Polygonatum sibiricum polysaccharides regulate chondrocyte inflammation and metabolism via S100A9 inhibition-mediated autophagy promotion to ameliorate osteoarthritis

Abstract

Background

Polygonatum sibiricum (P. sibiricum) polysaccharides (PSP), the main bioactive component in the roots of the medicinal and edible plant P. sibiricum, have been shown to exhibit strong anti-inflammatory activity. However, the role of PSP in alleviating osteoarthritis (OA)-related cartilage degeneration and the underlying mechanism—particularly its association with autophagy—remain elusive. This study aimed to investigate how PSP influences autophagy in the cartilage and chondrocytes, and to clarify the mechanisms involved.

Methods

The molecular weight of PSP was measured using high-performance gel permeation chromatography (HPGPC), and the monosaccharide composition was analyzed using 1-phenyl-3-methyl-5-pyrazolone (PMP)-HPLC. Chondrocytes overexpressing p65/ S100 calcium-binding protein A9 (S100A9) were generated through the use of lentiviral vectors for gene overexpression. Autophagosomes of chondrocytes were detected by transmission electron microscope, and the expression levels of proteins associated with chondrocytes was identified through WB, immunofluorescence and ELISA. In vivo, the C57BL/6 mice model of OA was created by means of partial medial meniscectomy. Overexpressed lentivirus was injected into the joint cavity and transfected to articular cartilage, constructing an S100A9 overexpression model.

Results

The results demonstrated that PSP can ameliorate interleukin-1β (IL-1β)-induced metabolic dysregulation and inflammatory responses in the chondrocyte extracellular matrix by enhancing chondrocyte autophagy, thereby restoring the impaired autophagy induced by IL-1β. RNA-seq analysis revealed that PSP inhibited the expression of S100A9 in IL-1β-induced chondrocytes. Further mechanistic evidence indicated that PSP promoted chondrocyte autophagy via the S100A9/nuclear factor κB (NF-κB) signaling pathway, thus alleviating chondrocyte inflammation and matrix metabolic disorders.

Conclusions

Both in vivo and in vitro evidence supports that PSP promotes chondrocyte autophagy by suppressing the S100A9/NF-κB pathway to achieve the outcome of OA improvement. This discovery highlights the potential of PSP as a valuable natural plant-derived compound in OA treatment.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12906-026-05467-3.

Introduction

Osteoarthritis (OA) leads to chronic pain and dysfunction, which substantially affect the standard of living for patients [1, 2]. The increasing aging of the global population is leading to a rise in both the health and economic impacts of OA. Statistical reports indicate that in 2020, around 595 million people across the world suffered from OA, which made up 7.6% of the entire population. It is projected that the number of individuals affected by OA could increase to 1.045 billion by 2050 [3]. The pathological mechanism of OA is highly complex and involves the interplay of multiple factors, including aging, inflammation, metabolic alterations, and genetic predispositions [4]. The precise pathogenesis of OA remains elusive, however, and this has hindered the development of therapeutic approaches. Current oral medications for the treatment of OA, including non-steroid anti-inflammatory drug, steroids, and opioids, show some efficacy, but they are often accompanied by serious adverse effects that limit their long-term use [5]. A safer and more effective alternative treatment is therefore urgently needed.

Medicinal plants, owing to their broad biological activities and low toxicity, have attracted increasing research interest in the treatment or prevention of OA, and a large body of evidence has demonstrated their therapeutic potential against OA [6]. Polygonatum sibiricum (P. sibiricum) is a versatile herbal plant known for its multifaceted therapeutic benefits and minimal adverse effects on human health; its rhizomes, valued for both their edible and medicinal properties, are highly regarded. P. sibiricum is rich in bioactive constituents, including polysaccharides, saponins, flavonoids and alkaloids, which confer a wide range of health benefits and highlight its considerable potential for nutritional and pharmaceutical applications. Numerous studies have shown that P. sibiricum possesses significant therapeutic potential in enhancing immune function, exerting anti-inflammatory and antioxidant effects, improving bone density, and delaying aging [7]. P. sibiricum polysaccharides (PSP), the principal active components of its mature rhizomes, exhibit a broad spectrum of biological functions, among which anti-inflammatory activity is one of the most well-documented [8, 9]. It has been reported that PSP strongly suppresses the inflammatory response in chondrocytes triggered by interleukin-1β (IL-1β), potentially through inhibition of the nuclear factor κB (NF-κB) signaling pathway [10]. Furthermore, Kuang et al. demonstrated that PSP inhibited the expression of inflammatory cytokines in the cartilage of OA mice and reduced the secretion of inflammatory factors by OA chondrocytes [11]. In vivo experiments have found that PSP alleviates lung pathological changes and reduces the levels of inflammatory factors in bronchoalveolar lavage fluid of lipopolysaccharide (LPS) -induced acute lung injury in mice and rats [12, 13]. Liu et al. further demonstrated through in vitro experiments that the main biological mechanism of PSP anti-inflammatory effect is the inhibition of the toll-like receptor 4 (TLR4)/NF-κB pathway [12]. Zhou et al. demonstrated that PSP could effectively block the activation of the TLR4-MAPK/NF-κB signaling pathway triggered by LPS, thereby inhibiting the polarization of macrophages towards the pro-inflammatory M1 type and promoting their transformation towards the anti-inflammatory M2 type, ultimately achieving a significant anti-inflammatory effect [13]. In addition, PSP was also found to alleviate intervertebral disc degeneration in rats. The mechanism of action might be that PSP can effectively inhibit the expression of inflammatory factors in nucleus pulposus cells induced by H₂O₂ [14]. As OA is a chronic inflammatory joint disease, numerous studies have demonstrated that PSP exhibits potent anti-inflammatory effects both in vivo and in vitro. Therefore, we hypothesize that PSP may serve as adjuvants or substitutes for OA patients. Our previous studies have demonstrated that PSP exerts anti-inflammatory, pro-matrix synthesis, and anti-catabolic effects on knee OA chondrocytes, which may be attributed to its inhibition of toll-like receptor 2 expression and subsequent suppression of NF-κB signaling pathway activation [11]. This leads us to believe that PSP has potentially valuable in the intervention of OA. However, the involvement of PSP in other biological processes in OA remains unclear.

