Targeting FABP4 to Inhibit AGEs-RAGE/NF-κB Signalling Effectively Ameliorates Nucleus Pulposus Dysfunction and Angiogenesis in Obesity-Related Intervertebral Disc Degeneration

Abstract

Intervertebral disc degeneration (IVDD) is a primary contributor to low back pain, posing significant social and economic burdens. Increasing evidence shows that obesity contributes to IVDD, yet the underlying mechanisms remain elusive. Here, we firstly revealed a causal correlation between obesity and IVDD via a two-sample mendelian randomization analysis and identified fatty acid-binding protein 4 (FABP4) as the potential regulator to associate IVDD and obesity. Elevated FABP4 expression promoted extracellular matrix (ECM) disequilibrium and angiogenesis to exacerbate IVDD progression. Genetically knocking out or pharmacologically inhibiting FABP4 in high-fat diet-induced mice alleviated IVDD. Mechanistically, obesity activated the mammalian target of rapamycin complex 1 (mTORC1), which upregulated FABP4 expression, leading to the accumulation of advanced glycation end-products (AGEs) in intervertebral disc tissue. AGEs further activated the NF-κB signalling pathway, exacerbating ECM degradation and neovascularization. Conversely, rapamycin-mediated inhibition of mTORC1 suppressed FABP4 expression in nucleus pulposus cells (NPCs), alleviating IVDD in vivo. Collectively, our findings reveal a critical role of the obesity-induced mTORC1-FABP4 axis in ECM degradation and angiogenesis during IVDD progression. Targeting FABP4 may represent a promising therapeutic strategy for IVDD in obese individuals.

Keywords: ECM homeostasis; FABP4; angiogenesis; intervertebral disc degeneration; mendelian randomization; obesity; signal pathway; therapeutic target.

FIGURE 1

Obesity increased the risk of IVDD (A) The impact of genetically correlated BMI and obesity‐related traits on IVDD was assessed using scatter plot calculations employing methods such as Inverse Variance Weighting (IVW), MR‐Egger, Simple Mode, Weighted Median, and Weighted Mode. (B) The overall symmetry of the funnel plot indicates that there is no publication bias or other systematic bias in the MR study results. (C) H&E staining of the IVD tissue from mice in different groups. (D) SOFG staining of the IVD tissue from mice in different groups. (E) Histological score of the IVD tissue from mice in different groups, including morphology, cellularity, and the bonder between NP and AF tissue (n = 5). (F) IHC staining for ACAN and MMP3 the IVD tissue from mice in different groups. (G and H) Quantitation results of the IHC staining for ACAN and MMP3 the IVD tissue from mice in different groups (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 2

FABP4 played a critical role in mediating obesity‐related IVDD. (A) RT‐qPCR analysis of the expression of FABP4 in NPCs under lipotoxic condition (n = 3). (B) RT‐qPCR analysis of the expression of FABP4 in IVD tissue from obesity and none‐obesity mice (n = 3). (C) IHC staining for FABP4 in IVD tissue from obesity and none‐obesity mice. (D) Quantitation result of IHC staining for FABP4 in IVD tissue from obesity and none‐obesity mice (n = 5). (E) IF staining for the expression of FABP4, COL2A1, and MMP3 in NPCs under lipotoxic condition with or without IL‐1β. (F–H) Quantitation results of the IF staining for the expression of FABP4, COL2A1, and MMP3 in NPCs under lipotoxic condition with or without IL‐1β (n = 5). (I) Degeneration evaluated by H&E, SOFG, and IHC staining of IVD tissue in WT and FABP4‐KO mice from sham and IVDD group, respectively. (J) Histological score of IVD tissue in WT and FABP4‐KO mice from sham and IVDD group, respectively (n = 5). (K and L) Quantitation results of IHC staining for ACAN and MMP3 of the IVD tissue in WT and FABP4‐KO mice from sham and IVDD group, respectively (n = 5). (M) IF staining for COL2A1 in IL‐1β‐induced NPCs with or without silencing FABP4. (N) Quantitation result of IF staining for COL2A1 in IL‐1β‐induced NPCs with or without silencing FABP4 (n = 5). (O) IF staining for MMP3 in IL‐1β‐induced NPCs with or without silencing FABP4. (P) Quantitation result of IF staining for MMP3 in IL‐1β‐induced NPCs with or without silencing FABP4 (n = 5). (Q) Degeneration evaluated by H&E, SOFG, and IHC staining of IVD tissue in blank and FABP4‐overexpressed mice from sham and IVDD group, respectively. (R) Histological score of IVD tissue in blank and FABP4‐overexpressed mice from sham and IVDD group, respectively (n = 5). (S and T) Quantitation results of IHC staining for ACAN and MMP3 of the IVD tissue in blank and FABP4‐overexpressed mice from sham and IVDD group, respectively (n = 5). (U) IF staining for COL2A1 and MMP3 in IL‐1β‐induced NPCs with or without the administration of recombinant FABP4. (V and W) Quantitation results of the IF staining for COL2A1 and MMP3 in IL‐1β‐induced NPCs with or without the administration of recombinant FABP4 (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

