Experimental animals
Healthy male Sprague-Dawley (SD) rats, weighing 180-200 grams, were used to establish the model used in this experiment. The experimental animals used to extract HFMSCs were male SD rats aged 8-10 days. We purchased experimental rats from the Animal Experimental Center of the Second Affiliated Hospital of Harbin Medical University, China. The animals were kept in a 12/12 hour light-dark cycle chamber 22±2 ℃. We fed rats with standard laboratory rodent diets and provided them with free access to water. However, experimental rats were fasted for 12 hours before surgery. The experimental design and methods complied with ethical guidelines and standards, and were approved by the Ethics Committee of Harbin Medical University (approval number: SYDW2019-226).
Experimental design
We divided the experimental animals into three groups. They were the sham group (n=5), IR group (n=5) and IR+HFMSCs group (n=5) (Fig. 1A). We established small intestinal IR injury model by clamping and opening the superior mesenteric artery (SMA). In the sham group, only laparotomy was performed, the SMA was isolated, and the abdomen was closed two and a half hours later. In the IR group, the SMA was clamped for 30 minutes, and reperfusion was applied for 2 hours, followed by the injection of 1 ml of saline into the tail vein. Rats in the IR+HFMSCs group were subjected to intestinal IR via operation, and the same volume of a suspension containing 1×106 ml-1 PKH67-labelled HFMSCs was injected into the tail vein. Previous experiments have proven that the proportion of MSCs that home to the small intestine after IR is higher on the 7th day than before or after the 7th day [18, 19]. After 7 days, immunofluorescence, immunohistochemistry, Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the therapeutic effect of HFMSCs.
Small intestinal IR injury model establishment
The classic model of small intestinal IR injury was achieved by clamping the SMA [8] (Fig. 1B). After comparing and evaluating the IR modelling times used in previous studies, 30 min of SMA clamping and 2 hours of reperfusion were used in this study [20, 21]. The rats were anaesthetized by intraperitoneal injection of a compound anaesthetic composed of 4.25% chloral hydrate and 0.886% sodium pentobarbital (0.3 ml/100 g). After approximately 5-10 min, the rats lost consciousness, and were then fixed in the supine position on an operating plate. After the skin on the abdominal surgical area had been prepared and disinfected, a longitudinal incision of approximately 5cm was made on the midline of the abdomen. Sterile forceps were used to separate the SMA, and an atraumatic vascular clamp was used to clamp the SMA root for 30 min. During this period, sterile gauze soaked with saline was used to cover the surgical incision on the experimental animals to keep it moist. The vascular clamp was opened 30 min later, and the incision was sutured, and the abdominal cavity was closed 2 hours later. During modelling, a small amount of warm saline was intermittently injected into the abdominal cavity intermittently to avoid transient hypovolemic reaction, and thermostatic pads were placed under the rats to maintain the body temperature at 37±5 ℃.
Small intestinal IR injury model evaluation
We compared the control group (n=5) and IR group (n=5) based on the following three indicators to judge the success of model establishment. First, the lengths of the small intestine in the control and IR groups were compared. Rats were sacrificed after modelling, and the length of the small intestine was measured. Second, haematoxylin-eosin (HE) staining of the intestinal tract over a length of approximately 5 cm at 15cm above the distal ileum was performed. More specifically, HE staining was carried out as follows: small intestines were dried with filter paper, fixed with 4% paraformaldehyde and then paraffin sectioned for approximately 4 μm thick. The sections were placed in a mixture of haematoxylin and eosin for even staining, and then placed in a constant temperature oven at approximately 60 ℃. After that the sections were placed into warm (40 ℃) water for washing, blow dried, and treated with xylene to complete dewaxing and clearing. Then the sections were sealed with neutral gum. Five pathological sections from each rat were selected for microscopic observation and photography with an optical microscope (Nikon Corp., Tokyo, Japan), and the microscopic fields of the sections were evaluated by calculating Chiu's score [22], a score specifically used to evaluate the degree of damage to the small intestinal mucosa. We invited two pathologists to score the sections in a double-blind manner. Third, we judged the height and area of small intestinal villi under one field of vision in each section. We selected five villi from the visual field and calculated their average villi height and area with Image-Pro Plus 6.0 and WPS Excel software.