Autophagy is essential to in the biological processes that enable cells to maintain homeostasis [15]. Defects in the regulation of chondrocyte autophagy have been observed in both human OA cases and mouse OA models [16]. Studies have shown that activation of autophagy can alleviate mitochondrial dysfunction in human chondrocytes, and autophagy can be upregulated to protect mice from OA by inducing loss of mammalian target of rapamycin (mTOR) function [17, 18]. Thus, inducing autophagy in chondrocytes could serve as a possibly efficient therapy for OA. Li et al. found that the extract of P. sibiricum could exert anti-cancer activity by promoting autophagy in Ishikawa cells [19]. A study demonstrated that PSP elicits autophagic responses in endometrial cancer cells [20]. However, up to now, no studies have explicitly shown that PSP can induce autophagy in chondrocytes.

Considering the advantageous role of autophagy OA, we proposed that PSP might stimulate chondrocyte autophagy, thus suppressing the development of OA. This research examined the influence of PSP on IL-1β-induced autophagy levels in chondrocytes, as well as its specific impact on autophagy in the cartilage of mice with OA induced by destabilization of the medial meniscus (DMM). The purpose of this research was to assess how PSP influences chondrocyte autophagy and to explore the mechanisms involved.

Methods

Molecular weight detection

The PSP used in this experiment was sourced from Shanghai Yuanye Biotechnology Co., Ltd. PSP uniformity and relative molecular mass were analyzed using high-performance gel permeation chromatography (HPGPC). The experimental setup utilised dextran standards with molecular weights of 1, 5, 12, 25, 50 and 150 kDa. A calibration curve was plotted, and molecular weight values were calculated using a linear regression equation.

Monosaccharide composition detection

The composition of polysaccharides was analyzed using high-performance liquid chromatography (HPLC). Samples containing polysaccharides were hydrolyzed with trifluoroacetic acid for 4 h. Upon evaporation of the solvent, the residue was redissolved in distilled water to prepare a polysaccharide hydrolysate. The hydrolysate was then mixed with a 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution and sodium hydroxide solution, derivatized in a water bath, terminated with dilute hydrochloric acid after the reaction, extracted with chloroform, filtered through a membrane filter to obtain the sample for analysis, and subsequently analyzed by HPLC. Qualitative and quantitative analysis of monosaccharide types was performed by comparing the retention times and peak areas of monosaccharides in the sample with those of monosaccharide standards. The standard monosaccharide mixture includes D-galactose (D-Gal), L-fucose (L-Fuc), D-arabinose (D-Ara), D-glucose (D-Glc), D-rhamnose (D-Rha), D-xylose (D-Xyl), D-mannose (D-Man), D-fructose (D-Fru), D-galacturonic acid (D-GalA), D-glucuronic acid (D-GlcA), D-ribose (D-Rib), and D-mannuronic acid (D-ManA).

Cell culture, characterization and processing

Human primary articular chondrocytes (Procell Technology, China) were cultured in a constant temperature incubator at 37 °C and maintained at full humidity. Immunofluorescence staining was carried out to validate the expression of collagen II, which helped to confirm the identity of the chondrocytes. For the experiments, third-passage chondrocytes were selected and pre-treated with PSP for a duration 2 h, followed by exposure to 10 ng/mL IL-1β (PeproTech EC, UK) for 24 h.

Cell counting kit-8 (CCK-8) assay

The digested cells were seeded and cultured in 96-well plates at 5000 cells per well. After addition of drug treatment 2, fresh culture medium was replaced and 20 µL CCK-8 (Beyotime Biotechnology, China) solution was mixed for every 200 mL of culture medium. Incubate 96-well plates in an incubator for 2 h to ensure that there are no bubbles in the wells. Set the absorbance value of the microplate reader (Thermofisher, USA) to 450 nm and calculate the cell activity of each group.

Transmission electron microscope (TEM)

Cells were harvested, centrifuged at 300 × g for 5 min, washed twice with PBS, and the cell was collected. The cell was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at 4 °C, followed by post-fixation with 1% osmium tetroxide in the same buffer for 1 h at room temperature. After buffer rinses, the fixed specimen was dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%; 10 min each), followed by two changes of 100% acetone. The dehydrated samples were infiltrated and embedded in epoxy resin and polymerized at 60 °C for 48 h. Ultrathin Sects.  (60–80 nm) were cut with an ultramicrotome and collected on copper grids. The sections were first stained with 2% uranyl acetate in saturated ethanol solution for 8 min in the dark, washed three times with 70% ethanol and three times with ultrapure water, then stained with 2.6% lead citrate solution for 8 min in a CO₂-free environment, followed by three rinses with ultrapure water. After gentle blotting with filter paper, the grids were air-dried overnight at room temperature. Imaging was performed using a TEM (Hitachi, Japan), and images were acquired for analysis.

