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Title: [Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice].
Author: Wu D, Wang C, Li T, Wang H, Hu JH, Peng X.
Journal: Zhonghua Shao Shang Za Zhi; 2021 Sep 20; 37(9):811-820. PubMed ID: 34645146.
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. 目的： 建立高效的人肠三叶因子（ITF）重组表达及纯化策略，观察重组人ITF（rhITF）对烧伤小鼠肠黏膜损伤与修复的作用并探讨其机制。 方法： 采用实验研究方法。采用新型酵母表达载体pGAPZαA和酵母菌X33重组表达ITF，并通过金属螯合亲和层析与阴、阳离子交换层析法纯化蛋白，行非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及蛋白质印迹法鉴定rhITF。rhITF与胃蛋白酶溶液、胰蛋白酶溶液按体积比1∶1混合，分别作用0.5、1.0、1.5、2.0 h和1.0、2.0、4.0 h后行非还原SDS-PAGE，分析rhITF的稳定性。按随机数字表法将105只6~8周龄BALB/c雄性小鼠分为假伤组（30只）、单纯烧伤组（45只）、烧伤+rhITF组（30只）。将单纯烧伤组和烧伤+rhITF组小鼠背部造成30%体表总面积Ⅲ度烧伤，假伤组小鼠模拟致假伤，烧伤+rhITF组小鼠伤后灌胃1 mg/kg rhITF，其余2组小鼠伤后给予等量生理盐水。伤后24 h，取15只单纯烧伤组小鼠，制备烧伤血清，用于细胞实验。伤后3、5、7 d，每组各取10只小鼠处死后取小肠组织，苏木精-伊红染色观察肠黏膜病理变化，分光光度法和酶联免疫吸附测定法测定肠组织二胺氧化酶（DAO）及乳酸脱氢酶（LDH）活性。取3批人结直肠腺癌HT-29细胞，分为阴性对照组、25 μg/mL rhITF组、50 μg/mL rhITF组（样本数为3），正常对照组、烧伤血清组、烧伤血清+rhITF组（样本数为3），CK869抑制剂组、CK666抑制剂组和溶剂对照组（样本数为2），分别进行相应处理，Transwell实验观察培养12 h后细胞移行。另取2批HT-29细胞，每批细胞均分为正常对照组、烧伤血清组和烧伤血清+rhITF组（样本数均为6），培养24 h后，蛋白质印迹法检测细胞腺苷酸活化蛋白激酶（AMPK）、磷酸化AMPK（p-AMPK）、Ras相关C3肉毒杆菌毒素底物1（Rac1）及肌动蛋白相关蛋白2/3（Arp2/3）复合亚基1B（ARPC1B）蛋白表达，活化磁珠下拉实验检测细胞Rac1活性。对数据行析因设计方差分析、单因素方差分析、SNK检验。 结果： 每升发酵液获得rhITF 82.35 mg，蛋白纯度高达98%，且具有良好的抗原特异性。rhITF在胃蛋白酶和胰蛋白酶溶液中较稳定，与胰蛋白酶溶液作用2.0 h后仍有45%残余，与胃蛋白酶溶液作用4.0 h后仍有90%残余。假伤组小鼠伤后各时间点肠黏膜未见充血、水肿，单纯烧伤组小鼠伤后各时间点肠黏膜主要病理表现为充血、水肿、糜烂及出血。烧伤+rhITF组小鼠伤后3、5 d肠黏膜主要以充血、水肿为主，伤后7 d肠黏膜水肿和充血减轻。与单纯烧伤组比较，假伤组、烧伤+rhITF组小鼠伤后3、5、7 d肠组织DAO和LDH活性显著升高（P<0.05或P<0.01）。培养12 h后，25 μg/mL rhITF组细胞移行数为（58±12）个，显著多于阴性对照组的（16±5）个（P<0.01），显著少于50 μg/mL rhITF组的（123±9）个（P<0.05）。培养12 h后，烧伤血清组细胞移行数为（60±13）个，显著少于正常对照组的（143±11）个和烧伤血清+rhITF组的（138±8）个（P<0.05）。培养12 h后，溶剂对照组细胞移行数为（155±9）个，明显多于CK666抑制剂组的（33±5）个、CK869抑制剂组的（28±5）个（P<0.01）。培养24 h后，烧伤血清组细胞AMPK、Rac1蛋白表达量与正常对照组、烧伤血清+rhITF组相近（P>0.05），p-AMPK蛋白表达量明显高于正常对照组、烧伤血清+rhITF组（P<0.05或P<0.01），ARPC1B蛋白表达量明显低于正常对照组、烧伤血清+rhITF组（P<0.05）。培养24 h后，烧伤血清组细胞Rac1活性明显低于正常对照组、烧伤血清+rhITF组（P<0.05或P<0.01）。 结论： 本研究获得的rhITF具有较高的纯度和超强的稳定性，能耐受极端pH和蛋白酶水解，减轻烧伤小鼠肠黏膜损伤。rhITF能通过抑制AMPK磷酸化维持Rac1-Arp2/3活性，促进肠上皮细胞移行，加速肠黏膜修复。.

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