Yun Teng,Yixue Huang,Hao Yu,Cenhao Wu,Qi Yan,Yingjie Wang,Ming Yang,Haifeng Xie,Tianyi Wu,Huilin Yang,Jun Zou
Nimbolide targeting SIRT1 mitigates intervertebral disc degeneration by reprogramming cholesterol metabolism and inhibiting inflammatory signaling
Nimbolide,SIRT1,Intervertebral disc degeneration,Nucleus pulposus,Macrophage polarization,Cholesterol
Inflammation, abnormal cholesterol metabolism, and macrophage infiltration are involved in the destruction of the extracellular matrix of the nucleus pulposus (NP), culminating in intervertebral disc degeneration (IDD). Whether nimbolide (Nim), a natural extract, can alleviate IDD is unclear. In this study, we demonstrated that Nim promotes cholesterol efflux and inhibits the activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by activating sirtuin 1 (SIRT1) in nucleus pulposus cells (NPCs) during inflammation. Thus, Nim balanced matrix anabolism and catabolism of NPCs. However, the inhibition of SIRT1 significantly attenuated the effects of Nim. We also found that Nim promoted the expression of SIRT1 in RAW 264.7, which enhanced the proportion of M2 macrophages by facilitating cholesterol homeostasis reprogramming and impeded M1-like macrophages polarization by blocking the activation of inflammatory signaling. Based on these results, Nim can improve the microenvironment and facilitate matrix metabolism equilibrium in NPCs. Furthermore, in vivo treatment with Nim delayed IDD progression by boosting SIRT1 expression, modulating macrophage polarization and preserving the extracellular matrix. In conclusion, Nim may represent a novel therapeutic strategy for treating IDD.
Nimbolide targeting SIRT1 mitigates intervertebral disc degeneration by reprogramming cholesterol metabolism and inhibiting inflammatory signaling

Inflammation, abnormal cholesterol metabolism, and macrophage infiltration are involved in the destruction of the extracellular matrix of the nucleus pulposus (NP), culminating in intervertebral disc degeneration (IDD). Whether nimbolide (Nim), a natural extract, can alleviate IDD is unclear. In this study, we demonstrated that Nim promotes cholesterol efflux and inhibits the activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by activating sirtuin 1 (SIRT1) in nucleus pulposus cells (NPCs) during inflammation. Thus, Nim balanced matrix anabolism and catabolism of NPCs. However, the inhibition of SIRT1 significantly attenuated the effects of Nim. We also found that Nim promoted the expression of SIRT1 in RAW 264.7, which enhanced the proportion of M2 macrophages by facilitating cholesterol homeostasis reprogramming and impeded M1-like macrophages polarization by blocking the activation of inflammatory signaling. Based on these results, Nim can improve the microenvironment and facilitate matrix metabolism equilibrium in NPCs. Furthermore, in vivo treatment with Nim delayed IDD progression by boosting SIRT1 expression, modulating macrophage polarization and preserving the extracellular matrix. In conclusion, Nim may represent a novel therapeutic strategy for treating IDD.