FABP4 contributed to dysfunction and ECM unbalance in NPCs via activating AGEs/RAGE/NFκB signalling pathway. (A) KEGG analysis of the NPCs with or without the administration of rFABP4 (n = 3). (B) KEGG analysis of the IVD tissue from WT and KO mice (n = 5). (C) ELISA result of AGEs level in the supernatant of the NPCs treated by rFABP4 in a dose‐dependent manner (n = 5). (D) ELISA result of AGEs level in the supernatant of the NPCs under lipotoxic condition with or without the treatment of rFABP4, FPS‐ZM1 (an inhibitor for RAGE), and JSH23 (an inhibitor for p65) (n = 5). (E) IF staining for COL2A1, MMP3, and p65 in NPCs under lipotoxic condition with or without the treatment of rFABP4, FPS‐ZM1, and JSH23. (F–H) Quantitation results of the IF staining for COL2A1, MMP3, and p65 in NPCs under lipotoxic condition with or without the treatment of rFABP4, FPS‐ZM1, and JSH23 (n = 5). (I) IF staining for RAGE, p‐p65, COL2A1, and MMP3 in WT and KO mice from sham or IVDD groups under HFD condition, respectively. (J) Quantitation results of the IF staining for RAGE, p‐p65, COL2A1, and MMP3 in WT and KO mice from sham or IVDD groups under HFD condition, respectively (n = 5). (K) IF staining for COL2A1, MMP3, and p65 in NPCs treated by AGEs with or without the treatment of FPS‐ZM1 and JSH23. (L and M) Quantitation results of the F staining for COL2A1, MMP3, and p65 in NPCs treated by AGEs with or without the treatment of FPS‐ZM1 and JSH23 (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Obesity‐induced expression of FABP4 was regulated by the mTORC1 pathway. (A) RT‐qPCR for the expression of FABP4, ACAN, COL2A1, MMP3, and MMP13 in NPCs under lipotoxic condition treated with or without rapamycin (a specific inhibitor of the mTOR pathway) (n = 3). (B) ELISA result of FABP4 and AGEs levels under lipotoxic condition treated with or without rapamycin (n = 5). (C–F) IF staining for FABP4, COL2A1, MMP3, PS6, and p65 in NPCs under lipotoxic condition treated with or without rapamycin. (G) Quantitation results of the IF staining for FABP4, COL2A1, MMP3, and PS6 in NPCs under lipotoxic condition treated with or without rapamycin (n = 5). (H) Quantitation results of the IF staining for nucleus translocation of p65 in NPCs under lipotoxic condition treated with or without rapamycin (n = 5). (I) Degeneration and mTORC1 pathway activation evaluated by H&E, SOFG, and IHC staining of IVD tissue in sham, IVDD, and IVDD+ rapamycin groups, respectively. (J) Histological score of IVD tissue in sham, IVDD, and IVDD+ rapamycin groups, respectively (n = 5). (K) Quantitation results of the IHC staining for COL2A1, ACAN, and PS6 in sham, IVDD, and IVDD+ rapamycin groups, respectively (n = 5). (L and M) IF staining and quantitation results for FABP4 in NPCs treated with or without MHY1485 (an activator for mTOR pathway). N and O: IF staining and quantitation results for COL2A1 and MMP3 in NPCs treated with or without MHY1485 (an activator for mTOR pathway). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

