Polyprezinc prevents NSAID-related small intestinal mucosal injury
- Categories:Stomach healthy
- Time of issue:2020-12-02
Polyprezinc prevents NSAID-related small intestinal mucosal injury
- Categories:Stomach healthy
- Time of issue:2020-12-02
Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) can damage the stomach and duodenum. With the advent of capsule endoscopes and double-balloon endoscopes, more and more NSAID-related small intestinal mucosal injuries have been diagnosed, but no specific drugs have been developed for small intestinal mucosal injuries. In clinical trials, prostaglandin E1 (PGE1) derivatives, misoprostol and antacids, including proton pump inhibitors (PPI) are commonly used to prevent and treat NSAID-related small intestinal mucosal damage. However, these drugs can cause many adverse reactions and reduce patient compliance. For example, misoprostol can cause diarrhea. Multiple studies have confirmed the effectiveness of metronidazole on small intestinal mucosal damage. However, the combination of metronidazole and NSAID can lead to the emergence of resistant strains. Therefore, the development of small intestinal injury specific drugs that can be taken safely for a long time, such as mucosal protective drugs, is of great significance for the treatment of small intestinal mucosal injury. Polyprezinc (PZ) is a chelate composed of zinc ions and L-carnosine. It is a gastric mucosal protective agent. It is widely used in the stomach because of its powerful reactive oxygen species (ROS) quenching effect. Treatment of ulcers and gastritis. In this study, the rat small intestinal epithelial cell line RIE-1 was used as the research object to investigate the mechanism of indomethacin (IND)-induced small intestinal mucosal damage and the protective effect and mechanism of polyprezinc.
1 Materials and methods
1.1 Intestinal epithelial cell line
Rat intestinal epithelial cell line (RIE-1), cultured in Dulbecco's modified Eagle's medium (DMEM)/F12, supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin and 1% Streptomycin. The cells are cultured in humidified air containing 5% CO2 at 37°C, and seed implantation is performed when the cell monolayer becomes sub-fluid.
1.2 Cell viability
Use MTT's WST-8 detection kit to measure cell viability. The cells were seeded in a 96-well plate and cultured for 0-48h. Wash the cells twice with PBS, and add WST-8 solution. Incubate at 37°C for 1 h, and read the optical density at 450 nm.
After was labeled, the effect of indomethacin on cell apoptosis was observed by fluorescence microscope. After pretreatment with indomethacin for 24 hours, the cells were incubated with 10μg/ml HO342 dye at 37°C for 15min, and with 10μg/ml PI at 37°C for 10min. Observe the double stained cells with an inverted fluorescence microscope. Living cells with cell membrane function, living cells and early apoptotic cells absorb blue dye (HO342). Apoptosis is characterized by morphology and analyzed by concentrated chromatin. Cells stained in red (PI) are late apoptotic (chromatin condensation) or necrotic cells. Quantitative assessment of apoptosis was performed with the apoptosis detection ELISA kit. After incubating with indomethacin for 24 or 48 hours in the presence or absence of PZ, Zn and L-carnosine, remove the supernatant (to remove necrotic cells), lyse the cells, centrifuge, and detect the supernatant with an ELISA kit . Read the optical density at 405nm with a microplate reader.
1.4 Separate cell lysate into cytoplasm and mitochondria
Using the mitochondrial/cytoplasmic fractionation kit, the cytoplasm and the mitochondrial compartment are separated by ultracentrifugation. The cells were collected, washed, resuspended in 19 cytosolic extraction buffers, and homogenized with a grinder. The homogenate was centrifuged at 700g and 4°C for 10min, the supernatant was centrifuged at 10000g and 4°C for 30min, and then the supernatant was used as cytoplasm. The obtained pellet was resuspended in 19 mitochondrial extraction buffer for mitochondrial evaluation.
