Effect of regulating macrophage polarization phenotype on intervertebral disc degeneration

Abstract

Background: Macrophages are the only inflammatory cells that can penetrate the closed nucleus pulposus and their polarization plays an important role in intervertebral disc degeneration (IVDD). This paper attempted to investigate the pathogenesis of IVDD by altering the polarization state of macrophages.

Methods: Macrophage RAW264.7 cells were induced by interferonγ (IFN-γ) and lipopolysaccharide (LPS). The polarization of RAW264.7 cells was estimated by western blot and immunofluorescence. The expressions of inflammatory factors were detected by ELISA. Subsequently, RAW264.7 cells were treated with different concentrations of minocycline (Mino) and sinomenine (Sino), followed by the assessment of cell viability with cell counting kit-8 kit. Then, RAW264.7 cell culture medium was collected for the culture of human nucleus pulposus cells (NPCs). Toluidine blue staining and type II collagen staining were applied to assay the level of type II collagen. The cell apoptosis, oxidative stress, and nitric oxide (NO) level were appraised by TUNEL, oxidative stress kits and NO kit, respectively. Western blot was employed to test the levels of apoptosis- and oxidative stress-related proteins.

Results: IFN-γ and LPS could induce M1 polarization of RAW264.7 cells. Mino and Sino could reduce the polarization of RAW264.7 cells toward M1. M1-polarized medium inhibited LPS-induced activity, inflammation, and damage of NPCs, which were enhanced by Mino and Sino in medium.

Conclusion: M1 polarization of macrophages promoted LPS-induced inflammation and damage of NPCs.

Keywords: M1 polarization; inflammation; intervertebral disc degeneration; macrophages.

Figure 1

Induction of polarization of macrophages. (A) Western blot detected the expressions of iNOS and Arg‐1. (B and C) IF detected the expressions of iNOS and Arg‐1. (D) ELISA was used to detect the expressions of TNF‐α, IL‐6 and IL‐10. ***p < .001 versus Control. Arg‐1, arginase‐1; IF, Immunofluorescence; IL‐6, interleukin‐6; IL‐10, interleukin‐10; iNOS, inducible nitric oxide synthase; TNF‐α, tumor necrosis factor‐α.

Figure 2

Mino and Sino inhibited macrophage transformation to M1 type. (A and B) CCK‐8 detected the cell viability. ^# p < .05, ^### p < .001 versus IFN‐γ + LPS (M1). (C and D) Western blot detected the expressions of iNOS and Arg‐1. (E and F) IF detected the expressions of iNOS and Arg‐1. (G) ELISA was used to detect the expressions of TNF‐α, IL‐6 and IL‐10. *p < .05, ***p < .001 versus Control; ^# p < .05, ^## p < .01, ^### p < .001 versus IFN‐γ + LPS (M1). Arg‐1, arginase‐1; CCK‐8, cell counting kit; IF, Immunofluorescence; IFN‐γ, interferonγ; IL‐6, interleukin‐6; IL‐10, interleukin‐10; iNOS, inducible nitric oxide synthase; TNF‐α, tumor necrosis factor‐α.

Figure 3

M1 macrophages promoted the expressions of type II collagen and Aggrecan in LPS‐induced myeloid cells. (A) CCK‐8 detected the cell viability. Type II collagen staining (B) and toluidine blue staining (C) detected the secretion levels of Type II collagen. *p < .05 versus control; ^# p < .05 versus LPS. CCK‐8, cell counting kit; LPS, lipopolysaccharide.

Figure 4

M1‐type macrophages promoted LPS‐induced apoptosis of myeloid cells and oxidative stress injury. (A and B) TUNEL assay detected the apoptosis. (C) The expressions of ROS, MDA and SOD were detected by the corresponding kits. (D) NO detection kit detected the level of NO. ***p < .001 versus Control; ^### p < .001 versus LPS; ${}^{@@@}p$ < .001 versus LPS + M1 medium. LPS, lipopolysaccharide; MDA, malonaldehyde; NO, nitric oxide;  ROS, reactive oxygen species; SOD, superoxide dismutase.

Figure 5

Expressions of related proteins. (A) Western blot detected the expressions of Bax, Bcl‐2 and caspase‐3. (B) Western blot detected the expressions of MMP2, MMP9 and iNOS. *p < .05, **p < .01, ***p < .001 versus Control; ^# p < .05, ^## p < .01 versus LPS; ^@ p < .05, ${}^{@@}p$ < .01, ${}^{@@@}p$ < .001 versus LPS+ M1 medium. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide.