Resveratrol increases nucleus pulposus matrix synthesis through activating the PI3K/Akt signaling pathway under mechanical compression in a disc organ culture

Abstract

Disc nucleus pulposus (NP) matrix homeostasis is important for normal disc function. Mechanical overloading seriously decreases matrix synthesis and increases matrix degradation. The present study aims to investigate the effects of resveratrol on disc NP matrix homeostasis under a relatively high-magnitude mechanical compression and the potential mechanism underlying this process. Porcine discs were perfusion-cultured and subjected to a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days in a mechanically active bioreactor. The non-compressed discs were used as controls. Resveratrol was added along with culture medium to observe the effects of resveratrol on NP matrix synthesis under mechanical load respectively. NP matrix synthesis was evaluated by histology, biochemical content (glycosaminoglycan (GAG) and hydroxyproline (HYP)), and expression of matrix macromolecules (aggrecan and collagen II). Results showed that this high-magnitude mechanical compression significantly decreased NP matrix content, indicated by the decreased staining intensity of Alcian Blue and biochemical content (GAG and HYP), and the down-regulated expression of NP matrix macromolecules (aggrecan and collagen II). Further analysis indicated that resveratrol partly stimulated NP matrix synthesis and increased activity of the PI3K/Akt pathway in a dose-dependent manner under mechanical compression. Together, resveratrol is beneficial for disc NP matrix synthesis under mechanical overloading, and the activation of the PI3K/Akt pathway may participate in this regulatory process. Resveratrol may be promising to regenerate mechanical overloading-induced disc degeneration.

Keywords: matrix; mechanical compression; nucleus pulposus; resveratrol.

Figure 1. Histological observation.

Alcian Blue staining of nucleus pulposus (NP) tissue; magnification = 200×, scale = 100 μM, n=3. Data are expressed as mean ± SD. *: significant difference (P<0.05) between two groups. #: significant difference (P<0.05) compared with the 1.3 MPa group.

Figure 2. Gene expression analysis.

Real-time PCR analysis of nucleus pulposus (NP) matrix biosynthesis-related molecules (SOX-9, aggrecan, and collagen II). Data are expressed as mean ± SD. *: significant difference (P<0.05) between two groups. #: significant difference (P<0.05) compared with the 1.3 MPa group.

Figure 3. Immunohistochemical observation.

Immunohistochemical staining of nucleus pulposus (NP) matrix macromolecules (aggrecan and collagen II) and its quantification; magnification = 200×, scale = 100 μM, n=3. Data are expressed as mean ± SD. *: significant difference (P<0.05) between two groups. #: significant difference (P<0.05) compared with the 1.3 MPa group.

Figure 4. PI3K/Akt pathway activity and protein expression of matrix anabolism-related molecules.

Western blot analysis of the activity of the PI3K/Akt pathway (A) and protein expression of nucleus pulposus (NP) matrix biosynthesis-related molecules (SOX-9, aggrecan, and collagen II) (B); magnification = 200×, scale = 100 μM, n=3. Data are expressed as mean ± SD. *: significant difference (P<0.05) between two groups. #: significant difference (P<0.05) compared with the 1.3 MPa group.

Figure 5. Biochemical content measurement.

Measurement of biochemical content within the nucleus pulposus (NP) tissue. (A) Glycosaminoglycans (GAG) content analysis and (B) hydroxyproline (HYP) content analysis. Data are expressed as mean ± SD. *: significant difference (P<0.05) between two groups. #: significant difference (P<0.05) compared with the 1.3 MPa group.