1.5 Oxidative stress assessment
After labeling live cells with redox sensitive fluorescent probes, RedoxSensor Red CC-1 and mitochondria, the effect of indomethacin on ROS production in RIE-1 cells was observed with a laser scanning confocal microscope. Incubate for 30 min with or without 200 μM indomethacin and 100 μM PZ. The cells were then incubated with 1 μM RedoxSensor Red CC-1 and 1 μM MitoTracker Green FM at 37°C for 10 min. In addition, the oxidative stress in the cell was evaluated by another method of detecting S-oxidized protein. Incubate RIE-1 cells with or without 200μM indomethacin and 100μM PZ for 3h, then add 100μM Bt-Cys to RIE-1 cells and incubate for 30min, and then prepare whole cell lysates or isolated lysates. Transfer the protein to the nitrocellulose membrane. After transfer, the membrane was incubated in a blocking agent at room temperature for 30 min. Then the membrane was incubated with horseradish peroxidase (HRP)-conjugated streptavidin (Invitrogen) or rabbit polyclonal anti-actin antibody as an internal standard at room temperature for 1 h. Anti-tubulin antibodies or anti-cytochrome c oxidase subunit IV (COX4) antibodies were used to determine whether each lysate was cytoplasm or mitochondria, respectively. After washing, the bound HRP was detected by enhanced chemiluminescence and visualized with VersaDoc 5000MP.
1.6 Detection of apoptosis-related proteins by Western blotting
The cleavage products were separated by SDS-PAGE and transferred to nitrocellulose membrane. After transfer, incubate for 30 min in a blocking agent at room temperature. Then incubate with anti-cytochrome c antibody or anti-smac/DIABLO antibody or anti-caspase-3 antibody at room temperature for 1 h, and then use secondary anti-rabbit IgG antibody for ECL culture. ImageJ software was used for quantitative analysis.
1.7 Statistical analysis
Data are expressed as mean±SEM. Use paired t test or t test for statistical analysis. P<0.05 is statistically significant.
2 Results and analysis
2.1 Indomethacin induces RIE-1 cell apoptosis in a time and dose-dependent manner
RIE-1 cells were incubated with indomethacin at a concentration of 0-400μM for 24 hours, and found that indomethacin induced apoptosis in a concentration-dependent manner. Indomethacin-induced apoptosis also occurs in a time-dependent manner. After indomethacin treatment for 12 hours, cell apoptosis was most obvious.
2.2 Indomethacin induces apoptosis-based apoptosis
Use HO342 and PI to stain RIE-1 cells and observe the apoptosis mode under a fluorescence microscope. After indomethacin was incubated with RIE-1 cells for 24 hours, the changes in induced apoptosis are shown in Figure 2B. Nucleus condensation and the formation of apoptotic bodies, which are signs of apoptotic cells; this change is not the case in control cells. obvious. Indomethacin group RIE-1 cells appeared early and late apoptosis. Very few primary necrotic cells were seen in both groups.
Quantitative analysis of indomethacin-induced apoptosis by enzyme-linked immunosorbent assay. As shown in Figure 3, the proportion of apoptotic cells in the medium group slowly increased in a time-dependent manner. However, the proportion of apoptotic cells in the indomethacin (200μM) group increased significantly from 3h (P<0.05 at 3h and 6h, P<0.01 after 9h).
2.3 PGE2 treatment failed to attenuate indomethacin-induced apoptosis
Non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin have a certain degree of side effects on the digestive system because they have an inhibitory effect on cyclooxygenase (COX), and cyclooxygenase can catalyze the formation of arachidonic acid into PG . Therefore, PG derivatives are clinically used to prevent gastrointestinal diseases and ulcers induced by NSAIDs. In order to clarify the role of COX in indomethacin-induced apoptosis of RIE-1 cells, PGE2 was used, and it was found that PGE2 could not protect RIE-1 indomethacin-induced apoptosis. This result indicates that indomethacin induces apoptosis in RIE-1 through a mechanism independent of COX.
2.4 Polyprezinc protects RIE-1 cells from indomethacin-induced apoptosis
Previous studies have shown that indomethacin induces important apoptosis in differentiated CaCo2 cells (immortalized human colonic epithelial cell line, which exhibits the phenotype of the small intestine based on differentiation), and α-tocopherol through its powerful antioxidant The effect significantly reduces this apoptosis. In view of the above results, it is hypothesized that indomethacin-induced apoptosis is caused by COX-dependent and ROS-dependent signal transduction. The mucosal protective drug PZ has a strong ROS inactivation effect, so the effect of PZ on indomethacin-induced apoptosis of RIE-1 cells was studied. The results showed that higher concentrations of PZ can significantly inhibit indomethacin-induced RIE-1 cell apoptosis (Figure 5). Indomethacin reduced cell viability by 58.7±3.5%; however, PZ (50 and 100 μM) inhibited the indomethacin-induced cell viability reduction by 80.9±4.9% and 99.8±7.9%, respectively. Compared with indomethacin alone, the difference is statistically significant. There is no significant difference in cell viability between using PZ alone (1-100μM) and using medium alone.