Lower back pain (LBP) is a common clinical condition. Intervertebral disc degeneration (IDD) is the leading contributor to LBP etiology, accounting for at least 40% of all LBP cases. IDD has become one of the top three causes of disability in developed countries and causes a heavy socioeconomic burden, the number of affected people is increasing worldwide. Currently, conservative, and surgical treatments are mainly adopted for patients with LBP caused by IDD. Conservative treatment includes medication, massage, physiotherapy, and bed rest. Surgical treatment includes open and minimally invasive procedures such as discectomy and/or spinal fusion,. However, the pathophysiological process of IDD cannot be interfered with using either approach, which aims to relieve pain or other symptoms. Biological therapy for IDD has been introduced as a treatment method that may fundamentally solve this problem. The intervertebral disc (IVD), which is mainly composed of the nucleus pulposus (NP) and annulus fibrosus (AF), is an important component of the vertebral structure,. Its physiological functions mainly include participation in spinal flexion, extension, and rotation, maintaining the height of the intervertebral space, and bearing the load and dispersing stress,. The IVD is thought to originate from the NP. The extracellular matrix (ECM) of NP made up of collagen II and aggrecan, decreases during IDD. IDD is always accompanied by upregulation of metalloproteinases (MMPs), such as MMP3 and MMP13, which are thought to be responsible for ECM breakdown. Although the exact pathogenesis of IDD is not fully understood. Inflammation has always been an important factor that induces degradation of the ECM in IVD and accelerates IDD. The nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways are closely related to the release of inflammatory cytokines, and their activation promotes the translation and transcription of MMPs or a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), which aggravate IDD. Therefore, inflammatory signaling remains a crucial target for IDD treatment13, 14, 15. Cholesterol is a small lipid molecule containing a cyclopentanone drophenanthrene skeleton, which participates in significant physiological activities in the body. Recent studies have also reported that elevated levels of total cholesterol (TC) and low-density lipoprotein (LDL) in the serum increase the risk of developing IDD. Apolipoprotein E (ApoE) is involved in the transformation and metabolism of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL). ApoE-knockout rabbits exhibit symptoms of elevated cholesterol levels, atherosclerosis, and IDD. The nucleus pulposus cells (NPCs) extracted from these rabbits overexpress inflammatory cytokines and catabolic factors that damage the ECM. Yan et al. demonstrated that cholesterol causes pyroptosis in NPCs, suggesting that cholesterol may promote inflammation. Therefore, the role of cholesterol in IDD development cannot be ignored. Macrophages, a type of immune cell, have been identified in IVD. Recent studies have reported that different phenotypes of macrophages through polarization are involved in the pathological changes in IDD,. Some cytokines and other proteins from M2-like macrophages contribute to repairing the ECM in IDD, and these cytokines from M1-like macrophages worsen ECM loss. However, the transformation of M1 macrophages inevitably requires the activation of inflammatory pathways, such as MAPK and NF-κB. Cholesterol metabolism has also been proven to be a participant in macrophages polarization especially M2-like macrophagaes. Therefore, inhibition of inflammatory signals and regulation of cholesterol metabolism also helps to adjust the ratio of M1 to M2 macrophages to improve the NP environment. Nimbolide (Nim) extracted from the neem tree (Azadirachta indica) is a limonoid belonging to a class of highly oxidized tetranortriterpenoids. Nim has been reported to have various biological functions, including antibacterial, antitumor, antidiabetic, antirheumatic arthritis, etc.26, 27, 28. Among them, the antitumor effect of Nim is particularly prominent. Nim has almost no side effects and toxicity to the human body while exerting its chemopreventive tumor effect or inhibiting tumor invasion and angiogenesis. In addition, Nim can inhibit inflammation and reduce the accumulation of cholesterol in cells,. However, no study has confirmed whether Nim can protect NP or how Nim exerts these effects. Therefore, in this study, we explored whether Nim can inhibit inflammatory signals and improve cholesterol metabolism in NPCs and macrophages by promoting the activation of sirtuin 1 (SIRT1) and enhancing its expression. Nim may directly restore matrix metabolism damaged by inflammation and cholesterol accumulation in NPCs. However, it may also effectively prevent the polarization of M1-phenotype macrophages and boost M2-phenotype macrophages, creating a microenvironment beneficial to the secretion of the ECM. Therefore, Nim may be a candidate drug for the treatment of IDD.

RAW 264.7 macrophages (CL-0190) and mouse nucleus pulposus cells (mNPCs) (CP-M146) were purchased from Procell (Wuhan, China). mNPCs were cultured in specialty medium at 37 °C with 5% CO2. LPS (1 μg/mL) was used for the mNPCs degeneration model. Complete Dulbecco's modified Eagle's medium (DMEM) (100 U/mL penicillin, 100 mg/mL streptomycin and 10% FBS) was used to culture RAW 264.7 macrophages. LPS (100 ng/mL) and interleukin-4 (IL-4) (20 ng/mL) were applied to induce M1-like and M2-like macrophage polarization. EX-527 (10 μmol/L) were used to inhibit the activity of SIRT1. When RAW 264.7 macrophage density reached 60%, LPS, IL-4 were added to the culture medium after macrophages were pretreated with or without Nim due to different groups. After 24 h, the medium containing LPS, IL-4 or Nim (5 μmol/L) was discarded and the macrophages were treated with the culture medium for another 24 h, which was collected after centrifugation at 1000 × g and was considered as macrophage culture medium (CM). The NPCs under normal or LPS-stimulation condition were cultured in mixed medium (50% specialty medium and 50% CM from different groups of macrophages) for further experiments.