ELISA

Cell culture supernatant was collected, centrifuged for 10 min, and the resulting supernatant was used for detection. The kit was equilibrated to room temperature for 60 min, after which a standard curve was constructed. The test solutions were added to the wells, the plate was sealed, and incubated at 37 °C for 2 h. The liquid in the wells was then discarded, detection antibody was added, and the plate was incubated at 37 °C for 1 h. The plate was subsequently washed five times; HRP-conjugated working solution was added and incubated at 37 °C for 30 min in the dark. After washing, 100 µL of chromogenic substrate solution was added to each well, and the plate was incubated at room temperature for 15 min in the dark. Stop solution was added, and the absorbance at 450 nm was measured using a microplate reader within 10 min. The concentration of the target protein in each sample was calculated from the standard curve. The kit is sourced from Jingmei Biotechnology: interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α).

Western blot (WB)

After total protein extraction, protein concentrations were determined using a BCA assay, and samples were diluted to ensure equal protein loading across groups. The proteins were then mixed with 5× SDS loading buffer, boiled for 5 min, and stored at − 80 °C until use. Equal amounts of protein were separated by SDS-PAGE on an appropriate percentage polyacrylamide gel and subsequently transferred onto a PVDF membrane pre-activated with ethanol. The membrane was blocked with 5% non-fat dry milk in TBST for 2 h at room temperature, followed by incubation with the primary antibody at 4 °C for approximately 15 h. After three washes with TBST (10 min each), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 90 min at room temperature. The membrane was washed again three times with TBST, and immunoreactive bands were visualized using enhanced chemiluminescence substrate and detected with an imaging system(Bio-Rad, USA). Primary antibodies acquired from Affinity: anti-matrix metalloproteinase 13 (MMP13), anti-p65, anti-p-p65, anti-i kappaB kinase α (IκBα), anti-p-IκBα, anti-collagen II, anti-protein light chain 3 (LC3) and anti-p62. The Primary antibodies acquired from Proteintech: anti-S100 calcium-binding protein A9 (S100A9).

Immunofluorescence (IF)

Cell-seeded coverslips were rinsed three times with pre-cooled PBS (30 s each) and then fixed with 4% paraformaldehyde for 15 min at room temperature. Following fixation, the coverslips were washed thoroughly with PBS (three times, 5 min each) and permeabilized with Triton X-100 for 10 min at room temperature. After permeabilization, the coverslips were washed again three times with PBS (5 min each) and blocked with 5% BSA blocking solution for 60 min at room temperature in the dark. The primary antibody was then evenly applied onto the coverslip surface and incubated overnight at 4 °C in a humidified chamber. The coverslips were washed three times with PBS, and fluorophore-conjugated secondary antibody was added and incubated for 60 min at room temperature in the dark. Following three additional PBS washes, nuclei were counterstained with 1 µg/mL DAPI aqueous solution for 5 min at room temperature. Finally, the coverslips were mounted onto clean glass slides with anti-fade mounting medium and left to cure overnight at room temperature in the dark. The samples were observed and imaged using a fluorescence microscope (Leitz, Germany). Primary antibodies acquired from Affinity: anti-MMP13, anti-collagen II, anti-p65, anti-LC3 and anti-p62. Primary antibodies acquired from Proteintech: anti-S100A9.

qRT-PCR and RNA-Seq

Total RNA was extracted from chondrocytes using TRIzol (Invitrogen). After determining the RNA concentration and purity, cDNA was synthesized from RNA using reverse transcriptase and primers. The qPCR reaction system was prepared and amplified in a real-time fluorescence quantitative PCR instrument. The relative expression of the target gene was calculated by the 2^−ΔΔCt method. The details are shown in Table S1.

RNA sequencing (Illumina 3000) was conducted by Shanghai Ouyi Biotechnology Co., Ltd., based on the total RNA extracted in this study. Briefly, after the constructed cDNA library passed quality control, high-throughput sequencing was performed using the Illumina sequencing platform. Raw sequencing data were subjected to quality control using FastQC. Clean reads were then aligned to the reference genome to determine gene expression levels and perform differential gene expression analysis. Subsequently, functional enrichment analyses (GO and KEGG) were carried out to identify potential target genes.

Lentivirus transduction

Overexpression of S100A9 and p65 was achieved in vitro. The lentiviral titer used in this study was 1 × 10⁸ TU/mL. Prior to formal transduction, the optimal multiplicity of infection (MOI) was determined to be 20 by testing a gradient of MOIs. Chondrocytes were seeded into culture plates and cultured until they reached 30–50% confluence. The medium was removed, fresh medium was added, and lentivirus was slowly added at an MOI of 20 together with 6 µg/mL polybrene. 24 h post-infection, the virus-containing supernatant was replaced with fresh complete medium. GFP expression was monitored daily under a fluorescence microscope. When the transduction efficiency exceeded 80% and the cell monolayer reached 60–70% confluence, the culture medium was replaced with medium containing 2 µg/mL puromycin, and the cells were cultured for 48 h to select stably transduced cells. Parts of cells were harvested to confirm the gene overexpression effect via WB and immunofluorescence analysis. Concurrently, the successfully transfected cells were preserved at -80 °C for later experimental use. The lentivirus supplied by Hanheng Bioscience (Shanghai, China): overexpression lentivirus Hblv-h-S100A9-3×flag-ZsGreen-PURO (Lv-S100A9), overexpression lentivirus Hblv-h-p65-3×flag-ZsGreen-PURO (Lv-p65) and negative control lentivirus Hblv-h-ZsGreen-PURO (Lv-NC).

