Sesamin-mediated high expression of BECN2 ameliorates cartilage endplate degeneration by reducing autophagy and inflammation

Transcriptome analysis molecular mechanism of Sesamin on LPS-induced ATDC5 degeneration

A total of 54 up-regulated and 63 down-regulated genes were identified using the cutoff criteria: p-values less than 0.05 and fold change greater than 2, as shown in the volcano map (Figure 1A). The top 50 DEGs were shown in the heat map (Figure 1B). Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to analyze the function of DEGs. In Figure 1C, the top 10 KEGG pathways of DGEs were mainly enriched in neuroactive ligand-receptor interaction, complement and coagulation cascades, vascular smooth muscle contraction, tight junction, glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate, linoleic acid metabolism, arachidonic acid metabolism, PPAR signaling pathway, hypertrophic cardiomyopathy, and dilated cardiomyopathy. The results of the top 10 Gene Ontology (GO) pathways of DEGs in Figure 1D showed changes in biological processes (BP), cell component (CC) and molecular function (MF).

Figure 1. Transcriptome analysis molecular mechanism of Sesamin on LPS-induced ATDC5 degeneration. (A) volcano map of mRNA expression profiles. (B) The top50 of differentially expressed genes (DGEs). (C) The top 10 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of DEGs. (D) The top 10 enriched Gene Ontology (GO) pathway of the DEGs sorted by significance in biological process (BP), cellular component (CC) and molecular function (MF).

BECN2 overexpression decreased the apoptosis of LPS-induced ATDC5 cells by inhibiting autophagy and inflammation

Based on transcriptome sequencing results, we found that BECN2 was significantly overexpressed in the Sesamin treated degeneration group, suggesting that Sesamin may participate in the degeneration of intervertebral cartilaginous endplate by regulating the BECN2 RT-PCR was used to verify the expression of BECN2, and the results in Figure 2A showed that Sesamin significantly increased BECN2 expression in LPS-induced ATDC5 cells (p = 0.042). ADmax-BECN2 adenovirus was used to overexpress the BECN2, and the overexpression efficiency was detected, as illustrated in Figure 2B–2D, indicating that ADmax-BECN2 significantly increased the expression of BECN2 in mRNA and protein levels (p < 0.001, p = 0.001, respectively).

Figure 2. The effect of BECN2 overexpression on the function of LPS-induced ATDC5 degeneration. (A) The mRNA level of BECN2 was detected by RT-PCR in LPS-induced ATDC5 cells. (B) The effect of ADmax-BECN2 on the mRNA expression of BECN2 was detected by RT-PCR. (C) The protein level of BECN2 was evaluated by western blot. (D) The protein expression of BECN2 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (E) CCK-8 was used to assess cell viability. (F, G) Cell apoptosis was detected by Muse. (H) The immunofluorescence staining of LC3 was used to evaluate the autophagy level (green signals represent LC3, blue signals represent DAPI, scale bar: 10 μm). (I, J) Autophagy-related protein expression levels were determined by immunoblotting. (K, L) The expression of IL-1β, Caspase1 and IL-18 were assessed by western blot. Relative protein expression was qualified by ImageJ software, GAPDH was used as the internal control, respectively. All data represent mean ± SD. All in vitro experiments were repeated three times independently. * p < 0.05, ** p < 0.01, and *** p < 0.001.

We examined the effects of ADmax-BECN2 on cell viability, apoptosis and autophagy. And as shown in Figure 2F, 2G, the overexpression of BECN2 promoted cell viability (p < 0.001) (Figure 2E) and inhibited cell apoptosis (p < 0.001). The immunofluorescence results showed that ATDC5 cells formed fewer autophagosomes in the LPS+ADmax-BECN2 group than in the LPS+ADmax-NC group (Figure 2H). Moreover, the overexpression of BECN2 inhibited the expression of LC3II/LC3I and p-mTOR induced by LPS (p < 0.001, p < 0.001, respectively). However, the expression of P62 was increased in the LPS+ADmax-BECN2 group compared to the LPS+ADmax-NC group (p = 0.017) (Figure 2I, 2J). Additionally, the expression levels of inflammation related protein IL-1β, Caspase 1 and IL-18 were detected by immunoblotting, and the results indicated that LPS-induced high expression of inflammatory cytokines was inhibited by BECN2 overexpression (p < 0.001, p = 0.004, p < 0.001, respectively) (Figure 2K, 2L). Taken together, BECN2 overexpression could significantly inhibit apoptosis and autophagy induced by LPS in ATDC5 cells.