The two-dimensional structure of Nim was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and then the three-dimensional structure of Nim was drawn using ChemBio 3D to optimize the conformation. The crystal structure of SIRT1 (PDB ID: 5BTR, 4I5I) were downloaded from Protein Data Bank (https://www.rcsb.org/) and were pretreated by the Protein Preparation Wizard module in Schrödinger software. Receptor Grid Generation program was used for setting the most suitable Enclosing box to perfectly wrap the natural ligand structure, and obtain the active pocket of the protein on this basis. The molecular docking between ligand and receptor were underwent in Glide module and the molecular mechanics generalized Born surface area (MM/GBSA) were calculated. The binding sites were visualized by PyMOL.

The RAW 264.7 macrophages and NPCs were lysed in Trizol (Invitrogen, CA, USA). Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reverse Transcription Kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer's instructions. SYBR Green Master Mix reagent (Vazyme, Nanjing, China) combined with the forward and reverse primers (Supporting Information Table S1) were used for performing RT-qPCR to measure the mRNA expression of genes. The Gapdh gene was used to normalize other genes, and the values were calculated using the 2–ΔΔCt method.

All experimental procedures were approved by the Laboratory Animal Center of Soochow University (202007A039). Briefly, 20 male Sprague–Dawley (SD) rats (400 ± 20 g) were randomly divided into four groups: sham surgery (sham), Nim injection (Nim), saline injection (saline), and degeneration. Each group included five rats. The Co9/10 vertebral gaps were positioned using radiography. The rats were anesthetized with Zoletil 50 (50 mg/kg) and xylazine hydrochloride (3 mg/kg). Then, the Co9/10 IVDs were punctured using 20G injection needles in the Nim, saline, and degeneration groups and punctured using 33G injection needles in the sham group. The needle tips were inserted to the tail, then rotated by 180° and still for 30 s. The degenerated group was punctured without any injection. The Nim and saline groups were injected with 2 μL Nim (3 mg/mL) or saline to the disc, respectively, using a 33G needle three times a week for 4 weeks.

One-way ANOVA was performed to analyze data from three or more groups, which are presented as mean ± standard deviation (SD). The Kruskal–Wallis test was performed to analyze non-parametric data. Differences were considered statistically significant at P < 0.05. The detailed description of other materials and methods are available in the Supporting Information.

The chemical structure of Nim is shown in the Supporting Information Fig. S1A. The CCK-8 assay demonstrated that Nim had no effect on the proliferation of NPCs at the tested concentrations (2.5, 5 μmol/L) (Fig. S1B). RT-qPCR results show that Nim effectively inhibited mRNA expression of genes related to matrix anabolism (Mmp13, Mmp3, and Adamts5) (Fig. 1A). Western blotting results show that the protein levels of these genes also decreased (Fig. 1B). The influence of inflammation on downregulating the mRNA expression and protein level of genes [Col2a1, Acan, and SRY-related HMG box 9 (Sox9)] involved in matrix synthesis was reversed by Nim (Fig. 1A and B). However, Nim (5 μmol/L) did not directly upregulate or downregulate mRNA expression and protein level of matrix-related genes in normal NPCs. The immunofluorescence assay also reveals that Nim repressed inflammation-induced destruction of matrix metabolism (Fig. S1C). We explored cholesterol metabolism in NPCs and found that inflammation accelerated the accumulation of intracellular cholesterol according to Filipin III staining and the cholesterol kit (Fig. 1C and D). Accumulation of cholesterol may also exacerbate imbalances in matrix metabolism (Supporting Information Fig. S2A and S2B). Nim enhanced the transcription of ATP-binding cassette transporter A1 (Abca1) and ATP-binding cassette transporter G1 (Abcg1), and increased their protein levels which are linked to cholesterol efflux (Fig. 1E and F). Additionally, we detected the activation of inflammatory signals by Western blot analysis. The results show that Nim blocked the phosphorylation of P65 and IκBα, which are activated by LPS in NPCs. Meanwhile, the phosphorylation levels of extracellular regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and P38 in the MAPK pathway were decreased by Nim in a dose-dependent manner (Fig. 1F). Because of the blockade of inflammatory pathways, the expression of the inflammatory indicator cyclooxygenase-2 (COX2) and nitric oxide synthase (iNOS) also decreased in NPCs (Fig. 1F). Immunofluorescence analysis clearly show that Nim delayed LPS-induced translocation of P65 into the nuclei of NPCs and reduced iNOS expression (Fig. 1G).

Previous study reminded us that SIRT1 may be a candidate target for Nim. So, we performed molecular docking to predict the affinity between Nim and SIRT1. Molecular docking results show the docking of Nim with two crystal structures of SIRT1 and that Nim was more tightly bound to the crystal structure 5BTR (−47.72 kcal/mol) than 4I5I (−9.26 kcal/mol) (Fig. 2A, Supporting Information Fig. S3A). The XP GScore and MM-GBSA based energy between Nim or native ligand and 5BTR or 4I5I were listed in Supporting Information Table S2. Nim forms a hydrogen bond with residues THR209 and ASN226 of SIRT1 protein (5BTR), respectively (Fig. 2A). Based on this, Nim could promote the activation of SIRT1 (Fig. S3B). At the same time, we found that Nim could enhance the transcription of Sirt1 in NPCs (Fig. S3C). The protein level of SIRT1 in NPCs under inflammation increased according to the Western blotting and immunofluorescence results (Fig. 2B and C). We used the SIRT1-siRNA (siSIRT1) to silence its expression in NPCs and NC-siRNA (siNC) was used as control (Fig. S3D). We observed that the protective effects of Nim on the NPCs were significantly decreased, including the balance of matrix metabolism, the promotion of cholesterol efflux, and the inhibition of inflammatory pathways (Fig. 2D). Similarly, inhibition of SIRT1 activity with EX-527 had the same effect (Fig. 2E).

We observed that in RAW 264.7 macrophages, Nim upregulated the mRNA expression of genes related to the M2 phenotype, such as arginase 1 (Arg1), resistin-like molecule α (Fizz1), Cd206, and interleukin-10 (Il10), and increased the secretion of IL-10 (Fig. 3A and B). CD206-positive macrophages were significantly increased in the Nim group compared to those in IL-4 group according to flow cytometric analysis (Fig. 3C). We found that Nim significantly enhanced the protein level of SIRT1 in macrophages induced by IL-4 (Supporting Information Fig. S4A). Benefitting from this, Nim significantly promoted the reprogramming of cholesterol homeostasis induced by IL-4, especially increased the mRNA expression of Abca1, Abcg1 and cholesterol 25-hydroxylase (Ch25h) (Fig. S4B). Fluorescence microscopy show that stimuli from Nim significantly lowered the fluorescence intensity of filipin III staining in cells (Fig. S4C). We also found that Nim stimulation significantly reduced the intracellular cholesterol content (Fig. S4D). When macrophages were treated with EX-527, IL-4 and Nim, it was found that the nimbolide-promoting effect had been fully reversed (Fig. S4E). At the same time, RT-qPCR results show that Nim reduced the transcription of M1-phenotype associated genes, such as interleukin-1β (Il1b), tumor necrosis factor-α (Tnfa), interleukin-6 (Il6), and inducible nitric oxide synthase (Nos2) (Fig. 3D). The secretion of TNF-α and IL-6 in the medium also decreased in the Nim group (Fig. 3E). RAW 264.7 macrophages stained with CD86 and CD206 were counted using flow cytometry, and the results suggest that Nim restricted LPS-stimulated M1 macrophage polarization and increased M2 macrophage polarization, indicating that Nim promoted the alteration of the M1 to M2 phenotype (Fig. 3F). Similarly, SIRT1 levels in LPS-stimulated macrophages were also increased by Nim (Supporting Information Fig. S5A). Western blotting results show that Nim also decreased p-P65, p-IκBα, p-ERK1/2, p-JNK, and p-P38 expressions in RAW 264.7, indicating that Nim may inhibit inflammatory signaling in macrophages (Fig. S5B). Owing to the blockade of inflammatory signaling, iNOS and COX-2 levels also decreased in M1-like macrophages (Fig. S5B). Additionally, immunofluorescence results illustrate that Nim reduced the translocation of P65 into the nuclei of macrophages (Fig. S5C). However, when macrophages treated with LPS and Nim were pretreated with EX-527, the effect of Nim was also significantly restricted (Fig. S5D).