Animals and models

The experimental animals were 36 six-week-old male C57BL/6 mice. Maintain environmental conditions within a temperature range of 23–25 °C and with a relative humidity of 60%. Following a one-week acclimatization period during which no abnormalities were observed, the experimental procedures commenced. In this study, DMM was employed to create a mouse model for OA [21]. Based on the principles of animal welfare and experimental feasibility, all mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) before euthanasia. After confirming that the mice were completely anesthetized, cervical dislocation was used for euthanasia. All C57BL/6 mice required for the experiment were ordered and obtained from the Animal Experiment Center of Hunan University of Chinese Medicine [Animal License No. SCXK (Hunan, 2019–0004)]. The Ethics Committee of Hunan University of Chinese Medicine approved the animal experiments conducted in this study, Ethical code number: LLBH-202,304,060,001.

S100A9-overexpression mice

To induce S100A9 overexpression in vivo, 10 µL (1 × 10⁸ TU/mL) of S100A9-overexpressing lentivirus was injected into the mouse knee joint. Intra-articular injections were administered on the first day post-modeling and repeated daily for two consecutive days. S100A9 protein levels in cartilage tissue were evaluated by WB and immunohistochemistry to verify successful overexpression. The 18 mice were divided into four groups: model, Lv-NC, Lv-S100A9, PSP, PSP + Lv-NC, and PSP + Lv-S100A9 groups.

Immunohistochemistry (IHC) of tissues

Mouse knee joint tissue sections were baked, dewaxed, and then hydrated using a gradient of ethanol. The slices were immersed in a sodium citrate buffer solution to facilitate antigen retrieval. Appropriate amount of serum was added and incubated for 30 min at room temperature. The primary antibody was added and incubated at 4 °C in a humid environment for 12 h. After thorough washing, the secondary antibody was then introduced and incubated for 30 min. Diaminobenzidine staining solution was added, and the slides were observed under a microscope. Image J software was used for data analysis of the acquired image results.

Histomorphological analysis of cartilage tissue

Knee joint cartilage specimens were fixed in 4% paraformaldehyde (pH 7.4) at 4 °C for 24 h, decalcified with 10% ethylenediaminetetraacetic acid for 2–3 weeks (changing the solution every 3 days), dehydrated with graded ethanol, cleared with xylene, paraffin-embedded, sectioned into 5-µm-thick slices, and mounted on poly-L-lysine-coated slides. After dewaxing, rehydration, and rinsing with distilled water, sections were stained with 0.1% hematoxylin for 5 min, differentiated with 1% acetic acid for 10 s, stained with 0.1% Safranin O for 10 min, rapidly dehydrated and cleared, mounted with neutral binder, and finally observed under a microscope. The degree of cartilage damage in mouse knee joints was quantitatively assessed using the histopathological scoring criteria established by the Osteoarthritis Research Society International (OARSI) [22].

Statistical analysis

To examine the difference in means between two independent groups, the t-test was employed. For comparisons involving multiple groups, one-way ANOVA was used, followed by Tukey’s post hoc analysis. For measures that did not satisfy the assumptions of normality or homogeneity of variance, the rank sum test was employed. The Kruskal-Wallis H test was used to compare nonparametric data. A p below 0.05 was regarded as suggesting statistically significant results. All data were derived from at least three biological replicates. All statistical analyses were performed with the aid of SPSS software (version 26.0; IBM, USA).

Result

Composition analysis of PSP

As shown in Fig. 1A, PSP exhibits two distinct peaks in its molecular weight distribution, with corresponding average molecular weights (Mw) of 8.73 kDa and 1.95 kDa, accounting for 41.52% and 58.48% of the total, respectively. This suggests that the PSP used in this study is a heterogeneous polysaccharide. Monosaccharide composition analysis indicated that PSP is primarily composed of Rha, Ara, Gal, Glc, Man, GalA and GlcA, with a molar ratio of 1:1.54:6.38:30.62:3.15:3.43:1.81 (Fig. 1B and C).

Fig. 1.