Furthermore, in order to explore the mechanism of BECN2 inhibiting LPS-induced ATDC5 degeneration, western-blot and immunofluorescence were used to detect the protein expression of autophagosome and inflammasome. The expression of autophagosome component proteins ATG14, VPS34 and GASP1 were showed in Figure 3A, 3B. Compared to LPS+ADmax-NC group, overexpressed BECN2 inhibited the expression of these proteins by a fold of 0.43,0.52 and 0.88 (p < 0.001, p = 0.008, and p = 0.045, respectively). The results of immunofluorescence in Figure 3C–3F were consistent with WB. Meanwhile, we observed that BECN2 overexpression significantly decreased the expression of NLRP3, NLRC4, NLRP1, and AIM2 by 0.65-fold (p = 0.005), 0.66-fold (p = 0.005), 0.77-fold (p = 0.004), and 0.63-fold (p = 0.004), respectively (Figure 3G, 3H). This result was verified by immunofluorescence, indicating that BECN2 may attenuate the degeneration of LPS-induced ATDC5 by inhibiting the formation of the inflammasome (Figure 3I–3M). These results indicated that BECN2 may protect chondrocyte degeneration by inhibiting autophagy and inflammation.

Figure 3. Increased BECN2 attenuated the autophagy and inflammation of LPS-induced ATDC5 degeneration. (A) Western blot was employed to detect the expression of ATG14, VPS34 and GASP1. (B) The protein expression of ATG14, VPS34 and GASP1 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (C) ATG14, VPS34 and GASP1 expression were determined by immunofluorescence staining (scale bar: 100μm). (D–F) Average optical density was calculated by ImageJ software. (G) Western blot was employed to detect the expression of NLRP3, NLRC4, NLRP1 and AIM2. (H) The protein expression of NLRP3, NLRC4, NLRP1 and AIM2 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (I) NLRP3, NLRC4, NLRP1 and AIM2 expression were determined by immunofluorescence staining (scale bar: 100μm). (J–M) Average optical density was calculated by ImageJ software. All data represent mean ± SD. All in vitro experiments were repeated three times independently. * p < 0.05, ** p < 0.01, and *** p < 0.001.

BECN2 knockdown increased the apoptosis of ATDC5 cells by promoting autophagy and inflammation

In order to further elucidate the role of BECN2 in inhibiting chondrocyte degeneration of Sesamin, we constructed the knockdown double-stranded siRNA. Interference efficiency was detected, and the optimal effect was observed with siRNA-BECN2-3 among the three interference sequences. The RT-PCR and WB results in Figure 4A–4C showed that siRNA-BECN2-3 decreased the expression of BECN2 mRNA and protein to levels 0.07-fold (p = 0.001) and 0.69-fold (p < 0.001) lower than that of siRNA-NC group. The results in Figure 4D showed that siRNA-BECN2 significantly decreased the cell viability to level 0.41-fold compared to siRNA-NC group. We noted that down-expressed BECN2 significantly promoted cell apoptosis, and the apoptosis rate reached 1.16% in Figure 4E, 4F (p = 0.031). The results were shown in immunofluorescence Figure 4G. Down-regulation of BECN2 increased the number of speckled autophagosomes. The results of autophagy related protein expression were shown in Figure 4H, 4I. The expression of p-mTOR and P62 were weakened by down-expressed BECN2 to levels 0.68-fold (p = 0.002) and 0.42-fold (p < 0.001) compared to siRNA-NC group. In contrast, the down-regulation of BECN2 resulted in an increase in the expression of LC3 II/LC3 I and total mTOR by 1.19-fold (p = 0.024) and 1.78-fold (p < 0.001), respectively. In addition, the expression of IL-1β, Caspase 1 and IL-18 were significantly increased induced by BECN2 knockdown to level 1.38-fold (p = 0.019), 1.31-fold (p = 0.009), and 1.39-fold (p < 0.001) in Figure 4J, 4K, respectively. These results suggested that BECN2 knockdown promoted the degeneration of ATDC5 chondrocytes.

