The efficacy of bone marrow mesenchymal stem cells on rat intestinal immune-function injured by ischemia/reperfusion

Abstract

Background: Transplantation of bone marrow mesenchymal stem cells (BMSCs) has a promising therapeutic efficiency for varieties of disorders caused by ischemia or reperfusion impairment. It has been shown that BMSCs can mitigate intestinal ischemia/reperfusion (I/R) injuries, but the underlying mechanism is still unclear. This study aimed at investigating the efficacy of BMSCs on the immune function of intestinal mucosal microenvironment after I/R injuries.

Methods: Twenty adult Sprague-Dawley rats were randomly assigned to a treatment or a control group. All the rats underwent superior mesenteric artery clamping and unclamping. In the treatment group, BMSCs were implanted into the intestine of ten rats by direct submucosal injection whereas the other ten rats in the control group were injected with the same volume of saline. On the fourth and seventh day after BMSCs transplantation, intestinal samples were examined for the CD4 (CD4-positive T-lymphocytes)/CD8 (CD8-positive T-lymphocytes) ratio of the bowel mucosa via flow cytometry, and for the level of Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Interleukin-6 (IL-6) via ELISA. Paneth cell counts and Secretory Immunoglobulin A (SIgA) level were examined via immunohistochemical (IHC) analysis. Real time PCR (RT-PCR) was used to detect the expression levels of tumor necrosis factor-α (TNF-α) and trypsinogen (Serine 2) (PRSS2) genes. White blood cell (WBC) count was measured by manual counting under the microscope.

Results: The CD4/CD8 ratio in the treatment group was significantly lower compared with that in the control group. The concentration of IL-2 and IL-6 was lower in the treatment group compared with the control group, while the level of IL-4 is the reverse between the two groups. The number of Paneth cells in intestinal mucosa increased significantly, while the level of SIgA in intestinal mucosa decreased significantly, after BMSCs transplantation. The gene expression levels of TNF-α and PRSS2 in intestinal mucosa of treatment group were significantly lower than those of control group. The WBC count in the treatment group was significantly lower than that in the control group.

Conclusion: We identified immune-relevant molecular changes that may explain the mechanism of BMSCs transplantation efficacy in alleviating rat intestinal immune-barrier after I/R.

Keywords: Intestinal mucosal immune barrier; Ischemia reperfusion injury; Mesenchymal stem cells; Repair mechanism.

Fig. 1

The counts and morphology of Paneth cells which is stained by H&E. The number of Paneth cells in the control group (a) was significantly lower than that in the treatment group (b). The repair effect of BMSCs was also observed in the morphology (Villus length and crypt depth) of intestinal mucosa.

Fig. 2

The morphology of BMSCs in passage 3 generation (a–f). The cell states are different at different growth stages. Most BMSCs are typical spindle-shaped, fibroblast-like cells, and a small number of BMSCS may be approximately circular.

Fig. 3

Purity identification of BMSCs. After completing isotype control for CD45 and CD95 (b, d), we obtained the ratio of CD90 (+)CD45(−) cells in passage 3 generation BMSC was more than 98% (a, c), indicating that most of the cells obtained were bone mesenchymal stem cells.

Fig. 4

Effects of BMSCs on CD4⁺ lymphocytes (a), CD8⁺ lymphocytes (b), IL-2 (c), IL-4 (d), IL-6 (e) and CD4/CD8 ratio (f) in rats intestinal mucosa. Number 1–20 refers to 20 rats from the control group and the treatment group in sequence. Number 1–5, 6–10, 11–15, 16–20 is the control group rats sampled on day 4 and day 7, treatment group rats sampled on day 4, day 7. The data were reported as mean ± SD or median, values were compared by T test or U test. *Significant difference, P < 0.05; **significant difference, P < 0.01; ***significant difference, P < 0.001.

Fig. 5

Effects of BMSCs on Paneth cell (a) and SigA (b). We observed Paneth cells and SIgA with microscope, measured the amount of SIgA by mean gray scale and determined the number of Paneth cells by direct counting method. Numbers refers to different samples. For Pan's cells: Number 1–9, 10–18, 19–27, 28–36 is the control group rats sampled on day 4 and day 7, treatment group rats sampled on day 4, day 7. For SIga: Number 1–10, 11–20, 21–30, 31–40 is the control group rats sampled on day 4 and day 7, treatment group rats sampled on day 4, day 7. The data were reported as mean ± SD, values were compared by T test. *Significant difference, P < 0.05; **significant difference, P < 0.01; ***significant difference, P < 0.001.

Fig. 6

SIgA was measured by IHC, compared with the control group-10 (a), the content of SIgA in treatment group-17 (b) decreased significantly. The data were reported as median, values were compared by U test.