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Experimental study on treatment of ischemic colitis in rats with Ralston

Experimental study on treatment of ischemic colitis in rats with Ralston

  • Categories:Stomach healthy
  • Author:
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  • Time of issue:2021-01-19
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(Summary description)

Experimental study on treatment of ischemic colitis in rats with Ralston

(Summary description)

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2021-01-19
  • Views:0
Information

Objective: To explore the repair effect and possible mechanism of polaprezinc (PZ) on colonic mucosal injury in rats with ischemic colitis (IC).

Methods: 40 rats were randomly divided into three groups: experimental group (laparotomy + laser irradiation + PZ treatment) with 24 rats, control group (laparotomy + laser irradiation) with 12 rats and blank group with 4 rats. The experimental group was given 3000mg / L PZ solution 3ml every day, and the rest was given free water. After modeling, the control group and the blank group only took water freely. On the 5th day after operation, the rats were killed and the gross appearance of colonic mucosa was observed by naked eye. The degree of colonic mucosal injury was scored under microscope. The expression of HSP70 and NF - κ B P50 in colonic mucosa was evaluated by immunohistochemistry.

Results: the visible colon injury in the experimental group was significantly lower than that in the control group (16.67% vs 58.33%, P < 0.05). The colonic mucosal erosion, inflammation and lymphocyte infiltration in the experimental group were lighter than those in the control group (P < 0.05). The colon injury score of the experimental group (1.125 ± 1.262) was significantly lower than that of the control group (2.750 ± 1.138) (P < 0.01). The expression of HSP70 in the experimental group was significantly higher than that in the control group (P < 0.01), and the expression of NF - κ B P50 in the experimental group was significantly lower than that in the control group (P < 0.01).

Conclusion: rileson can reduce the inflammation and erosion of colonic mucosa in IC model rats, and promote the repair of colonic mucosa injury; its mechanism may be through promoting the expression of HSP70, reducing the expression of NF - κ B P50 and inhibiting its activity.


preface

Ischemic colitis (IC) refers to the intestinal ischemic injury caused by various causes of insufficient blood supply to the colon. It is the most common intestinal vascular disease and one of the most common causes of lower gastrointestinal bleeding in the elderly. Mild cases can relieve themselves, and severe cases can cause intestinal necrosis, suppurative peritonitis and even life-threatening. Our previous analysis of 79 patients with IC showed that patients with ulcer had longer hospitalization time. At present, there is no known drug that can promote the repair of ischemic intestinal mucosal injury.

Relaxant (PZ) is the first zinc containing mucosal protective agent. It is a chelate composed of zinc and l-muscle titanium. It has the functions of mucosal protection, immune regulation, anti-oxidation, maintaining cell stability, promoting granulation tissue regeneration and healing of gastric ulcer. The purpose of this study is to explore the repair effect and possible mechanism of Ralston on intestinal mucosal injury in rats with IC, so as to provide a beneficial choice for the prevention and treatment of IC in clinic.

1. Materials and methods

1.1 animals and drugs

Forty SD rats, weighing 200 ± 10g, were reared in a standard animal laboratory with standard ambient temperature (20-24 ℃) and humidity, and alternate light and dark cycles for 12 hours. The raw material powder of ruilaisheng (jupri zinc granules) is from Boda Weiye Pharmaceutical Co., Ltd.

1.2 experimental grouping

Forty rats were randomly divided into three groups: experimental group (laparotomy + laser irradiation + PZ treatment) with 24 rats, control group (laparotomy + laser irradiation) with 12 rats and blank group with 4 rats. Rats in the experimental group were given 3000mg / L PZ 3ml daily after modeling, and normal water was taken. The control group and blank group were fed with free water after modeling.

