Salvianolic acid A facilitates cartilage repair in knee osteoarthritis model rats by promoting WDR5 expression

Abstract

Background

The aim of this study was to investigate the regulation of Salvianolic acid A (SAA) on the chondrogenic differentiation of bone mesenchymal stem cells (BMSCs), and its effect on cartilage repair in knee osteoarthritis (KOA) model rats and the action mechanism.

Methods

Immunohistochemistry was performed to detect collagen type II (COL2A1), MMP13 and caspase-3 (CASP3) expression in cartilage tissues, and Safranin-O/Fast Green staining for cartilage damage. Alcian blue staining was performed to measure chondrogenic differentiation of BMSCs. Chondrocyte apoptosis was detected by using flow cytometry.

Results

SAA treatment significantly attenuated cartilage damage in KOA model rats in a dose-dependent manner, and inhibited chondrocyte apoptosis induced by IL-1β in a dose-dependent manner. Moreover, SAA treatment promoted chondrogenesis-related proteins (COL2A1 and Aggrecan) expression and inhibited catabolism-related proteins (MMP13 and MMP3) expression both in the cartilage tissues from KOA model rat and in the IL-1β-treated chondrocytes. WD repeat domain 5 (WDR5) was a downstream target of SAA, and it facilitated chondrogenic differentiation of BMSCs derived from KOA model rats (KOA-BMSCs). Importantly, the inhibition of SAA treatment to the apoptosis and catabolism of chondrocyte and the promotion of SAA treatment to chondrogenic differentiation of KOA-BMSCs were rescued by silencing WDR5.

Conclusion

Overall, SAA treatment could facilitate cartilage repair via inhibiting the apoptosis and catabolism of chondrocyte and promoting chondrogenic differentiation of KOA-BMSCs by promoting WDR5 expression. Our data suggested that SAA may a potential drug for the treatment of KOA.

Highlights

• •Salvianolic acid A attenuates cartilage damage in KOA rat.
• •Salvianolic acid A inhibits IL-1β-induced chondrocyte apoptosis and catabolism.
• •WDR5 is a downstream target of Salvianolic acid A.
• •WDR5 promotes chondrogenic differentiation of KOA-BMSCs.
• •Salvianolic acid A performs its function via promoting WDR5 expression.

1. Introduction

Knee osteoarthritis (KOA) is a chronic degenerative joint disorder characterized by progressive cartilage degeneration, bone loss, subchondral bone sclerosis and cartilage calcification or mineralisation, and is the most common cause of pain and disability in elderly patients [1,2]. An epidemiological study showed that more than 300 million people in the world were afflicted by osteoarthritis, and KOA accounts for 75 % of the patients with osteoarthritis [3]. There is currently no effective cure for KOA, although nonpharmacologic (for example, exercise and biomechanical intervention, education, weight management, and walking canes) and pharmacologic (for example, paracetamol, non-steroidal anti-inflammatory drugs and glucosamine) therapy methods are generally effective at relieving pain and improving physical function, but they cannot solve the pathologic process fundamentally [[4], [5], [6]]. Chondrocytes play a key role in maintaining the normal function of articular cartilage. Emerging evidence suggests that reducing the apoptosis of chondrocytes to limit the deterioration of chondrocytes is an effective way to treat KOA. In recent years, the chondrogenic differentiation of bone mesenchymal stem cells (BMSCs) has been proven to be a promising treatment for KOA [7]. Inducing the differentiation of BMSCs to chondrocytes is a significant issue that needs to be resolved at present.

Danzikangxi granules is a traditional Chinese medicine, and it is mainly composed of Danshen Root, Rehmannia glutinosa, Dragon's Blood, Achyranthes bidentata Blume, Malaytea Scurfpea Fruit, Doubleteeth Pubescent Angelica, Bajitian, Ramulus Taxilli, Ground Beetle, Root of Frankincense and White Peony. Danzikangxi granules has been reported to be effective in reducing apoptosis of chondrocytes in KOA animal model, and in improving KOA, while the mechanism of action of it is unknown [8,9]. We have analyzed the active ingredients of Danzikangxi granules through BATMAN database. Among the active ingredients, salvianolic acid A (SAA) has been reported to reduce IL-1β-induced apoptosis in chondrocytes in vitro and in KOA model mouse via regulating NF-κB and MAPK signaling pathway [10], indicating that SAA may has great potential for the development of novel drugs to treat KOA.

WD repeat domain 5 (WDR5) is an important component of SET1/MLL histone-methyltransferase complex. WDR5 is widely involved in various biological activities and plays a crucial role in the development of many disorders, mainly cancers [11]. While the function and action mechanism of WDR5 in the occurrence and development of KOA not yet been reported. Francesca Gori and Marie B Demay have found that the expression of WDR5 (also known as BIG-3) was increasing during the chondrogenic differentiation of chondroprogenitor cell line ATDC5 [12], suggesting that WDR5 may participate in chondrogenic differentiation. In the present study, we found that WDR5 is a potential downstream target of SAA using ChEMBL database.

Firstly, we verified the therapeutic function of SAA at different doses in KOA and the inhibition of SAA at different doses to IL-1β-induced catabolism and apoptosis in chondrocytes. Secondly, we investigated whether WDR5 is a downstream target of SAA and mediates the regulation of SAA to catabolism and apoptosis of chondrocytes. Finally, we clarified whether WDR5 promotes the chondrogenic differentiation of the BMSCs derived from KOA model rats, and ensued whether SAA facilitates BMSCs differentiation into chondrocytes by facilitating WDR5 expression. Our experiments would beneficial develop novel idea for the treatment of KOA, and our findings provide a solid evidence base for a novel therapeutic target.

2. Methods and materials

2.1. Animal experiments

Healthy male SD rats (2-month-old; weighing at 180–220 g) were purchased from Beijing HFK Bioscience Co.Ltd. (Beijing, China). After adaptive feeding for 1 week in a room with 12 h light/dark cycle, the rats were divided randomly into five groups (n = 6 for each group): Sham group (administration with the same amount of normal saline via oral gavage), KOA group (single intra-articular injection of 2.0 mg monosodium iodoacetate into the bilateral knee joint of rats, and monosodium iodoacetate dissolved in 40 μl 0.9 % normal saline), KOA + DMSO group (administration with the same amount of DMSO via oral gavage and the same amount of monosodium iodoacetate), KOA + SAA-10 (administration with 10 mg/kg/d SAA for 1 month via oral gavage and the same amount of monosodium iodoacetate) and KOA + SAA-20 (administration with 20 mg/kg/d SAA for 1 month via oral gavage and the same amount of monosodium iodoacetate). Monosodium iodoacetate was purchased from Sigma, St. Louis (MO, USA). SSA was purchased from Winherb (Shanghai, China).

Animal experiments were conducted in accordance with the internationally accepted ethical guidelines. Ethical approval was obtained from the ethics committee of Hunan University of Chinese Medicine (APPROVAL NUMBER LLBH-202311070015). We have performed animal experiments depends on the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health (2011).

2.2. Safranin-O/Fast Green staining

One month after SAA treatment, all rats were anesthetized with 40 mg/kg sodium pentobarbital via intraperitoneal injection. Then, fresh knee samples were dissociated from rats, and were stored in 4 % paraformaldehyde. The knee tissues were embedded in paraffin, and then were cut into thin slices with 5-μm-thick. Safranin-O/Fast Green staining Kit (Solarbio, Beijing, China) was obtained for Safranin-O/Fast Green staining according to the manufacture's instruction. Finally, the Osteoarthritis Research Society International (OARSI) score was evaluated to analyze severity of cartilage degeneration.

2.3. Immunohistochemistry

Immunohistochemistry was carried out to analyze the expression level of Collagen type II (COL2A1), Matrix metalloproteinases 13 (MMP13) and Caspase-3 (CASP3) in the knee cartilage samples. The paraffin slices were dewaxed and rehydrated, and were treated with antigen retrieval using EDTA solution (pH 9.0; Servicebio, Wuhan, China). After that, the slices were incubated with 3 % hydrogen peroxide solution for removing endogenous peroxidase for 25 min at room temperature, and subsequently were blocked with 3 % BSA for 30 min at room temperature. The slices next were maintained with the primary antibodies against COL2A1 (1:100; Abcam, UK), MMP13 (1:100; Abcam) and CASP3 (1:100; Abcam) at 4 °C overnight, and next day, the slices were incubated with the HRP-marked secondary antibodies (Abcam) for 1 h at room temperature. Finally, diaminobenzidine-peroxidase substrate and hematoxylin solution (Solarbio) were maintained with the slices to observe the complexes of antibody-antigen. Three fields were randomly selected from each slice for counting the proportion of positive cells.

