Hyperoside ameliorates TNF‑α‑induced inflammation, ECM degradation and ER stress‑mediated apoptosis via the SIRT1/NF‑κB and Nrf2/ARE signaling pathways in vitro

Abstract

Intervertebral disc degeneration (IDD) is the main pathogenesis of numerous cases of chronic neck and back pain, and has become the leading cause of spinal‑related disability worldwide. Hyperoside is an active flavonoid glycoside that exhibits anti‑inflammation, anti‑oxidation and anti‑apoptosis effects. The purpose of the present study was to investigate the effect of hyperoside on tumor necrosis factor (TNF)‑α‑induced IDD progression in human nucleus pulposus cells (NPCs) and its potential mechanism. The activity and apoptosis of NPCs were detected by Cell Counting Kit‑8 and flow cytometry analyses, respectively. The expression of interleukin (IL)‑6 and IL‑1β was detected with ELISA kits. Western blotting was used to detect the expression levels of proteins. The results showed that hyperoside effectively alleviated TNF‑α‑induced NPC apoptosis, and hyperoside treatment inhibited the upregulation of inducible nitric oxide synthase, cyclooxygenase‑2, IL‑1β and IL‑6 in TNF‑α‑stimulated NPCs. Compared with the findings in the TNF‑α group, the intervention of hyperoside attenuated the upregulated expression of aggrecan and collagen II, and downregulated the expressions of matrix metalloproteinase (MMP) 3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5. In addition, hyperoside upregulated sirtuin‑1 (SIRT1) and nuclear factor E2‑related factor 2 (Nrf2) protein expression, and inhibition of SIRT1 or Nrf2 signaling reversed the protective effect of hyperoside on TNF‑α‑induced NPCs. In summary, hyperoside ameliorated TNF‑α‑induced inflammation, extracellular matrix degradation, and endoplasmic reticulum stress‑mediated apoptosis, which may be associated with the regulation of the SIRT1/NF‑κB and Nrf2/antioxidant responsive element signaling pathways by hyperoside.

Keywords: endoplasmic reticulum stress; extracellular matrix degradation; hyperoside; inflammation; intervertebral disc degeneration; sirtuin‑1/NF‑κB and nuclear factor E2‑related factor 2/antioxidant responsive element signaling.

Figure 1.

Hyperoside enhances TNF-α-induced viability of HNPCs. (A) Chemical structure formula of hyperoside. (B) The effect of different concentrations (0, 10, 20 and 50 µM) of hyperoside on viability of HNPCs. (C) Hyperoside on TNF-α-induced HNPCs viability. ***P<0.001 vs. control and ^##P<0.01 vs. TNF-α. HNPCs, human nucleus pulposus cells.

Figure 2.

Hyperoside inhibits TNF-α-induced endoplasmic reticulum stress-mediated apoptosis in HNPCs. (A) Flow cytometry was used to detect the effect of hyperoside on TNF-α-induced apoptosis of HNPCs. (B) The expression levels of Bcl-2, Bax, GRP78, p-PERK, ATF6, CHOP and caspase 12 proteins were detected by western blot analysis. ***P<0.001 vs. control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. TNF-α. HNPCs, human nucleus pulposus cells; GRP, glucose-regulated protein; p-PERK, ATF, activating transcription factor 6; p-, phosphorylated; PERK, protein kinase RNA-like ER kinase; CHOP, C/EBP homologous protein.

Figure 3.

Hyperoside reduces TNF-α-induced inflammation in human nucleus pulposus cells. (A and B) The expression level of inflammatory cytokines (A) IL-1β and (B) IL-6 were detected by ELISA. (C) Western blotting was performed to detect the expression levels of COX-2 and iNOS. ***P<0.001 vs. control; ^##P<0.01 and ^###P<0.001 vs. TNF-α. COX, cyclooxygenase; iNOS, inducible nitric oxide synthase.

Figure 4.

Hyperoside inhibits TNF-α-induced degradation of ECM in human nucleus pulposus cells. The expression of ECM degradation related proteins (Aggrecan, Collagen II, MMP3, MMP13 and ADAMTS5) was detected by western blot analysis. ***P<0.001 vs. control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. TNF-α. ECM, extracellular matrix; MMP, matrix metalloproteinase.

Figure 5.

Hyperoside regulates the SIRT1/NF-κB and Nrf2/ARE signaling pathways. Western blot analysis was used to detect the expression of SIRT1/NF-κB and Nrf2/ARE pathway-related proteins (SIRT1, p-NF-κB, NF-κB, Nrf2, HO-1 and NQO1). ***P<0.001 vs. control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. TNF-α. SIRT, sirtuin; Nrf, nuclear factor E2-related factor 2; ARE, antioxidant responsive element; p-, phosphorylated; HO, heme oxygenase; NQO1, NAD(P)H quinone dehydrogenase.

Figure 6.

EX527 and ML385 reverse the inhibitory effect of hyperoside on TNF-α-induced endoplasmic reticulum stress-mediated apoptosis of human nucleus pulposus cells. (A) Cell viability and (B) apoptosis were detected by Cell Counting Kit-8 and flow cytometry, respectively. (C) The expression levels of Bcl-2, Bax, GRP78, p-PERK, ATF6, CHOP and caspase 12 proteins were detected by western blotting. ***P<0.001 vs. control; ^###P<0.001 vs. TNF-α; ⁺P<0.05, ⁺⁺P<0.01 and ⁺⁺⁺P<0.001 vs. TNF-α + hyperoside. GRP, glucose-regulated protein; p-, phosphorylated; PERK, protein kinase RNA-like ER kinase; ATF, activating transcription factor 6; CHOP, C/EBP homologous protein.

Figure 7.

EX527 and ML385 reverse the inhibitory effect of hyperoside on TNF-α-induced inflammation and ECM degradation of human nucleus pulposus cells. (A and B) The expression level of inflammatory cytokines (A) IL-1β and (B) IL-6 were detected by ELISA. (C) Western blotting was performed to detect the expression levels of COX-2 and iNOS. (D) The expression of ECM degradation-related proteins (Aggrecan, Collagen II, MMP3, MMP13 and ADAMTS5) was detected by western blotting. ***P<0.001 vs. control; ^###P<0.001 vs. TNF-α; ⁺P<0.05, ⁺⁺P<0.01 and ⁺⁺⁺P<0.001 vs. TNF-α + hyperoside. ECM, extracellular matrix; COX, cyclooxygenase; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase.