We obtained 12 NP tissue samples from patients who underwent surgery at the Department of Orthopedics, Lanzhou University Second Hospital, in 2025. During their lumbar IVD surgery, NP tissue was removed and transported to the laboratory. The samples were divided into the following two groups: a mild degeneration group (Grade II), which included NP tissues from three males and three females aged 22–46 years (mean age: 32.9 years), and a severe degeneration group (Grade V), which included NP tissues from three males and three females aged 47–69 years (mean age: 60 years). Exclusion criteria included history of spinal tumors, tuberculosis, or infectious diseases, consistent with previous studies [20].

For rat NP cell isolation, 4–6 weeks old Sprague–Dawley rats were deeply anesthetized via inhalation of 3–4% isoflurane until loss of pedal reflex, followed immediately by cervical dislocation as a secondary physical method to ensure definitive euthanasia [21], in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). NP tissues were harvested from the IVD and digested with 0.5% type II collagenase (BS164; Biosharp, Hefei, Anhui, China) at 37 °C for 2 hours. The resulting cell suspension was cultured into DMEM/F-12 medium (C11330500BT, Gibco, Thermo Fisher Scientific, Suzhou, Jiangsu, China) supplemented with 10% FBS (FSP500, ExCell Bio, Suzhou, Jiangsu, China) and maintained in a humidified incubator at 37 °C with 5% CO₂ atmosphere. Upon reaching 80–90% confluency, cells were subcultured employing 0.25% trypsin–EDTA solution (T1300, Solarbio, Beijing, China). The cultured rat NP cells were validated for the NP cell–specific marker aggrecan by immunofluorescence staining (Supplementary Material) and tested negative for mycoplasma contamination. An oxidative stress environment was established in vitro by exposing the NP cells to the indicated concentrations of tert-butyl hydroperoxide (TBHP) (458139; Sigma-Aldrich, St. Louis, MO, USA) for 48 h. All in vitro experiments were independently performed three times.

Total RNA was extracted with RNAiso Plus Reagent (9109, Takara Bio Inc., Kusatsu, Japan) and assessed for purity and concentration using NanoDrop 2000. After genomic DNA removal, the first-strand complementary DNA (cDNA) was synthesized using the Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit (H4509060, Yeasen Biotech, Shanghai, China). Quantitative polymerase chain reaction (PCR) proceeded on a QuantStudio 3 instrument (Thermo Fisher Scientific, Waltham, MA, USA) with Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) kit (11202ES08, Yeasen Biotech, Shanghai, China) following the manufacturer’s protocol. Relative expression was calculated by 2^–ΔΔCt method with $\beta$-actin as reference. Primers were synthesized by Tsingke Biotechnology (Beijing, China) (Table 1).

NP cells were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) supplemented with protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (ST506, Beyotime, Shanghai, China) and phosphatase inhibitors (P1091, Beyotime, Shanghai, China). Protein concentrations were spectrophotometrically measured via bicinchoninic acid (BCA) method (PC0020, Solarbio, Beijing, China). Equal amounts of protein were electrophoretically separated on polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with 5 % skim milk or BSA. Membranes were subsequently probed with primary antibodies targeting SIRT7 (1:1000, 12994-1-AP, Proteintech, Wuhan, Hubei, China), matrix metalloproteinase-3 (MMP3) (1:4000, 66338-1-Ig, Proteintech, Wuhan, Hubei, China), phospho-p65 (p-p65, Ser536, 1:1000, 3033, CST, Danvers, MA, USA), total p65 (1:1000, 8242, CST, Danvers, MA, USA), or $\beta$-actin (1:5000, YM8343, Immunoway, Suzhou, Jiangsu, China) overnight at 4 °C, followed by HRP-conjugated goat anti-rabbit IgG (1:5000, GB23303, Servicebio, Wuhan, Hubei, China) or HRP-conjugated goat anti-mouse IgG (1:5000, GB23301, Servicebio, Wuhan, Hubei, China) incubation for 1 h. Bands were visualized with ECL (36208ES60, Yeasen, Shanghai, China) and imaged using a Chemiluminescence System (Surwit, Hangzhou, Zhejiang, China). Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to $\beta$-actin.

Intracellular ROS accumulation was evaluated utilizing dihydroethidium (DHE) fluorescent probe (S0064S; Beyotime, Shanghai, China). Following loading with 10 µM DHE for 30 min, NP cells underwent triple PBS washes prior to immediate assessment of DHE-derived fluorescence via flow cytometric analysis (BD Biosciences, Franklin Lakes, NJ, USA) or inverted fluorescence microscopy (Zeiss, Jena, Thuringia, Germany).

SIRT7-overexpressing lentiviruses (Lv-SIRT7) and a control lentivirus (Lv-NC) were purchased from GeneChem (Shanghai, China). At ~30% confluency, NP cells were transfected with Lv-SIRT7 (MOI = 50). Medium was changed after 48 h. SIRT7 overexpression was verified by qRT-PCR and Western blotting.

$\mathrm{\Delta }$$\mathrm{\Psi }$m was measured using the MitoTracker Deep Red 633 fluorescent dye (C1997S, Beyotime, Shanghai, China). Briefly, NP cells were stained with 5 µM MitoTracker Deep Red 633 for 30 min at 37 °C in darkness, washed twice with PBS, and immediately analyzed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

After exposure to TBHP, NP cells were stained with 2 µM Hoechst 33342 (C1025, Beyotime, Shanghai, China) for 15 min at 37 °C in darkness. They were then rinsed thrice with PBS and observed under an inverted fluorescence microscope (Zeiss, Jena, Thuringia, Germany). Apoptosis was identified based on nuclear condensation, fragmentation, and intense blue fluorescence.

NP cells plated in 24-well or confocal dishes underwent fixation with 4% paraformaldehyde for 20 min, followed by permeabilization using 0.5% Triton X-100 and blocking with 10% goat serum for 1 h. Overnight probing at 4 °C was conducted employing primary antibodies targeting aggrecan (1:200, DF7561, Affinity Bioscience, Changzhou, Jiangsu, China), collagen II (1:200, 28459-1-AP, Proteintech, Wuhan, Hubei, China), matrix metalloproteinase-13 (MMP13) (1:200, YT2796, Immunoway, Suzhou, Jiangsu, China), Bax (1:200, 60267-1-Ig, Proteintech, Wuhan, Hubei, China), Bcl-2 (1:200, 68103-1-Ig, Proteintech, Wuhan, Hubei, China), cleaved caspase-3 (1:200, 9664, CST, Danvers, MA, USA), or p65 (1:400, 8242, CST, Danvers, MA, USA). Subsequent to triple PBS rinses, specimens were exposed to cyanine 3- conjugated goat anti-rabbit IgG (1:200, GB21303, Servicebio, Wuhan, Hubei, China) or cyanine 3-conjugated goat anti-mouse IgG (1:200, GB21301, Servicebio, Wuhan, Hubei, China) for 1 h and nuclei were labeled with DAPI. Microscopic examination was carried out utilizing inverted fluorescence or confocal microscopy (Zeiss, Jena, Thuringia, Germany). For quantitative analysis, fluorescence intensity was measured using ImageJ software. Images were acquired under identical exposure settings across all groups. For NF-$\kappa$B nuclear translocation assay, the nuclear-to-cytoplasmic fluorescence intensity ratio was calculated. For other immunofluorescence analyses, the integrated fluorescence intensity was normalized to the cell number to obtain mean fluorescence intensity. Images were captured from at least three randomly selected fields per sample, and experiments were repeated independently three times.

The rat model of IVDD was established according to a previously published protocol [22]. Twenty-eight 8-week-old male Sprague–Dawley rats (obtained from the Laboratory Animal Center of Lanzhou University) were randomly allocated into four groups: Sham, IVDD, IVDD + Lv-NC, and IVDD + Lv-SIRT7. The rats were sedated with 2% isoflurane (R510-22-10, RWD Life Science, Shenzhen, Guangdong, China) and subsequently anesthetized with 40 mg/kg intraperitoneal pentobarbital (SCRC 69020180, Sinopharm Chemical Reagent Shanghai Co., Ltd, China). Following sterile exposure of the Co7/8 disc, the NP was punctured with a 21G needle parallel to the CEP (360° rotation, 60 s), then injected with 3 µL of Lv-NC or Lv-SIRT7 (titer: 1 $\times$ 10⁹ TU/mL) using a microliter syringe. The operator performing the surgeries was blinded to the group assignment. At the end of the experiment, the animals were euthanized using the method described in Section 2.2.

MRI was conducted to evaluate the impact of SIRT7 on disc degeneration. At the eighth week post-operation, T2-weighted sagittal images were acquired using a uMR 9.4T small-animal MRI scanner (United Imaging Life Science Instrument, Wuhan, Hubei, China) via fast-spin echo (TR/TE = 3000/40 ms; matrix, 224 $\times$ 85; FOV, 17 $\times$ 45 mm; NEX, 4; and slice thickness, 0.5 mm contiguous).

Human NP and rat IVD samples were preserved in 4% paraformaldehyde, with rat samples additionally decalcified prior to processing. Following dehydration and paraffin embedding, 4 µm sections were stained with: hematoxylin and eosin (H&E) (G1120, Solarbio, Beijing, China) for general tissue architecture and cellularity; Safranin O/Fast Green (G1371, Solarbio, Beijing, China) and Alcian blue (G1560, Solarbio, Beijing, China) for matrix proteoglycans; Masson staining (G1346, Solarbio, Beijing, China) for collagen and fibrosis. Protocols followed manufacturer specifications, with Olympus microscopy used for imaging.

Paraffin sections of human NP and rat IVD samples underwent dewaxing, rehydration, and citrate-based antigen retrieval. Peroxidase quenching utilized 3% hydrogen peroxide (10 min) prior to overnight primary antibody binding at 4 °C: SIRT7 (1:200, DF6161, Affinity Bioscience, Changzhou, Jiangsu, China), aggrecan (1:100, DF7561, Affinity Bioscience, Changzhou, Jiangsu, China), collagen II (1:200, ab307674, Abcam, Cambridge, UK), MMP3 (1:200, ab52915, Abcam, Cambridge, UK), Bax (1:400, 60267-1-Ig, Proteintech, Wuhan, Hubei, China), or p65 (1:400, 8242, CST, Danvers, MA, USA). Immunoreactivity was visualized using an IHC detection kit (KIT-9710, Maixin Biotech Co., Fuzhou, Fujian, China) and DAB peroxidase substrate kit (KIT-0038, Maixin Biotech Co., Fuzhou, Fujian, China) per manufacturer instructions. For quantitative analysis, at least three fields within the NP region were randomly selected from each section, and positively stained cells (brown, DAB) and total cells (hematoxylin counterstain) were counted in each field. The percentage of positive cells was calculated as (positively stained cells / total cells) $\times$ 100%, and the mean value across all examined fields was used for statistical analysis.

Quantitative results represent mean $\pm$ standard deviation (SD) from $\ge$3 independent trials. Statistical Analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Intergroup comparisons used two-tailed Student’s t-test (two groups) or one-way analysis of variance (ANOVA) with Tukey’s post-hoc test (multiple groups). Statistical significance was defined as p 0.05.

SIRT7 expression and its association with IVDD were evaluated using human NP tissue of varying degenerative severity. Pfirrmann classification by T2-weighted MRI (Fig. 1A) was histologically confirmed through H&E, safranin O/fast green, and Alcian blue staining (Fig. 1B) and validated via IHC staining for aggrecan, collagen II, and MMP3 (Fig. 1C–F). IHC analysis revealed significantly lower SIRT7 levels in severe versus mild NP degeneration (Fig. 1C,G), indicating SIRT7 expression decline during disease progression.

