Introduction

Intervertebral disc degeneration (IDD) is an age-related degenerative disease and the main cause of various spinal degenerative diseases such as cervical spondylosis, lumbar disc herniation, and lumbar spinal stenosis. Due to the aging of the population, the prevalence of spinal degenerative diseases is increasing year by year, which not only seriously affects the quality of life of patients, but also brings a great health burden to the society [1]. However, so far the treatment of IDD has not achieved satisfactory results.

Cellular senescence is usually defined as irreversible cell cycle arrest due to replication stress and senescence [2], and the senescence-associated secretory phenotype (SASP) is an important feature of cellular senescence, which secretes a series of cytokines such as pro-inflammatory factors, growth factors, chemokines and proteases [3, 4]. The main feature of IDD is the degradation of the extracellular matrix (ECM), and NPCs, as the main functional cells of IDD, are essential to maintain the homeostasis of ECM [5]. However, senescent NPCs were accompanied by the reduction in Collagen-2, the main component of the ECM, which induced the occurrence of IDD [6], so one of the most important features of IDD is the senescence of NPCs. It has been reported earlier that the degree of cellular senescence was closely related to the IDD grade [7, 8]. Therefore, it is of great significance to explore the mechanism of senescence of NPCs for the exploration of IDD progression.

Myricetin (MYR) is a naturally occurring flavonoid compound widely found in fruits, vegetables and nuts [9]. Several studies have reported that MYR had antioxidant, antiviral, antibacterial and anticancer effects [9, 10]. Studies found that myricetin had anti-photoaging effect [11] and prevented ethanol-induced inflammatory damage [12], and could reduce the occurrence of death by inhibiting the secretion of inflammatory cytokines [13]. However, its anti-aging potential in NPCs has not been intensively investigated so far.

In this study, we identified 421 differentially expressed genes (DEGs) by RNA sequencing (RNA-seq) technology in hydrogen peroxide (HO)-induced senescent NPCs treated with MYR. The Gene Ontology (GO) database was used to enrich the DEGs into the corresponding pathways. Furthermore, we obtained the serine protease inhibitor clade E member 1 (SERPINE1) with the highest log2FC abs, which belongs to one of the serine protease inhibitor family members by Venn diagram. It was finally confirmed that MYR inhibited the senescence of NPCs by regulating the expression of SERPINE1.

Material and methods

Cell culture and transfection

NPCs isolated from mild IDD patients (Pfirrmann grade I–II) were a gift from First Affiliated Hospital, ** genes in aging and inflammation-related genes (Fig. 4A) and found a total of 7 overlap** genes. The expression of the top 5 overlap** genes was then verified by qPCR, and the results proved that the expression trends of these five DEGs were consistent with the RNA-seq results (Fig. 4B). More importantly, SERPINE1 was used for the following studies since SERPINE1 had the highest log2FC abs among the top 5 overlap** genes. Next, we interfered with SERPINE1 expression in NPCs, and the results proved that silencing SERPINE1 was successful (Fig. 4C). Subsequently, by SA-β-gal staining, we found that silencing SERPINE1 reduced the percentage of SA-β-Gal-positive cells (Fig. 4D). Furthermore, by Western blot, we found that silencing SERPINE1 inhibited senescence markers (p21, p16) expression (Fig. 4E). Finally, ELISA was used to detect the effect of silencing SERPINE1 on the secretion of SASP pro-inflammatory factors (IL-6 and IL-8), the results confirmed that silencing of SERPINE1 inhibited the increased expression levels of IL-6 and IL-8. Taken together, the results suggest that silencing SERPINE1 inhibits HO-induced cellular senescence.

Fig. 4
figure 4

Silencing of SERPINE1 regulates HO-induced senescence in NPCs. A Venn diagram analysis of overlap** genes in aging and inflammation-related genes. B qPCR verifies the expression of the top 5 overlap** genes. C The silencing effect of SERPINE1 was detected by qPCR. D SA-β-gal staining was used to detect the level of cellular senescence in two groups of NPCs treated with 100 μM HO for 24 h. E Western blot was used to detect the expression levels of senescence markers (p21, p16) in HO-treated NPCs in si-NC and si-SERPINE1 groups. F The expression levels of IL-6 and IL-8 of NPCs treated with 100 μM HO for 24 h were detected by ELISA. *P < 0.5 VS si-NC group

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Overexpression of SERPINE1 inhibits the anti-aging effect of MYR

At the end of the experiment, we explored the effect of SERPINE1 on the anti-aging effect of MYR. First, we verified the transfection efficiency of SERPINE1 overexpression in NPCs. qPCR results showed that SERPINE1 was overexpressed successfully (Fig. 5A). Subsequent SA-β-gal staining (Fig. 5B) and Western blot (Fig. 5C) results confirmed that overexpression of SERPINE1 reversed the MYR-induced decrease in the percentage of SA-β-Gal-positive cells and expression of p16 and p21. Similarly, ELISA results showed that overexpression of SERPINE1 inhibited the expression of MYR-regulated IL-6 and IL-8 (Fig. 5D). Taken together, the results indicated that overexpression of SERPINE1 inhibited the anti-aging effect of MYR.