Molecular weight and monosaccharide composition of PSP. A Elution curve of PSP in HPGPC. B Chromatograms of monosaccharide standards. C Chromatograms of PSP. The peaks correspond to the following monosaccharide standards: (1) Fuc, (2) Rha, (3) Ara, (4) Gal, (5) Glc, (6) Xyl, (7) D-Man, (8) Fru, (9) Rib, (10) GalA, 11. GlcA, 12. ManA

PSP promotes IL-1β-induced chondrocyte autophagy

Chondrocytes were treated with PSP at gradient concentrations (0, 50, 100, 250, 500, and 1000 µg/mL) first, and the optimal concentration of PSP for chondrocyte intervention was identified to be 500 µg/mL (Fig. 2D). TEM observations revealed that the number of autophagy-associated vesicles was significantly reduced in IL-1β-induced chondrocytes compared to untreated controls. Notably, PSP treatment significantly restored the number of autophagy-associated vesicles (Fig. 2E). The expression of p62 is generally recognized to be negatively correlated with autophagic activity [23]. WB and IF analyses in our study demonstrated that PSP reversed the IL-1β-induced upregulation of p62 expression in chondrocytes (Fig. 2F, G, I and K). Additionally, LC3-II is a critical component of autophagosomes, and its protein expression in chondrocytes was shown to be markedly inhibited by IL-1β, an effect that was partially rescued by PSP administration (Fig. 2F, H, J and L) (Fig. S1A).

Fig. 2.

Effect of PSP on IL-1β-induced chondrocyte autophagy. A The intervention process of cells. B Representative images of chondrocyte morphology in the control and model groups. C Representative image of chondrocyte collagen II IF identification. D Changes in chondrocyte viability following 24 h of exposure to varying concentrations of PSP. E Transmission electron microscopy of autophagic vesicles in chondrocytes. F-H WB detection image and semi-quantitative analysis of p62 and LC3-II protein levels. I-L IF detection image and semi-quantitative analysis of p62 and LC3-II protein levels. (n = 3). *p < 0.001, **p < 0.01, ***p < 0.05

PSP regulates inflammation and metabolism in chondrocytes by promoting autophagy

To further investigate whether PSP modulates inflammation and metabolic alterations in OA model chondrocytes by promoting autophagy, we used an autophagy inhibitor, i.e., 3-methyladenine (3MA), to selectively block autophagic responses in chondrocytes. Cells were assigned to three groups: the IL-1β group received IL-1β alone for 24 h. The IL-1β + PSP group was pretreated with PSP for 2 h, followed by co-treatment with IL-1β and PSP for 24 h. The IL-1β + PSP+3MA group was pretreated with PSP plus 3MA (1 mg/mL) for 2 h, followed by treatment with IL-1β and PSP in the absence of 3MA for 24 h. The results showed that 3MA treatment significantly suppressed PSP-induced upregulation of collagen II protein (Fig. 3A and B) and LC3-II expression (Fig. 3A and D) (Fig. S1B). Additionally, 3MA reversed the PSP-mediated downregulation of MMP13 (Fig. 3A and C) and p62 (Fig. 3A and E) levels in chondrocytes by suppressing autophagy. Consistently, IF analysis confirmed these findings (Fig. 3H-O). Moreover, autophagy inhibition significantly attenuated the ability of PSP to reduce IL-1β-induced secretion of IL-6 and TNF-α in chondrocytes (Fig. 3F and G).

Fig. 3.

PSP regulates IL-1β-induced inflammation and metabolism in chondrocytes by promoting autophagy. A-E WB detection image and semi-quantitative analysis of p62 and LC3-II protein levels. F, G TNF-α and IL-6 secretion by chondrocytes was assayed using ELISA. H-O IF detection image and semi-quantitative analysis of p62 and LC3-II protein levels. (n = 3). *p < 0.001, **p < 0.01, *** p < 0.05

PSP modulates autophagic responses in chondrocytes by inhibiting NF-κB

Accumulating evidence indicates that activation of the NF-κB signaling pathway plays an inhibitory role in the cellular autophagy process [24, 25]. To determine whether PSP promotes chondrocyte autophagy by inhibiting NF-κB, we established NF-κB p65-overexpressing chondrocytes (Fig. 4A-D). WB analysis revealed that p65 overexpression in chondrocytes markedly suppressed the PSP-induced upregulation of LC3-II expression (Fig. 4H and J) (Fig. S1C) and reversed the PSP-mediated downregulation of IL-1β-induced p62 protein levels in chondrocytes (Fig. 4H and I). We then detected the expression of p62 and LC3 in chondrocytes via IF. Consistent with the WB results, p65 overexpression was shown to attenuate the effects of PSP in inhibiting the p62 expression and promoting the LC3 expression (Fig. 4E-G).

Fig. 4.

PSP modulates IL-1β-induced autophagic response in chondrocytes by inhibiting NF-κB. A-D WB and IF detection of p65 protein expression in lentivirus transfected chondrocytes. E-G IF detection image and semi-quantitative analysis of p62 and LC3-II protein levels. H-J WB detection image and semi-quantitative analysis of p62 and LC3-II protein levels. (n = 3). *p < 0.001, **p < 0.01, ***p < 0.05

PSP promotes chondrocyte autophagy by inhibiting the S100A9/NF-κB signaling pathway

