Ginkgetin Alleviates Intervertebral Disc Degeneration by Inhibiting Apoptosis, Inflammation, and Disturbance of Extracellular Matrix Synthesis and Catabolism via Inactivation of NLRP3 Inflammasome

ABSTRACT Background Apoptosis, inflammation, and the extracellular matrix (ECM) synthesis and catabolism are compromised with intervertebral disc degeneration (IDD). Ginkgetin (GK) has been demonstrated to alleviate several diseases; however, its effect on IDD remains unknown. Methods The nucleus pulposus cells (NPCs) were stimulated with interleukin (IL)-1β to construct the IDD models in vitro. Rats were used for the construction of the IDD models in vivo via the fibrous ring puncture method. The effect and mechanism of GK on IDD were determined by cell counting kit-8 (CCK-8), flow cytometry, western blot, real-time quantitative polymerase chain reaction (RT-qPCR), enzyme‑linked immunosorbent assay (ELISA), hematoxylin and eosin (HE) and safranine O staining, and immunohistochemistry (IHC) assays, respectively. Results GK increased the cell viability and upregulated the expressions of anti-apoptosis and ECM synthesis markers in NPCs treated with IL-1β. GK also decreased apoptosis rate, and downregulated the expressions of proteins related to pro-apoptosis, ECM catabolism, and inflammation in vitro. Mechanically, GK reduced the expression of nucleotide binding oligomeric domain like receptor protein 3 (NLRP3) inflammasome-related proteins. Overexpression of NLRP3 reversed the effect of GK on the proliferation, apoptosis, inflammation, and ECM degradation in IL-1β-induced NPCs. Moreover, GK attenuated the pathological manifestations, inflammation, ECM degradation, and NLRP3 inflammasome expression in IDD rats. Conclusion GK suppressed apoptosis, inflammation, and ECM degradation to alleviate IDD via the inactivation of NLRP3 inflammasome.


Introduction
Low back pain (LBP) is a common degenerative syndrome of the musculoskeletal system with high morbidity and disability rates. LBP has become a significant medical problem and loaded a huge economic burden worldwide (Chou 2021;Hartvigsen et al. 2018;Kyu et al. 2018). Over 80% of adults experience from LBP at some point during life, and approximately 10% of these suffers will advance chronic disability (Ma et al. 2019). The etiology of

Animals
Male Sprague-Dawley rats (four or eight weeks-old) were provided by Vital River (Beijing, China) and acclimated to the standard laboratory conditions for one week. Rats were freely supplied with rodent chow and water with a 12 hours/12 hours light-dark cycle and 40%-60% the relative humidity at (25 ± 2) °C. All the procedures were strictly based on the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the Animal Research Ethics Committee of the First Affiliated Hospital of XiaMen University (Approval number: XMYY-2020KY013).

Isolation and culture of NPCs
The NPCs were isolated according to the previous description (Bai et al. 2022). In brief, NP tissues were separated from lumbar IVD of SD rats with 4 weeks old, and then were cut into pieces with the size of 1 mm 3 . Pieces were incubated with 0.25% trypsin and type II collagenase at 37°C for 4 h. Subsequently, the mixture was filtered through a 200-mesh strainer and centrifuged to harvest NPCs. Cells were cultured in DMEM/F12 medium (PM150310A, Procell, Wuhan, China) with 10% fetal bovine serum (FBS, 164210, Procell) at 37°C with 5% CO 2 . NPCs used in the subsequent assays were the second passage.

Cell counting kit-8 (CCK-8) assay
NPCs were plated into 96-well plates with 1 × 10 4 cells per well. After the different treatments as describe above, 10 µl CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added into each well and cultured for 2 h at 37°C. The absorbance was read at 450 nm with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Flow cytometry
NPCs were sowed into 6-well plates with an inoculation density of 1 × 10 6 cells/well, and administrated with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) (CA1020, Solarbio) in the dark for 30 min. The apoptosis of NPCs was determined on a FACScan flow cytometry with the CellQuest software (BD Biosciences, NJ, USA). The apoptosis rate was quantified by the summation of the ratio in quadrants 2 (Q2, top right) and Q3 (low right).

Real-time quantitative PCR (RT-Qpcr)
Total RNA was extracted from NPCs using TRIzol reagent (15596026, Thermo Fisher Scientific). Total RNA (1 μg) was reversely transcribed into cDNA with a PrimeScript RT reagent Kit (RR047A, Takara, Dalian, China) based on the operating manual. RT-qPCR was conducted by using TB Green TM Premix Ex TaqTM II (Tli RNaseH Plus) (RR820A, Takara) on the A PIKORed 96 (Thermo Fisher Scientific). The PCR amplification conditions were 94°C for 10 min, 94°C for 10 s and 58°C for 45 s for 40 cycles. GAPDH was acted as the internal reference. The expressions of genes were calculated with the comparative threshold cycle method (2 −△△CT method), in which ΔΔCT = ΔCT treatment -ΔCT control and ΔCT = Ct target -Ct reference . Primer sequences were listed in Table 1.

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of TNF-α and IL-6 in sera or culture supernatants were measured by using Rat IL-6 ELISA KIT (SEKR-0005, Solarbio) and Rat TNF-α ELISA KIT (SEKR-0009, Solarbio) based on the operation instruction. The absorbance was determined at 450 nm using a microplate reader (Thermo Fisher Scientific).

Animal model of IDD
SD rats with 8-weeks-old were constructed an IDD model in vivo by a fibrous ring puncture approach (Bai et al. 2022;Jeong et al. 2010). Briefly, the rat tail disc (Co4-5) was located on the coccygeal vertebrae. A puncture needle (26 G) pierced the entire annulus of the fibre via the skin of the tail, and held in the disc for 60 s. Eight rats were contained in each group based on the previous study (Bai et al. 2022). However, after modeling, some rats developed complications such as infections and arrhythmias, so only six rats were included in each group. Rats were randomly divided into sham, IDD and IDD+GK (n = 6). Rats in IDD and IDD+GK group were modeled as the above description, and rats in sham group were not treated. Rats in IDD+GK group were immediately intraperitoneally administrated with 100 mg/kg GK (Pan et al. 2019) once a day after the surgery, while rats in sham and IDD group were intraperitoneally received with the same volume of saline. Following the treatments for eight weeks, rats were sacrificed with the intraperitoneal administration of sodium pentobarbital (100 mg/kg). The tail venous blood was collected, and tails and tail disc samples were also harvested for the following assays.

Histopathologic staining
Tail disc samples were fixed in 10% neutral-buffered formalin (G2161, Solarbio) overnight and treated with gradient alcohol for the dehydration, xylene for the clearance, and paraffin for embeddedness. Paraffin sections (5 µm) were treated with hematoxylin and eosin (H&E) and safranine O staining. The images were captured by a light microscopy (Olympus, Tokyo, Japan) and determined with the Image-Pro Plus 6.0 software (Media Cybernetics, USA). The histologic scores were quantified by a grading scale according to the manifestations of morphology of the nucleus pulposus, cellularity of the nucleus pulposus, morphology of anular fibrosus, cellularity of the anular fibrosus and endplates. The histological score of normal was 5, moderate degeneration was 6 to 11, and severe degeneration was 12 to 14 (Mao et al. 2011).

Statistical analysis
All the cell experiments were repeated for three times, and all the animal experiments were repeated for five times. All results were shown as mean ± standard deviation (SD). Data were determined by the SPSS 20.0 software (IBM, Armonk, New York, USA). Statistical differences were analyzed with one-way analysis of variance (ANOVA) followed by LSD post hoc Bonferroni test. P < .05 was defined as significant difference.

GK increased proliferation and inhibited apoptosis in IL-1β-induced NPCs
To investigate the effect of GK (Figure 1a) in the progression of IDD, NPCs were incubated with various concentrations of GK (2.5, 5, 10, 20, 40, and 80 μM) to detect the toxicity of GK on the NPCs. As shown in Figure 1b, no influence of GK varied from 2.5 to 40 μM was found on the cell viability of NPCs, while 80 μM of GK significantly decreased the cell viability of NPCs. Thus, GK varied from 2.5 to 40 μM was further used to explore the effect of GK on IL-1β-induced NPCs. IL-1β stimulation prominently reduced the cell viability of NPCs, which was notably rescued with treatment of GK varied from 5 to 40 μM ( Figure 1c). Therefore, three different concentrations of GK, including 10, 20, and 40 μM GK, were chosen for the subsequent assays, and represented as low, middle and high concentration of GK, respectively. Administration of GK (10, 20 and 40 μM) observably counteracted the IL-1β-induced apoptosis rate of NPCs (Figure 1d,e). Besides, administration of GK (10, 20, and 40 μM) markedly neutralized IL-1β-induced the relative protein expression of cleaved caspase 3 and bax, while restored IL-1β-induced the relative protein level of bcl-2 of NPCs ( Figure 1f). Collectively, GK promoted proliferation and reduced apoptosis in IL-1βinduced NPCs.

