Efficacy of Juanbi capsule on ameliorating knee osteoarthritis: a network pharmacology and experimental verification-based study

Background : The purpose of the study was to investigate the active ingredients and potential biochemical mechanisms of Juanbi capsule in knee osteoarthritis based on network pharmacology, molecular docking and animal experiments. Methods : Chemical components for each drug in the Juanbi capsule were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, while the target proteins for knee osteoarthritis were retrieved from the Drugbank, GeneCards, and OMIM databases. The study compared information on knee osteoarthritis and the targets of drugs to identify common elements. The data was imported into the STRING platform to generate a protein-protein interaction network diagram. Subsequently, a “component-target” network diagram was created using the screened drug components and target information with Cytoscape software. Common targets were imported into Metascape for GO function and KEGG pathway enrichment analysis. AutoDockTools was utilized to predict the molecular docking of the primary chemical components and core targets. Ultimately, the key targets were validated through animal experiments. Results : Juanbi capsule ameliorated Knee osteoarthritis mainly by affecting tumor necrosis factor, interleukin1β, MMP9, PTGS2, VEGFA, TP53, and other cytokines through quercetin, kaempferol, and β-sitosterol. The drug also influenced the AGE-RAGE, interleukin-17, tumor necrosis factor, Relaxin, and NF-κ B signaling pathways. The network pharmacology analysis results were further validated in animal experiments. The results indicated that Juanbi capsule could decrease the levels of tumor necrosis factor-α and interleukin-1β in the serum and synovial fluid of knee osteoarthritis rats and also down-regulate the expression levels of MMP9 and PTGS2 proteins in the articular cartilage. Conclusion : Juanbi capsule may improve the knee bone microstructure and reduce the expression of inflammatory factors of knee osteoarthritis via multiple targets and multiple signaling pathways.


Background
Knee osteoarthritis (KOA) is the predominant form of arthritis, and it is the primary cause of disability and diminished quality of life among middle-aged and elderly individuals [1].Its pathological changes encompass cartilage degeneration, subchondral bone remodeling, intra-articular osteophyte formation, synovitis, ligament relaxation, contracture, and other related factors [2].The prevalence of KOA is on the rise due to the aging of society [3].Currently, the management of KOA primarily centers on the reduction of inflammation, alleviation of pain, and preservation of joint function.In Western medicine, the primary treatment for early-stage KOA is drug therapy, which includes non-steroidal anti-inflammatory drugs, glucosamine, and corticosteroids.Surgical intervention, such as knee replacement, is considered for advanced stages [4].Given the limitations of Western medicine, including its focus on singular effects, symptom management rather than addressing underlying causes, and potential for serious long-term side effects, there is a critical need to identify a safe and sustainable treatment approach that is both safe and efficacious.
KOA is a prevalent condition in the orthopedic department of traditional Chinese medicine (TCM) and is classified under the "arthralgia syndrome" category of traditional Chinese medicine [5,6].The pathogenesis of KOA involves the depletion of essential substances in the liver and kidneys, which results in an inability to meet the nutritional needs of musculoskeletal activities and metabolism in the large joints, such as the knee and ankle.This leads to decreased immunity in these parts, making them vulnerable to the invasion of external pathogenic substances such as wind, cold, dampness, and heat (wind, cold, dampness, and heat are causes of disease resulting from abnormal changes in the natural climate that exceed the body's ability to adapt), which can lead to the blockage of muscles, channels, joints, and the onset of KOA.Therefore, traditional Chinese medicine theory holds that the pathogenesis of KOA is based on the depletion of essential substances in the liver and kidneys, and the invasion of external pathogens is the contributing factor.
The Juanbi capsule (JBJN) (Preparation code: Z20190007, batch number: 2019S00026) is a traditional Chinese medicine preparation produced and used in the Affiliated Hospital of Shaanxi University of Chinese Medicine.This preparation is based on the principle of "tonifying kidney and replenishing qi (qi refers to the various energies of the organs and tissues in the human body)" in the treatment of OA by Professor Li Kanyin, an expert in Chinese medicine doctor specializing in bone injuries, and it is backed by decades of clinical experience.The formulation consists of nine Chinese herbs, which are Yinyanghuo (Epimedii Folium), Roucongrong (Cistanches Herba), Huangqi (Astragali Radix), Danggui (Angelicae Sinensis Radix), Gusuibu (Drynariae Rhizoma), Shudihuang (Rehmanniae Radix Praeparata), Niuxi (Achyranthis Bidentatae Radix), Shaoyao (Paeoniae Radix Alba), and Gancao (Glycyrrhizae Radix et Rhizoma).The function of JBJN is to maintain the liver and kidneys with sufficient intrinsic energy, enhance blood circulation throughout the body, and promote the metabolism of musculoskeletal cells.It is used to alleviate inflammatory symptoms associated with arthralgia syndrome, such as pain, swelling, and limitation of joint movement.
Epidemiological evidence indicates that the prevalence of OA is higher in postmenopausal women and that estrogen deficiency resulting from ovarian dysfunction is a significant factor [7].According to traditional Chinese medicine, postmenopausal women with estrogen deficiency often exhibit a constitution characterized by low internal energy in the liver and kidney, weakened body immunity, and poor blood circulation.Therefore, the treatment of OA in postmenopausal women should focus on nourishing the intrinsic energy of the liver and kidneys, and improving blood circulation throughout the body.Our previous clinical and basic research has confirmed that the JBJN effectively alleviates the pain and swelling symptoms of KOA, particularly in postmenopausal middle-aged and elderly women with arthritis [8,9].Therefore, we established a new method of inducing estrogen deficiency-induced OA by ovariectomizing and Hulth to examine the therapeutic effects of JBJN on the development of KOA in a rat model with kidney deficiency.
In order to conduct a comprehensive investigation of JBJN, it is essential to elucidate its mechanism of action.In this study, network pharmacology was used to investigate the active ingredients, targets, and mechanisms of JBJN in the treatment of KOA.Subsequent to this, experiments involving animals were carried out to further confirm the effectiveness of the treatment and to offer valuable insights for clinical application and further research of JBJN.

Collection of chemical components and prediction of JBJN
The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) is a digital information platform that disseminates the primary constituents of traditional Chinese medicine [10].The active compounds in JBJN were retrieved from the TCMSP database for the purposes of this study.Compounds with an oral bioavailability of 30% or higher and a drug-likeness score of 0.18 or greater are considered to exhibit enhanced pharmacological effects and are thus selected for further analysis as potential active compounds.The compounds searched under these conditions were the candidate components, after which the target proteins corresponding to the candidate components were identified and categorized.Utilizing the UniProt data platform (https://www.uniprot.org/),the species was set to "Homo sapiens", following which the target proteins of JBJN were inputted, and the corresponding gene names for each protein were searched, thereby revealing the target proteins of JBJN.

Construction of a chemical component-target network of JBJN
Cytoscape 3.9.1 software was utilized to construct the chemical component-target network of JBJN in section 2.1.Based on the Degree of connectivity, Betweenness, and Closeness, the primary chemical components in JBJN were identified.

Highlights
An

Model establishing, grouping, and treating
The rats were randomly divided into three groups: a normal control group (Control), a KOA model control group (Model), and a JBJN treatment group, with 8 rats in each group.The Hulth method and bilateral ovariectomy were used to induce KOA rat models with kidney deficiency in the Model and JBJN groups [11,12].After 6 weeks, 3 rats were randomly selected from the model group and the JBJN group for micro-CT scanning of the knee joint.The joint space on the model side was narrower than that on the healthy side, and subchondral sclerosis or osteophyte formation indicated the successful establishment of the KOA model.After successfully establishing the model, the rats in the JBJN group were administered JBJN by gavage, while the rats in the Control and Model groups were given an equivalent amount of distilled water by gavage.The three groups were treated for 6 weeks.
The clinically administered oral dose of JBJN for humans (70 kg) was 127 g/d, and according to the human-rat body surface area dose conversion method, the equivalent dose of rats was 6.3 times that of humans.Thus, a rat was given equivalent doses of crude drug of JBJN of 11.43 g/kg daily.