Fig. 1.

SIRT7 expression is markedly downregulated in degenerated NP tissues from humans and rats. (A) Representative T2-weighted MRI scans illustrating mild (Pfirrmann Grade II) and severe (Pfirrmann Grade V) disc degeneration in patients. (B) Histological characterization of human NP specimens employing H&E, Safranin O/fast green, and Alcian blue staining. Scale bar: 20 µm. (C) IHC staining of aggrecan, Collagen II (Col II), MMP3, and SIRT7 in degenerative human NP tissue. Scale bar: 20 µm (main images) and 5 µm (insets). (D) Quantitative analysis of aggrecan immunoreactivity in degenerative human NP tissue. (E) Quantitative analysis of Col II immunoreactivity in degenerative human NP tissue. (F) Quantitative analysis of MMP3 immunoreactivity in degenerative human NP tissue. (G) Quantitative analysis of SIRT7 immunoreactivity in degenerative human NP tissue. (H) Typical T2-weighted MRI images captured from rats at 8 weeks post-needle puncture-induced IVDD surgery. (I) Histopathological evaluation of rat NP tissue utilizing H&E, Safranin O/fast green, and Masson’s trichrome staining. Scale bar: 20 µm. (J) IHC detection of aggrecan, Col II, MMP3, and SIRT7 expression patterns in degenerated rat NP tissue sections. Scale bar: 20 µm (main images) and 5 µm (insets). (K) Quantitative analysis of positive staining rates for aggrecan in degenerative rat NP tissue. (L) Quantitative analysis of positive staining rates for Col II in degenerative rat NP tissue. (M) Quantitative analysis of positive staining rates for MMP3 in degenerative rat NP tissue. (N) Quantitative analysis of positive staining rates for SIRT7 in degenerative rat NP tissue. Quantitative results represent mean $\pm$ SD. Human samples: n = 6 (Grade II) and n = 6 (Grade V). Rat: n = 7 per group. The comparison between the two groups was conducted using Student’s t-test. *p 0.05, **p 0.01. SIRT7, sirtuin 7; NP, nucleus pulposus; IHC, immunohistochemistry; H&E, hematoxylin and eosin; MMP3, matrix metalloproteinase-3; IVDD, intervertebral disc degeneration; SD, standard deviation; MRI, magnetic resonance imaging.

These findings were verified in a puncture-induced rat IVDD model. Eight weeks post-surgery, MRI revealed reduced T2 signal intensity in punctured versus sham discs (Fig. 1H). Histological assessment via H&E, safranin O/fast green, and Masson staining revealed disrupted NP architecture, proteoglycan depletion, and fibrosis (Fig. 1I). Concurrent IHC analysis indicated decreased aggrecan, collagen II, and SIRT7 levels alongside elevated MMP3 (Fig. 1J–N). Together, these data suggest SIRT7 downregulation during IVDD.

ROS are considered a major pathological driving factor of IVDD. TBHP, an exogenous ROS donor, is widely used to establish cell models of IVDD in vitro [23]. To induce oxidative stress, NP cells were exposed to indicated concentrations of TBHP for 48 h. Intracellular ROS levels were subsequently quantified by flow cytometry following DHE labeling, revealing dose-dependent ROS accumulation (Fig. 2A,B). The qRT-PCR analysis demonstrated that 75 µM TBHP markedly suppressed collagen II expression (Fig. 2C) while upregulated MMP13 expression (Fig. 2D) in NP cells, thereby confirming the degenerative phenotype. Following validation of the cellular model, transcript level analysis revealed that SIRT7 expression was significantly downregulated in NP cells treated with 50 and 75 µM TBHP (Fig. 2E). These in vitro findings align with prior in vivo observations, collectively indicating decreased SIRT7 expression in NP cell degeneration. Accordingly, 75 µM TBHP was selected for subsequent experiments.

Fig. 2.