GK reduced the ECM degradation in IL-1β-induced NPCs
To investigate the effect of GK on the ECM degradation, the transcriptional and translational expressions of ADAMTS5, MMP13, Collagen II and Aggrecan were examined in IL-1β-induced NPCs. The relative mRNA and protein levels of ADAMTS5 and MMP13 were observably upregulated, but the level of Collagen II and Aggrecan were markedly decreased in IL-1β-treated NPCs (Figure 2a,b). However, treatment of GK (10, 20, and 40 μM) prominently reversed these alterations in IL-1β-induced NPCs (Figure 2a,b). Thus, GK dampened the ECM degradation in IL-1β-induced NPCs.

GK attenuated inflammation in IL-1β-induced NPCs
Furthermore, the concentrations of IL-6 and TNF-α in cell supernatants and the relative protein levels of IL-6 and TNF-α in NPCs were significantly increased in IL-1β-treated NPCs (Figure 3a-c). Administration of GK (10, 20, and 40 μM) markedly reduced the concentrations and relative protein expressions of IL-6 and TNF-α in IL-1β-induced NPCs (Figure 3a-c). These results indicated that GK inhibited inflammation in IL-1β-induced NPCs.

GK suppressed apoptosis, ECM degradation, and inflammation of NPCs via the inactivation of NLRP3 inflammasome
As figured in Figure 4a, the relative protein expression levels of NLRP3, cleaved caspase 1/pro-caspase 1 and cleaved IL-1β/pro-IL-1β were markedly increased in IL-1β-induced NPCs, which were significantly neutralized with the treatment of GK (10, 20 and 40 μM). Moreover, the overexpression of NLRP3 (Figure 4b) prominently reduced GK-induced cell viability of IL-1β-treated NPCs (Figure 4c), and inverse results were indicated in the apoptosis rate ( Figure 4d). Besides, overexpression of NLRP3 significantly enhanced the GK-induced the relative protein levels of NLRP3, cleaved caspase 1/pro-caspase 1 and cleaved IL-1β/pro-IL-1β in IL-1β- induced NPCs (Figure 4e). In addition, the overexpression of NLRP3 markedly increased the GK-induced the relative levels of ADAMTS5 and MMP13 proteins, while diminished the GK-induced the relative protein expressions of Collagen II and Aggrecan in IL-1β-induced NPCs (Figure 4f,g). Furthermore, GK significantly reduced the IL-1β-induced concentrations of IL-6 and TNF-α in cell supernatants, which were recovered with the NLRP3 overexpression ( Figure 4h). These outcomes revealed that GK restrained apoptosis, ECM degradation, and inflammation of NPCs through the inactivation of NLRP3 inflammasome.

GK relieved IDD in rats
More importantly, the effect of GK on IDD was investigated in a rat model. Pathological results showed that the NPCs numbers were decreased with the disordered and ruptured arrangement of annulus fibrous in IDD rats compared with the sham rats, which were distinctly alleviated with the GK injection ( Figure 5a). Correspondingly, administration of GK significantly declined the histological score of IDD rats (Figure 5a). Treatment of GK prominently decreased the serum concentrations of IL-6 and TNF-α in IDD rats ( Figure 5b). Additionally, injection of GK markedly decreased the relative expressions of ADAMTS5 and MMP13 proteins, and increased the relative expression of Collagen II and Aggrecan proteins in IDD rats (Figure 5c). Also, the IHC results showed that the level of NLRP3 was significantly enhanced in IDD rats, which was markedly offset by the GK administration ( Figure 5d). Besides, the relative protein levels of NLRP3, cleaved caspase 1/ pro-caspase 1 and cleaved IL-1β/pro-IL-1β were elevated in IDD rats, which were markedly counteracted with the GK administration ( Figure 5e). Thus, GK alleviated IDD in rats with suppression of inflammation, ECM degradation, and the expression of NLRP3 inflammasome.