Figure 4. The effect of BECN2 knockdown on the function of ATDC5. (A) The effect of siRNA-BECN2 on the mRNA expression of BECN2 was detected by RT-PCR. (B) The protein level of BECN2 was evaluated by western blot. (C) The protein expression of BECN2 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (D) CCK-8 was used to assess cell viability. (E, F) Cell apoptosis was detected by Muse. (G) The immunofluorescence staining of LC3 was used to evaluate the autophagy level (green signals represent LC3, blue signals represent DAPI, scale bar: 10 μm). (H, I) Autophagy-related protein expression levels were determined by immunoblotting. (J, K) The expression of IL-1β, Caspase1 and IL-18 were assessed by western blot. Relative protein expression was qualified by ImageJ software, GAPDH was used as the internal control, respectively. All data represent mean ± SD. All in vitro experiments were repeated three times independently. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Furthermore, the western blot analysis in Figure 5A, 5B showed that ATG14, VPS34 and GASP1 expression were successfully promoted to levels 2.58-fold (p <0.001), 1.78-fold (p = 0.002) and 2.41-fold (p <0.001) by BECN2 siRNA. The immunofluorescence results in Figure 5C–5F showed that knockdown of BECN2 significantly increased the fluorescence signals of ATG14, VPS34, and GASP1 (p = 0.026, p = 0.003, p = 0.037, respectively). Similarly, BECN2 knockdown also increased the expression levels of NLRP3, NLRC4, AIM2, and NLRP1 by 2.14-fold (p = 0.003), 2.73-fold (p < 0.001), 1.56-fold (p < 0.001), and 4.64-fold (p < 0.001) respectively (Figure 5G, 5H). Subsequent immunofluorescence staining of NLRP3, NLRC4, NLRP1 and AIM2 showed that down-regulation of BECN2 by siRNA obviously promoted the formation of autophagosomes (p = 0.003, p = 0.011, p = 0.015, and p = 0.012, respectively) (Figure 5I–5M). These data indicated that BECN2 knockdown may promote chondrocyte degeneration by increasing autophagy and inflammation.

Figure 5. Decreased BECN2 activated the autophagy and inflammation of ATDC5. (A) Western blot was employed to detect the expression of ATG14, VPS34 and GASP1. (B) The protein expression of ATG14, VPS34 and GASP1 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (C) ATG14, VPS34 and GASP1 expression were determined by immunofluorescence staining (scale bar: 100μm). (D–F) Average optical density was calculated by ImageJ software. (G) Western blot was employed to detect the expression of NLRP3, NLRC4, NLRP1 and AIM2. (H) The protein expression of NLRP3, NLRC4, NLRP1 and AIM2 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (I) NLRP3, NLRC4, NLRP1 and AIM2 expression were determined by immunofluorescence staining (scale bar: 100μm). (J–M) Average optical density was calculated by ImageJ software. All data represent mean ± SD. All in vitro experiments were repeated three times independently. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Sesamin ameliorates LDD in vivo

Moreover, the protective effect of Sesamin on cartilage endplate degeneration after 4 weeks of surgery was further confirmed by Safranin O-fast green staining, as shown in Figure 6A. Compared with sham group, we found that the number of cartilage cells was reduced, the staining degree was decreased, the cartilage cells were replaced by connective tissue, and cartilaginous endplate was collapsed in LDD group, while Sesamin treatment attenuated these degenerations. Meanwhile, cartilage endplate tissues were collected and examined the expression of ADAMTS5, Collagen II and MMP13 using RT-PCR, western blot and immunohistochemistry. The western blot results in Figure 1B, 1C showed significantly higher levels of ADAMTS5 (p < 0.001) and MMP13 (p < 0.001) in the puncture-induced rat model. However, under Sesamin treatment, the expression of ADAMTS5 (p = 0.011) and MMP13 (p = 0.036) was reduced. The protein level of Collagen II was obviously increased under Sesamin treatment compared to the LDD model (p = 0.019). The results were also confirmed by immunohistochemistry in Figure 1D–1G. These data suggested that Sesamin may alleviate the degeneration of rat cartilage endplate caused by acupuncture.

