Carnosine zinc-stabilizes the integrity of the small intestine and promotes intestinal repair
- Categories:Stomach healthy
- Time of issue:2020-12-02
Carnosine zinc-stabilizes the integrity of the small intestine and promotes intestinal repair
- Categories:Stomach healthy
- Time of issue:2020-12-02
In recent years, the value of natural medicine in preventing and treating diseases has attracted much attention. Zinc carnosine (ZnC) is a synthetic chelate, as a "zinc supplement", has added value to stomach health. Theoretically, compared with pure zinc supplementation, the combination of carnosine and zinc can produce a synergistic effect and provide more benefits to the body.
In order to fully study the potential biological significance of ZnC, this article explores the protective and repair effects of ZnC on various types of gastrointestinal injuries through a series of in vitro, in vivo and clinical studies.
Materials and Method
All chemicals were purchased from Sigma (Pool, Dorset, UK). ZnC was provided by Lonza Nutrition (USA).
All animal experiments were approved by the local animal ethics committee, and corresponding licenses were obtained in accordance with the 1986 Animal Procedure Law of the Ministry of the Interior. The clinical trial was approved by the local ethics committee and complied with national requirements.
In vitro and in vivo experiments take experimental conditions as factors, and use single factor or two factor analysis of variance to analyze the data. In clinical research, patients, time and study group were used as factors to perform analysis of variance. p<0.05 has a significant difference, and the t-test based on the residuals and degrees of freedom obtained from the analysis of variance is used for individual comparison.
Study A: In vitro repair effect of ZnC
One of the fastest repair responses after tissue injury is the migration of surviving cells to the denuded area to rebuild the integrity of the epithelium. However, it is very difficult to study this effect in humans or animals, and cell culture models are usually used as a surrogate marker for the migration-promoting effect.
1, cell migration
The cell migration experiment uses human colon cancer cell line HT29 and canine kidney epithelial cell line MDCK. The cells were cultured in DMEM containing 10% fetal bovine serum, 37°C, 5% CO2, and fused in a six-well plate. The serum was removed and ZnC, equimolar zinc sulfate and equivalent bovine serum albumin (BSA) concentrations were added at a concentration of 1–1000 μM (to analyze non-specific protein effects). The proliferation inhibitor mitomycin C (5 mg/mL) was added to the monomolecular membrane pretreated with ZnC to determine whether the damage depends on cell proliferation. Take micrographs with an inverted microscope and digital camera to determine the migration rate of the damage. The results are expressed as the average (standard mean error (SEM)) of three independent experiments.
2, cell proliferation
The cell proliferation experiment uses HT29, MDCK and rat small intestinal epithelial cell (RIE) cell lines. The cells were cultured in DMEM containing glutamine and 10% fetal calf serum. After removing the serum, the effects of ZnC, zinc sulfate and BSA (analysis of non-specific protein effects) were tested. In order to evaluate the rate of cell DNA synthesis, [³H]-thymidine (2μCi/well) was added 24h after the test factor was added to evaluate cell proliferation.
1, ZnC promotes cell migration and heals injuries
"In HT29 cells and MDCK cells, ZnC induces cell migration in a dose-dependent manner (Figure 1). When the concentration is greater than 10 μM, ZnC induces cell migration to promote wound healing, and the maximum rate appears at 100 μM (about 3 times the baseline rate). When the concentration is >100 μM, ZnC will not further increase the migration rate. Equimolar concentrations of zinc sulfate and bovine serum albumin have no significant effect on the migration rate. Consistent with the expected results, the presence of mitomycin C did not affect the migration rate induced by ZnC.
2, ZnC promotes cell proliferation and heals injuries
ZnC induced a dose-dependent increase in [³H]-thymidine in HT29 cells. The maximum effect occurs at 100 μM. Adding equimolar zinc sulfate, [³H]-thymidine increased trace elements (about 26%) may be due to the presence of zinc. The addition of equimolar BSA did not promote cell proliferation. The results of MDCK and RIE cell lines are similar.
Study B: The repairing effect of ZnC in vivo
Use animal models to evaluate the repair effects of ZnC on stomach and small intestine injuries to obtain more biological information about ZnC.