HFMSC isolation and culture
At 8-10 days after the SD rats were sacrificed, the skin tissue at the whiskers was cut off and then cut into smaller strips along the area of hair follicles, with a row of hair follicles visible in each skin tissue sample, which was convenient for subsequent tissue digestion and HFMSC extraction. After repeated washing with phosphate-buffered saline (PBS), the skin tissue strips were placed in 0.1% type I collagenase (Sigma Aldrich, St. Louis, MO, USA) at 4 ℃ in the dark for digestion for 15 hours to more easily completely detach the hair follicles from the tissue. After sufficient digestion, the tissue was placed under a stereomicroscope (SZX7, Olympus, Japan), and the hair follicles were separated by microscopic tweezers and cultured in 24-well plates at 37 ℃ and 5% CO2. The HFMSC medium consisted of DMEM/F12 (Sigma Aldrich, St. Louis, MO, USA), foetal bovine serum (ScienCell, San Diego, California, USA) and penicillin/streptomycin (Beyotime, Shanghai, China) at a ratio of 90:10:1. We observed the growth of adherent cells every day, and the culture medium was changed once every 1-2 days. The nonadherent cells were also discarded along with the discarded culture medium. When the primary cells covered 80% of the pore plate (approximately 2 weeks), the cells were passaged, and the second-passage cells were stained and injected. A flow chart is shown in Fig. 3A. All the above protocols were carried out in a sterile environment.
HFMSC identification and staining
We evaluated the differentiation ability of HFMSCs based on whether they could be induced to undergo osteogenic and adipogenic differentiation. HFMSCs were cultured in osteogenic and lipogenic differentiation media for 14 days. Then Oil Red O (Sigma Aldrich, St. Louis, MO, USA) and Alizarin Red S (Sigma Aldrich, St. Louis, MO, USA) were used for HFMSC staining.
Flow cytometry was used by our research group to determine whether the HFMSCs extracted by the above methods could express the MSC markers CD29 and CD90; the HFMSCs expressed hardly any CD31 and CD45 [15, 23]. Therefore, HFMSCs were successfully extracted with this method.
Second-passage HFMSCs were stained with the green fluorescent cell membrane dye PKH67 (Sigma Aldrich, St. Louis, MO, USA). HFMSC nuclei were stained with DAPI (Beyotime, Shanghai, China). Cell staining was completed according to the instructions for the reagent. The stained HFMSCs were viewed using fluorescence microscopy (BX51, Olympus, Japan).
Immunofluorescence
After the experimental animals had been sacrificed, the small intestinal tissues were taken and washed with saline, and the surface was then dried with filter paper to remove moisture. The tissue blocks were cut into approximately 1 cm3 pieces and placed in an embedding box with optimal cutting temperature (OCT) compound wrapping the tissue. After that, the embedding box was slowly placed in liquid nitrogen to freeze, and placed in a -80 ℃ freezer until use. A frozen slicer was used to cut the frozen tissue into 5-μm slices. 0.1% Triton X-100 was dropped on the sections to permeabilize cell. Then, the sections were sealed with goat serum (ABS933, Absin, Shanghai, China) at 37 ℃ for 30 min, and primary antibodies were added and incubated at 4 ℃ overnight. The primary antibodies used were anti-PCNA antibody (1:400, 13110, Cell Signaling Technology, Boston, USA), anti-Bmi1 antibody (1:100, ab126783, Abcam, Cambridge, UK), and anti-SOX9 antibody (5 μg/ml, ab185966, Abcam, Cambridge, UK). On the second day, goat anti-rabbit secondary antibody (SA00013-4, Proteintech, Wuhan, China) was added to the sections and incubated at 37 ℃ for 1 hour in the dark. Then, DAPI was added to stain the nuclei for 5 min. After that, images were taken by laser scanning confocal microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany). The confocal cross-section fluorescence intensity was analysed with Origin 2017 and ImageJ software.