Figure 6. Sesamin ameliorated degeneration of cartilage endplate in puncture-induced rat model. (A) Representative images of the degenerative disc at 4 weeks post surgery by Safranin O-fast green staining (4X, scale bar: 200μm, 20X, scale bar: 5μm). (B) The protein level of ADAMTS5, Collagen II and MMP13 was evaluated by western blot. (C) The protein expression of ADAMTS5, Collagen II and MMP13 were determined using ImageJ software, GAPDH was used as the internal control, respectively (n=3). (D) ADAMTS5, Collagen II and MMP13 staining were detected (scale bar: 20 μm). (E–G) The positive rate was calculated by ImageJ software. All data represent mean ± SD. Three independent samples were used for statistics in all in vivo experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Additionally, immunohistochemistry was used to assess the expression of Bax, Bcl-2, IL-1β and IL-18 in Figure 7. In LDD model, the protein level of Bax was obviously promoted (p < 0.001), and Bcl-2 was inhibited (p < 0.001), these results were reversed by Sesamin treatment (p = 0.006, and p = 0.003, respectively) (Figure 7B, 7C). The results in Figure 7D, 7E showed that significantly higher positive rates of IL-1β (p < 0.001) and IL-18 (p = 0.002) were seen in the puncture-induced rat model, implying the activation of inflammation in LDD model group, however, Sesamin treatment significantly lowered the protein levels of IL-1β (p = 0.012) and IL-18 (p = 0.012). These results suggested that Sesamin regulated cartilage endplate degeneration through inflammation.

Figure 7. Apoptosis was inhibited by Sesamin treatment in puncture-induced rat model. (A) Bax, BCL-2, IL-1β and IL-18 were detected by immunohistochemistry (scale bar: 20 μm). (B–E) Relative positive rates were calculated by ImageJ software. All data represent mean ± SD. Three independent samples were used for statistics in all in vivo experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Cell culture and cell injury model

The murine chondrogenic ATDC5 cells were obtained from the BeNa Culture Collection in Suzhou, China. Cells were cultured in DMEM/F12 medium (Hyclone, USA) supplemented with 10% (v/v) FBS (Hyclone, USA) and 1% (v/v) Penicillin-Streptomycin-Glutamine (Gibco, USA) in 50 cm² cell culture flask. Flask was placed in a humidified incubator with 5% CO₂ at 37° C. Lipopolysaccharide (LPS) was used to establish a cell injury model, and a concentration of 10 μg/ml LPS was selected for further experiments based on our previous research.

Transcriptome sequencing

To understand the effect of Sesamin on the cartilage endplate degeneration, two groups were conducted, the LPS induced-ATDC5 group and LPS induced-ATDC5 treated with Sesamin (1 μmol/l) (MedChemExpress, USA) group. The cellular RNA was extracted for transcriptome analysis at Shanghai Genesky Biotechnology Company in Shanghai, China. The quality and quantity of the RNA used to generate the RNA sequencing libraries were evaluated using an Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). The RNA library and RNA sequencing were performed using the Illumina HiSeq platform (Illumina, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from ATDC5 or cartilage endplate tissues using the TRIzol reagent (Invitrogen, USA). cDNA was synthesized from 1.0 μg of total RNA using the PrimeScript™ II 1^st Strand cDNA Synthesis Kit (TaKaRa, Japan). QRT-PCR was performed by the ABI 7500 Sequence Detection System (Applied Biosystems, USA). The relative gene expression, normalized to GAPDH, was calculated using the 2^-ΔΔCt formula. The primers used for qPCR are listed as follows. BECN2, Forward: 5’-TGCAACTAGACGACCAGCTC-3’, Reverse: 5’-CAGAGTACTCGACCCTGTGC-3’. GAPDH, Forward: 5’-CATCACTGCCACCCAGAAGACTG-3’, Reverse: 5’-ATGCCAGTGAGCTTCCCGTTCAG-3’.

