[Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice].

Abstract

Objective: To establish an efficient human intestinal trefoil factor (ITF) recombinant expression and purification strategy and to observe the effect of recombinant human ITF (rhITF) on intestinal mucosal injury and repair in burned rats and to explore the mechanism. Methods: The experimental research method was applied. New yeast expression vector pGAPZαA and yeast X33 were used to express recombinant ITF. The protein was purified by metal chelation affinity chromatography and anion and cation exchange chromatography. The rhITF was identified by non-reductive sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting. The rhITF was mixed with pepsin solution and trypsin solution in a volume ratio of 1∶1, respectively. After mixed with pepsin solution for 0.5, 1.0, 1.5, 2.0 h and trypsin solution for 1.0, 2.0, 4.0 h, the stability of rhITF was analyzed with non-reductive SDS-PAGE. One hundred and five male BALB/c mice aged 6-8 weeks were divided into sham injury group (n=30), burn alone group (n=45), and burn+rhITF group (n=30) according to the random number table. Mice in burn alone group and burn+rhITF group were inflicted with 30% total body surface area full-thickness burn on the back, while mice in sham injury group were simulated with burn. After burn, mice in burn+rhITF group were intragastrically administered with rhITF of 1 mg/kg, while mice in the other two groups were given the same amount of normal saline. At post injury hour 24, 15 mice in burn alone group were collected to prepare burn serum, which was used in the cell experiment. On post injury day (PID) 3, 5, and 7, 10 mice in each group were sacrificed to collect the small intestinal tissue. The pathological changes of the intestinal mucosa were observed by hematoxylin-eosin staining, and the activities of diamine oxidase (DAO) and lactic dehydrogenase (LDH) in the intestinal tissue were determined by spectrophotometry and enzyme linked immunosorbent assay. Three batches of human colorectal adenocarcinoma HT-29 cells were taken and divided into negative control group, 25 μg/mL rhITF group, 50 μg/mL rhITF group (n=3), normal control group, burn serum group, burn serum+rhITF group (n=3), and CK869 inhibitor group, CK666 inhibitor group, solvent control group (n=2), respectively, which were dealt with the corresponding treatment. After 12 h of culture, the migration of cells were observed by Transwell experiment. Another 2 batches of HT-29 cells were taken and each batch of cells were divided into normal control group, burn serum group, and burn serum+rhITF group (n=6). After 24 h of culture, the protein expressions of adenosine monophosphate activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK), Ras related C3 botulinum toxin substrate 1 (Rac1), and actin-related protein 2/3 (Arp 2/3) complex, subunit 1B (ARPC1B) in the cells were detected by Western blotting, and the Rac1 activity of the cells was detected by activated magnetic bead pull-down test. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Student-Newman-Keuls test. Results: Totally 82.35 mg rhITF was gathered from per litre of fermentation broth with protein purity up to 98%, and the rhITF had good antigenicity. The rhITF was stable in pepsin solution and trypsin solution, with 45% rhITF remained after 2.0 h in trypsin solution, and there was 90% rhITF remained after 4.0 h in pepsin solution. At each time point post injury, no hyperemia, or edema was observed in intestinal mucosa of mice in sham injury group, the main pathological manifestations of intestinal mucosa in mice of burn alone group were hyperemia, edema, erosion, and hemorrhage, and the main manifestations of intestinal mucosa of mice in burn+rhITF group were hyperemia and edema on PID 3 and 5, which were alleviated on PID 7. Compared with those of burn alone group, the activities of DAO and LDH in intestinal tissue of mice in sham injury group and burn+rhITF group were significantly increased on PID 3, 5, and 7 (P<0.05 or P<0.01 ). After 12 h of culture, the number of cell migration in 25 μg/mL rhITF group was 58±12, which was obviously more than 16±5 in negative control group (P<0.01) and obviously less than 123±9 in 50 μg/mL rhITF group (P<0.05). After 12 h of culture, the number of cell migration in burn serum group was 60±13, which was significantly less than 143±11 in normal control group and 138±8 in burn serum+rhITF group (P<0.05). After 12 h of culture, the number of cell migration in solvent control group was 155±9, which was significantly more than 33±5 in CK666 inhibitor group and 28±5 in CK869 inhibitor group (P<0.01). After 24 h of culture, the protein expressions of AMPK and Rac1 of cells in burn serum group were close to those of normal control group and burn serum+rhITF group (P˃0.05), the protein expression of p-AMPK of cells in burn serum group was significantly higher than that of normal control group and burn serum+rhITF group, respectively (P<0.05 or P<0.01), and the protein expression of ARPC1B of cells in burn serum group was significantly lower than that of normal control group and burn serum+rhITF group (P<0.05). After 24 h of culture, the Rac1 activity of cells in burn serum group was significantly lower than that in normal control group and burn serum+rhITF group, respectively (P<0.05 or P<0.01). Conclusions: The rhITF obtained in this study has high purity and super stability, which can resist extreme pH and hydrolysis of protease and can relieve intestinal mucosal damage in burned mice. The rhITF can promote the migration of intestinal epithelial cells and accelerate the repair of intestinal mucosa through inhibiting phosphorylation of AMPK to maintain Rac1-Arp2/3 activity.