1. The effect of ZnC on rat gastric injury model
2 male SD rats (225-275g), fed in a standard way. The rats randomly received one of the following factors: 1 mL of saline (negative control), ZnC (1 and 5 mg/mL) and epidermal growth factor (EGF) (25 mg/mL, positive control). Add 2% hydroxypropyl methylcellulose to the gavage fluid to delay gastric emptying. After intragastric administration for 30 minutes, indomethacin (20mg/kg) was injected subcutaneously. Three hours after the indomethacin administration, the rats were sacrificed, the stomach was taken, and the pH value in the stomach was measured with a miniature pH electrode; the stomach was inflated with 4 mL of 10% neutral buffered formalin. The specimens were randomly coded, and the macroscopic damage was assessed using a dissecting microscope (610) and a reference grid. Make paraffin sections, observe under a microscope, and evaluate the degree of damage. The score ranges from 0 to 4: 0=no damage, 1=1 small corrosion (<0.5 mm), 2=2 small or 1 large corrosion (>0.5) mm), 3 = 2 or more large areas of erosion, 4 = ulcers extend to the muscularis mucosa, the higher the score, the more severe the damage.
2, the effect of ZnC on the growth and damage of the small intestine of mice
4 groups of mice (n=6 per group), 2 groups were supplemented with zinc (40 mg/mL) in drinking water for 7 days, and the other 2 groups drank tap water. According to previous studies, indomethacin-induced small intestine damage was the most serious at 12h, so indomethacin (85mg/kg) or vehicle control was injected subcutaneously 12h before sacrifice. One hour before sacrifice, the mice were intraperitoneally injected with bromodeoxyuridine (BrdU; 50 mg/kg) to assess the intestinal proliferation.
3. Morphological evaluation
Using the pre-calibrated traction tube to trace the villus contour, evaluate the villus height and width of 4 microanatomical samples, and measure the villus height and width by tracing method. Each animal was evaluated with 20 villi and the average value was used for analysis of variance (ANOVA).
4. Diffusion assessment
Tissue sections were stained with BrdU and counterstained with hematoxylin. Observe the slide until you find a directional crypt. Record the number of mitotic cells/crypts marked by each animal. The average number of labeled cells is used in the analysis of variance.
1, ZnC improves indomethacin-induced gastric injury
The average macroscopic gastric injury score of rats pretreated with indomethacin and vehicle control was 46(7)mm²/stomach. After ZnC pretreatment, the degree of gastric injury was significantly reduced, and it was dose-dependent. Rats receiving 1 mg/mL zinc decreased the degree of gastric damage by 38%, and rats receiving 5 mg/mL zinc decreased by 75%.
The results of the microhistological scoring system are similar to those of the naked eye. Administration of 5 mg/mL ZnC has a protective effect on the stomach, which is roughly similar to the protective effect of EGF on the stomach at 25 mg/mL.
2, ZnC improves indomethacin-induced small intestine injury
"Under the condition of no injury (no indomethacin treatment), ZnC pretreatment does not affect the growth of the small intestine or colon (measured by wet weight, villus height and BrdU incorporation). The control group had typical morphological changes of shortening and dulling of villi, while the ZnC group had no obvious morphological changes.
Indomethacin caused a decrease in villus height by about 1/3 (p<0.01), while in mice co-treated with ZnC, only slight villus shortening (about 14%) was observed. After pretreatment with indomethacin, it increased the wet weight of the small intestine in the control group, but did not increase the wet weight of the small intestine in the ZnC group. The results of BrdU staining showed that the proliferation of jejunal gland cells in the control group and ZnC group mice increased with the increase of the indomethacin dose, but was not affected by the ZnC combination.
Study C: The effect of ZnC on indomethacin-induced intestinal permeability changes
Through the quantitative analysis of the non-mediated absorption of at least two sugars of different sizes to assess the permeability of the intestinal tract, providing sensitive indicators for intestinal injury. Lactulose was used as a disaccharide probe, and rhamnose and mannitol were used as two alternative monosaccharide probes for low permeability formulations. The "mannitol peak" in urine samples is sometimes obscured by the overlap of other urine components, so only lactulose and rhamnose data are provided. In order to maintain consistency between studies (including osmotic pressure), the same mixture as before was used, but again, only the lactulose:rhamnose ratio was presented. This method is reasonable and has been widely used to assess bowel disease caused by non-steroidal anti-inflammatory drugs (NSAIDs).
After fasting overnight, the subjects drank a standardized sugar solution containing 5g lactulose, 2g mannitol and 1g rhamnose dissolved in 450mL water (osmotic pressure 69 mOs). After 1 hour, the subjects drank plenty of water to ensure adequate urine output. Collect and pool urine in the next 5h, and record the total volume. Centrifuge to remove coarse debris, and freeze the supernatant at 220°C for later use.
Use high performance liquid chromatography (HPLC) to separate various sugars and determine the sugar content.