Fig. 5
figure 5

Overexpression of SERPINE1 affects the anti-aging effect of MYR. A qPCR was used to examine the effect of SERPINE1 overexpression. B SA-β-gal staining was performed to detect senescence levels in three groups of NPCs with 100 μM HO alone for 24 h or co-treated with 100 μM HO for 24 h and 10 μM MYR for 48 h. C Western blot was used to detect the expression levels of p21 and p16 in three groups of NPCs with 100 μM HO alone or co-treated with 100 μM HO and 10 μM MYR. D ELISA was used to detect the expression levels of IL-6 and IL-8 in three groups of NPCs with 100 μM HO alone or co-treated with 100 μM HO for 24 h and 10 μM MYR for 48 h

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Discussion

IDD is one of the ancient and common clinical diseases, and cellular senescence is the key inducement of IDD pathogenesis. In the present study, we found that MYR could effectively inhibit HO-induced cellular senescence and the secretion of inflammatory factors of NPCs by regulating SERPINE1.

The causes of cellular senescence are mainly divided into two categories, replicative aging and stress-induced premature aging. The former is a programmed death process, while the latter is under the action of some sub-lethal emergencies such as hyperoxia, HO, ultraviolet rays cells age in advance. HO can directly damage DNA and cause premature cell aging [16], so in this study HO was selected as an agent for inducing senescence in NPCs. The concentration of HO used for aging induction of NPCs was not consistent in previous studies [17,18,19], and the major considered parameter was the cell viability, which was generally at 80–90% [17, 18]. Consistently, the concentration (100 μM) we used for aging induction resulted in a ~ 80% viability of NPCs. With the increase in HO dose, cell viability reduced gradually, and only 40% of NPCs was alive after treated with 500 μM HO. Generally, the concentration of HO caused ~ 50% of cell viability was used to induce apoptosis of NPCs [20, 21].

MYR is a flavonol compound with various pharmacological activities such as anti-inflammatory and analgesic, anti-tumor, hypoglycemic, and liver protection [9]. MYR shows abundant resource advantages and huge potential utilization value. Multiple effects of MYR have been reported to depend on the therapeutic dose [22]. Cell viability assay indicated that the higher dose of MYR (40 and 80 μM) significantly inhibited cell growth, while the lower dose groups (10 and 20 μM) showed no cytotoxicity on NPCs. We also found that lower dose of MYR (10 and 20 μM) could not significantly promote the growth of NPCs, but they played positive growth effects on HO-treated NPCs. We inferred that the injured NPCs could not growth as normal, and the potential anti-aging and anti-inflammation effects of MYR helped the injured NPCs to gradually return to a better state. In addition, the positive growth effect of MYR on normal NPCs may be observed by extending the treatment time to 72 h, as a higher viability was observed in 10 μM group treated for 48 h. Further senescent phenotype detection results indicated that MYR could inhibit the senescence of NPCs.

Inflammatory response has always been an important factor affecting the occurrence and development of various degenerative diseases including IDD. It is difficult for the normal immune inflammatory response to act on the intervertebral disc tissue under physiological conditions. However, when disc herniation occurs, herniated disc tissue causes pain responses through the action of inflammatory factors [23]. Previous studies have demonstrated that MYR could inhibit the expression of inflammatory factors [13]. Consistently, we found that MYR inhibited the expression of inflammatory factors IL-6 and IL-8 secreted by HO-induced senescent NPCs.

Next, RNA-seq was used to further explore DEGs in senescent NPCs to uncover the key genes affecting NPC aging. We found a total of 260 up-regulated genes and 161 down-regulated genes compared with the control group. In the GO enrichment analysis of DEGs, DEGs were mainly enriched in the "CXCR chemokine receptor binding" terms, a previous report demonstrated that loss of CXCR7 expression leads to cellular senescence [24]. It indicated that DEGs may play a role by participating in CXCR chemokine receptor binding. At the same time, we searched the GO terms related to aging (aging, senescence) and inflammation (inflammat-), respectively, and a total of 8 related GO biological processes were screened. FOXO4 was enriched in the GO term of "aging", and it has been proved to be involved in IDD [28] and has been found to show pro-angiogenic, growth and migration stimulation and anti-apoptotic activities in recent years [29]. It is also confirmed to be the most reliable biological and prognostic marker for various cancers [30, 31]. More importantly, it is reported that SERPINE1 has showed important regulating role in aging [32, 33] and is also able to regulate inflammatory damage [34], but its role in the senescence of NPCs is still unknown. In this study, we found that the expression of SERPINE1 was significantly decreased in senescent NPCs cotreated with HO and MYR, and interfering with SERPINE1 expression could inhibit the secretion of inflammatory factors in NPCs, which was consistent with previous reports [34]. In addition, studies have shown that SERPINE1 can promote the expression of STAT3 signaling pathway [35], and studies have found that the aging of NPCs was closely related to STAT3 signaling pathway [36]. Therefore, we speculated that SERPINE1 may regulate the aging of NPCs by regulating the STAT3 signaling pathway.

qPCR validation also indicated that EDNRB’s validated log2 FC (abs) value was second only to that of SERPINE1. It is reported that EDNRB is closely correlated with hair graying with aging [37], and deletion of EDNRB leads to delayed development of neural crest cells [38]. Another noteworthy verified DEG is BCL6, which has been shown to regulate cellular senescence [39]. In addition, BCL6 is reported to be a potent inhibitor to suppress the senescence of mouse fibroblasts, and it could induce cyclin D1 expression thus bypassing the senescence response downstream of p53 [40]. The upregulation of the two candidates might also be involved in the anti-aging effect of MYR, which is worth for further studies.

Overall, the present study found that MYR was able to alleviate HO-induced senescence of NPCs by regulating the expression of SERPINE1 in vitro, providing a promising candidate molecule to reverse the senescence of NPCs in vivo. The study also identified and verified other candidate DEGs in MYR treating group, which helps to further investigate the multiple mechanisms of MYR.