2.5 The protective effect of polyprezinc benefits from Zn
In order to further evaluate the protective effect of PZ on indomethacin-induced apoptosis, PZ and its components Zn and L-carnosine were studied separately. Cell apoptosis was assessed by ELISA. As shown in Figure 6, Zn significantly reduced indomethacin-induced cell apoptosis to almost the same extent as PZ. But L-carnosine did not inhibit indomethacin-induced apoptosis. The protective effect of zinc combined with L-carnosine on indomethacin-induced apoptosis is almost equivalent to that of PZ and Zn, which indicates that the protective effect of PZ on indomethacin-induced apoptosis of RIE-1 cells benefits from Zn. Figure 6 PZ and its subcomponent Zn protect IND-induced apoptosis
2.6 Polyprezinc reduces indomethacin-induced ROS production in RIE-1 cells
According to reports, in the RGM1 cell line, ROS is related to indomethacin-induced apoptosis. Therefore, the redox-sensitive fluorescent probe RedoxSensor Red CC-1 was used to check whether indomethacin induced ROS production in RIE-1 cells and whether PZ inhibited ROS production. As shown in Figure 7, the mitochondria of RIE-1 cells in all groups were stained with MitoTracker Green FM. However, compared with cells incubated with indomethacin alone, the fluorescence intensity of RedoxSensor Red CC-1 was significantly increased in cells incubated with medium alone or with PZ alone. This indicates that indomethacin induced an increase in ROS (O2-, HO or H2O2) in cells, and this increase was inhibited by adding PZ, indicating that PZ has a ROS inactivation effect.
2.7 Polyprezinc reduces indomethacin-induced oxidative stress
"Because it is reported that ROS can modify intracellular molecules, such as proteins and lipids, the effect of indomethacin and/or PZ-induced RIE-1 cells on the production of ROS-modified proteins was evaluated. Among the amino acid residues of protein, cysteine residues are most vulnerable to ROS attack and form S-oxidized protein. Bt-Cys can be used as a molecular probe for detecting S-oxidized protein, and this oxidation can be reversed by using chemical reducing agents (such as DTT). As shown in Figure 8A (left 4 lanes), indomethacin significantly increased several S-thiolated proteins compared with the results of the control group. However, PZ inhibited indomethacin-induced S-thiolated protein production, indicating that PZ has a quenching effect on indomethacin-stimulated ROS in RIE-1 cells. When treated with DTT (right 4 lanes), these S-oxidized proteins could not be detected, indicating that these bands are specific to and correspond to S-oxidized proteins. In addition, indomethacin-induced S-oxidized protein production in the mitochondrial component of RIE-1 cells was more pronounced than in the cytoplasmic component (Figure 8B). This finding supports our data that mitochondria are the main site of ROS production in indomethacin-stimulated RIE-1 cells (Figure 7). Mitochondria are the main source of ROS production in RIE-1 cells stimulated by indomethacin. These results indicate that PZ can protect RIE-1 cells from indomethacin-induced apoptosis through its ROS quenching effect.
2.8 Polyprezinc inhibits indomethacin-induced cytochrome c and smac/DIABLO release and caspase-3 activation
"According to the above results, it seems that indomethacin-induced ROS produces S-oxidized proteins in the mitochondria, and these abnormal proteins cause the activation of certain apoptosis-inducing factors. Therefore, we examined cytochrome c and smac/DIABLO, which are the main pro-apoptotic factors in mitochondria. As shown in Figure 9A and B, PZ inhibited the release of indomethacin-induced cytochrome c and smac/DIABLO from the mitochondria to the cytoplasm. In addition, the cleaved caspase-3 (activation) induced by indomethacin acts downstream of cytochrome c and smac/DIABLO (apoptosis) and is inhibited by PZ (Figure 9C), which indicates that the protective effect of PZ depends on ROS quenching. Inactivation and inhibition of the release of cytochrome c and smac/DIABLO into the cytoplasm and caspase-3 activation.
"In short, the non-steroidal anti-inflammatory drug (NSAID) indomethacin (IND) induces apoptosis of small intestinal epithelial cells by generating ROS, releasing cytochrome c and smac/DIABLO, and activating caspase-3. Polyprezinc protects RIE-1 cells by inhibiting ROS production and mitochondrial protein S oxidation. Relaisheng (polyprezinc particles) protects small intestinal epithelial cells from indomethacin-induced apoptosis through its ROS quenching effect, and can be used to prevent NSAID-related small intestinal mucosal damage.
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