Discussion
Pathogenesis, such as apoptosis, the ECM synthesis and catabolism and inflammation, has been demonstrated to be strongly associated with the progression of IDD (Alpantaki et al. Figure 5. GK ameliorated IDD in rats. SD rats were constructed an IDD model using a fibrous ring puncture method in vivo. 100 mg/kg GK was intraperitoneally administrated into IDD rats once a day after the operation. Following the treatments for eight weeks, rats were sacrificed, the tail venous blood, tails and tail disc samples were also harvested. (a) Tail disc samples were subjected to H&E and safranine O staining and the histological grading scale was scored. Black arrows in H&E staining indicated the location of the lesion, and black arrows in safranine O staining indicated the typical areas of cartilage. (b) the serum concentrations of IL-6 and TNF-α were measured by ELISA. (c) the relative protein expressions of ADAMTS5, MMP13, collagen II and aggrecan were examined by western blot. The data were normalized with GAPDH. (d) the level of NLRP3 was quantified by IHC. Black arrows indicated the positive staining. (E) the relative protein expressions of NLRP3, cleaved IL-1β, pro-IL-1β and cleaved caspase 1 and pro-caspase 1 were determined by western blot. The data were normalized with GAPDH. **P < .01 vs. sham; #P < .05 vs. IDD.

