the cell culture medium. Real-time blotting annexin apoptosis detection method was used to detect Annexin V positive cells. The vertical axis shows the luminous intensity (E) of the untreated control group. Values ​​represent the mean ±S.E. #P <0.05; **or ## P <0.01 (* vs. control, #vs. CdCl₂ alone)

  in conclusion

  Polyprezinc can inhibit the apoptosis of lung epithelial cells induced by CdCl₂, revealing that Polyprezinc can significantly inhibit cadmium-induced apoptosis of lung epithelial cells by inhibiting cadmium-dependent ROS production. In addition, studies have found that the antioxidant effect of polyprezinc is mediated by the induction of metallothionein. At the same time, polyprezinc may also treat respiratory diseases such as COPD and ARDS.

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