RESUMO
Objective:To explore the mechanism of epidural scar tissue hyperplasia induced by erythrocyte rupture and release of interleukin-33 (IL-33) after laminectomy in mice.Methods:In the zoological experiment, the operation group (Laminectomy) and the sham operation group were set, and HE staining and Masson staining were performed to test for blood accumulation in the operation area after laminectomy in mice. Then 12 wild-type mice with 6-8 week old were selected and divided into 4 groups: the sham operation group, the operation group (normal saline control), the pure red blood cell intervention operation group, the whole blood intervention operation group. The normal saline (100 mg/kg) was injected into the postoperative area. The red blood cells or whole blood with the same volume were injected into the postoperative area in the pure red blood cell intervention group and the whole blood intervention group. The postoperative recovery of mice in each group was observed. The levels of fibronectin in the postoperative scar tissues of mice in four groups were detected by western blot technology, and the degree of postoperative epidural scar hyperplasia was directly observed by immunohistochemistry. In the cytological experiment, the wild-type mouse erythrocyte normal saline group, the control group of IL-33 knockout mouse erythrocyte normal saline, the wild-type mouse erythrocyte lysis group, and the IL-33 knockout mouse erythrocyte lysis group were set. The levels of IL-33 in the red blood cells of four groups were detected by western blot. Then, a blank wild-type mouse erythrocyte control group, a wild-type mouse relative to the control group (only secondary antibody added to test for non-specific binding), a wild-type mouse erythrocyte group and an IL-33 knockout mouse erythrocyte group (to test for antigen specificity of the primary antibody) were set. Immunofluorescence staining was performed on the erythrocytes of four groups and the level of IL-33 was detected by flow cytometry.Results:HE staining and Masson staining after laminectomy showed that there was blood stasis in the local incision area of mice in the operation group. The epidural scar hyperplasia in the incision area of mice after whole blood or red blood cells intervention was higher, especially in the whole blood intervention group. IL-33 expression was almost undetectable in the wild-type erythrocyte normal saline control group, the IL-33-knockout erythrocyte normal saline control group, and the IL-33-knockout erythrocyte lysis group, while significant IL-33 expression was detectable in the wild-type erythrocyte lysis group. Immunofluorescence staining showed that IL-33 was expressed in and on the erythrocyte membrane of wild-type mice, while non-specific expression of IL-33 or a very small amount of IL-33 was almost undetectable in the other three groups. The immunofluorescence intensities of IL-33 in the four groups were 0.62±0.41, 60.17±4.39, 16.78±7.43 and 0.61±0.03, respectively ( F=281.90, P<0.001). The expression of IL-33 in the erythrocyte group of wild-type mice was the highest ( P<0.05). According to the results of flow cytometry, except for the trace amount of IL-33 detected in the wild-type mouse erythrocyte group, the expression of IL-33 in the other three groups was basically 0. The ratios of fibronectin to β-actin in the modeling area of the four groups gradually increased, and the ratios were 0.79±0.09, 1.26±0.23, 1.79±0.05 and 2.29±0.58, respectively, and the differences were statistically significant ( F=12.86, P=0.002). Fibronectin in the operation area of the three operation groups (normal saline control group, red blood cell intervention group and whole blood intervention group) was significantly higher than that of the sham operation group. The immunohistochemical staining results of fibronectin in the modeling area of the four groups were the same as those in western blot experiment. The average optical density values of fibronectin in each group were 0.09±0.01, 0.18±0.01, 0.22±0.01 and 0.24±0.01, respectively, and the difference was statistically significant ( F= 210.7, P<0.001). Conclusion:There is indeed blood accumulation in the surgical area after laminectomy in mice, and it can aggravate the hyperplasia of epidural scar tissue. Erythrocyte is the main component in blood, and there is a large amount of IL-33 expression in the inner and outer membrane of erythrocyte membrane. The mechanism of promoting the proliferation of epidural scar tissue may be related to the release of IL-33 by erythrocyte lysis.
RESUMO
BACKGROUND@#Recent evidence suggests that IL-33, a novel member of the IL-1b family, is involved in organ fibrosis. However, the roles of IL-33 and its receptor ST2 in epidural fibrosis post spine operation remain elusive. @*METHODS@#A mouse model of epidural fibrosis was established after laminectomy. IL-33 in the wound tissues post laminectomy was measured with Western blotting, ELISA and imaging. The fibroblast cell line NIH-3T3 and primary fibroblasts were treated with IL-33 and the mechanisms of maturation of fibroblasts into myofibroblasts were analyzed. To explore roles of IL-33 and its receptor ST2 In vivo, IL-33 knockout (KO) and ST2 KO mice were employed to construct the model of laminectomy. The epidural fibrosis was evaluated using H&E and Masson staining, western-blotting, ELISA and immunohistochemistry. @*RESULTS@#As demonstrated in western blotting and ELISA, IL-33 was increased in epidural wound tissues post laminectomy. The immunoflurosence imaging revealed that endothelial cells (CD31 + ) and fibroblasts (a-SAM +) were major producers of IL-33 in the epidural wound tissues. In vitro, IL-33 promoted fibroblast maturation, which was blocked by ST2 neutralization antibody, suggesting that IL-33-promoted-fibroblasts maturation was ST2 dependent. Further, IL-33/ ST2 activated MAPK p38 and TGF-β pathways. Either p38 inhibitor or TGF-β inhibitor decreased fibronectin and a-SAM production from IL-33-treated fibroblasts, suggesting that p38 and TGF-β were involved with IL-33/ST2 signal pathways in the fibroblasts maturation. In vivo, IL-33 KO or ST2 KO decreased fibronectin, a-SMA and collagen deposition in the wound tissues of mice that underwent spine surgery. In addition, TGF-β 1 was decreased in IL-33 KO or ST2 KO epidural wound tissues. @*CONCLUSION@#In summary, IL-33/ST2 promoted fibroblast differentiation into myofibroblasts via MAPK p38 and TGF-β in a mouse model of epidural fibrosis after laminectomy.