Up-regulation of CREB-1 regulates tendon adhesion in the injury tendon healing through the CREB-1/TGF-β3 signaling pathway

Abstract

Aim: To explore the mechanism of the healing of tendon tissue and anti-adhesion, and to discuss the role of the transforming growth factor-β3 (TGF-β3)/cAMP response element binding protein-1 (CREB-1) signaling pathway in the healing process of tendons.

Method: All mice were divided into four groups of 1, 2, 4, and 8 weeks respectively. Each time group was divided into four treatment groups: the amplification group, the inhibition group, the negative group, and the control group. When the tendon injury model was established, the CREB-1 virus was injected into the tendon injury parts. A series of methods such as gait behaviourism, anatomy, histological examination, immunohistochemical examination and collagen staining were employed to assess the tendon healing and the protein expression of TGF-β3, CREB-1, Smad3/7 and type I/III collagen (COL-I/III). CREB-1 virus was sent to tendon stem cells to assess the protein expression of TGF-β1, TGF-β3, CREB-1, COL-I/III by methods such as immunohistochemistry and Western blot.

Results: The amplification group showed better gait behaviourism than the inhibition group in the healing process. The amplification group also had less adhesion than the negative group. Hematoxylin-eosin (HE) staining of tendon tissue sections showed that the number of fibroblasts in the amplification group was less than the inhibition group, and the immunohistochemical results indicated that the expression of TGF-β3, CREB-1, and Smad7 at each time point was higher than the inhibition group. The expression of COL-I/III and Smad3 in the amplification group was lower than the inhibition group at all time points. The collagen staining indicated that the ratio of type I/III collagen in the amplification group was higher than the negative group at 2,4,8 week. The CREB-1 amplification virus could promote the protein expression of TGF-β3, CREB-1 and inhibit the protein expression of TGF-β1 and COL-I/III in the tendon stem cells.

Conclusion: In the process of tendon injury healing, CREB-1 could promote the secretion of TGF-β3, so as to promote the tendon healing and have the effect of anti-adhesion in tendons. It might provide new intervention targets for anti-adhesion treatment of tendon injuries.

Keywords: Adhesion2; CREB-15; Healing3; TGF-β34; Tendon1.

Fig. 1

The culture and identification of tendon stem cells: a Tendon stem cells P2, day 1. b Tendon stem cell P2, day 4. c Tendon stem cell P2, day 7. The red arrow points to the tendon stem cells. d, e, f The flow cytology detection result of Tendon stem cell P2

Fig. 2

CatWalk gait analysis results: a Pattern diagram of gait analysis; b Result of step cycle. The result showed that the step cycle in the inhibition group was significantly longer than the other group at all study time points; c Result of average speed. The result showed that the average speed was significantly lower than the other experimental groups at all study time points. “ amp ” means amplification group, “inh”means inhibition group, “neg” means negative group, “con” means control group. Data were analyzed by $\overline{\mathrm{x}}$ ± s (n = 10). *: signifificant difference, P < 0.05

Fig. 3

The gross healing and HE stain condition. a The general situation of adhesion around tendon. b HE staining microscope observation. The black arrow points to the fibroblasts. c Statistical results of tendon adhesion grading. The result showed that the amplification group have less adhesion than the other groups. d HE staining section fibroblast cell count. The result showed that the number of cells in amplification group was significantly lower than the other group, and the inhibition group was significantly higher than the other groups.“ + ” means amplification group, “-”means inhibition group, “N” means negative group, “C” means control group. scale bars: 50 um, *: compared with the negative and control group, p < 0.05, #: compared with the inhibition group, P < 0.05

Fig. 4

The immunohistochemical results and quantitation of Smad3 and Smad7. a, c Results and quantitation of Smad3. The result showed that the amplification group expressed less Smad3, and the inhibition group expressed more. b, d Results and quantitation of Smad7. The result showed that the amplification group expressed more Smad7, and the inhibition group expressed less. “ + ” means amplification group, “-”means inhibition group, “N” means negative group, “C” means control group. scale bars: 200 um, *:compared with the negative and control group, P < 0.05, #: group compared with the inhibition group, P < 0.05

Fig. 5

The immunohistochemical results and quantitation of COL-I and COL-III. a, c Results and quantitation of COL-I. The result showed that the amplification group expressed less COL-I, and the inhibition group expressed more. b, d Results and quantitation of COL-III. The result showed that the amplification group expressed less COL-III, and the inhibition group expressed more. “ + ” means amplification group, “-”means inhibition group, “N” means negative group, “C” means control group. scale bars: 200 um, *:compared with the negative and control group, P < 0.05, #: group compared with the inhibition group, P < 0.05

Fig. 6

The result of Sirius red staining. (× 100) The result showed that the proportion of COL-III to COL-I in the amplification group was lower than the other groups, and the inhibition group was higher than the other groups.“ + ” means amplification group, “-”means inhibition group, “N” means negative group, “C” means control group. scale bars: 400 um, *:compared with the negative and control group, P < 0.05, #: group compared with the inhibition group, P < 0.05

Fig. 7

Result of Cellular immunohistochemistry. The result showed that the CREB, TGF-β3 and Smad7 expressed a high level in the amplification group, and the Smad3, COL-I and COL-III highly expressed in the inhibition group. The result of RT-qPCR are presented in Supplementary material. (Supplement Fig. 1) “ + ” means amplification group, “-”means inhibition group, “N” means negative group, “C” means control group. scale bars: 50 um, *: compared with the negative and control group, P < 0.05, #: compared with the inhibition group, P < 0.05

Fig. 8

The WB result of protein expression: a Protein bands for TGF-β1/3, CREB-1, COL-I/III and GAPHD by western blotting assay. b, c Relative protein expressions of TGF-β1/3, CREB-1, COL-I/III in tendon stem cell by western blotting assay. Expression was normalized to GAPDH. The gel were cut before hybridizing with the antibody. The markers in the blots has been cropped and the original blots are presented in Supplementary material. (Supplement Fig. 2) The result showed that the CREB-1 and TGF-β3 highly expressed in the amplification group, and the TGF-β1, COL-I and COL-III highly expressed in the inhibition group. “ + ” means amplification group, “-”means inhibition group, “N” means negative group, “C” means control group. *: compared with the negative and control group, P < 0.05, #: compared with the inhibition group, P < 0.05