1.3 the rat model was established by photochemical method

The model of ischemic colitis was established by laser irradiation from a frequency doubled semiconductor laser (wavelength 532nm). SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 3ml / kg. The colon was found by laparotomy. The femoral vein was injected with 5 mg / kg of propofol. At 5 minutes after injection, laser was used to irradiate the serosal surface of the distal mesocolon to protect the surrounding intestine from laser. The diameter of the laser spot was 2.34cm, and the output power of the laser was 550MW. After irradiation for 5min, the abdominal cavity was flushed with 40000 units of gentamicin sulfate, and the abdominal cavity was closed. The rats were sent to the clean room to continue feeding after anesthesia.

1.4 specimen collection

All rats were killed on the 5th day after operation, and fasted 24 hours before death. The colon tissue corresponding to the irradiated mesangium was immediately spread on a glass plate pre placed on ice. The color of the mucosa, the extent of mucosal damage, and the surrounding mucosa were observed and photographed. Then, they were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained with he. The pathological results of intestinal mucosa were observed under light microscope, and Chiu score was performed by double-blind method. Scoring criteria: 0: normal intestinal mucosa; 1: enlargement of the subcutaneous space at the top of villi with capillary congestion; 2: further enlargement of subepithelial space with moderate separation of upper cortex and lamina propria; 3: a large number of epithelial layers separated from both sides of villi with partial destruction of the top of villi; 4: destruction of villi with exposure of dilated capillaries and increase of cells in lamina propria; 5: increase of cells in lamina propria Layer destruction with bleeding and ulceration.

1.5 immunohistochemistry

The specimens were routinely made into paraffin sections. PBS (pH = 7.4) was used to wash for 3 min × 3 times, heat repair for 2 min, drip 3% hydrogen peroxide, and incubate for 10 min at room temperature to block the activity of endogenous peroxidase. PBS was rinsed for 3 min × 3 times, and diluted primary antibody (NF - κ B P50 diluted concentration 1:50; HSP70 diluted concentration 1:300) was dripped at 4 ℃ overnight. PBS was rinsed for 3 min × 3, and the ready to use super polymer reagent was dripped and incubated at room temperature for 10-15 min. After washing with PBS for 3 min × 3, DAB solution was added and observed under microscope for 3-5 min. the dyeing reaction was terminated by washing with tap water. Light re staining with hematoxylin and bluish after washing. Gradient alcohol dehydration, transparent xylene, neutral gum seal.

1.6 the expression of HSP70 and NF - κ B P50 was analyzed by semi quantitative method

The immunohistochemical results of HSP70 were analyzed by two senior medical residents in double-blind. Ten visual fields were randomly selected. According to the proportion of positive cells under high power microscope, the range of grade 1 staining was less than or equal to 25%, grade 2 staining was 26% - 75%, and grade 3 staining was more than or equal to 76%. At the same time, according to the staining intensity of the cells in the sections, grade 1 showed no staining, grade 2 showed light staining and grade 3 showed deep staining. The final score is the multiplication of the two grades of each specimen, < 3 is negative and ≥ 3 is positive.

1.7 statistical analysis

SPSS 11.5 statistical software was used for analysis. The count data were analyzed by X? Test, chi square method was used for pairwise comparison, and Kruskal Wallis test was used for grade data. P < 0.05 was statistically significant.

2 results and analysis

2.1 general manifestations of experimental rats

After laparotomy and laser irradiation, all rats showed anorexia, lazy movement, curly and dull hair, increased defecation frequency and loose stool. Among the 24 rats in the experimental group, 5 rats had a little blood in the stool on the first day after operation, and no visible blood silk on the third day. In the control group, 4 rats had a little blood in their stools on the first day after operation, and 3 rats had blood in their stools until the end of the experiment on the fifth day. There was no blood in the stool in the blank group.

2.2 gross appearance of colonic mucosa

Among the 24 rats in the experimental group, 4 (16.67%) had visible damage to the colonic mucosa. Among the 12 rats in the control group, 7 (58.33%) had visible mucosal damage, and the difference between the two groups was statistically significant (x 2 = 6.545, P = 0.011). No visible mucosal damage was found in the blank group (Fig. 1-2).

