Polyprezinc | Prevent endotoxin shock
- Categories:Stomach healthy
- Time of issue:2020-12-03
Polyprezinc | Prevent endotoxin shock
- Categories:Stomach healthy
- Time of issue:2020-12-03
lipopolysaccharide (LPS) is the main component of the outer membrane of gram-negative (G¯) bacteria. It plays an important role in the occurrence of septic shock of G¯ bacteria. The similar manifestation of septic shock in animals after injection of LPS is called endotoxin shock. LPS is a substance that causes endotoxic shock and an effective activator of macrophages. As we all know, LPS can induce the production of pro-inflammatory mediators such as nitric oxide (NO) and cytokines such as tumor necrosis factor-α (TNF-α), resulting in endotoxic shock. In addition, studies have shown that LPS can promote the production of cytokines and NO in macrophages, microglia and endothelial cells. LPS is recognized by TLR-4 on the surface of host cells and activates NF-κB after signal transduction. NF-κB can regulate the transcription of various inflammation-related proteins including cytokines.
Polaprezinc (Polaprezinc, PZ) is a chelate composed of zinc and L-carnosine, and is often used clinically to treat gastric ulcers. Previous in vivo and in vitro experiments have revealed that polyprezinc can prevent gastric mucosal damage caused by various stimuli. This protective effect is mostly derived from its antioxidant activity, membrane stabilization and stimulation of mucus production.
In this study, we investigated whether polyprezinc can inhibit LPS-induced endotoxic shock and LPS-mediated macrophage activation, and explored whether the inhibition of polyprezinc on macrophages is related to the induction of heat shock proteins. And how polyprezinc prevents LPS-mediated endotoxic shock.
Materials and Method
Male ddY mice, 5 weeks old, 5-6 cages. Before the experiment, the animals were subjected to a 12h light/dark cycle under controlled temperature (23±3°C) and humidity (55±5%) conditions.
1.2 Experimental method
LPS (40mg/kg body weight, 6mL/kg) and solution (10% DMSO) were injected intraperitoneally. Polyprezinc (100mg/kg, 5mL/kg) was suspended in 0.5% sodium carboxymethyl cellulose solution, gavage 2h before LPS, and observe the survival of mice after 96h. At 6, 12, 18 and 24 hours after the LPS treatment, the mice were anesthetized with ether, blood was collected from the right ventricle with a heparin syringe with a needle, and the samples were stored in liquid nitrogen.
1.3 Determination of serum zinc concentration
Use atomic absorption spectrophotometry to measure serum zinc concentration. Serum zinc concentration was determined 1, 2, 4 and 8 hours after intragastric administration of polyprezinc (10, 50 and 100 mg/kg body weight).
1.4 Cell culture and treatment
Obtain the original 264 mouse macrophage cell line from the Riken BRC cell bank. In a humidified incubator at 37°C, 10% heat-inactivated FCS, 2mM glutamine and antibiotics (100 units/mL penicillin, 100μg/mL streptomycin) ) In DMEM. After washing with medium to remove non-adherent cells, the adherent cells were exposed to LPS (100 ng/L and 1 μg/mL) for 24 hours at 37°C, 5% CO2/95% air. At 6 and 2h before LPS administration, polyprezinc, zinc sulfate and carnosine were added to the medium.
1.5 NO detection
Use the kit spectrophotometric method to measure the plasma NO2/NO3 level, the nitrite in the medium is used as an indicator of NO production.
1.6 Detection of TNF-α
Use enzyme-linked immunosorbent assay (ELISA) kit to measure the level of TNF-α in plasma and culture medium.
1.7 Western blot
The content of HSP70 and NF-κB p65 was determined by Western blotting, and Western blotting was quantified by Scion image.
1.8 Real-time PCR
Use RNeasy® mini kit to extract total RNA from lung tissue, use QuantiFast SYBR® green RT-PCR kit to perform reverse transcription and PCR in a single tube, and use 7900HT fast real-time PCR system to detect the relative content of all mRNAs.
1.9 Lung Histopathology
The lung tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H-E). In order to estimate the degree of damage, the paraffin section was observed under an optical microscope.
1.10 Protein determination
With bovine serum albumin as the standard, the protein content was determined by Bradford method.