Immunohistochemistry
Paraffin sections of the small intestine were prepared after fixation with 4% paraformaldehyde for 12 hours. The sections were placed in an 80 ℃ thermostat and heated overnight. After that, xylene and alcohol was used for gradient dewaxing, 3% H2O2 was used to inactivate endogenous peroxidase, and ethylenediamine tetraacetic acid (EDTA) was used for antigen retrieval. Then, the sections were sealed with bovine serum albumin (BSA) to block nonspecific binding, and primary antibody was added and incubated with the sections at 4 ℃ overnight. The primary antibodies used were anti-PCNA antibody (1:400, 13110, Cell Signaling Technology, Boston, USA), anti-Bmi1 antibody (1:200, ab126783, Abcam, Cambridge, UK), and anti-SOX9 antibody (1:1000, ab185966, Abcam, Cambridge, UK). On the second day, the sections were incubated with goat anti-rabbit secondary antibody (8114, Cell Signaling Technology, Boston, USA) at 37 °C for 1 hour without light. The sections were washed again with PBS, and the chromogenic substrate diaminobenzidine (DAB) (Zhongshan Golden Bridge Biotechnology, Beijing, China) was dropped on the sections. Staining was terminated when the positive cells were deeply stained but the background was unstained or lightly stained, as determined by viewing under a microscope. The sections were redyed with haematoxylin, fully rinsed with water, dehydrated with gradient alcohol and xylene, then sealed with the appropriate resin, dried, and photographed under an optical microscope (Nikon Corp., Tokyo, Japan). Semiquantitative analysis was performed with Image-Pro Plus 6.0 software.
Western blotting
Intestinal samples were thoroughly ground in liquid nitrogen and cleaved on ice for 5 min with an ultrasonic cell crusher (VCX130, Sonics, Connecticut, USA). The cleaved tissue was placed in a centrifuge tube and centrifuged with a rotational speed of 12000 ×g at 4 ℃ for 20 min. The concentration of protein in the supernatant after centrifugation was measured by the BCA method. The protein solution was heated and denatured at 100 ℃ for 5 min. The solution was removed, quickly placed on ice, and then stored at -20 ℃. Protein samples were transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was sealed with milk for 90 min. The PVDF membrane was cleaned twice with TBST for 5 min each and incubated with primary antibodies at 4 ℃ overnight. The primary antibodies used were as follows: anti-ANG1 antibody (1:500, A7877, ABclonal, Wuhan, China), anti-VEGF antibody (1:500, A12303, ABclonal, Wuhan, China), anti-IGF1 antibody (1:500, A11985, ABclonal, Wuhan, China), anti-Bcl-2 antibody (1:500, T40056, Abmart, Shanghai, China), anti-Bax antibody (1:1000, T40051, Abmart, Shanghai, China), and anti-β-actin antibody (1:1000, ab8226, Abcam, Cambridge, UK). On the second day, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 45 min at room temperature. Then, the PVDF membrane was soaked in enhanced chemiluminescence solution, and images were captured with a gel imaging system. Image analysis was performed with ImageJ software.
ELISA
The supernatant of the tissue lysis solution was obtained by the method used in Western blotting described above, and the concentrations of malondialdehyde (MDA), superoxide dismutase (SOD) and diamine oxidase (DAO) were detected with the following ELISA kits: MDA detection kit (CEA597Ge, Cloud-Clone Corp., Wuhan, China), SOD detection kit (SES134Ra, Cloud-Clone Corp., Wuhan, China), and DAO detection kit (SEA656Ra, Cloud-Clone Corp., Wuhan, China). All procedures were performed in accordance with the kit’s instructions, and the concentration of the index to be measured in the sample was finally calculated.
Statistical analysis
GraphPad Prism (8.3.0) statistical software was used for statistical analysis. The numerical data are expressed as the mean ± standard deviation (SD). Comparisons of differences between two groups were performed by t-test. One-way analysis of variance (ANOVA) was used for comparisons of differences among multiple groups. Differences for which P < 0.05 were considered statistically significant.