SIRT7 is downregulated in TBHP-treated NP cells. (A) Flow cytometry histograms of DHE fluorescence after 48 h TBHP treatment. (B) Quantitative analysis of relative intracellular ROS levels, expressed as fold change in DHE mean fluorescence intensity relative to the control group. (C) qRT-PCR results for Col II in NP cells. (D) qRT-PCR results for MMP13 in NP cells. (E) qRT-PCR results for SIRT7 in NP cells. Values represent mean $\pm$ SD from three independent experiments. One-way ANOVA with Tukey’s test. *p 0.05, **p 0.01. TBHP, tert-butyl hydroperoxide; DHE, dihydroethidium; ROS, reactive oxygen species; qRT-PCR, quantitative real-time polymerase chain reaction; ANOVA, analysis of variance.

ECM homeostasis disruption underlies IVDD pathogenesis. To elucidate SIRT7’s regulatory role in matrix metabolism, lentiviral-mediated SIRT7 overexpression was established in NP cells. The qRT-PCR (Fig. 3A) and Western blotting were performed to verify efficient SIRT7 upregulation in NP cells (Fig. 3B,C). Following oxidative challenge with TBHP, transcriptional profiling revealed substantial repression of aggrecan and collagen II alongside MMP3 induction, while SIRT7 overexpression reversed these TBHP-induced alterations (Fig. 3D–F). At the protein level, SIRT7 attenuated MMP3 accumulation (Fig. 3G,H), enhanced collagen II deposition (Fig. 3I,J), and diminished MMP13 immunoreactivity (Fig. 3K,L). Collectively, these findings suggest that SIRT7 upregulation preserves matrix integrity under oxidative stress, thereby retarding degenerative progression.

Fig. 3.

SIRT7 overexpression attenuates TBHP-induced ECM degradation in NP cells. (A) SIRT7 transcript levels following lentiviral delivery evaluated by qRT-PCR. (B) Representative Western blot showing SIRT7 protein expression in transfected NP cells. (C) Quantification of SIRT7 protein levels normalized to loading control. (D) Aggrecan mRNA quantification by qRT-PCR. (E) Col II gene expression assessed through qRT-PCR. (F) MMP3 mRNA quantification by qRT-PCR. (G) Representative Western blot of MMP3. (H) Relative MMP3 protein quantification by Western blot. (I) Representative immunofluorescence staining images of Col II. Scale bar: 20 µm. (J) Quantitative immunofluorescence analysis of Col II in NP cells. (K) Representative immunofluorescence staining images of MMP13. Scale bar: 20 µm. (L) Quantitative immunofluorescence analysis of MMP13. Values represent mean $\pm$ SD from three independent experiments. Between-group differences analyzed by Student’s t-test (two groups) or one-way ANOVA with Tukey’s post-hoc test (multiple groups). *p 0.05, **p 0.01. –, untreated; +, treated. ECM, extracellular matrix; Lv-NC, control lentivirus; Lv-SIRT7, SIRT7-overexpressing lentivirus.

DHE staining showed that TBHP significantly increased intracellular ROS levels, whereas SIRT7 overexpression effectively inhibited TBHP-induced increase in ROS (Fig. 4A,B). Increased ROS triggers a decrease in $\mathrm{\Delta }$$\mathrm{\Psi }$m, which suggests an early apoptotic event. $\mathrm{\Delta }$$\mathrm{\Psi }$m was measured using Mito-Tracker Deep Red 633, and flow cytometry revealed that TBHP depolarized $\mathrm{\Delta }$$\mathrm{\Psi }$m, but SIRT7 overexpression significantly inhibited this effect (Fig. 4C,D). Apoptotic morphology characterized by nuclear condensation, fragmentation, and intense blue fluorescence was observed by Hoechst 33342 staining. The results showed that TBHP promoted NP cell apoptosis, while SIRT7 overexpression markedly inhibited this effect (Fig. 4E,F). qRT-PCR and immunofluorescence analyses of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax indicated that TBHP increased the Bax/Bcl-2 ratio, whereas SIRT7 overexpression significantly decreased this ratio (Fig. 4G–J). Similarly, immunofluorescence analysis of cleaved caspase-3 showed that TBHP increased cleaved caspase-3 levels, whereas SIRT7 overexpression markedly decreased these levels (Fig. 4K,L). Altogether, these data demonstrate that SIRT7 overexpression attenuates TBHP-induced oxidative stress and apoptosis in degenerative NP cells.