3. Preliminary research
In order to determine the reproducibility of the experimental results, a single person conducted a 4-day permeability study (while not receiving any treatment and non-steroidal anti-inflammatory drugs). These samples are analyzed to determine the intra-patient variability and give a coefficient of variation of approximately 15%. In addition, 4 measurements were performed on a single sample to determine the within-group variation, and a coefficient of variation of 8% was given. These coefficients of variation are similar to the results of previous studies. In order to study whether ZnC affects permeability under basic conditions, 4 subjects performed preliminary permeability evaluation, and then evaluated again after taking ZnC for 7 consecutive days.
4. Main research
10 subjects (24-40 years old, 5 males and 5 females), did not take non-steroidal anti-inflammatory drugs, and no diseases that may affect intestinal permeability, such as celiac disease or intestinal surgery. During the 1 week before the start of the test and throughout the test period, the subjects did not drink alcohol or take any non-steroidal anti-inflammatory drugs, including aspirin. Each subject performed a total of 6 permeability assessments (Figure 5). Each group includes three groups of samples on day -2, 0, and 7. The samples collected on day -2 and day 0 are all "baseline" analysis. After collecting urine on day 0, the subjects took the test drug containing zinc (37.5 mg) and placebo capsules twice a day for 7 days.
After a 7-day course of treatment, a third urine collection was performed. After a 2-week washout period, the subject repeated the protocol with another test capsule. In addition, on the last 5 days of the study (days 2-7), the subjects received treatment with non-steroidal anti-inflammatory drugs (indomethacin 50 mg, 3 times a day;
1, elution curve
The peak times of mannitol, rhamnose and lactulose are 1.5min, 3min and 4.5min, respectively. Standards of monosaccharides and mixed sugars with a concentration of 0.05-50 mg/mL showed a good separation effect.
2, ZnC does not affect the permeability of the small intestine
Subjects not taking non-steroidal anti-inflammatory drugs taking ZnC had no effect on small intestinal permeability (the L:R ratio before ZnC treatment was 0.49 (0.18), and at the end of treatment it was 0.52 (0.17)).
3, ZnC restores indomethacin-induced changes in intestinal mucosal permeability
All 10 subjects completed the study without violating the protocol. When taking indomethacin (control group), 2 out of 10 subjects experienced mild upper abdominal discomfort, but completed the entire course of treatment. During the ZnC (+ indomethacin) treatment, there were no adverse reactions and no side effects due to the intake of ZnC. At the beginning of each study group and at the first and second (baseline) assessments within the group, the baseline permeability values were similar. In the control group, the permeability of indomethacin to the small intestine increased approximately 3 times (p<0.01v baseline value). When subjects took ZnC, indomethacin did not significantly increase intestinal permeability.
"In summary, zinc carnosine (ZnC) can stimulate cell migration and proliferation in vitro, thereby accelerating the healing of injuries. At the same time, in vivo experiments have shown that ZnC can effectively reduce stomach and small intestinal injuries. In addition, if the patient takes a certain dose of ZnC, it can prevent the increase in intestinal permeability caused by the clinical standard dose of non-steroidal anti-inflammatory drug indomethacin. Stabilize the integrity of the small intestine and promote intestinal repair.
Non-steroidal anti-inflammatory drugs (aspirin and indomethacin) are extremely harmful to the digestive tract. Among them, indomethacin causes damage to the gastrointestinal tract through a variety of mechanisms, including reducing mucosal prostaglandin levels, reducing mucosal blood flow, Activate neutrophils and stimulate apoptosis. Patients taking non-steroidal anti-inflammatory drugs, 10-30% have peptic ulcers, and they are usually asymptomatic. In addition, up to 70% of patients taking non-steroidal anti-inflammatory drugs have bowel droop, which is related to low blood flow and protein loss. Although PPI provides a strong acid inhibitory effect and effectively reduces the risk of gastric and duodenal ulcers, it has no effect on small intestine damage. The prostaglandin analogs have a protective effect on the small intestine, but they can cause serious adverse reactions, such as diarrhea. Boda Weiye Ruilaisheng (Polyprezinc Granules) is a chelate composed of zinc ions and L-carnosine. It is a brand-new "mucosal protective agent" that is clinically used to treat gastric ulcers. Studies have shown that polyprezinc can significantly improve the small intestinal mucosal damage induced by low-dose aspirin, which greatly compensates for the shortcomings of PPI and prostaglandin analogs. For patients who take long-term non-steroidal drugs, combined use of polyprezinc can Significantly reduce drug-related small intestinal mucosal damage, and is safe and non-toxic. At the same time, polyprezinc's unique endogenous protection and repair mechanism can effectively overcome the "treatment blind spot" of the small intestinal mucosa, and provide a safe and effective treatment option for non-steroidal drug-related small intestinal mucosal injury.
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