To identify how macrophages with different polarized states influence NPCs, we collected RAW 264.7 macrophage conditioned medium exposed to various treatments, including CMM0 (Raw 264.7 with no treatment), CMLPS, CMI-4, CMLPS+Nim, and CMI-4+Nim. We then treated NPCs with different macrophage media in normal or inflammatory (LPS) environment to detect the expression of genes and proteins associated with ECM synthesis and degradation. Under normal conditions, CMM0, CMI-4, and CMI-4+Nim exhibited no clear effect on the matrix synthesis of NPCs. However, CMLPS significantly impaired the matrix metabolism of NPCs by increasing the expressions of degraded enzymes-related genes and decreasing the matrix synthesis-related genes. Compared to the CMLPS group, the destructive effect of CMLPS+Nim on the ECM was significantly weakened (Fig. 4A and B). Nevertheless, in the inflammatory environment, compared with the CTRL group, the mRNA expressions of Col2a1, Sox9, and Acan in NPCs were upregulated by co-culturing with CMI-4 and CMI-4+Nim, which were further downregulated in the CMLPS and CMLPS+Nim groups (Fig. 4D). Western blotting results suggest that the protein levels of these genes were keeping with their mRNA expressions (Fig. 4E). In contrast, CMI-4 and CMI-4+Nim remarkably downregulated the transcription of Mmp13, Mmp13, and Adamts5 and protein levels of MMP13, MMP3 and ADAMTS5 compared to the CTRL group, and treatment of NPCs with CMLPS and CMLPS+Nim markedly elevated mRNA expressions and protein levels of these genes (Fig. 4D and E). Under inflammatory conditions, CMI-4+Nim was better at facilitating matrix synthesis in NPCs than CMI-4, and CMLPS+Nim did less damage to the ECM than CMLPS did (Fig. 4D and E). The immunofluorescence results show COLII and MMP13 expression in different groups, which is consistent with the Western blotting results (Fig. 4C and F).

To evaluate the therapeutic effect of Nim in vivo, we constructed a rat caudal IDD model by using acupuncture. This model was divided into degenerative, Nim, saline, and sham groups. The rats were injected intradiscally with 2 μL Nim (3 mg/mL) in the Nim group and 2 μL saline in the saline group three times a week for 4 weeks. The MRI results are shown in Fig. 5A. Degeneration was characterized by darkening (decreasing T2 signal) and narrowing of the intervertebral space in the saline and degeneration groups. The experimental disc in the Nim group showed a stronger T2 signal than those in the saline and degeneration groups. However, no significant statistical difference between Nim and degeneration groups is shown according to the Pfirrmann score in Fig. 5C. In the 4th week, H&E and Safranin O staining showed a clear boundary between the NP and AF in the Nim group, but the number of NPCs and ECM was lower than that in the sham group (Fig. 5B). According to the qualified analysis, the Nim group had a lower degree of degeneration than the saline and degeneration groups. This indicate that Nim had a therapeutic effect in retarding the progression of IDD (Fig. 5D). Immunohistochemical staining results reveal that more SIRT1-positive cells were present in the IVDs of Nim group than these in other three groups (Fig. 5E). Larger amounts of M1-like macrophages (CD86-positive cells) were detected in the IVD in the saline and degeneration groups than in the Nim and sham groups and they exhibited a tendency to infiltrate inward through the AF (Fig. 5E). Additionally, there were more M2-like macrophages (CD206-positive cells) in the Nim group, which were distributed in almost all layers of the AF (Fig. 5E). In contrast, the number of CD206-positive macrophages in the saline and degenerated groups was significantly lower than that in the Nim group (Fig. 5E). Our results indicate that Nim regulated macrophage polarization in vivo. Simultaneously, we observed protection of the ECM of the rat NP by Nim and found that compared with the degenerated group, the group injected with Nim contained fewer MMP13-positive cells and a larger COLII-positive area (Fig. 5E). These results suggest that Nim retarded the progression of IDD in vivo.