RNA-seq was performed to identify potential therapeutic targets after PSP intervention in chondrocytes, with IL-1β-stimulated chondrocytes as the model group and IL-1β-stimulated chondrocytes treated with PSP as the treatment group. A total of 15,026 and 14,811 genes were detected in the model and treatment groups, respectively. Based on the criteria of a p < 0.01 and a fold change threshold of 2.5, 178 differentially expressed genes (DEGs) were identified. Compared to the model group, 166 DEGs were downregulated and 12 were upregulated in the treatment group (Fig. 5A and B). GO enrichment analysis showed that “inflammatory response”, “plasma membrane” and “transmembrane signaling receptor activity” were the top-ranked functional categories among the three Level 1 GO terms (Fig. 5C). KEGG enrichment analysis revealed that the DEGs were most enriched in “Carbohydrate metabolism”, “Infectious disease: bacterial”, “Signal transduction”, “Immune system”, and “Transport and catabolism” (Fig. 5D and E). Given that PSP exerts a regulatory effect on chondrocyte inflammation and catabolism by inhibiting autophagy, we selected 27 DEGs from the GO term “inflammatory response” and 20 DEGs from the KEGG category “Transport and catabolism”, combining them into a set of DEGs of interest (Fig. 5F). After eliminating duplicate genes, there were 39 DEGs of interest in total, among which the top 10 genes with the highest p value were further selected for qRT-PCR validation, and S100A9 was identified as the target gene with the most significant expression change in the PCR results (Fig. 5G). WB analysis confirmed that the S100A9 protein expression was upregulated in IL-1β-induced chondrocytes compared to the control group, and PSP intervention reversed its expression in chondrocytes (Fig. 5H and I).

Fig. 5.

RNA-seq was used to explore the therapeutic target after PSP intervention in chondrocyte. A Common gene screening process. B Volcano plots of differentially expressed genes (DEGs) between Model group and Treatment group. C GO classification chart. D Bar chart of KEGG pathway classification. E Bubble chart illustrating the functional enrichment of KEGG pathways. F Heat mapping of 39 genes screened according to GO and KEGG analysis. G The top 10 ranked (by P-value) genes among the 39 genes screened were selected for qRT-PCR analysis. H, I WB detection image and semi-quantitative analysis of S100A9 protein levels. *p < 0.001, **p < 0.01

To further confirm whether PSP regulates chondrocyte autophagy by inhibiting the S100A9/NF-κB signaling pathway, we evaluated the effect of PSP on chondrocyte autophagy by constructing S100A9-overexpressing chondrocytes (Fig. 6A-D). WB results showed that S100A9 overexpression counteracted the PSP-mediated downregulation of p-p65 and p-IκBα levels in IL-1β-induced chondrocytes (Fig. 6E, H and I). Additionally, S100A9 overexpression weakened the ability of PSP to reduce the IL-1β-induced p62 protein expression and increase the LC3-II protein expression in chondrocytes (Fig. 6E, J and K) (Fig. S1D). Moreover, S100A9 overexpression also significantly attenuated the ability of PSP to promote the collagen II protein expression and inhibit the MMP13 protein expression in chondrocytes (Fig. 6E-G). ELISA results showed that S100A9 overexpression attenuated the effect of PSP in reducing levels of inflammatory factors (IL-6 and TNF-α) in IL-1β-induced chondrocytes (Fig. 6L and M).

Fig. 6.

PSP regulates IL-1β-induced autophagic response through inhibition of S100A9/NF-κB signaling pathway. A-D WB and IF detection of S100A9 protein expression in lentivirus transfected chondrocytes. E-K WB detection image and semi-quantitative analysis of collagen II, MMP13, p62, LC3-II, p65, p-p65, IκBα and p-IκBα protein levels. L, M TNF-α and IL-6 secretion by chondrocytes was assayed using ELISA. (n = 3). *p < 0.001, **p < 0.01, ***p < 0.05

PSP promotes autophagy in cartilage of DMM-induced knee OA mice by inhibiting S100A9

We further investigated how PSP modulates the autophagy responses of chondrocytes in vivo. Specifically, DMM-induced OA mice were set as the model group, while mice receiving sham operation with physiological saline treatment via gavage were set as the control group. Then, an experimental group was established by treating DMM-induced OA mice with PSP via gavage (initial PSP treatment started 3 days before DMM modeling, once daily, continuing for 4 weeks after DMM surgery) (Fig. 7A). Four weeks after DMM surgery, the cartilage tissues in the model group exhibited disorganization and severe damage, while the damage to the articular cartilage tissues was obviously improved in the experimental group (Fig. 7B). WB results showed that the LC3 II expression was significantly downregulated, whereas the p62 expression was significantly upregulated in the cartilage tissue of the model group (Fig. 7C-E) (Fig. S1E). PSP intervention significantly increased the LC3-II expression in OA mice (Fig. 7C and E). The expression of p62 protein in the articular cartilage of OA mice pretreated with PSP was significantly higher than that in OA mice without PSP treatment (Fig. 7C and D). Additionally, the expression of S100A9 in the cartilage tissue of the model group was significantly higher than that in the control group, and PSP treatment significantly inhibited the expression of S100A9 protein (Fig. 7C and F). Consistent with these findings, IHC results revealed similar trends (Fig. 7G-J).

Fig. 7.