1.11 Statistical evaluation
Data are expressed as mean ± standard error (SE). Use Student’t test and one-way analysis of variance to analyze the changes in variables of different analysis methods. Survival studies were analyzed by chi-square test. p<0.05 has significant difference.
Results and analysis
2.1 The effect of polyprezinc on serum zinc concentration
In order to determine the optimal treatment time and dose of polyprezinc, serum zinc concentration was measured 1, 2, 4, and 8 h after polyprezinc (10, 50, and 100 mg/kg body weight) pretreatment (Figure 1). Serum zinc levels increased in a dose-dependent manner, reaching a maximum 1 hour after administration, and then decreased in a time-dependent manner.
2.2 The effect of polyprezinc on the survival rate of mice after LPS
"96h survival rate dropped to 20% after LPS treatment. Pretreatment with polyprezinc (100mg/kg) 2h and 1h before LPS treatment can significantly increase the survival rate of mice to 80% and 55%, respectively. Although the serum zinc level in the polyprezinc treatment group was higher than that in the polyprezinc treatment group 1 h before LPS effect, the survival rate of the mice in the polyprezinc treatment group was better than that of the polyprezinc treatment group 2 hours before the LPS effect. high.
2.3 The effect of polyprezinc on plasma NO and TNF-α levels in mice after LPS
24h after LPS treatment, plasma NO level increased, polyprezinc pretreatment can significantly inhibit LPS-mediated NO production.
TNF-α level also increases with time, reaching a maximum at 18h after LPS action, and decreasing at 24h. Polyprezinc pretreatment can significantly inhibit LPS-mediated TNF-α production.
2.4 The effect of polyprezinc on lung histology in mice after LPS
LPS can induce lung histological changes in mice, such as inflammatory cell infiltration, perivascular edema, and obvious alveolar hemorrhage. Compared with the control group and the polyprezine treatment group, the pretreatment with polyprezinc can significantly improve the lungs. damage.
2.5 The effect of polyprezinc on the expression of HSP70 in lung tissue of mice after LPS
"Although polyprezinc is known to be an effective HSP inducer, polyprezinc does not enhance the induction of lung HSP70 after the action of LPS.
2.6 The effect of polyprezinc on iNOS gene expression in mouse lung tissue after LPS
6h after LPS treatment, the level of iNOS mRNA in lung tissue increased by about 20 times. Treatment with polyprezinc 2h before LPS effect can significantly reduce iNOS gene expression; the metallothionein-I (MT-I) and MT-II mRNA levels in the LPS treatment group and the polyprezinc treatment group 2h before LPS treatment did not Significant differences, but MT-I and MT-II gene expression increased significantly in both groups.
2.7 The effect of polyprezinc on the production of NO and TNF-α in 264 cells mediated by LPS
RAW 264 cells treated with polyprezinc (100μM) 6 h before LPS treatment can significantly inhibit the production of NO and TNF-α 24 h after LPS treatment.
2.8 The effect of polyprezinc on LPS-mediated NF-κB activation in 264 cells
It is known that LPS induces the expression of iNOS and inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IFN-γ by activating NF-κB. Western blot analysis whether polyprezinc inhibits LPS-induced NF in 264 cells -κB nuclear shift. LPS (100ng/mL) caused the transport of NF-κB p65 to the nucleus in a time-dependent manner. Pretreatment with polyprezinc for 6 hours before LPS significantly inhibited nuclear transport at 24 hours after LPS.
2.9 The effect of zinc sulfate and carnosine on LPS-mediated NO production in 264 cells
Polyprezinc is a chelate composed of zinc and L-carnosine. We want to study the effect of zinc and L-carnosine alone on the production of NO in 264 cells induced by LPS. Zinc sulfate can inhibit the production of NO in cells after 24 hours of LPS treatment, and it is dose-dependent. However, L-carnosine cannot inhibit the production of NO.
Lipopolysaccharide (LPS) causes endotoxin shock, multiple organ failure (MOF) is the direct cause of death from endotoxemia, and acute lung injury (ALI) is an important cause of MOF morbidity and mortality. Studies have shown that polypu Rezinc can protect the acute lung injury caused by LPS and significantly improve the survival rate of mice. Polyprezinc can resist LPS-mediated internalization by inhibiting the activation of NF-κB and inducing pro-inflammatory products such as NO and TNF-α. Toxin shock.
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