FABP4 promoted angiogenesis to exacerbate IVDD under lipotoxic condition. (A) Volcano plot of the DEGs of HFD‐induced IVDD from WT and KO mice. (B) Heart map of the DEGs of HFD‐induced IVDD from WT and KO mic (n = 5). (C) GO analysis of the DEGs of HFD‐induced IVDD from WT and KO mice. (D) KEGG analysis of the DEGs of HFD‐induced IVDD from WT and KO mice. (E) ELISA result of the VEGF level of the IVD tissue in WT and KO mice (n = 5). (F) ELISA result of the VEGF level of NPC degeneration model with or without FABP4 silencing or FPS‐ZM1 (n = 5). (G) IF staining for CD31 and EMCN in WT and KO mice from sham or IVDD groups under HFD condition, respectively. (H and I) Quantitation results of the IF staining for CD31 and EMCN in WT and KO mice from sham or IVDD groups under HFD condition, respectively (n = 5). (J) Tube formation assay of HUVECs in different groups. (K and L) Branch points and capillary length of HUVECs in different groups (n = 5). (M and N) Representative images and quantitation results of the scratch wound of HUVECs in different groups (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 6

Blocking the interaction of VEGF and VEGFR2 alleviated FABP4‐induced angiogenesis and IVDD (A) Degeneration and angiogenesis evaluated by H&E, SOFG, and IHC staining of IVD tissue from sham, IVDD, IVDD+rFABP4, and IVDD+rFABP4 + Ki8751 groups, respectively. (B) Histological score of IVD tissue from sham, IVDD, IVDD+rFABP4, and IVDD+rFABP4 + Ki8751 groups, respectively (n = 5). (C and D): Quantitation results of IF staining for CD31 and EMCN of IVD tissue from sham, IVDD, IVDD+rFABP4, and IVDD+rFABP4 + Ki8751 groups, respectively (n = 5). (E and F) Quantitation results of IHC staining for COL2A1 and MMP3 of IVD tissue from sham, IVDD, IVDD+rFABP4, and IVDD+rFABP4 + Ki8751 groups, respectively (n = 5). (G) Tube formation assay of HUVECs in different groups. (H and I) Branch points and capillary length of HUVECs in different groups (n = 5). (J and K) Representative images and quantitation results of the scratch wound of HUVECs in different groups (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 7

Inhibition of FABP4 alleviated obesity‐related IVDD via restoring ECM balance and inhibiting angiogenesis (A) Representative MRI images of IVD in sham, IVDD, and IVDD+BMS groups, respectively. (B) Degeneration evaluated by H&E, SOFG, and IHC staining of IVD tissue from sham, IVDD, and IVDD+BMS groups, respectively. (C–H) Histological score of IVD from sham, IVDD, and IVDD+BMS groups, respectively (n = 5). (I) Quantitation results of IHC staining for COL2A1, ACAN, and MMP3 of IVD from sham, IVDD, and IVDD+BMS groups, respectively (n = 5). (J–L) IF staining for COL2A1, MMP3, CD31, and EMCN of IVD from sham, IVDD, and IVDD+BMS groups, respectively. (M) Quantitation results of IF staining for COL2A1, MMP3, CD31, and EMCN of IVD from sham, IVDD, and IVDD+BMS groups, respectively (n = 5).

FIGURE 8

A diagram of the molecular mechanism in this study.