Cell transfection

For transient transfection, double-stranded siRNA oligonucleotides targeting BECN2 and negative control RNAs (siRNA-NC) were synthesized by Genepharma (Shanghai, China). ATDC5 cells were transfected with siRNA using Lipofectamine RNAiMax reagent (Invitrogen, USA). For adenovirus transfection, ADMax-NC and ADMax-BECN2 were constructed by the Shanghai Genepharma Company (Genepharma, China). The BECN2 interfering sequences were as follows: siRNA-BECN2-1: 5’- GAAGCUGACAGUCAGAACUTT-3’ (sense), 5’- AGUUCUGACUGUCAGCUUCTT-3’ (antisense), siRNA-BECN2-2: 5’- CCUGGCCAAUACAAUUGGATT-3’ (sense), 5’- UCCAAUUGUAUUGGCCAGGTT-3’ (antisense), siRNA-BECN2-3: 5’- CACUCAAGUUCAUGCUUAUTT-3’ (sense), 5’- AUAAGCAUGAACUUGAGAGUGTT-3’ (antisense). Negative control siRNA: 5’- UUCUCCGAACGUGUCACGUTT-3’ (sense), and 5’- ACGUCGACACGUUCGGAGAATT-3’ (antisense).

Cell viability

For CCK-8 assays, treated cells were placed in quintuplicate in 96-well plates and cultured in DMEM/F12 medium containing 10% FBS for 24 h. Following the manufacturer’s instructions, the Cell Counting Kit-8 (MedChemExpress, USA) assay was introduced to measure cell viability at an optical density of 450nm.

Cell apoptosis

Muse® Cell Analyzer (Millipore, USA) was adopted to assess the apoptosis in ATDC5 as per the manufacturer’s instructions. After trypsinization, the cells were harvested and subsequently washed twice with PBS. 100 μl of the Muse Annexin V and Dead Cell Reagent (Millipore, USA) was added to each sample, followed by incubation for 20 min at room temperature in the dark. The percentages of the four cell subpopulations were distinguished as follows: lower-left: viable cells, lower-right: cells in the early stages of apoptosis, upper-right: cells in the late stages of apoptosis, and upper-left: cells that have died via necrosis instead of apoptotic pathway.

Western blotting

For total protein extraction, ATDC5 or cartilage endplate tissues from each group were lysed using RIPA lysis buffer (Beyotime, China). The proteins were separated by electrophoresis and transferred to a PVDF membrane (Millipore, USA). After being blocked with 5% skim milk for 1 h at room temperature, the blot was incubated with a specific primary antibody overnight. Subsequently, the HRP-conjugated secondary antibody was used to conjugate the specific primary antibody. ECL reaction images were acquired and observed using the Touch Imager XLI (e-BLOT, China). GAPDH or β-actin was adopted as an internal reference, and ImageJ v1.47 software (National Institutes of Health, USA) was applied to analyze the protein band images. The antibodies used for the western blot analysis are listed as follows. Anti-MMP13 (1:1000, 18165-1-AP, Proteintech), anti- MMP3 (1:1000, 17873-1-AP, Proteintech), anti-IL-1 Beta (1:1000, 16806-1-AP, Proteintech), anti--mTOR (1:1000, 66888-1-Ig, Proteintech), anti-LC3 (1:1000, 14600-1-AP, Proteintech), anti-P62/SQSTM1 (1:1000, 18420-1-AP, Proteintech), anti-Beclin 2 (1:500, NB110-60984SS, Novus), anti-NLRP3 (1:1000, 19771-1-AP, Proteintech), anti-ATG14 (1:1000, 19491-1-AP, Proteintech), anti-Phospho-NLRC4 (Ser533) (1:1000, AF3580, Affinity), anti-VPS34 (1:1000, 12452-1-Ap, Proteintech), anti-Caspase 1 (1:1000, 22915-1-AP, Proteintech), anti-IL-18 (1:1000, 10663-1-AP, Proteintech), anti-GPRASP1 (1:1000, 20020-1-AP, Proteintech), anti-NLRP1 (1:1000, DF13187, Affinity), anti-Phospho-mTOR (Ser2448) (1:1000, #5536, Cell Signaling Technology), anti-AIM2 (1:500, 122335, ZEN Bioscience), anti-GAPDH (1:10000, 60004-1-Ig, Proteintech), anti-ADAMTS5 (1:1000, DF13268, Affinity), anti-Collagen II (1:1000, Bs-0709R, Bioss) antibody. And secondary antibodies involved HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:20000, SA00001-2, Proteintech), and HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:20000, SA00001-1, Proteintech).