2.4. Isolation of primary chondrocytes and cell culture

The knee cartilage samples of SD rats (male, five-days-old) were separated under a sterile environment. After removing the cartilage membrane and fascia and washing the cartilage tissues utilizing sterile PBS, cartilage tissues were cut into pieces, which was digested with 1.5 mg/ml Pronase K (Sigma–Aldrich) for 60 min at 37 °C followed by 1.2 mg/ml collagenase II (Sigma–Aldrich) for 60 min at 37 °C. After that, the primary chondrocytes were resuspended and inoculated into 25-cm² culture flasks. Primary chondrocytes were cultured in DMEM medium (Gibco, Paisley, UK) supplemented with 10 % fetal bovine serum (Clark Bioscience, Claymont, DE, USA) and antibiotics. The culture flasks were placed in an incubator with 5 % CO₂ and the cells were cultured at 37 °C. Primary chondrocytes were used for subsequent experiments from passages 1 to 2.

Primary chondrocytes were treated with 0, 5, 10, 15, 20, 25, and 30 μM SAA for 24 h, or treated with 20 μM SAA for 0, 12, 24, 48, and 96 h. 10 ng/ml IL-1β was used to induce cell injury in primary chondrocytes. In addition, the specific siRNA of WDR5 (si-WDR5), negative control siRNA (si-NC), the plasmid expressing WDR5 and the empty plasmid were obtained from GenePharma (Shanghai, China), and were transfected into primary chondrocytes using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

2.5. CCK-8 assay

Primary chondrocytes were treated with 0, 5, 10, 15, 20, 25, and 30 μM SAA for 24 h, or treated with 20 μM SAA for 0, 12, 24, 48, and 96 h, then, cell viability of the primary chondrocytes was determined using CCK-8 assay kit (Solarbio). Briefly, primary chondrocytes were seeded into 96-well plate at a density of 5000 cells/well. After SAA treatment, 10 μl CCK-8 solution was added into each well and the plates were incubated for 3 h at 37 °C. The OD value of each well was measured using a microplate reader (Thermo Fisher Scientific) at a wavelength of 450 nm.

2.6. Isolation of BMSCs and cell culture

BMSCs were separated from the femurs of healthy SD rats or KOA rats according to the previous literature [13]. The BMSCs at passage 2 were cultured in the OriCell MSC growth medium (RAWMX-90011, Cyagen Biosciences Inc., USA) containing 10 % fetal bovine serum and 1 % penicillin-streptomycin. To evaluate the chondrogenic differentiation ability of the healthy rats-derived BMSCs (Health-BMSCs) and the KOA rats-derived BMSCs (KOA-BMSCs), the chondrogenic differentiation medium was utilized to change the growth medium. After incubation for 14 days, the cells were fixed and stained with Alcian blue.

2.7. Alcian blue staining

Health-BMSCs and KOA-BMSCs were inoculated into 6-well plates at a density of 1 × 10⁵ cells/well, and after incubation with chondrogenic differentiation medium for 14 days or transfection of plasmid expressing WDR5, the BMSCs were fixed with 4 % paraformaldehyde and washed once with PBS. Subsequently, the chondrocytes derived from BMSCs were stained with 1 % Alcian blue (Sigma–Aldrich) for 30 min at room temperature, and then were washed once with 0.1 % HCl solution. Finally, the cells were visualized using a light microscope (Nikon Corporation).

2.8. RT-qPCR

Total RNA was extracted from chondrocytes and knee cartilage samples by Trizol reagent (Thermo Fisher Scientific, Waltham, USA), and then were reverse-transcribed to synthesize cDNA using a reverse transcription kit (Promega, Wisconsin, USA) according to the manufacturer's instruction. Subsequently, qRT-PCR was performed utilizing the SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) as the manufacturer's protocol. The gene expression levels were analyzed utilizing the 2^−ΔΔCt method. GAPDH was considered as the internal control.

The primers sequences for COL2A1, Aggrecan, MMP13, MMP3, CASP3, WDR5, SRY-related high-mobility group box 9 (SOX9) and GAPDH as follows: COL2A1: 5′-GCCAGGATGCCCGAAAATTAG-3′ (F) and 5′-GTCACCTCTGGGTCCTTGTTC-3′ (R); Aggrecan: 5′-CTAGCTGCTTAGCAGGGATAACG-3′ (F) and 5′-GATGACCCG CAGAGTCACAAAG-3′ (R); MMP13: 5′-AGCAGGTTGAGCCTGAACTGT-3′ (F) and 5′-GCAGCACTGAGCCTTTTCACC-3′ (R); MMP3: 5′-ACTCCCTGGGACTCT ACCAC-3′ (F) and 5′-GGTACCACGAGGACATCAGG-3′ (R); CASP3: 5′-GTATGCT TACTCTACCGCACCC-3′ (F) and 5′-CAGGGAGAAGGACTCAAATTC C-3′ (R); SOX9: 5′-AGCACAAGAAAGACCACCCC-3′ (F) and 5′-CGCCTTGAAGATGGCG TTAG-3′ (R); GAPDH: 5′-GGCAAGTTCAACGGCACAG-3′ (F) and 5′-CGCCAGT AGACTCCACGACA-3′ (R).

2.9. Western blot

Primary chondrocytes, knee cartilage samples and KOA-BMSCs were lysed with RIPA buffer (Solarbio) on the ice for 15 min, which containing 1 % phosphatase and protease inhibitors. After that, lysate mixtures were further lysed utilizing an ultrasonic disruptor, and the BCA protein assay kit (Solarbio) was utilized to examine the protein concentration. Subsequently, protein samples (15 μg per well) were separated on a SDS-PAGE gels, and then were transferred into PVDF membranes (Millipore, USA). Afterwards, the membranes were blocked with 5 % skim milk at room temperature for 1 h, followed by incubation with corresponding primary antibodies against COL2A1 (1:2000; Abcam), MMP13 (1:2000; Abcam), CASP3 (1:2000; Abcam), Bax (1:2000; Abcam), Bcl-2 (1:2000; Abcam), and WDR5 (1:2000; Abcam) at 4 °C overnight. The membranes were maintained with secondary antibodies for 1 h at room temperature. At last, an enhanced chemiluminescence reagent kit (Solarbio) were used to visualize the protein bands.

2.10. Flow cytometry

Primary chondrocytes were digested with 0.25 % trypsin (Thermo Fisher Scientific) and inoculated into 6-well cell culture plates at a density of 2 × 10⁶ cells/well. After SAA treatment or cell transfection for 48 h, the apoptosis of primary chondrocytes was determined through flow cytometry using the Annexin V FITC apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions. Briefly, cells were incubated with 5 μl Annexin V-FITC and 10 μl propidium iodide (PI) in each well for 15 min at room temperature in the dark. After that, cell fluorescence was detected using a flow cytometer with FlowJoSoftware 10 (FlowJo LLC).

2.11. ChEMBL database

ChEMBL (https://www.ebi.ac.uk/chembl/) is a manually curated database of bioactive molecules with drug-like properties. It brings together chemical, bioactivity and genomic data to aid the translation of genomic information into effective new drugs. We predicted the potential targets of SAA using ChEMBL (version 27) database. The list of these targets was shown in Table 1.

Table 1.

The targets of SAA predicted using ChEMBL (version 27) database.