In our study, we found that Nim stabilizes NPC function from different aspects. The activation of inflammatory pathways, particularly the NF-κB and MAPK pathways, has an important effect on matrix metabolism,,. NF-κB can enhance the activity of promoters of matrix-degrading enzymes to promote their transcription, whereas NF-κB inhibits the promoters of SOX9 and COL2A1 to inhibit their transcription,. Our study showed that Nim had an excellent inhibitory effect on the NF-κB pathway. Similarly, the MAPK pathway, which includes P38, ERK, and JNK, is involved in the matrix regulation of NPCs and can promote the transcription of ECM degradation genes in the early stage of inflammation,,. Meanwhile, our study found that Nim could significantly inhibit the phosphorylation of P38, ERK, and JNK, which may be a method for the reduction of matrix-degrading enzymes. LPS also interferes with cholesterol metabolism and accumulates cholesterol in the cells. However, few studies have explored the effects of inflammation on cholesterol levels in NPCs. LPS significantly increased the cholesterol content of NPCs. We amplified the damage caused by cholesterol accumulation in NPCs by adding water-soluble cholesterol and found that cholesterol also damaged the matrix metabolism of NPCs. Pyroptosis is an important mechanism through which cholesterol causes cellular inflammation,. Yan et al. discovered that cholesterol can induce pyroptosis in NPC, which increases the secretion of inflammatory factors and damages the ECM in IDD. However, Nim effectively inhibited this effect induced by LPS. Further investigation revealed that Nim effectively increased the expression of ABCA1 and ABCG1, which are important proteins that promote cholesterol efflux, are the first steps in reverse cholesterol transport. In chondrocytes, studies have found that the expression of ABCA1 and ABCG1 in patients with osteoarthritis (OA) is significantly decreased, which hinders cholesterol metabolism. Some miRNAs, such as miR-33, regulate transcription by binding to the non-coding region of ABCA1, and inhibiting the expression of miR-33 can effectively alleviate OA. ABCA1 and ABCG1 also have significant anti-inflammatory effects. Nim may enhance its anti-inflammatory effects on NPCs by increasing the expression of ABCA1 and ABCG1. SIRT1 is a sirtuin that regulates cellular activities by deacetylating substrates. Our further study suggests that SIRT1 is an important target for Nim. We docked Nim with the two crystal structures of SIRT1 and found that Nim had a stronger binding force with SIRT1 (5BTR). However, the binding force to the screening site for SIRT1 (4I5I) inhibitors was extremely weak. We found that Nim can strongly bind to SIRT1 and interact with the active sites THR209 and ASN226 to promote the activation of SIRT1 and enhance its deacetylation activity, which is similar to the binding of resveratrol to SIRT1. In addition, Nim can promote the transcription of SIRT1, thereby increasing the protein level of SIRT1 in NPCs during inflammation. LPS can post-transcriptionally regulate the expression of SIRT1 and decrease SIRT1 protein levels by affecting the expression of intracellular miRNAs such as miR-34a and miR-146a. Nim also probably alters the miRNAs expression in NPCs to upregulate SIRT1 protein levels, which need further investigation. We used siSIRT1 or EX-527 to inhibit SIRT1 and found that both significantly weakened the protective effect of Nim on NPCs. The anti-inflammatory function of SIRT1 has been confirmed by many studies. On the one hand, the anti-inflammatory effect of SIRT1 comes from the direct deacetylation of P65 and c-JUN. On the other hand, it can promote the deacetylation of Peroxisome-proliferator-activated receptorγcoactivator-1α (PGC-1α) and forkhead box O (FOXO), and enhance the mitochondrial biological activity and antioxidant capacity of cells, thereby reducing the activation of inflammatory signals by reactive oxygen species induced by inflammation. At the same time, the regulatory effects of SIRT1 on cholesterol cannot be ignored. It can deacetylate LXRs (liver X receptors), enhance the transcription of downstream ABCA1 and ABCG1, and reduce the accumulation of intracellular cholesterol. Macrophages have been shown to exist as immune cells in IVD. Through flow cytometry and immunofluorescence