PSP promotes DMM-induced autophagic response in knee cartilage of OA mice. A Flowchart of animal experiments. B Safranin O-fast green staining image of knee joint cartilage(n = 6). C-F WB detection image and semi-quantitative analysis of S100A9, p62 and LC3-II protein levels. G-J IHC detection image and semi-quantitative analysis of S100A9, p62 and LC3-II protein levels. (n = 3). *p < 0.001, **p < 0.01, ***p < 0.05. F: femur; T: tibia; M: meniscus; FC: femur cartilage; TC: tibia cartilage

PSP promotes cellular autophagic responses in knee cartilage of OA mice through suppression the S100A9/NF-κB pathway

To further verify the mechanism by which PSP regulates autophagy in chondrocytes in vivo, we injected a S100A9-overexpressing lentivirus into the knee joints of mice to achieve specific overexpression of S100A9 in articular cartilage. The experimental procedure is shown in Fig. 8A. WB and IHC analyses revealed that DMM-induced mouse knee cartilage exhibited significant S100A9 overexpression (Fig. 8B-E). WB results demonstrated that S100A9 overexpression in mouse articular cartilage counteracted the effect of PSP in reducing both cartilage p-p65 and p-IκBα levels. Additionally, the effect of PSP in reducing p62 in cartilage and increasing LC3-II protein was partially counteracted by the overexpression of S100A9 (Fig. 8F-J) (Fig. S1F). IF analysis revealed that S100A9 overexpression impaired the ability of PSP to inhibit p65 protein nuclear translocation (Fig. 8K). IHC analysis demonstrated that S100A9 overexpression impaired the ability of PSP to reduce p62 protein expression and enhance LC3-II protein expression in cartilage (Fig. 8L-N).

Fig. 8.

PSP promotes autophagic responses by inhibiting the S100A9/NF-κB pathway in OA mice. A Flowchart of animal experiments. B-E WB and IHC detection of S100A9 protein expression in lentivirus transfected articular cartilage. F-J WB detection image and semi-quantitative analysis of p62, LC3-II, p65, p-p65, IκBα and p-IκBα protein levels. K immunofluorescence detection image of p65 protein. L-N Immunohistochemistry detection of p62 and LC3-II protein expression in articular cartilage. (n = 3). F: femur; T: Tibia; M: meniscus. (n = 3). * P < 0.001, ** P < 0.01

Discussion

Autophagy is not only a fundamental mechanism for cellular self-protection but also a crucial component in maintaining cell survival. Previous studies have shown that as OA progresses, the autophagic capacity of chondrocytes often decreases, and it has been speculated that this reduction may promote cartilage degeneration and exacerbate disease progression [26]. Another study indicated that in the late stages of OA, chondrocytes exhibit reduced autophagy and enhanced apoptosis [27]. Mechanistically, reduced autophagy may lead to the accumulation of damaged organelles and macromolecules within chondrocytes, thereby impairing chondrocyte survival and promoting age-related OA progression [28]. Therefore, promoting chondrocyte autophagy has become a potential approach for treating OA. IL-1β induction is a commonly used method for preparing OA chondrocyte models. In terms of inhibiting chondrocyte autophagy, IL-1β has been demonstrated to induce chondrocyte autophagy dysfunction and promote apoptosis in vitro [29], while IL-1β receptor antagonists can restore chondrocyte autophagy and mitigate extracellular matrix degradation [30]. In the present study, using chondrocytes as an experimental model, we found that PSP significantly activated the autophagic response in IL-1β-induced chondrocytes. Furthermore, PSP enhanced chondrocyte anabolism and suppressed catabolism and inflammation by promoting autophagy.

During the formation of autophagosomes, cytosolic LC3-I is conjugated to phosphatidylethanolamine through a ubiquitin-like conjugation reaction that involves Atg7 (functioning as the ubiquitin-activating enzyme) and Atg3 (acting as the ubiquitin-conjugating E2 enzyme variant), resulting in the generation of the lipidated form of LC3, known as LC3-II [31]. Therefore, when autophagy is impaired, autophagosomes decrease and LC3 expression is reduced. Our research noted a reduction in LC3-II expression in chondrocytes stimulated by IL-1β, whereas treatment with PSP reversed the decline in LC3-II expression in these cells. SQSTM1/p62 serves as a multifunctional protein that interacts with LC3 in autophagic vesicles, functioning as a selective autophagy adaptor. It ultimately facilitates the delivery of ubiquitinated substrates to the autophagic lysosome for degradation [31]. SQSMT1/p62 is essential for the removal of ubiquitinated proteins from the cytoplasm during autophagy, and its levels are typically inversely correlated with autophagic activity [32]. In this study, we observed that the expression levels of p62 in the articular cartilage of OA model mice and in chondrocytes from OA models were significantly increased compared to the blank control group. Treatment with PSP significantly reduced the expression of p62 in cartilage and chondrocytes.

Currently, therapies targeting autophagy in cartilage are being explored as promising new targets for OA treatment [33]. In vivo studies have demonstrated that rapamycin can alleviate cartilage damage in OA model mice by enhancing autophagy via the inhibition of mTORC1 [34]. In addition, another study found that intra-articular injection of parathyroid hormone-(1–34) can activate autophagy in chondrocytes and alleviate cartilage degeneration [35]. Takayama et al. induced OA in the knees of mice using DMM and found that joint cartilage autophagy-related markers were activated after intra-articular injection of rapamycin, resulting in improved joint cartilage degeneration [36]. Similarly, we used DMM to construct a mouse OA model and found that the articular cartilage in the knee OA model showed weakened autophagy activity, which was partially restored in the cartilage after PSP intervention. These results indicate that PSP protects cartilage by promoting autophagy in OA articular chondrocytes in mice.

The NF-κB pathway not only regulates cellular inflammatory responses and cell survival, but also influences the process of autophagy through multiple mechanisms [37, 38]. Extensive evidence indicates that NF-κB interacts with autophagy during the pathogenesis of OA [39, 40]. Wang et al. found that blocking IL-1β can restore autophagy in chondrocytes and reduce extracellular matrix degradation [41]. Liu et al. demonstrated that vitamin D inhibits chondrocyte NF-κB phosphorylation and nuclear translocation, by modulating the AMPK/mTOR signaling pathway, it enhances autophagy and exerts a protective effect [42]. Yao et al. reported that cepharanthine restores chondrocyte autophagy by suppressing the activation of the NF-κB pathway, thereby reducing cartilage degradation and protecting against OA [39]. Moreover, Li et al. revealed that kojic acid alleviates OA-related inflammation by inhibiting the activated NF-κB and PI3K/AKT/mTOR signaling pathways, consequently restoring impaired chondrocyte autophagy [43]. Our study indicates that NF-κB acts as an upstream signaling molecule regulating chondrocyte autophagy, and the mechanism by which PSP improves IL-1β-induced impairment of chondrocyte autophagy may be related to its inhibition of the NF-κB signaling pathway.

S100A9, a member of the S100 protein family, as a Ca2 + binding protein, has been proven to mediate the activation and maintenance of inflammation [44, 45]. In the articular cartilage of human knee OA patients, the expression of S100A9 is significantly elevated, and the serum expression level of S100A9 is positively correlated with the formation of OA osteophytes [46, 47]. Our experiments found that the expression level of S100A9 in chondrocytes induced by IL-1β was higher than that in the control group, which is consistent with the research results of Zreiqat et al. [48]. S100A9 has been found to induce the production of inflammatory factors by macrophages in arthritis through activating the NF-kappa B signaling pathway [49]. In addition, inhibiting S100A9 can improve pain related to inflammation [50]. Our research has found that PSP can inhibit the expression of S100A9 in chondrocytes and improve the inflammatory response and matrix metabolism of OA chondrocytes.

Limitations of this study: (1) Some studies have shown that rapamycin can inhibit the activation of NF-κB in chondrocytes by activating autophagy, thereby delaying the progression of OA inflammation [51]. This suggests that there is crosstalk between autophagy and the NF-κB signaling pathway. However, we only investigated whether PSP can influence autophagy through the NF-κB pathway and did not clarify whether PSP can regulate the NF-κB pathway through autophagy to intervene in OA; (2) Additionally, besides chondrocytes, other cell types, such as synovial cells and macrophages, also significantly involved in the development of OA [52]. This research focused solely on the impact of PSP on chondrocytes, and its effects on other cell types remain to be further explored; (3) The quality of PSP selected for this study was inconsistent. To better elucidate the biological functions of polysaccharides, future studies should focus on purifying and characterizing such polysaccharides to eliminate structural heterogeneity and advance their translational applications; (4) This study focused on comparisons across treated groups, further investigation is needed to fully characterize basal autophagy levels and PSP-modulated autophagic responses in chondrocytes under IL-1β stimulation; (5) In addition, further evaluation of the improvement in cartilage phenotype after PSP treatment and establishment of a causal relationship between autophagy regulation and OA disease modification are needed.

Conclusion

In this study, we demonstrated for the first time that PSP can reverse the IL-1β-induced inhibition of chondrocyte autophagy and counteract the progression of OA by suppressing S100A9/NF-κB signaling pathway activation (Fig. 9). Our findings highlight PSP as a promising natural plant-based agent for the prevention and treatment of articular cartilage degeneration in OA.

Fig. 9.

The mechanism diagram

Abbreviations

3MA

3-methyladenine

CCK-8

Cell Counting Kit-8

DMM

Destabilization of the medial meniscus

ELISA

Enzyme-linked immunosorbent assay

HPGPC

High performance gel permeation chromatography

IF

Immunofluorescence

IHC

Immunohistochemistry

IL-1β

Interleukin-1β

IκBα

I kappaB kinase α

LC3

Protein light chain 3

LPS

Lipopolysaccharide

MMP13

Matrix metalloproteinase 13

mTOR

Mammalian target of rapamycin

Mw

Molecular weights

NF-κB

Nuclear factor kappa-B

OA

Osteoarthritis

PI3K

Phosphoinositide 3-kinase

PSP

Polygonatum sibiricum Redoute polysaccharides

S100A9

S100 calcium-binding protein A9

TEM

Transmission electron microscope

WB

Western blot

Authors’ contributions

Shida Kuang contributed to in vivo and in vitro experiments, analyzed the data, prepared figures 1-9 and wrote the main manuscript text. Qiyou Ding contributed to in vitro experiments and data curation. Li Lin contributed to in vitro experiments and analyzed the data. Linhu Li contributed to critically revised the manuscript. Zhewen Liu contributed to analyzed the data. Qinghu He contributed to conceptualization and supervision. Qingfeng Du contributed to project administration, funding acquisition and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Funding

This work was supported by Scientific and Technological Innovation 2030-Major Project (2024ZD0523400). Major Program of health Science and Technology of Guangzhou (2024A031007). Innovation Team Project of Guangdong General Colleges and Universities (2022KCXTD003).

Data availability

The RNA sequencing data is available in the NCBI GEO database under the SRA accession number PRJNA1105975.

Declarations

Ethics approval and consent to participate

The Ethics Committee of Hunan University of Chinese Medicine approved the animal experiments conducted in this study, Animal License No. SCXK (Hunan, 2019-0004), Ethical code number: LLBH-202304060001.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.