Target	Target Pref. Name	Confidence 70 %	Confidence 80 %	Confidence 90 %	Activity Threshold
CHEMBL2902	Dihydrofolate reductase	Inactive	Inactive	Inactive	6
CHEMBL2725	Beta-lactamase	Active	Active	Both	6
CHEMBL5062	Coagulation factor X	Empty	Empty	Active	6.5
CHEMBL1947	Thyroid hormone receptor beta-1	Empty	Empty	Inactive	7
CHEMBL2157850	Ubiquitin carboxyl-terminal hydrolase 7	Inactive	Inactive	Inactive	6
CHEMBL226	Adenosine A1 receptor	Inactive	Inactive	Inactive	7
CHEMBL4618	Leukotriene A4 hydrolase	Inactive	Inactive	Inactive	6
CHEMBL1075145	Transitional endoplasmic reticulum ATPase	Empty	Inactive	Both	6
CHEMBL1827	Phosphodiesterase 5A	Empty	Inactive	Inactive	6
CHEMBL4793	Dipeptidyl peptidase IX	Empty	Empty	Inactive	6
CHEMBL3229	Anandamide amidohydrolase	Inactive	Inactive	Inactive	6
CHEMBL5880	Interleukin-2	Active	Active	Active	6
CHEMBL4552	Peripheral-type benzodiazepine receptor	Empty	Inactive	Inactive	6
CHEMBL2888	Metabotropic glutamate receptor 3	Empty	Inactive	Both	7
CHEMBL1907596	Neuronal acetylcholine receptor; alpha4/beta2	Inactive	Inactive	Inactive	5
CHEMBL2334	Caspase-3	Inactive	Inactive	Inactive	6
CHEMBL5498	Muscarinic acetylcholine receptor M3	Empty	Inactive	Both	7
CHEMBL4625	Apoptosis regulator Bcl-X	Empty	Empty	Both	5
CHEMBL233	Mu opioid receptor	Empty	Inactive	Inactive	7
CHEMBL1991	Inhibitor of nuclear factor kappa B kinase beta subunit	Empty	Empty	Inactive	7.5
CHEMBL2283	Carbonic anhydrase II	Active	Active	Active	6
CHEMBL5077	Butyrylcholinesterase	Empty	Empty	Inactive	6
CHEMBL2056	Dopamine D1 receptor	Inactive	Inactive	Inactive	7
CHEMBL4828	Synaptic vesicular amine transporter	Inactive	Inactive	Inactive	6
CHEMBL231	Histamine H1 receptor	Inactive	Inactive	Inactive	7
CHEMBL219	Dopamine D4 receptor	Inactive	Inactive	Inactive	7
CHEMBL2487	Beta amyloid A4 protein	Empty	Empty	Inactive	6
CHEMBL1824	Receptor protein-tyrosine kinase erbB-2	Inactive	Inactive	Inactive	7.5
CHEMBL3969	Carbonic anhydrase VB	Empty	Empty	Inactive	6
CHEMBL1907594	Neuronal acetylcholine receptor; alpha3/beta4	Empty	Inactive	Both	5
CHEMBL2652	Phosphodiesterase 2A	Inactive	Inactive	Inactive	6
CHEMBL3202	Prolyl endopeptidase	Empty	Empty	Both	6
CHEMBL3571	Cannabinoid CB1 receptor	Inactive	Inactive	Inactive	7
CHEMBL5896	Lysine-specific demethylase 4A	Inactive	Inactive	Inactive	6
CHEMBL1907595	Neuronal acetylcholine receptor; alpha4/beta4	Empty	Inactive	Inactive	5
CHEMBL1825	TNF-alpha	Empty	Empty	Inactive	6
CHEMBL3798	Calcitonin gene-related peptide type 1 receptor	Inactive	Inactive	Inactive	7
CHEMBL218	Cannabinoid CB1 receptor	Inactive	Inactive	Inactive	7
CHEMBL224	Serotonin 2a (5-HT2a) receptor	Inactive	Inactive	Inactive	7
CHEMBL6031	Histone-lysine N-methyltransferase, H3 lysine-9 specific 5	Inactive	Inactive	Inactive	6
CHEMBL4801	Caspase-1	Empty	Empty	Inactive	6
CHEMBL2189121	GTPase KRas	Empty	Inactive	Inactive	6
CHEMBL4430	Cytochrome P450 17A1	Empty	Empty	Both	6
CHEMBL3313832	Lysine-specific demethylase 4B	Inactive	Inactive	Inactive	6.5
CHEMBL2111432	PI3-kinase p110-delta/p85-alpha	Inactive	Inactive	Inactive	6
CHEMBL4408	Phosphodiesterase 8B	Empty	Empty	Inactive	6
CHEMBL3429	Estrogen-related receptor alpha	Empty	Empty	Both	7
CHEMBL1941	Histamine H2 receptor	Empty	Empty	Both	7
CHEMBL208	Progesterone receptor	Inactive	Inactive	Inactive	7
CHEMBL220	Acetylcholinesterase	Inactive	Inactive	Inactive	6
CHEMBL4150	Enoyl-acyl-carrier protein reductase	Inactive	Inactive	Both	6
CHEMBL234	Dopamine D3 receptor	Inactive	Inactive	Inactive	7
CHEMBL1809	Dihydrofolate reductase	Empty	Empty	Both	6
CHEMBL1835	Thromboxane-A synthase	Inactive	Inactive	Inactive	6
CHEMBL3788	Serine/threonine-protein kinase PLK4	Inactive	Inactive	Inactive	7.5
CHEMBL1821	Muscarinic acetylcholine receptor M4	Inactive	Inactive	Inactive	7
CHEMBL4018	Neuropeptide Y receptor type 2	Inactive	Inactive	Inactive	7
CHEMBL2327	Neurokinin 2 receptor	Inactive	Inactive	Inactive	7
CHEMBL1907591	Neuronal acetylcholine receptor; alpha4/beta4	Active	Active	Active	5
CHEMBL2695	Focal adhesion kinase 1	Empty	Inactive	Inactive	7.5
CHEMBL2326	Carbonic anhydrase VII	Empty	Active	Active	6
CHEMBL3776	Caspase-8	Inactive	Inactive	Inactive	6
CHEMBL1944495	Proteasome subunit beta type-9	Inactive	Inactive	Inactive	6
CHEMBL3004	Multidrug resistance-associated protein 1	Empty	Empty	Empty	6
CHEMBL4780	Acetylcholinesterase	Empty	Empty	Both	6
CHEMBL4794	Vanilloid receptor	Empty	Empty	Inactive	5
CHEMBL235	Peroxisome proliferator-activated receptor gamma	Inactive	Inactive	Inactive	7
CHEMBL1983	Serotonin 1d (5-HT1d) receptor	Empty	Inactive	Inactive	7
CHEMBL1798	Cysteinyl leukotriene receptor 1	Empty	Empty	Inactive	7
CHEMBL1968	Epoxide hydrolase 1	Empty	Empty	Inactive	6
CHEMBL5932	LIM domain kinase 2	Inactive	Inactive	Both	7.5
CHEMBL3199	Acetylcholinesterase	Inactive	Inactive	Inactive	6
CHEMBL1942	Alpha-2b adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL237	Kappa opioid receptor	Inactive	Inactive	Inactive	7
CHEMBL223	Alpha-1d adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL1822	Inosine-5′-monophosphate dehydrogenase 1	Inactive	Inactive	Inactive	6
CHEMBL3012	Phosphodiesterase 7A	Inactive	Inactive	Inactive	6
CHEMBL3589	Adenosine kinase	Inactive	Inactive	Inactive	6
CHEMBL5113	Orexin receptor 1	Inactive	Inactive	Inactive	7
CHEMBL5891	Protein arginine N-methyltransferase 3	Inactive	Inactive	Inactive	6
CHEMBL3991	Coagulation factor VII	Empty	Active	Active	6
CHEMBL1163125	Bromodomain-containing protein 4	Empty	Active	Active	6
CHEMBL4768	Acetylcholinesterase	Empty	Active	Both	6
CHEMBL4973	Excitatory amino acid transporter 2	Empty	Empty	Both	6
CHEMBL222	Norepinephrine transporter	Inactive	Inactive	Inactive	6
CHEMBL236	Delta opioid receptor	Empty	Inactive	Inactive	7
CHEMBL4608	Melanocortin receptor 5	Inactive	Inactive	Inactive	7
CHEMBL1957	Insulin-like growth factor I receptor	Empty	Inactive	Both	7.5
CHEMBL4191	Monoglyceride lipase	Empty	Inactive	Inactive	6
CHEMBL3371	Serotonin 6 (5-HT6) receptor	Inactive	Inactive	Inactive	7
CHEMBL3403	Butyrylcholinesterase	Inactive	Inactive	Inactive	6
CHEMBL2034	Glucocorticoid receptor	Inactive	Inactive	Inactive	7
CHEMBL3464	Nitric oxide synthase, inducible	Empty	Active	Both	6
CHEMBL286	Renin	Inactive	Inactive	Inactive	6
CHEMBL4860	Apoptosis regulator Bcl-2	Empty	Active	Both	5
CHEMBL251	Adenosine A2a receptor	Inactive	Inactive	Inactive	7
CHEMBL4653	Dipeptidyl peptidase IV	Inactive	Inactive	Inactive	6
CHEMBL245	Muscarinic acetylcholine receptor M3	Inactive	Inactive	Inactive	7
CHEMBL1075132	Heat shock protein 75 kDa, mitochondrial	Empty	Active	Both	6
CHEMBL1850	Dopamine D5 receptor	Inactive	Inactive	Inactive	7
CHEMBL2007625	Isocitrate dehydrogenase [NADP] cytoplasmic	Inactive	Inactive	Inactive	6
CHEMBL325	Histone deacetylase 1	Inactive	Inactive	Inactive	6
CHEMBL3048	Nitric-oxide synthase, brain	Inactive	Inactive	Inactive	6
CHEMBL2424	Glyoxalase I	Empty	Empty	Active	6
CHEMBL319	Alpha-1a adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL3706	ADAM17	Inactive	Inactive	Inactive	6
CHEMBL3060	Glycine transporter 2	Empty	Empty	Empty	6
CHEMBL1628481	Apelin receptor	Empty	Empty	Inactive	7
CHEMBL2140	Tryptophan 2,3-dioxygenase	Empty	Inactive	Inactive	6
CHEMBL4531	Galectin-3	Inactive	Inactive	Inactive	6
CHEMBL3510	Carbonic anhydrase XIV	Empty	Inactive	Inactive	6
CHEMBL2163176	Lysine-specific demethylase 5C	Empty	Inactive	Both	6
CHEMBL4530	Coagulation factor XIII	Inactive	Inactive	Inactive	6
CHEMBL1795101	Peptide deformylase	Empty	Empty	Inactive	6
CHEMBL4040	MAP kinase ERK2	Inactive	Inactive	Inactive	7.5
CHEMBL2431	Serine/threonine-protein kinase AKT2	Empty	Inactive	Both	7.5
CHEMBL2366504	Structural capsid protein	Inactive	Inactive	Inactive	6
CHEMBL278	Integrin alpha-4	Empty	Empty	Both	6.5
CHEMBL244	Coagulation factor X	Inactive	Inactive	Inactive	6
CHEMBL4685	Indoleamine 2,3-dioxygenase	Empty	Empty	Both	6
CHEMBL287	Sigma opioid receptor	Inactive	Inactive	Inactive	6
CHEMBL4478	Voltage-gated N-type calcium channel alpha-1B subunit	Empty	Inactive	Inactive	5
CHEMBL3471	Human immunodeficiency virus type 1 integrase	Empty	Inactive	Inactive	6
CHEMBL2035	Muscarinic acetylcholine receptor M5	Inactive	Inactive	Inactive	7
CHEMBL3459	Serotonin 1b (5-HT1b) receptor	Empty	Inactive	Both	6.5
CHEMBL4336	Prostanoid EP3 receptor	Empty	Empty	Both	7
CHEMBL1293197	Acidic mammalian chitinase	Empty	Inactive	Both	6
CHEMBL3991501	Isocitrate dehydrogenase [NADP], mitochondrial	Empty	Inactive	Inactive	6
CHEMBL4644	Melanocortin receptor 3	Inactive	Inactive	Inactive	7
CHEMBL246	Beta-3 adrenergic receptor	Empty	Inactive	Inactive	7
CHEMBL252	Endothelin receptor ET-A	Empty	Empty	Empty	7
CHEMBL3922	Methionine aminopeptidase 2	Empty	Empty	Both	6
CHEMBL332	Matrix metalloproteinase-1	Empty	Empty	Inactive	6
CHEMBL326	Alpha-1d adrenergic receptor	Empty	Empty	Active	7
CHEMBL4718	MAP kinase-interacting serine/threonine-protein kinase MNK1	Empty	Empty	Active	7.5
CHEMBL1795117	Histone-lysine N-methyltransferase, H3 lysine-79 specific	Inactive	Inactive	Inactive	6
CHEMBL5163	Sodium channel protein type III alpha subunit	Active	Active	Active	5
CHEMBL1795116	Protein arginine N-methyltransferase 5	Inactive	Inactive	Inactive	6
CHEMBL3710	Prostanoid EP3 receptor	Empty	Inactive	Inactive	7
CHEMBL5413	Metastin receptor	Empty	Empty	Inactive	7
CHEMBL5375	Hepatitis C virus NS5B RNA-dependent RNA polymerase	Empty	Empty	Both	6
CHEMBL333	Matrix metalloproteinase-2	Inactive	Inactive	Inactive	6
CHEMBL2397	Acetyl-CoA carboxylase 1	Active	Active	Both	6
CHEMBL2208	MAP kinase-activated protein kinase 2	Empty	Empty	Both	7.5
CHEMBL3880	Heat shock protein HSP 90-alpha	Empty	Empty	Both	6
CHEMBL4123	Neurotensin receptor 1	Inactive	Inactive	Inactive	7
CHEMBL1926	Dihydrofolate reductase	Empty	Empty	Both	6
CHEMBL284	Dipeptidyl peptidase IV	Inactive	Inactive	Inactive	6
CHEMBL5017	Serotonin 4 (5-HT4) receptor	Inactive	Inactive	Inactive	7
CHEMBL280	Matrix metalloproteinase 13	Inactive	Inactive	Inactive	6
CHEMBL3884	Sodium/glucose cotransporter 2	Empty	Inactive	Both	6
CHEMBL3108639	Cat eye syndrome critical region protein 2	Empty	Both	Both	6
CHEMBL2366517	Protease	Empty	Empty	Inactive	6
CHEMBL3933	Prostanoid DP receptor	Inactive	Inactive	Inactive	7
CHEMBL3072	Androgen receptor	Inactive	Inactive	Inactive	7
CHEMBL6136	Lysine-specific histone demethylase 1	Empty	Inactive	Inactive	6
CHEMBL2095194	Coagulation factor VII/tissue factor	Empty	Empty	Active	6
CHEMBL4523	Serine/threonine-protein kinase PIM2	Empty	Inactive	Inactive	7.5
CHEMBL2147	Serine/threonine-protein kinase PIM1	Inactive	Inactive	Inactive	7.5
CHEMBL256	Adenosine A3 receptor	Inactive	Inactive	Inactive	7
CHEMBL3649	Xanthine dehydrogenase	Inactive	Inactive	Inactive	6
CHEMBL242	Estrogen receptor beta	Inactive	Inactive	Inactive	7
CHEMBL4683	Fibroblast activation protein alpha	Inactive	Inactive	Inactive	6
CHEMBL3305	Testis-specific androgen-binding protein	Inactive	Inactive	Inactive	6.5
CHEMBL2966	Adenosine deaminase	Active	Active	Active	6
CHEMBL1741208	NACHT, LRR and PYD domains-containing protein 3	Empty	Empty	Both	6
CHEMBL5979	Alkaline phosphatase, tissue-nonspecific isozyme	Inactive	Inactive	Both	6
CHEMBL2094128	Cyclin-dependent kinase 2/cyclin A	Inactive	Inactive	Inactive	7.5
CHEMBL268	Cathepsin K	Inactive	Inactive	Inactive	6
CHEMBL254	Phosphodiesterase 4A	Inactive	Inactive	Inactive	6
CHEMBL1841	Tyrosine-protein kinase FYN	Inactive	Inactive	Inactive	6.5
CHEMBL5366	Poly [ADP-ribose] polymerase 2	Empty	Empty	Both	6
CHEMBL4078	Acetylcholinesterase	Inactive	Inactive	Inactive	6
CHEMBL1795139	Transmembrane protease serine 6	Inactive	Inactive	Inactive	6
CHEMBL3501	Cholecystokinin A receptor	Inactive	Inactive	Inactive	7
CHEMBL4508	Glutaminyl-peptide cyclotransferase	Empty	Active	Active	6
CHEMBL1075269	Adenosine A3 receptor	Empty	Active	Both	7
CHEMBL2622	Aldose reductase	Empty	Empty	Both	6
CHEMBL335	Protein-tyrosine phosphatase 1B	Empty	Empty	Inactive	6
CHEMBL2434	Interleukin-8 receptor B	Empty	Empty	Inactive	7
CHEMBL321	Matrix metalloproteinase 9	Empty	Empty	Both	6
CHEMBL309	Muscarinic acetylcholine receptor M2	Inactive	Inactive	Both	7
CHEMBL5373	Cannabinoid CB2 receptor	Inactive	Inactive	Inactive	7
CHEMBL4086	Prostanoid EP4 receptor	Active	Active	Active	7
CHEMBL4657	Dipeptidyl peptidase VIII	Empty	Empty	Inactive	6
CHEMBL255	Adenosine A2b receptor	Empty	Empty	Inactive	7
CHEMBL269	Delta opioid receptor	Empty	Active	Active	7
CHEMBL5763	Cholinesterase	Inactive	Inactive	Inactive	6
CHEMBL2781	Sodium/hydrogen exchanger 1	Empty	Inactive	Both	6
CHEMBL3883328	Casein kinase II alpha'/beta	Inactive	Inactive	Both	6.5
CHEMBL3486	Dihydroorotate dehydrogenase	Inactive	Inactive	Inactive	6
CHEMBL4699	Isoprenylcysteine carboxyl methyltransferase	Empty	Inactive	Inactive	6
CHEMBL264	Histamine H3 receptor	Inactive	Inactive	Inactive	7
CHEMBL258	Tyrosine-protein kinase LCK	Inactive	Inactive	Inactive	7.5
CHEMBL1865	Histone deacetylase 6	Empty	Empty	Inactive	6
CHEMBL2411	Serotonin 3a (5-HT3a) receptor	Empty	Empty	Both	6.5
CHEMBL4074	Angiotensin-converting enzyme	Empty	Empty	Empty	6
CHEMBL2363	Dihydrofolate reductase	Empty	Empty	Both	6
CHEMBL338	Dopamine transporter	Inactive	Inactive	Inactive	6
CHEMBL2304404	Adenosine A1 receptor	Empty	Inactive	Both	7
CHEMBL2820	Coagulation factor XI	Empty	Empty	Inactive	6
CHEMBL3242	Carbonic anhydrase XII	Empty	Empty	Active	6
CHEMBL5141	Cytochrome P450 26A1	Empty	Empty	Inactive	6
CHEMBL311	Glutamate [NMDA] receptor subunit epsilon 2	Empty	Empty	Active	5
CHEMBL2176771	Complement factor D	Empty	Inactive	Both	6
CHEMBL3714079	G-protein coupled receptor 84	Empty	Active	Both	7
CHEMBL259	Melanocortin receptor 4	Inactive	Inactive	Inactive	7
CHEMBL4465	Acyl coenzyme A:cholesterol acyltransferase 2	Inactive	Inactive	Inactive	6
CHEMBL3478	Phosphodiesterase 5A	Empty	Active	Both	6
CHEMBL2000	Plasma kallikrein	Empty	Empty	Inactive	6
CHEMBL2955	Sphingosine 1-phosphate receptor Edg-5	Empty	Empty	Active	7
CHEMBL3038482	DNA gyrase	Empty	Empty	Empty	6
CHEMBL2016	Coagulation factor IX	Empty	Inactive	Inactive	6
CHEMBL2002	Inosine-5′-monophosphate dehydrogenase 2	Empty	Empty	Inactive	6
CHEMBL5023	p53-binding protein Mdm-2	Inactive	Inactive	Inactive	6
CHEMBL4329	Kappa opioid receptor	Inactive	Inactive	Inactive	7
CHEMBL1906	Serine/threonine-protein kinase RAF	Empty	Active	Active	7.5
CHEMBL3650	Fibroblast growth factor receptor 1	Inactive	Inactive	Inactive	7.5
CHEMBL3081	Aldose reductase	Inactive	Inactive	Both	6
CHEMBL4077	Melanocortin receptor 1	Inactive	Inactive	Inactive	7
CHEMBL1899	Serotonin 3a (5-HT3a) receptor	Inactive	Inactive	Inactive	5
CHEMBL1898	Serotonin 1b (5-HT1b) receptor	Empty	Empty	Active	7
CHEMBL4062	Calpain 1	Empty	Inactive	Inactive	6
CHEMBL1867	Alpha-2a adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL299	Protein kinase C alpha	Empty	Empty	Active	7.5
CHEMBL3321	Monoglyceride lipase	Empty	Empty	Both	6
CHEMBL1921666	DNA gyrase subunit B	Empty	Inactive	Both	6
CHEMBL3632452	Mucosa-associated lymphoid tissue lymphoma translocation protein 1	Empty	Empty	Both	6
CHEMBL1075317	WD repeat-containing protein 5	Empty	Active	Active	6
CHEMBL4462	NAD-dependent deacetylase sirtuin 2	Inactive	Inactive	Inactive	6
CHEMBL3655	Tyrosine-protein kinase SRC	Inactive	Inactive	Both	6.5
CHEMBL2096912	Protein farnesyltransferase	Active	Active	Both	6
CHEMBL2169736	Tyrosyl-DNA phosphodiesterase 2	Empty	Empty	Both	6
CHEMBL276	Muscarinic acetylcholine receptor M1	Active	Active	Active	7
CHEMBL3912	Carbonic anhydrase XIII	Inactive	Inactive	Inactive	6
CHEMBL4072	Cathepsin B	Empty	Empty	Both	6
CHEMBL302	Adenosine A2a receptor	Empty	Active	Active	7
CHEMBL3286	Urokinase-type plasminogen activator	Inactive	Inactive	Inactive	6
CHEMBL1889	Vasopressin V1a receptor	Inactive	Inactive	Inactive	7
CHEMBL1916	Alpha-2c adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL1914	Butyrylcholinesterase	Empty	Empty	Inactive	6
CHEMBL2094135	Gamma-secretase	Empty	Inactive	Inactive	5
CHEMBL1900	Aldose reductase	Empty	Active	Active	6
CHEMBL249	Neurokinin 1 receptor	Inactive	Inactive	Inactive	7
CHEMBL3130	PI3-kinase p110-delta subunit	Active	Active	Active	6
CHEMBL275	Phosphodiesterase 4B	Empty	Empty	Both	6
CHEMBL329	Type-1A angiotensin II receptor	Inactive	Inactive	Inactive	7
CHEMBL2414	C–C chemokine receptor type 4	Empty	Empty	Inactive	7
CHEMBL2372	Catechol O-methyltransferase	Empty	Active	Active	6.5
CHEMBL315	Alpha-1b adrenergic receptor	Empty	Empty	Inactive	7
CHEMBL5145	Serine/threonine-protein kinase B-raf	Inactive	Inactive	Inactive	7.5
CHEMBL2617	Tryptase beta-1	Empty	Active	Both	6
CHEMBL3247	HMG-CoA reductase	Empty	Empty	Inactive	6
CHEMBL3535	Phosphodiesterase 9A	Inactive	Inactive	Inactive	6
CHEMBL2830	Voltage-gated L-type calcium channel alpha-1C subunit	Empty	Active	Both	5
CHEMBL2176774	Bromodomain-containing protein 1	Inactive	Inactive	Inactive	6
CHEMBL1875	Serotonin 4 (5-HT4) receptor	Inactive	Inactive	Inactive	7
CHEMBL1849	Enoyl-[acyl-carrier-protein] reductase	Empty	Empty	Both	6
CHEMBL260	MAP kinase p38 alpha	Inactive	Inactive	Inactive	7.5
CHEMBL2575	Dihydrofolate reductase	Empty	Empty	Inactive	6
CHEMBL274	C–C chemokine receptor type 5	Inactive	Inactive	Inactive	7
CHEMBL5568	Proto-oncogene tyrosine-protein kinase ROS	Inactive	Inactive	Inactive	7.5
CHEMBL2150837	ATPase family AAA domain-containing protein 2	Inactive	Inactive	Both	6
CHEMBL248	Leukocyte elastase	Inactive	Inactive	Inactive	6
CHEMBL1901	Cholecystokinin A receptor	Inactive	Inactive	Inactive	7
CHEMBL3864	Protein-tyrosine phosphatase 2C	Empty	Empty	Both	6
CHEMBL1929	Xanthine dehydrogenase	Empty	Inactive	Inactive	6
CHEMBL2094108	Protein farnesyltransferase	Empty	Inactive	Both	6
CHEMBL3797017	Histone deacetylase 8	Empty	Empty	Empty	6
CHEMBL1907603	Glutamate NMDA receptor; GRIN1/GRIN2B	Empty	Empty	Both	5
CHEMBL3085613	DNA gyrase subunit B	Empty	Inactive	Inactive	6
CHEMBL4361	Induced myeloid leukemia cell differentiation protein Mcl-1	Empty	Empty	Both	6
CHEMBL3712907	Transmembrane domain-containing protein TMIGD3	Inactive	Inactive	Inactive	6
CHEMBL1966	Dihydroorotate dehydrogenase	Empty	Empty	Both	6
CHEMBL4822	Beta-secretase 1	Empty	Inactive	Inactive	6
CHEMBL5282	Cytochrome P450 2A6	Inactive	Inactive	Inactive	6
CHEMBL213	Beta-1 adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL3142	DNA-dependent protein kinase	Empty	Inactive	Inactive	7.5
CHEMBL5137	Metabotropic glutamate receptor 2	Empty	Active	Active	7
CHEMBL5122	Discoidin domain-containing receptor 2	Inactive	Inactive	Inactive	7.5
CHEMBL3023	Sphingosine kinase 2	Inactive	Inactive	Both	6
CHEMBL2329	Dihydrofolate reductase	Empty	Inactive	Both	6.5
CHEMBL3157	Bradykinin B2 receptor	Empty	Empty	Inactive	7
CHEMBL1250375	NADPH oxidase 4	Inactive	Inactive	Inactive	6
CHEMBL206	Estrogen receptor alpha	Empty	Inactive	Inactive	7
CHEMBL2189117	Polycomb protein EED	Inactive	Inactive	Inactive	6
CHEMBL2288	Peptidyl-prolyl cis–trans isomerase NIMA-interacting 1	Empty	Active	Both	6
CHEMBL1781	DNA topoisomerase I	Empty	Empty	Both	6
CHEMBL4835	l-lactate dehydrogenase A chain	Empty	Empty	Inactive	6
CHEMBL210	Beta-2 adrenergic receptor	Inactive	Inactive	Inactive	7
CHEMBL238	Dopamine transporter	Inactive	Inactive	Inactive	6
CHEMBL2424504	Lysine-specific demethylase 5A	Inactive	Inactive	Inactive	6
CHEMBL5493	Free fatty acid receptor 2	Inactive	Inactive	Inactive	6.5
CHEMBL2069156	Kelch-like ECH-associated protein 1	Empty	Empty	Both	6
CHEMBL402	HMG-CoA reductase	Inactive	Inactive	Inactive	6
CHEMBL1255150	G-protein coupled bile acid receptor 1	Inactive	Inactive	Inactive	7
CHEMBL4029	Interleukin-8 receptor A	Inactive	Inactive	Inactive	6.5
CHEMBL4015	C–C chemokine receptor type 2	Inactive	Inactive	Inactive	7
CHEMBL4940	l-lactate dehydrogenase B chain	Empty	Active	Active	6
CHEMBL1804	Somatostatin receptor 2	Inactive	Inactive	Inactive	7
CHEMBL211	Muscarinic acetylcholine receptor M2	Inactive	Inactive	Inactive	7
CHEMBL3137262	LSD1/CoREST complex	Empty	Empty	inactive	6
CHEMBL205	Carbonic anhydrase II	Empty	Empty	Inactive	6
CHEMBL4607	Angiotensin II type 2 (AT-2) receptor	Empty	Empty	Empty	6.5
CHEMBL3815	Squalene synthetase	Empty	Empty	Both	6
CHEMBL3356	Cytochrome P450 1A2	Inactive	Inactive	Inactive	6
CHEMBL1907601	Cyclin-dependent kinase 4/cyclin D1	Empty	Inactive	Inactive	7.5
CHEMBL4398	Purinergic receptor P2Y2	Empty	Active	Active	7
CHEMBL3385	MAP kinase ERK1	Inactive	Inactive	Inactive	7.5
CHEMBL3959	Quinone reductase 2	Inactive	Inactive	Both	6
CHEMBL3024	Serine/threonine-protein kinase PLK1	Inactive	Inactive	Inactive	7.5
CHEMBL4777	Neuropeptide Y receptor type 1	Empty	Inactive	Inactive	7
CHEMBL2104	P2X purinoceptor 4	Empty	Active	Both	5
CHEMBL3774298	Histone acetyltransferase KAT6A	Empty	Inactive	Inactive	6
CHEMBL1801	Plasminogen	Inactive	Inactive	Inactive	6
CHEMBL4979	Sodium/glucose cotransporter 1	Empty	Inactive	Inactive	6
CHEMBL1829	Histone deacetylase 3	Inactive	Inactive	Inactive	6
CHEMBL3780	Small conductance calcium-activated potassium channel protein 3	Inactive	Inactive	Inactive	6.5
CHEMBL3623	Quinone reductase 1)	Inactive	Inactive	Inactive	6
CHEMBL228	Serotonin transporter	Inactive	Inactive	Inactive	6
CHEMBL4414	Plasmepsin 2	Inactive	Inactive	Inactive	6
CHEMBL4372	Anthrax lethal factor	Empty	Inactive	Inactive	6
CHEMBL1907604	Glutamate NMDA receptor; GRIN1/GRIN2A	Inactive	Inactive	Inactive	5
CHEMBL4358	Arachidonate 15-lipoxygenase	Empty	Empty	Empty	6
CHEMBL2073	Tyrosine-protein kinase YES	Active	Active	Active	6.5
CHEMBL1977	Vitamin D receptor	Empty	Inactive	Inactive	7
CHEMBL3430892	Integrin alpha-V/beta-8	Empty	Active	Both	6.5
CHEMBL3594	Carbonic anhydrase IX	Inactive	Inactive	Inactive	6
CHEMBL4204	MAP kinase signal-integrating kinase 2	Active	Active	Both	7.5
CHEMBL4588	Matrix metalloproteinase 8	Empty	Empty	Both	6
CHEMBL1293267	G-protein coupled receptor 35	Empty	Inactive	Both	7
CHEMBL5457	Dihydrofolate reductase	Empty	Empty	Both	6
CHEMBL2489	Prostanoid EP4 receptor	Empty	Empty	Active	7
CHEMBL3797	Serine-protein kinase ATM	Inactive	Inactive	Inactive	7.5
CHEMBL4601	Tyrosine-protein kinase BRK	Inactive	Inactive	Both	6.5
CHEMBL217	Dopamine D2 receptor	Inactive	Inactive	Inactive	7
CHEMBL3807	T-cell protein-tyrosine phosphatase	Active	Active	Active	6
CHEMBL4198	Inhibitor of apoptosis protein 3	Empty	Inactive	Inactive	6
CHEMBL1792	Somatostatin receptor 5	Inactive	Inactive	Inactive	7

2.12. Statistical analysis

Statistical analyses were conducted with SPSS version 12.0 software. We calculated the means and standard deviations (SD) for all data. The difference between independent two groups was analyzed using Student's t test. The differences among three and mode groups were analyzed using One-way ANOVA followed by post hoc Tukey's test. P < 0.05 is considered statistically significant.

3. Results

3.1. SAA attenuated cartilage damage in a rat KOA model

Danzikangxi granules has been proved to be effective in KOA management [8,9]. In order to explore the mechanism by which Danzikangxi granules exerts its function, we have analyzed the active ingredients of the Danzikangxi granules using BATMAN database (Score cutoff = 0.89), and the active ingredients-targets-signaling pathway-diseases network has been obtained (Supplementary Fig. 1). We found that multiple components were associated with cell apoptosis-related signaling pathway, such as CASP3 and BCL2, and these signaling pathways were involved in the development of some cancers including breast cancer and colorectal cancer. While the relationship among these components, signaling pathway and KOA was not shown in the network. In recent years, the latent bioprotective capabilities of SAA in KOA has been found, which is one of the active ingredients of the Danzikangxi granules [10]. In our present study, uneven articular surface, hyperplasia of collagen fiber and cartilage calcification were observed in the cartilage tissues of KOA rat, SAA treatment at 10 mg/kg and 20 mg/kg dosages could improve the above damages in a dose-dependent manner (Fig. 1A). Meantime, the positive rate of chondrogenesis marker COL2A1 was significantly downregulated in the cartilage tissues of KOA rat, but this rate was partly rescued after 10 mg/kg or 20 mg/kg SAA treatment (Fig. 1B). The percentages of MMP13- and CASP3-positive chondrocytes were significantly elevated in the cartilage tissues of KOA rat, which were downregulated by SAA treatment at a dosage of 10 mg/kg and 20 mg/kg (Fig. 1C and D). Consistently, the results of RT-qPCR showed that the expression level of COL2A1 mRNA was lower in the cartilage tissues of KOA rat than the sham rat, which was reversed by 10 mg/kg and 20 mg/kg SAA treatment in a dose-dependent manner (Fig. 1E). The gene expression of Aggrecan, a chondrogenesis marker, was also downregulated in the cartilage tissues of KOA rat, and it was increased after SAA treatment in a dose-dependent manner (Fig. 1F). MMP13, MMP3 and CASP3 expression at the mRNA level was evaluated in the cartilage tissues of KOA rats, which was rescued by SAA treatment in a dose-dependent manner (Fig. 1G-I). These results indicated that SAA treatment could effectively improve cartilage damage, collagen deposition and loss of chondrocytes in KOA rats.

Fig. 1.

SAA improved cartilage damage in a rat KOA model. KOA rats were accepted with SAA treatment at a dosages of 10 mg/kg/d and 20 mg/kg/d for 4 weeks. Fresh knee cartilage tissues were taken. Then, (A) Safranin-O/Fast Green staining was performed to analyze cartilage tissue morphology and collagen deposition; (B) The rate of positive COL2A1 expression in chondrocytes was detected by immunohistochemistry; (C–D) The rate of positive MMP13 and CASP3 expression in chondrocytes was detected by immunohistochemistry. (E–I) RT-qPCR was performed to detect the mRNA expression levels of COL2A1, Aggrecan, MMP13, MMP3 and CASP3 in cartilage tissues. All experiments were repeated at least 3 biological replicates. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

3.2. SAA inhibited IL-1β-induced catabolism and apoptosis in chondrocytes

Primary chondrocytes were isolated from healthy rat, and were treated with SAA at a series dosages of 0, 5, 10, 15, 20, 25 and 30 μM. SAA intervention at dosages of 0, 5, 10, 15, 20 and 25 μM had no effect on the cell viability of chondrocytes, but 30 μM SAA treatment suppressed the cell viability of chondrocytes (Fig. 2A). Chondrocytes was treated with 20 μM SAA for 0, 12, 24, 48 and 96 h. The data showed that cell viability began to decrease at 96 h (Fig. 2B). Subsequently, chondrocytes were treated with 10 ng/ml IL-1β alone or in combination with 20 μM SAA for 48 h. The expression of COL2A1 mRNA and Aggrecan mRNA was suppressed in IL-1β-induced chondrocytes, and SAA treatment could promote both COL2A1 mRNA and Aggrecan mRNA expression in IL-1β-induced chondrocytes (Fig. 2C and D). Oppositely, the expression of MMP13 and MMP3 at the mRNA level was enhanced in IL-1β-induced chondrocytes, which was rescued by SAA treatment (Fig. 2E and F). At the protein level, COL2A1 expression was also inhibited but MMP13 expression was enhanced in IL-1β-induced chondrocytes, but SAA treatment could facilitate COL2A1 expression and suppress MMP13 expression in the cells (Fig. 2G-I). Overall, SAA treatment inhibited IL-1β-induced catabolism in chondrocytes. Furthermore, we also detected the level of chondrocyte apoptosis. CASP3 mRNA was found to be highly expressed in IL-1β-induced chondrocytes, but SAA treatment obviously inhibited the expression of it (Fig. 2J). As shown in Fig. 2K–M, the expression levels of CASP3 and Bax were upregulated in IL-1β-induced chondrocytes, but SAA treatment could reduce the expression of them. The expression level of Bcl-2 was downregulated IL-1β-induced chondrocytes, and SAA treatment could enhance the expression of it (Fig. 2K and N). Flow cytometry was also used to detect cell apoptosis, and the data indicated that the apoptosis rate of chondrocytes was higher in IL-1β-treated cells than that in control cells, but SAA combined induction with IL-1β obviously reduced cell apoptosis (Fig. 2O and P). SAA inhibited IL-1β-induced apoptosis in chondrocytes.

Fig. 2.

SAA treatment suppressed IL-1β-induced damages in chondrocyte. Primary chondrocytes were isolated from healthy rats and cultured in vitro. (A) Chondrocytes were treated with SAA for 24 h. The SAA concentrations were increasing from 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM–30 μM. CCK-8 assay was used to detect cell viability. (B) Chondrocytes were treated with SAA (25 μM) for 0, 12, 24, 48 and 96 h, and then CCK-8 assay was used to detect cell viability. Subsequently, primary chondrocytes were treated with IL-1β (10 ng/ml) alone or in combination with SAA (25 μM) for 48 h. (C–F) The expression of COL2A1, Aggrecan, MMP13, MMP3 at mRNA level was examined by RT-qPCR. (G–I) The expression of COL2A1 and MMP13 at protein level was detected by Western blot. (J) The expression of CASP3 at mRNA level was examined by RT-qPCR. (K–N) Western blot was carried out to detect the expression of CASP3, Bax and Bcl-2 at protein level. (O and P) Flow cytometry was performed to evaluate the rate of apoptotic cells. All experiments were repeated at least 3 biological replicates. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

3.3. SAA performed its protective function by promoting WDR5 expression

Base on the above results, we can know that SAA contributed cartilage repair in rat KOA model and suppressed IL-1β-induced damages in chondrocytes, while details in the mechanisms of its functions are remain unclear. Hence, we analyzed the potential targets of SAA using ChEMBL (version 27) database, and focused on the top 5 targets. Among the targets, WDR5 has been proven to promote chondrocyte differentiation [12], but its functions and action mechanism in KOA development remain unclear. Here, our data indicated that the expression of WDR5 at mRNA (Fig. 3A) and protein (Fig. 3B and C) levels was significantly downregulated in the cartilage tissues of KOA rat, while SAA treatment at a dosage of 10 mg/kg and 20 mg/kg could rescue the expression of WDR5 mRNA and WDR5 protein. In IL-1β-treated chondrocytes, the expression of both WDR5 mRNA (Fig. 3D) and WDR5 protein (Fig. 3E and F) was also found to be decreased, which was rescued by SAA treatment. The above data suggested that WDR5 participates in the development of KOA and it is a downstream target of SAA. To explore whether SAA performs its function via WDR5, the specific siRNA-3 was chosen for silencing WDR5 expression due to the highest efficiency of interference of it (Supplementary Fig. 2A). Our data indicated that SAA treatment-induced increase in both COL2A1 mRNA and Aggrecan mRNA expression in the chondrocytes treated with IL-1β was reversed after WDR5 silencing (Fig. 3G and H). SAA treatment-induced decrease in both MMP13 mRNA and MMP3 mRNA expression in the IL-1β-treated chondrocytes was also rescued after WDR5 silencing (Fig. 3I and J). At protein level, SAA treatment could facilitate COL2A1 expression and suppress MMP13 expression in IL-1β-treated chondrocytes, which was still rescued by silencing WDR5 (Fig. 3K–M). The inhibition of SAA to IL-1β-induced catabolism in chondrocytes was rescued by silencing WDR5. In addition, CASP3 mRNA (Fig. 3N) and CASP3 protein expression (Fig. 3O and P) were decreased in IL-1β-treated chondrocytes after SAA treatment, which was rescued by silencing WDR5. Inhibiting the expression of WDR5 also rescued SAA treatment-resulted downregulating in Bax and upregulating in Bcl-2 expression in IL-1β-treated chondrocytes (Fig. 3O, Q and R). Results of flow cytometry indicated that the downregulation of apoptosis rate after SAA treatment in IL-1β-treated chondrocytes was rescued by silencing WDR5 (Supplementary Fig. 2B). Overall, the inhibition of SAA to IL-1β-induced apoptosis in chondrocytes was rescued by silencing WDR5.

Fig. 3.

SAA treatment suppressed IL-1β-induced chondrocyte damages by targeting WDR5. KOA rats were accepted with SAA treatment at a dosage of 10 mg/kg/d and 20 mg/kg/d for 4 weeks. Fresh knee cartilage tissues were taken. Then, (A) the expression of WDR5 mRNA was assessed by RT-qPCR; (B–C) the expression of WDR5 protein was detected by Western blot. Primary chondrocytes were treated with IL-1β (10 ng/ml) alone or in combination with SAA (25 μM) for 48 h. Then, (D) RT-qPCR was performed to assess the expression of WDR5 mRNA, and (E–F) Western blot was carried out to evaluate the expression of WDR5 protein. In addition, IL-1β-treated chondrocytes were administrated with SAA alone or in combination with si-WDR5. (G–H) The COL2A1 mRNA and Aggrecan mRNA expression was measured by RT-qPCR. (I–J) The MMP13 mRNA and MMP3 mRNA expression was determined by RT-qPCR. (K–M) The COL2A1 and MMP13 expression at protein level was detected by Western blot. (N) Apoptosis-related CASP3 mRNA expression was assessed by RT-qPCR. (O–R) Western blot was performed to detect the expression of CASP3, Bax and Bcl-2 at protein level. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

3.4. SAA contributed to differentiation of BMSCs into chondrocytes by promoting WDR5 expression

Inducing BMSCs differentiation into chondrocytes and thus attenuating cartilage injury is one of the most effective ways to improve KOA. In the present experiments, we isolated primary BMSCs from healthy or KOA rats, and verified the chondrogenic differentiation ability of the BMSCs through Alcian blue staining (Fig. 4A). The expression of WDR5 mRNA could be significantly upregulated after transfection of the WDR5 expressing plasmid (Fig. 4B). Our data showed that increasing the expression of WDR5 obviously enhanced the differentiation of BMSCs derived from KOA rats into chondrocytes (Fig. 4C). The expression of chondrogenic differentiation markers (SOX9, COL2A1 and Aggrecan) was increased with WDR5 overexpression in the BMSCs derived from KOA rats (Fig. 4D-F). In summary, overexpression of WDR5 facilitated KOA-BMSCs differentiation into chondrocytes.

Fig. 4.

WDR5 facilitated KOA-BMSCs differentiation into chondrocytes. BMSCs were isolated from the cartilage tissues of healthy rats and KOA rats. (A) Alcian blue staining was carried to evaluate the chondrogenic differentiation ability of healthy-BMSCs and KOA-BMSCs. (B) RT-qPCR was performed to detect the expression of WDR5 mRNA in the chondrocytes transfected with WDR5 overexpression plasmid. In addition, WDR5 overexpression plasmid was transfected into the chondrocytes derived from KOA rats, then (C) Alcian blue staining was carried to evaluate the chondrogenic differentiation ability of KOA-BMSCs; (D–F) the expression of SOX9, COL2A1 and Aggrecan at mRNA level was detected by RT-qPCR. All experiments were repeated at least 3 biological replicates. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

Furthermore, our results demonstrated that SAA treatment significantly enhanced the chondrogenic differentiation ability of BMSCs derived from KOA rats, while the differentiation ability of BMSCs was weakened after WDR5 silencing (Fig. 5A). SAA treatment obviously promoted the expression of SOX9 mRNA, COL2A1 mRNA and Aggrecan mRNA in KOA-BMSCs, but they were rescued by silencing WDR5 (Fig. 5B-D). SOX9, COL2A1 and Aggrecan at protein level were also highly expressed in KOA-BMSCs after SAA treatment, which was reversed by silencing WDR5 (Fig. 5E-H). Overall, SAA treatment promoted the differentiation of KOA-BMSCs into chondrocytes via targeting WDR5.

Fig. 5.

Silencing of WDR5 could rescue the promotion of SAA to chondrogenesis of KOA-BMSCs. BMSCs derived from KOA rats were treated with SAA alone or SAA plus si-WDR5. (A) The chondrogenic differentiation ability of cells was examined by Alcian blue staining. (B–D) SOX9, COL2A1 and Aggrecan expression at mRNA level was measured by RT-qPCR. (E–H) SOX9, COL2A1 and Aggrecan expression at protein level was measured by Western blot. All experiments were repeated at least 3 biological replicates. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

4. Discussion

In animal experiments, we found that SAA treatment could effectively attenuate the damage of cartilage tissues in a rat KOA model. In cellular experiments, SAA treatment could suppress the damage of chondrocytes induced by IL-1β and promote the differentiation of BMSCs into chondrocytes. Importantly, SAA exerts its functions via targeting WDR5.

We discovered that SAA, a bioactive polyphenol, is one of the active ingredients of Danzikangxi granules. SAA has been proved to have multiple biological activities, such as anti-inflammation, anti-cancer, anti-apoptosis and anti-oxidant [14]. In the past 5 years, a few studies showed that SAA have therapeutic potential for KOA [10,15]. Our results agree with previous research findings based on experimental evaluations. The results demonstrated that broken articular surface, deposited extracellular matrix and loss of chondrocytes in the cartilage tissues of KOA model rats were improved after SAA treatment. 20 mg/kg/d dose of SAA was more effective than 10 mg/kg/d dose of SAA. In addition, the immunohistochemical results showed that COL2A1 expression was increased, but MMP13 and CASP3 expression was decreased in the cartilage tissues of KOA rat after SAA treatment. COL2A1 is one of the markers of chondrocytes and one of the components of cartilage matrix, its expression is increasing during the differentiation of BMSCs to chondrocytes [16]. MMP13 is one of the catabolic markers of chondrocytes and an extracellular proteolytic enzymes in degradation of cartilage [13]. It has been reported that MMP13 and CASP3 expression is upregulated in KOA model rat [17]. Moreover, the expression of cartilage protein Aggrecan was also increased and the expression of catabolic marker MMP3 was decreased in the cartilage tissues of KOA rat after SAA treatment. From these, SAA treatment effectively alleviated the loss and catabolism of chondrocytes in KOA. In cellular experiments, IL-1β-induced decreasing in COL2A1 and Aggrecan expression and increasing in MMP13 and MMP3 expression were inhibited by SAA treatment. IL-1β-induced increasing in apoptosis rate was also inhibited by SAA treatment. These were again confirmed the inhibition of SAA to chondrocytes apoptosis and catabolism.

The detailed molecular mechanism of action by which SAA performs its function remains unclear. Existing researches only confirm that SAA improve KOA through NF-κB and MAPK signaling pathway [10,15]. Here, our study presented a new mechanism of action of SAA. WDR5 was confirmed as a potential target of SAA by using ChEMBL data. Our data showed that WDR5 expression was increased in both KOA model rats and IL-1β-induced chondrocytes after SAA treatment. Importantly, the promotion of SAA treatment to chondrogenesis markers (COL2A1 and Aggrecan) and the inhibition of SAA treatment to cartilage degradation-related proteins (MMP13 and MMP3) were rescued by silencing WDR5. The inhibition of SAA treatment to cell apoptosis-related proteins (CASP3 and Bax) and the promotion of SAA treatment to anti-apoptotic protein Bcl-2 were also rescued by silencing WDR5. In summary, SAA inhibited apoptosis and catabolism of chondrocytes via facilitating WDR5 expression.

An increasing body of research has suggested that chondrogenic differentiation of BMSCs is an effective therapeutic strategy for KOA. Liu et al. have demonstrated that kartogenin treatment significantly enhance the chondrogenic differentiation of BMSCs, and kartogenin-treated BMSCs had a better effective in attenuating pain in osteoarthritis model [18]. Xu et al. have reported that inhibiting cytokine receptor-like factor 1 could against cartilage damage in osteoarthritis model mice via promoting the differentiation of BMSCs to chondrocytes [19]. Here, we found that increasing WDR5 could facilitate the differentiation of BMSCs derived from KOA model rats to chondrocytes. Same as COL2A1 and Aggrecan, SOX9 also is one of the markers of chondrogenesis. SOX9 is responsible for controlling the expression of COL2A1 and Aggrecan [20]. In the present study, the increased expression of COL2A1, Aggrecan and SOX9 proved again the promotion of WDR5 to chondrogenic differentiation of KOA-BMSCs. In addition, SAA treatment was proved to be helpful for the chondrogenic differentiation of KOA-BMSCs, but it was rescued by silencing WDR5.

5. Conclusion

In conclusion, our results indicated that SAA treatment could effective promote cartilage repair in KOA model rat. Mechanistically, on the one hand, SAA treatment inhibited chondrocytes apoptosis and catabolism via promoting WDR5 expression; on the other hand, SAA treatment enhance chondrogenic differentiation of BMSCs via promoting WDR5 expression. Our study may provide a novel idea for the treatment of KOA and new molecular mechanism of KOA development. Nevertheless, we are aware of several limitations of the study. First and foremost, the regulatory mechanism of SAA to WDR5 remains unclear. Second, WDR5 is critical for the methylation of H3K4 on chromatin catalyzed by the MLL1 complex, whether WDR5 participates in the H3K4 methylation of its downstream targets still is needed to be explored.

Ethics approval and consent to participate

Animal experiments were approved by the ethics committee of Hunan University of Chinese Medicine (APPROVAL NUMBER LLBH-202311070015). We have performed animal experiments depends on the Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed are included in this article. Further inquiries can be directed to the corresponding author.

Funding

This work was supported by the Natural Science Foundation of Changsha City (kq2208196), Hunan University of Chinese Medicine Scientific Research Fund Project (2021XJJJ055), the Scientific Research Project of Hunan Traditional Chinese Medicine (B2024078) and Natural Science Foundation of Hunan Province (2025JJ80903 and 2025JJ80920).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary figure 1.

Active ingredients of Danzikangxi granules-targets-signaling pathway-disorders network.

Supplementary figure 2.

SAA treatment inhibited IL-1β-induced chondrocyte apoptosis by targeting WDR5. (A) Three different siRNAs targeting WDR5 were transfected into chondrocytes, and the interference efficiency of them was ensured by RT-qPCR. (B) The BMSCs derived from KOA rats were treated with SAA alone or SAA plus si-WDR5. Flow cytometry was carried out to determine the rate of apoptotic chondrocytes. All experiments were repeated at least 3 biological replicates. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.