Fig. 4.

SIRT7 overexpression protects NP cells from oxidative stress and apoptosis induced by TBHP. (A) Representative fluorescence micrographs of intracellular ROS detected by DHE staining in Lv-NC and Lv-SIRT7 groups treated with or without 75 µM TBHP for 48 h. Scale bar: 50 µm. (B) Quantitative assessment of intracellular ROS level in (A). (C) Representative flow cytometry histograms of $\mathrm{\Delta }$$\mathrm{\Psi }$m detected with Mito-Tracker Deep Red 633. (D) Quantitative analysis of $\mathrm{\Delta }$$\mathrm{\Psi }$m fluorescence intensities in (C). (E) Representative Hoechst 33342 staining images showing apoptotic morphology (white arrows). Scale bar: 50 µm (main images) and 10 µm (insets). (F) Apoptosis rate calculated from Hoechst 33342 staining. (G) qRT-PCR analysis of Bax/Bcl-2 ratio. (H) Representative immunofluorescence of Bax. Scale bar: 20 µm. (I) Representative immunofluorescence of Bcl-2. Scale bar: 20 µm. (J) Quantitative analysis of Bax/Bcl-2 ratio by immunofluorescence. (K) Representative immunofluorescence of C-Cas-3. Scale bar: 20 µm. (L) Quantitative analysis of C-Cas-3 by immunofluorescence. Values represent mean $\pm$ SD from three independent experiments. One-way ANOVA with Tukey’s post-hoc test. *p 0.05, **p 0.01. –, untreated; +, treated. $\mathrm{\Delta }$$\mathrm{\Psi }$m, mitochondrial membrane potential; C-Cas-3, cleaved caspase-3.

Accumulating evidence has established NF-$\kappa$B as a well-characterized driver of apoptosis and ECM degradation in IVDD. Accordingly, targeted suppression of the NF-$\kappa$B pathway effectively attenuates these pathological processes [23, 24]. To explore whether SIRT7-mediated protection against apoptosis and matrix breakdown involves NF-$\kappa$B pathway modulation, we evaluated NF-$\kappa$B activation in TBHP-treated NP cells. p65 serves as a crucial component of the NF-$\kappa$B pathway, wherein its phosphorylation status and nuclear translocation from the cytoplasm are critical indicators for evaluating NF-$\kappa$B pathway activation state [9]. IHC staining of human specimens revealed higher nuclear p65 protein expression in severely degenerated discs than in mildly degenerated ones (Fig. 5A,B), confirming that NF-$\kappa$B activation correlates with IVDD severity. Subsequently, we performed Western blotting and immunofluorescence assays to investigate whether SIRT7 overexpression could inhibit NF-$\kappa$B activation induced by TBHP in NP cells. Western blotting showed that, compared with the control, TBHP had no effect on total p65 expression, but significantly increased phosphorylated p65 (p-p65) expression and the p-p65/p65 ratio, whereas SIRT7 overexpression inhibited these effects (Fig. 5C,D). Consistent with the results of Western blotting, immunofluorescence analysis revealed that TBHP induced p65 nuclear translocation, whereas SIRT7 overexpression attenuated it (Fig. 5E,F). Altogether, these results suggest that SIRT7 overexpression suppresses the NF-$\kappa$B pathway activation in TBHP-induced NP cells.

Fig. 5.

