Senolytic Combination Treatment Is More Potent Than Single Drugs in Reducing Inflammatory and Senescence Burden in Cells from Painful Degenerating IVDs

Abstract

Background: Low back pain is a global health problem directly related to intervertebral disc (IVD) degeneration. Senolytic drugs (RG-7112 and o-Vanillin) target and remove senescent cells from IVDs in vitro, improving tissue homeostasis. One drawback of using a single senolytic agent is the failure to target multiple senescent antiapoptotic pathways. This study aimed to determine if combining the two senolytic drugs, o-Vanillin and RG-7112, could more efficiently remove senescent cells and reduce the release of inflammatory factors and pain mediators in cells from degenerating human IVDs than either drug alone.

Methods: Preliminary data evaluating multiple concentrations of o-Vanillin and RG-7112 led to the selection of four treatment groups. Monolayer and pellet cultures of cells from painful degenerate IVDs were exposed to TLR-2/6 agonist. They were then treated with the senolytics o-Vanillin and RG7112 alone or combined. p16^ink4a, Ki-67, caspase-3, inflammatory mediators, and neuronal sprouting were assessed.

Results: Compared to the single treatments, the combination of o-Vanillin and RG-7112 significantly reduced the amount of senescent IVD cells, proinflammatory cytokines, and neurotrophic factors. Moreover, both single and combination treatments significantly reduced neuronal sprouting in rat adrenal pheochromocytoma (PC-12 cells).

Conclusions: Combining o-Vanillin and RG-7112 greatly enhanced the effect of either senolytic alone. Together, these results support the potential of senolytics as a promising treatment for IVD-related low back pain.

Keywords: cellular senescence; combination therapy; intervertebral disc degeneration; low back pain; senolytics; senotherapeutics.

Figure 1

RG-7112 reduces the expression of p16, TLR-2, and SASP factors following TLR-2 activation in IVD cells from patients with back pain and IVD degeneration. (A–D) Human IVD cells from painful degenerate IVDs in monolayer culture induced with Pam2CSK4 and treated with 5 µM RG-7112. qRT-PCRs were normalized to the baseline (cells from painful degenerate IVD not induced with Pam2CSK4 and not treated with RG-7112). (A) Gene expression of SASP factors (CCL2, CCL5, CCL7, CCL8, GM CSF, BDNF, NGF, TNF-α, TGF-β, CXCL1, CXCL8, and CXCL10). (B) Gene expression of senescence marker p16. (C) Gene expression of Toll-like receptors (TLR-1, TLR-2, TLR-4, and TLR-6). (D) All monolayer culture media were analyzed by Raybio Human Cytokine Array. TNF-α, IL-1β, IL-8 and NGF protein concentration were evaluated. Values are presented as fold change ± SD in (A–C) and mean ± SD in (D).* Indicates significance calculated using a one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (A–D) n = 5 donor samples per condition.

Figure 2

Identifying the lowest effective concentration of RG-7112 and o-Vanillin at which senolytic activity is preserved in monolayer culture. (A) Representative images of monolayer cultures stained with (a) p16, (b) Ki-67 and (c) caspase-3. (d–f) Magnified images of (a–c) with arrowheads representing positive (green) and negative (red) stained cells. Scale bars (A): (a–c) 200 µm and (d–f) 50 µm. Quantification of senolytic serial dilution experiments of (B) p16, (C) Ki-67 and (D) caspase-3 expression. Values are presented as mean ± SD in (B–D). * Indicates significance between treatment and control groups. All analyses were performed using a one-way ANOVA, n = 7 donor samples. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Combination treatment with o-Vanillin and RG-7112 results in additive apoptotic and proliferative activity in pellet cultures. (A) Timeline of pellet culture. (B) Representative images of pellets stained with (a) DAPI, (b) p16, (c) caspase-3, (d) p16 and caspase-3 merged staining, (e) p16, caspase-3 and DAPI merged staining. (f–j) Magnified images of (a–e) with arrowheads representing positive (yellow) and negative (red) stained cells. Scale bars (a–e) 100 µm and (f–j) 25 µm. (C) Representative images of pellet cultures stained with (a) DAPI, (b) Ki-67, and (c) DAPI and Ki-67 merged. (d–f) Represent magnified images of (a–c) with arrowheads representing positive (yellow) and negative (red) stained cells. Scale bars (a–c) 100 µm and (d–f) 25 µm. Quantification of pellet fluorescent staining of (D) p16, (E) Ki-67, (F) caspase-3 and (G) p16 and caspase-3 expression. (H) AlamarBlue assessed the evaluation of cell viability. (I) sGAG concentration measured by DMMB assay using day 12 pellet culture media. Values are presented as mean ± SD in (D–I). * Indicates significance between treatment (single or combination) and control groups. # Indicates significance calculated when combination treatment is compared to single treatment groups. All analyses were performed using a one-way ANOVA, n = 7 donor samples. * or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001, **** p < 0.0001.

Figure 4

Combination treatment with RG-7112 and o-Vanillin significantly decreases SASP factor release in pellet cultures. Pellet culture media were analyzed by Raybio Human Cytokine Array. The cytokine concentration of (A) IL-6, (B) IL-8, (C) IL-1β, (D) TNF-α and neurotrophic factors (E) NGF and (F) BDNF were evaluated. Values are presented as mean ± SD in (A–F). * Indicates significance between treatment (single or combination) and control groups. # Indicates significance calculated when combination treatment is compared to single-treatment groups. All analyses were performed using a one-way ANOVA, n = 7 donor samples. * or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001, **** or #### p < 0.0001.

Figure 5

Pellet media from RG-7112 and o-Vanillin combined treated pellets have lower levels of neurite sprouting and neurite growth gene expression in PC12 cells. (A) Gene expression of neurite growth factors (NF-L, Plaur, PIK-2, PVR and VGF) was evaluated in PC-12 cells cultured in day 12 pellet media for 48 h. qRT-PCRs were normalized to culture media from pellets that were not induced with Pam2CSK4 and no senolytic treatment. (B) Representative images of neurite sprouting with arrowheads indicating neurite sprouting (green) and no neurite sprouting (red). Scale bar 75 µm. (C) Quantification of PC-12 neurite sprouting. Values are presented as (A) fold change ± SD or (C) mean ± SD. All analyses were performed using a one-way ANOVA, n = 7 donor samples. * p < 0.05, ** or ## p < 0.01, *** p < 0.001, **** or #### p < 0.0001.