Immunofluorescence assay

ATDC5 cells for immunofluorescence staining were grown and handled on chamber slides. 4% formaldehyde and 0.2% Triton X-100 were used for cellular fixation and permeabilization. Subsequently, the cells were incubated with the specific primary antibody and fluorescently-labeled secondary antibodies. The immunofluorescence assay used the same specific primary antibody as the western blot, and the dilution ratio of the primary antibody was 1:50-1:200. The secondary antibodies are listed as below. Fluorescein (FITC) conjugated Affinipure Goat Anti-Mouse IgG (1:100, SA00003-1, Proteintech), Fluorescein (FITC)–conjugated Affinipure Goat Anti-Rabbit IgG (1:100, SA00003-1, Proteintech). The nuclei were stained with DAPI (Boster, China). Autophagosome were evaluated in the Leica TCS SP8 laser confocal fluorescence microscope (Leica, Germany), and the expression of proteins were observed by Nikon Ts2R inverted fluorescence microscope (Nikon, Japan). Fluorescence intensity was measured using ImageJ.

Animals and establishment of rat LDD injury model

Forty-eight adult male Sprague-Dawley rats (200±20g, 6-8 weeks old) were randomly divided into four groups: the control group, the sham group, the LDD group, and the LDD + Sesamin group. All rats were anaesthetized with an intraperitoneal injection of 10% chloral hydrate (40 mg/Kg). Rat disc degeneration model was constructed by performing parallel punctures using a 0.4mm needle at the cartilaginous endplate for 30 sec. Three segments of the disc were selected for puncture in each rat, including L3/4, L4/5, and L5/6. Two weeks post-surgery, the rats in the LDD+Sesamin group were treated with 80mg/Kg Sesamin by intragastric administration for two weeks, and normal saline was used in the sham and LDD groups. In accordance with the standard of Rat Health Guide (http://ratguide.com/), the rats were monitored daily to guarantee their well-being. Four weeks after puncture, all rats were sacrificed, and the cartilaginous endplate tissue was used for the test.

General histological and immunohistochemistry

Immunohistochemistry was performed to evaluate the expression and location of specific protein. Sections were incubated with primary antibodies against Bax (1:200, GB114122, Servicebio), IL-1β (1:100, GB11113, Servicebio), COL2 (1:200, GB11021, Servicebio), ADAMTS5 (1:100, TD13268M, Abcam), MMP13 (1:100, 18164-1-AP, Proteintech), Bcl-2 (1:100, 26593-AP, Proteintech), IL-18 (1:100, 10663-1-AP, Proteintech), and a secondary antibody. IHC was performed using the DAB solution and hematoxylin. Safranin-O/Fast Green staining is an approach used to assess the severity of cartilage injury. The Safranin-O/Fast Green staining kit (Servicebio, China) was utilized to stain and evaluate the distribution and composition of the extracellular cartilage matrix in the intervertebral disc.

Statistical analysis

GraphPad Prism v.8.4.0 software was used to calculate the experimental data. All measurement data were shown as mean ± standard deviation. Differences were analyzed using Student’s t-test for comparing two groups and one-way analysis of variance for comparing multiple groups. All experimental data were obtained through independently repeated experiments for three times.