SIRT7 overexpression inhibits NF-$\kappa$B pathway activation in degenerative NP cells. (A) Representative IHC images demonstrating p65 distribution in human degenerative NP specimens. Scale bar: 20 µm (main images) and 5 µm (insets). (B) Semiquantitative assessment of nuclear p65 immunoreactivity. (C) Immunoblot detection of p-p65 and total p65 protein levels. (D) Densitometric quantification of p-p65 normalized to total p65. (E) Confocal immunofluorescence visualization of p65 subcellular compartmentalization. Scale bar: 20 µm. (F) Quantitative evaluation of p65 nuclear/cytoplasmic fluorescence intensity ratio. All data expressed as mean $\pm$ SD. Human samples: n = 6 (Grade II) and n = 6 (Grade V). Cell experiments: n = 3 independent experiments. Between-group differences analyzed by Student’s t-test (two groups) or one-way ANOVA with Tukey’s post-hoc test (multiple groups). **p 0.01. –, untreated; +, treated. NF-$\kappa$B, nuclear factor kappa B; p-p65, phospho-p65 (Ser536).

We established an IVDD rat model via needle puncture to evaluate the therapeutic potential of SIRT7 in vivo. Twenty-eight Sprague–Dawley rats were randomly assigned to sham, IVDD, IVDD + Lv-NC, or IVDD + Lv-SIRT7 groups. With the exception of sham-operated animals, rats in the other groups were injected with PBS, Lv-NC, or Lv-SIRT7, respectively. Eight weeks later, T2-weighted MRI was performed, and the IVD was harvested for histological and IHC analyses. As shown in Fig. 6, SIRT7 overexpression significantly attenuated IVDD progression. IHC analysis revealed that SIRT7 expression was markedly upregulated in the NP tissue of the IVDD + Lv-SIRT7 group compared with that in the other groups (Fig. 6C,D), confirming the successful overexpression of SIRT7. MRI showed that the T2-weighted signal intensity was considerably preserved in the IVDD + Lv-SIRT7 group compared with that in the IVDD and IVDD + Lv-NC groups (Fig. 6A). Consistent with this, histological analysis by H&E, Safranin O/fast green, and Masson staining revealed severe NP collapse, proteoglycan loss, and fibrosis in the IVDD and IVDD + Lv-NC groups; however, these degenerative changes were significantly attenuated in the IVDD+Lv-SIRT7 group (Fig. 6B). Additionally, IHC for collagen II, MMP3, and Bax showed that collagen II expression was downregulated, whereas MMP3 and Bax expression were upregulated in the IVDD and IVDD + Lv-NC groups, indicating increased ECM degradation and NP cell apoptosis. SIRT7 overexpression reversed these pathological changes (Fig. 6C,E–G). Importantly, nuclear p65 protein levels were significantly lower in the IVDD + Lv-SIRT7 group than in the IVDD and IVDD + Lv-NC groups (Fig. 6C,H), indicating that SIRT7 inhibits NF-$\kappa$B activation. These data suggest that SIRT7 overexpression mitigates IVDD progression in vivo, and its protective effect may be associated with NF-$\kappa$B inhibition.

Fig. 6.

SIRT7 overexpression alleviates IVDD in rats by suppressing the NF-$\kappa$B pathway. (A) T2-weighted MRI acquired from experimental rats at eight weeks post-puncture. (B) Histological analysis of rat caudal disc specimens using H&E, Safranin O/fast green, and Masson staining. Scale bar: 20 µm. (C) Representative IHC images of SIRT7, Col II, MMP3, Bax, and p65. Scale bar: 20 µm. (D) Quantitative IHC analysis of SIRT7. (E) Quantitative IHC analysis of Col II. (F) Quantitative IHC analysis of MMP3. (G) Quantitative IHC analysis of Bax. (H) Quantitative IHC analysis of nuclear p65 protein expression. Values represent mean $\pm$ SD. Rat: n = 7 per group. Group comparisons employed one-way ANOVA followed by Tukey’s post-hoc test. **p 0.01. ns, not significant. –, untreated; +, treated.