experiments, we found that Nim significantly promoted the IL-4-induced polarization of M2 macrophages and inhibited the LPS-induced polarization of M1 macrophages. The mechanism by which Nim regulated macrophages polarization was explored in our study, and we found that SIRT1 was indispensable for Nim to regulate macrophage polarization. After pretreatment of macrophages with EX-527, Nim did not significantly activate the M2 phenotype or inhibit M1 phenotype macrophages. Macrophages in lyz2-Cre+/−; SIRT1flox/flox mice tended to express the M1 phenotype, whereas M2 phenotype was significantly attenuated. Undoubtedly, the inhibition of inflammatory pathways by SIRT1 is key to hindering the polarization of M1 macrophages. However, mechanism of SIRT1 regulating M2 macrophage polarization remained unclear. Our study suggests that SIRT1 may indirectly promote the polarization of M2 phenotype macrophages by regulating cholesterol metabolism. We found that cholesterol homeostasis in macrophages was reprogrammed by IL-4 and Nim, especially the expression of genes related to cholesterol efflux (ABCA1 and ABCG1) and genes related to cholesterol oxidation (CH25H), which were elevated. Li et al. observed that Ch25h-25-HC inhibited M1 phenotype macrophage expression while promoting M1 to M2 cell transformation. In addition, by promoting cholesterol efflux, it can also enhance the sensitivity of macrophages to IL-4, thereby promoting the activation of the AKT–STAT6 pathway. To explore the effects of Nim-treated macrophages on NPCs, we collected the supernatant of polarized macrophages and added it to the culture medium of NPCs, a method also used by Zhou et al. and Ni et al.. We found that Nim enhanced the ability of M2 macrophage supernatants to restore NPC function. Simultaneously, Nim attenuated the damage of the M1 macrophage supernatant to the matrix metabolism. Therefore, the influence of a microenvironment composed of macrophages on NPCs should not be ignored. Our experiments demonstrated that it is feasible to improve the function of NPCs by regulating macrophages using Nim. In in vivo experiments, we found that injection of Nim can effectively delay the degeneration of rat intervertebral discs by increasing the expression of SIRT1 in the NP tissue, promoting the transformation of M1 to M2 macrophages, and reducing the loss of NP extracellular matrix. However, our animal experiments had the following shortcomings. On the one hand, we did not inject shSIRT1 lentivirus into the intervertebral disc of rats or use SIRT1 knockout mice to further clarify that the target of Nim is SIRT1. On the other hand, we did not use conditional knockout mice to interfere with macrophage function to confirm that Nim can exert protective effects through macrophages. However, the extremely small size of the mouse intervertebral discs makes local injection difficult. Secondly, the intervertebral disc lacks vascular tissue, and it is difficult for drugs to reach the intervertebral disc through oral or intraperitoneal injection. This also makes it difficult to use a mouse model and this issue need further exploration and solution.

We demonstrated that Nim targeting SIRT1 facilitated matrix metabolism balance in NPCs during inflammation by promoting cholesterol homeostasis reprogramming and inhibiting the NF-κB and MAPK pathways. Additionally, Nim created an immune microenvironment conducive to matrix synthesis of NPCs by modulating macrophage polarization mediated by SIRT1. Therefore, Nim is expected to become a new drug for the treatment of IDD.

We thank the Center of Pharmaceutical Technology of Tsinghua University (Beijing, China) for the support of Schrödinger software and molecular docking. This study was supported by the 10.13039/501100001809National Natural Science Foundation of China (grant numbers 91849114, 82030068, and 82172506).

Jun Zou and Yun Teng designed the experiments. Yun Teng, Yixue Huang, and Hao Yu performed the cellular studies and prepared the manuscript. Yingjie Wang, Ming Yang, Cenhao Wu, and Haifeng Xie performed the data analysis. Qi Yan, Tianyi Wu, Yingjie Wang, Hao Yu, and Yixue Huang built the animal model and were responsible for animal experiments. Yun Teng, Hao Yu, and Jun Zou provided funding support and supervised the experiments.

The authors declare no conflicts of interest.