Quercetin relieves compression-induced cell death and lumbar disc degeneration by stabilizing HIF1A protein

Abstract

Background: Lumbar disc degeneration (LDD) is a prevalent condition characterized by the decreased viability and functional impairment of nucleus pulposus mesenchymal stem cells (NPMSCs). Shaoyao-Gancao decoction (SGD), a traditional Chinese medicine formula, has been used to treat LDD, but its active components and mechanisms are unclear.

Methods: An integrative network pharmacology and transcriptome analysis were conducted to identify bioactive compounds in SGD that could target LDD. NPMSCs were cultured under mechanical compression as a cellular model of LDD. A rat model of annulus fibrosus needle-puncture was established to induce intervertebral disc degeneration. The effects of quercetin, a predicted active component, on alleviating compression-induced NPMSC death and LDD were evaluated in vitro and in vivo.

Results: The analysis identified hypoxia-inducible factor 1-alpha (HIF1A) as a potential target of quercetin in LDD. HIF1A was upregulated in degenerated human disc samples and compression-treated NPMSCs. Quercetin treatment alleviated compression-induced oxidative stress, apoptosis, and loss of viability in NPMSCs by stabilizing HIF1A. The protective effects of quercetin were abrogated by HIF1A inhibition. In the rat model, quercetin ameliorated intervertebral disc degeneration.

Conclusion: Our study identified HIF1A as a protective factor against compression-induced cell death in NPMSCs. Quercetin, a bioactive compound found in the traditional Chinese medicine formula SGD, improved the survival of NPMSCs and alleviated LDD progression by stabilizing HIF1A. Targeting the HIF1A pathway through natural compounds like quercetin could represent a promising strategy for the clinical management of LDD and potentially other degenerative disc diseases.

Keywords: HIF1A; Lumbar disc degeneration (LDD); Quercetin; Shaoyao-Gancao decoction (SGD); nucleus pulposus mesenchymal stem cells (NPMSCs).

Fig. 1

Identification of potential protein targets of SGD formula. (A) A total of 93 active ingredients were obtained from SGD formula using TCMSP, BATMAN-TCM, and TCMID databases. The interaction network shows a total of 217 corresponding protein targets for the 93 active ingredients. (B) Venn diagram showing 51 common targets between 553 LDD-related genes and 217 protein targets of SGD formula. (C) The 51 overlapping targets corresponds 87 active components from SGD formula.

Fig. 2

Enrichment analysis of the candidate targets. (A) The top 5 hits of 1403 biological processes (BP), 27 cellular components (CC) and 77 molecular functions (MF) enriched in the candidate targets. (B) The top 10 ranked KEGG pathways.

Fig. 3

Interrogation of the core targets in the PPI network. The protein-protein interaction (PPI) network of the 51 drug-related disease targets, which consists of 51 nodes and 782 edges. (B) The top 10 nodes in the network. (C) The ranking of core targets in the network using various analysis modules (MNC, Degree, BottleNeck, EcCentricity, Closeness, Radiality, Betweenness, and Stress) in cytohubba. (D) The top 20 targets of the above-mentioned scoring modules were integrated to get 6 core targets: IL6, MMP9, JUN, HIF1A, ESR1 and SPP1. (E) Volcano blot showing the differentially expressed genes in LDD samples from GSE56081 and GSE167119 dataset.

Fig. 4

Molecular docking analysis Molecular docking results revealed (A) the interaction between HIF1A and quercetin, (B) the interaction between IL6 with paeoniflorin, and (C) the interaction between IL6 and quercetin. (D) Summary of the optimal model binding energy and the interaction force between the docking area of each pair.

Fig. 5

A cell pressure model reveals the up-regulation of HIF1A and down-regulation of IL6 A cell pressure model of nucleus pulposus mesenchymal stem cells (NPMSCs) was established by inducing 1.0 MPa compression for different duration. (A) qRT-PCR analysis of HIF1A and IL6 mRNA after different duration of compression. (B) Immunoblotting of HIF1A and IL6 protein after different duration of compression. (C) Cell viability examination by CCK-8 assay. (D) Apoptotic events were quantified by flow cymotetry method. N = 3 experiments. One-way ANOVA test was adopted with Tukey's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 6

Quercetin alleviates pressure-induced cell death in NPMSCs In the cell pressure model for 48 h induction, quercetin was applied at 5–20 μM. (A) The mRNA levels of IL6 and HIF1A were quantified by qRT-PCR. (B) The protein levels of IL6 and HIF1A were analyzed by immunoblotting. (C) Cell viability examination by CCK-8 assay. (D) Apoptotic events were quantified by flow cymotetry method. N = 3 experiments. One-way ANOVA test was adopted with Tukey's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 7

Quercetin stabilizes HIF1A protein in NPMSCs NPMSCs under the compression were treated with 20 μM quercetin in the presence or absence of 5 μM PX-478 (an HIF1A inhibitor). (A) The mRNA levels of HIF1A were quantified by qRT-PCR. (B) The protein levels of HIF1A were analyzed by immunoblotting. (C) Cycloheximide (CHX) was applied to block de novo protein synthesis in above conditions, and the remaining protein levels of HIF1A at different time points after CHX addition were analyzed by immunoblotting, with actin serving as a internal control. (D) Quantification of transcription activity of HIF1A in above experimental conditions. N = 3 experiments. One-way ANOVA test was adopted with Tukey's post hoc test in A, B, and D. Two-way ANOVA test was adopted with Tukey's post hoc test in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 8

The rescue effect of quercetin on pressure-induced oxidative stress and cell death depends on HIF1A protein NPMSCs under the compression were treated with 20 μM quercetin in the presence or absence of 5 μM PX-478 (an HIF1A inhibitor). (A) Cell viability examination by CCK-8 assay. (B) Apoptotic events were quantified by flow cymotetry method. (C) The measurement of cellular levels of reactive oxygen species (ROS) and (D) mitochondria-derived ROS. (E) Electron microscope analysis of mitochondrial phenotype, and the arrows indicates the changes of mitochondrial cirstea. N = 3 experiments. One-way ANOVA test was adopted with Tukey's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 9

Quercetin ameliorates LDD in the rat model A rat model of compression-induced LDD was induced for 4 weeks, and quercetin was administrated for 2 weeks in the drug intervention group after LDD induction. (A) H&E and (B) Safranin O-fast Green staining in intervertebral disc tissues. (C) Western blot analysis of HIF1A expression in intervertebral disc samples. N = 6 animals in each group. One-way ANOVA test was adopted with Tukey's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. S1

Original uncropped versions of blotting images for data in Fig. 5B.

Fig. S2

Original uncropped versions of blotting images for data in Fig. 6B.

Fig. S3

Original uncropped versions of blotting images for data in Fig. 7B.

Fig. S4

Original uncropped versions of blotting images for data in Fig. 7C.

Fig. S5

Original uncropped versions of blotting images for data in Fig. 9C.