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Fig. 1 laser irradiation on the mesangial serosa of rat colon

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Fig. 2 the mucosa of the control group was hyperemia and erosion, and the mucosa of the experimental group healed well

2.3 colonoscopic and pathological findings

In the control group, the epithelial and glandular structures on the surface of some intestinal mucosa disappeared, and fibrous tissue hyperplasia with infiltration of lymphocytes and plasma cells could be seen (Fig. 3). In the experimental group, the epithelial cells and proper glands on the surface of intestinal mucosa were arranged regularly, and lymphocytes and plasma cells were infiltrated in the stroma. Inflammatory degree of colonic mucosa in experimental group Compared with the control group, severe inflammation accounted for 17.67% (4 / 24) in the experimental group and 75.0% (9 / 12) in the control group, the difference was statistically significant (P < 0.05); mucosal erosion in the experimental group was 12.5% (3 / 24), which was significantly lower than 50.0% (6 / 12) in the control group, the difference was statistically significant (P < 0.05); lymphatic erosion in the experimental group was 17.67% (4 / 24) The cell infiltration was significantly lower than 75.0% (9 / 12) of the control group, the difference was statistically significant (P < 0.05); 45.83% (11 / 24) of the experimental group had submucosal edema, which was lower than 66.67% (8 / 12) of the control group, but the difference was not statistically significant (P > 0.05) (Table 1); there were 2 cases of colonic submucosal blood stasis in the control group, but no colonic submucosal blood stasis in the experimental group.

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Fig. 3 pathological picture of rat colonic mucosa (HE staining, 200 times magnification)

A: Control group: Rats with intestinal mucosal erosion;

B: Experimental group: mild inflammation of intestinal mucosa.

Table 1 Comparison of observation indexes of colonic mucosa under microscope in rats of each group

Cases (%)

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2.4 analysis of damage degree of colonic mucosa in rats

The degree of colonic mucosal injury in the experimental group was mainly 0 and 1, while that in the control group was mainly 2-4 (Table 2). The Chiu score of the experimental group (1.125 ± 1.262) was significantly lower than that of the control group (2.750 ± 1.138) (P = 0.001).

Table 2 distribution of damage degree of colonic mucosa in each group

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2.5 expression of HSP70 and NF - κ B P50 in rat colon

The expression of HSP70 in colonic mucosa of experimental group was significantly higher than that of control group, and the difference was statistically significant; the expression of NF - κ B P50 in colonic mucosa of experimental group was significantly lower than that of control group, and the difference was statistically significant (P < 0.05, table 3, figure 4-5).

Table 3 colon tissue of rats in each group

Comparison of HSP70 and NF - κ B P50 expression

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Fig. 4 expression of HSP70 in rat colonic mucosa (200 × magnification)

Note: A: experimental group; B: control group.

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Fig. 5 expression of NF - κ B P50 in rat colonic mucosa (200 fold magnification)

Note: A: experimental group; B: control group.

Discussion

Hsp70 is one of the main members of heat shock protein family. It plays an important role as a molecular chaperone in gastric mucosa and colon mucosa. The results showed that the expression of HSP70 in colonic mucosa of rats in rileson treatment group was significantly higher than that in control group. Rileson may accelerate the healing of colonic mucosa by inducing the expression of HSP70 in colonic mucosa of rats.

NF - κ B P50 is a transcription regulator. Its activation can regulate the transcription of many proinflammatory factors such as TNF - α, IL-1 β and IL-8, so it plays an important role in inflammation. The results showed that the expression of NF - κ B P50 in the treatment group was significantly lower than that in the model group, which may be related to the increased expression of HSP70 in rat colonic mucosa, the decreased expression of NF - κ B P50 and the inhibition of its activity.

In conclusion, rileson can reduce the inflammation and erosion of colonic mucosa in IC model rats, thus promoting the repair of intestinal mucosal injury. This may be related to the up regulation of HSP70 expression and the down regulation of NF - κ B P50 expression in ischemic intestinal mucosa. These results provide an experimental basis for the clinical application of mucosal protective agent (jupri zinc granules) in patients with ischemic colitis, and it is expected that the drug of choice for the prevention and treatment of ischemic colitis can be made.

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