2019
). GK is reported to alleviate myocardial ischemia-reperfusion injury by suppressing apoptosis and inflammation on myocardial ischemia-reperfusion injury, and inhibit inflammation and ECM deposition on high glucose-induced mesangial cell (Wei et al. 2021). However, whether GK mitigates IDD by attenuating apoptosis, ECM degradation, and inflammation is unknown. The models of IDD were constructed in NPCs stimulated with IL-1β and in rats through a fibrous ring puncture method in vitro and in vivo, respectively. GK promoted proliferation, and attenuated apoptosis, inflammation, and ECM degradation in IL-1β-induced NPCs. Mechanically, GK reduced the relative protein expression levels of NLRP3, cleaved caspase 1/pro-caspase 1 and cleaved IL-1β/pro-IL-1β in IL-1β-induced NPCs. Overexpression of NLRP3 significantly inverted the effect of GK in the proliferation, apoptosis, ECM degradation and inflammation in IL-1β-induced NPCs. Moreover, similar effect of GK was also confirmed in IDD rats. Taken together, GK suppressed apoptosis, inflammation, and ECM degradation to alleviate IDD via the inactivation of NLRP3 inflammasome. NPCs apoptosis is a pivotal pathological basis of IDD that participates in the development of IDD (Zhao et al. 2017). An increase in TUNEL-positive NPCs is observed in patients with degenerative discs compared with the healthy discs (Gruber and Hanley 1998). Bai et al. (2022) has shown an upregulation of cleaved caspase 3 and bax with a downregulation of bcl-2 in IL-1β-induced NPCs, as well as an enhancement of TUNELpositive cells in IDD rats. Along with these findings, our results consistently revealed that IL-1β incubation promoted apoptosis of NPCs, as indicated by an increase in the cleaved caspase 3 and bax expression and a diminishment in the bcl-2 level. Moreover, apoptosis can lead to the loss of NPCs numbers (Zhao et al. 2017) and NPCs depletion is the initiation and booster of the IDD progression (Vergroesen et al. 2015). Thus, a prominent decease in cell viability was discovered in IL-1β-induced NPCs in the current study. However, treatment of GK reversed these outcomes in IL-1β-induced NPCs. The pro-survival and antiapoptotic activities of GK have been demonstrated in a variety of disease models, such as Parkinson's disease  cerebral ischemia-reperfusion injury (Pan et al. 2019;Tian et al. 2019) and myocardial ischemia-reperfusion injury . Altogether, these results expounded that GK promoted proliferation and inhibited apoptosis in IDD.
The synthesis and catabolism of ECM are another significant pathogenesis involved in the progression of IDD, in which ECM degradation always occurs, thereby resulting in the dysregulation of the synthesis and catabolism of the ECM (Tao et al. 2016). ECM that mainly comprises of proteoglycans and collagen is highly organized in IVD, which is momentous to maintain the proper spine mechanics (Colombini et al. 2008). Collagen II and aggrecan, the primary ingredients of NP ECM, serve crucial roles in maintaining the osmotic pressure and elasticity of the disc . ADAMTS5 and MMP13 are proteolytic enzymes that play central roles in controlling the ECM catabolism . Here, the relative mRNA and protein expressions of ADAMTS5 and MMP13 were enhanced, and these of Collagen II and Aggrecan were downregulated in IL-1β-induced NPCs, which indicated an imbalance in the ECM synthesis and catabolism in IL-1β-induced NPCs, in line with the results shown in a recent report (Bai et al. 2022). Administration of GK reversed the IL-1β-induced the mRNA and protein expressions of ADAMTS5, MMP13, Collagen II and Aggrecan of NPCs, which demonstrated that GK attenuated the ECM degradation in IL-1β-induced NPCs. More importantly, GK also inverted the translational expressions of ADAMTS5, MMP13, Collagen II and Aggrecan of NPCs in IDD rats. Wei et al. (2021) revealed that GK dampens ECM deposition in high glucose-elicited mesangial cell. Therefore, all the results elucidated that GK restrained the ECM degradation in IDD.
Plenty of studies have revealed that inflammation is a pivotal inducer and contributor for the IVDD pathological development (Fu et al. 2020;Lin and Lin 2020;Ruiz-Fernández et al. 2019). Inflammatory regulators, including IL-1β and TNF-α, are upregulated in degenerative discs of humans and mammalian animals compared with the normal discs (Le Maitre et al. 2007). Thus, these inflammatory regulators are generally positively correlative with the pathological processes of IDD (Weiler et al. 2005). A series of agents that target to inflammation have been demonstrated to alleviate IDD, such as cardamonin (Xie et al. 2021) melatonin  and notoginsenoside R1 (Tang et al. 2021). Furthermore, the anti-inflammatory effect of GK has been also shown on skin inflammation (Kwak et al. 2002) cerebral ischemia/reperfusion injury ) myocardial ischemiareperfusion injury Zhang et al. 2018) diabetic nephropathy (Wei et al. 2021) and neuroinflammation Zeng et al. 2016). In the current study, GK reduced the concentrations and the relative protein expressions of IL-6 and TNF-α in IL-1β-induced NPCs, as well as the serum levels of IL-6 and TNF-α in IDD rats, which indicated that GK inhibited inflammation in IDD.
It is shown that NLRP3 inflammasome is widely activated in the process of IDD (Chao-Yang et al. 2021). The activation of NLRP3 inflammasome activates caspase 1, which results in the cleavages of pro-IL-1β and pro-IL-18 into active IL-1β and IL-18 to promote the inflammatory response (Guo et al. 2015). These inflammatory mediators further contribute to the development of IDD (Bai et al. 2020;Chen et al. 2015). It has been substantiated that the levels of NLRP3 and the downstream targets, including caspase-1 and IL-1β, are positively relevant in Pfirrmann IVDD grade (Chen et al. 2015). Our results illustrated that the levels of NLRP3, cleaved caspase 1/pro-caspase , and cleaved IL-1β/pro-IL-1β were enhanced both in IL-1β-induced NPCs and in IDD rats, which suggested that NLRP3 inflammasome was activated in IDD. GK decreased these promotions both in vitro and in vivo, which indicated that GK inactivated NLRP3 inflammasome in IDD. Moreover, the overexpression of NLRP3 significantly enhanced the GK-induced the relative protein levels of NLRP3, cleaved caspase 1/pro-caspase 1 and cleaved IL-1β/pro-IL-1β in IL-1β-induced NPCs, demonstrating that the overexpression of NLRP3 neutralized the effect of GK in inactivation of NLRP3 inflammasome in IL-1β-induced NPCs. The overexpression of NLRP3 counteracted the suppressive effect of GK on apoptosis, inflammation and ECM degradation in IL-1β-induced NPCs. Therefore, we concluded that it was the overexpression of NLRP3 that resulted in the activation of NLRP3 inflammasome, not the overexpression of NLRP itself, overrode the suppressive effect of GK on the apoptosis, ECM degradation and inflammation of NPCs. A review has summarized that activated NLRP3 inflammasome contributes to inflammation, ECM degradation, and apoptosis, thereby promoting the development of IDD (Chao-Yang et al. 2021). Collectively, GK dampened apoptosis, ECM degradation, and inflammation of NPCs via the inactivation of NLRP3 inflammasome.
In conclusion, our results elaborated that GK inhibited apoptosis, ECM degradation, and inflammation of NPCs, which was strongly associated with the inactivation of NLRP3 inflammasome. Nevertheless, several limitations need to be addressed in the following study. Except for apoptosis, ECM degradation, and inflammation, other pathogenesis, such as autophagy, oxidative stress and ferroptosis, are also closely involved in the progression of IDD. Thus, the effect of GK on the other pathogenesis can be explored in the subsequent studies. In addition, the direct role of NLRP3 inflammasome should be investigated in IDD rats to solidify our conclusion in the following assays. Moreover, further preclinical and clinical trials are still needed in the following study, which can promote the clinical translation and application. Briefly, our results identify an alternative agent for the IDD therapy and contribute to the development of the IDD treatment.

Disclosure statement
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Funding
The author(s) reported there is no funding associated with the work featured in this article.