Micro-CT scan
Three rats from each group were randomly selected to take the intact left knee joint, and Skyscan1276 was used for scanning with micro-CT equipment (Bruker, Belgium).After fixing the samples, the scanning voltage was set to 60 kV, and the scanning current was set to 200 μA.The knee joint was scanned along the long axis of the femur using a scanning thickness of 10 μm, a filter of AL0.5 mm, and an exposure time of 550 ms to obtain continuous plane micro-CT images.After scanning, 3D reconstruction processing software NRecon and Data Viewer were used to reconstruct and select, and CT Analyzer was used to quantitatively analyze the cartilage and subchondral bone areas in the same position in the image.The microstructure parameters of tibia trabecular bone were analyzed, including bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

ELISA detection
After intraperitoneal anesthesia with 1% sodium pentobarbital (4 mL/kg), blood samples were collected from the abdominal aorta and allowed to stand at 4 °C for two hours before centrifugation at 3500 rpm for fifteen minutes.Knee punctures were performed, and a sterile medical syringe was used to inject 1 mL of sterile PBS solution into the knee joint.Subsequently, the knee joint was exercised ten times, followed by the slow extraction of the joint fluid.The extracted fluid was then allowed to stand at 4 °C for one hour before being centrifuged at 3,500 rpm for twenty minutes.The concentrations of TNF-α and IL-1β in both serum and joint fluid were determined using an ELISA kit following the provided instructions.

Western blot detection
The articular cartilage samples from each group were frozen in liquid nitrogen for ten minutes.Then, 500 μL of RIPA and PMSF (in a 100:1 ratio) were added, and the samples were ground in a frozen grinder (−49 °C).After grinding, the cartilage was placed on ice for fifteen minutes to allow the protein to fully dissolve.Subsequently, the homogenate was centrifuged to obtain a solution of total proteins.Protein samples were prepared by adding a loading buffer to ensure consistent protein concentrations of 50 μg/10 μL for each group.The protein samples were separated using 10% SDS-PAGE and subsequently transferred onto the PVDF membrane.The membrane was then blocked with 5% skim milk for 2 hours, followed by overnight incubation with primary antibodies (MMP9 at a dilution of 1:2,000, PTGS2 at a dilution of 1:1,500) at 4 °C.After incubating with secondary antibodies for 90 minutes at room temperature, the films were developed using ECL chemiluminescence solution and then scanned using ImageJ software following exposure.β-actin served as an internal control.

Statistical analysis
All statistical analyses were conducted using the SPSS 26.0 software.The data were presented as mean ± standard deviation ( ± ).One-way ANOVA with Tukey's post hoc test was used for comparing multiple groups, a significance level of P < 0.05 was employed as the threshold to ascertain statistical significance.

Screening of chemical component
The TCMSP database was utilized to search for 167 chemical components of JBJN, including 23

Construction and analysis of chemical component-target network
The DrugBank and UniProt databases were utilized to search for 315 target proteins associated with the chemical components of JBJN.Subsequently, we obtained information about the drug composition, chemical components, and their respective relationships with targets.Using Cytoscape 3.9.1 software, a network illustrating the interactions between JBJN chemical components and targets was constructed.This network consisted of a total of 475 nodes, which included 9 drug nodes, 167 active compound nodes, and 315 target nodes, all connected by a total of 4,135 edges (Figure 1). Figure 1 shows that each chemical component interacts with multiple targets while each target corresponds to multiple chemical components simultaneously or independently affiliated with each traditional Chinese medicine.These findings indicate that JBJN exerts its effects on KOA through multiple components, targets, and pathways.
Notably, quercetin, kaempferol, beta-sitosterol, Stigmasterol, and luteolin emerged as the main active compounds based on their higher Degree values.

Target prediction and mutual PPI network construction
The DrugBank, GeneCards, and OMIM databases were utilized to search for 363 KOA targets.Subsequently, drug and disease targets were incorporated into the R software, resulting in 49 overlapping targets (Figure 2).Overlapping targets of JBJN and KOA were retrieved from the STRING databases and imported into the Cytoscape software platform to generate the PPI network diagram (Figure 3).This network comprises a total of 48 nodes and 603 edges.The nodes represent the target proteins, and the edges represent the interactions between different target proteins.According to Figure 3, TNF, IL6, IL1B, MMP9, PTGS2, VEGFA, TP53, and other target proteins are the central targets in this network.

Enrichment analysis of overlapping target by GO and KEGG
pathway GO enrichment analysis of the overlapping targets between JBJN and KOA was performed using the Metascape data platform.Through analysis, a total of 1,072 GO items representing potential targets of JBJN were obtained.Among them, 975 items were related to biological processes, 72 items were related to molecular functions, and 25 items were related to cellular components.The top 10 items in biological processes, cellular components, and MF were selected to create the bubble diagram (Figure 4A).The biological processes Involved in the treatment of KOA by JBJN mainly include regulation  of inflammatory response, cell migration, signal peptide activity, metabolism of reactive oxygen species, hormonal stress, and response to xenobiotic stimulation.The cellular components include the extracellular matrix, endoplasmic reticulum lumen, membrane raft, and outer membrane of the plasma membrane.Molecular functions include cytokine activity, serine endopeptidase activity, protease binding, integrin binding, oxidoreductase activity, and DNA binding transcription factor binding.
KEGG pathways were identified using the Metascape data platform, and signaling pathways that significantly influence the treatment of KOA by JBJN were analyzed.A total of 107 KEGG pathways with significant enrichment of potential targets of JBJN were obtained (P < 0.05).According to the significance arrangement, the top 30 items were selected and depicted in a bar chart (Figure 4B).The treatment of KOA by JBJN mainly involves targeting the cancer pathway, AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, Relaxin signaling pathway, NF-κB signaling pathway, Th17 cell differentiation, PI3K-Akt signaling pathway, HIF-1 signaling pathway, MAPK signaling pathway, and others.

Molecular docking
The primary chemical components (quercetin, kaempferol, β-sitosterol) identified in section 3.2 were docked with key targets (TNF, IL1B, MMP9, PTGS2, VEGFA, TP53) identified in section 3.3, and the results of the docking are presented in Table 2.The average affinity between each molecule was −6.942 kcal/mol, which was lower than −5 kcal/mol, indicating that the primary chemical components of JBJN exhibited a strong binding ability to the target protein [13].The results indicate that all the chemical components of JBJN penetrated deeply into the active site and formed a relatively stable conformation with the target receptor protein through hydrogen bonding.Some molecular docking binding modes are shown in Figure 5.

Assessment of the KOA rat model
The 2D images obtained from the micro-CT revealed that the knee  joint surface of rats in the normal control group was relatively smooth, with a normal joint space and no observed osteophytes.In contrast, the joint surface of the model control group 6 weeks after the operation appeared uneven, with hardened subchondral bone and the formation of osteophytes (Figure 6).

Effect of JBJN on knee joint in KOA rats with micro-CT
Micro-CT analysis of the subchondral bone structure revealed that, compared to the normal control group, the model control group exhibited a significantly narrower medial joint space and disordered microstructure of trabecular bone in the subchondral region, indicating the successful establishment of the animal model.The treatment with JBJN demonstrated an improvement in both the joint space and the trabecular meshwork structure of the subchondral bone (Figure 7A).To evaluate changes in the subchondral bone, the medial tibial

Effect of JBJN on the expression of inflammatory factors in KOA rats
The analysis of inflammatory factor levels in the serum showed that the expression levels of TNF-α and IL-1β were significantly lower in the JBJN group compared to the model control group (Figure 8A).Subsequently, the changes in expression of TNF-α and IL-1β in the joint fluid were detected to further confirm that the levels in the JBJN group were also significantly lower than those in the model control group (Figure 8B).

Effect of JBJN on the expression of MMP9 and PTGS2 in cartilage tissue
MMP9 and PTGS2 are common molecules in the inflammatory pathway, and they are also the primary target proteins of JBJN in the treatment of KOA.Western blot data confirmed that the protein levels of cartilage MMP9 and PTGS2 in the tissue of the JBJN treatment group were significantly lower than those of the model control group (Figure 9).

Discussion
Traditional Chinese medicine formulas are developed based on TCM theories and individual conditions, combining a variety of herbs to form the appropriate drug structure for the treatment of various medical conditions..This allows the drugs to produce synergistic and antagonistic effects and even generate new substances [14].Network pharmacology of TCM compounds explores the potential mechanisms of TCM treatment and disease prevention by studying the complex chemical components and multi-target, multi-pathway mechanisms of Chinese herbal medicine [15].The specific mechanism of JBJN in the treatment of KOA has not been thoroughly studied.Therefore, this study utilized network pharmacology to identify the active constituents, targets, and mechanisms of action responsible for JBJN's therapeutic effectiveness in treating KOA.Furthermore, the binding affinity between the key components of JBJN and target proteins was validated through molecular docking technology.Ultimately, animal  The results of network pharmacology showed that quercetin, kaempferol, β-sitosterol, sitosterol, and luteolin may be the main components of JBJN in the treatment of KOA.In osteoarthritis, quercetin has good anti-inflammatory and cartilage-protective effects [16].Quercetin has anti-inflammatory effects by inhibiting the NLRP3 signaling pathway, p38 activation, and endoplasmic reticulum stress, as well as inhibiting NO, TNF-α, and IL-1β [17,18].Qiu et al. discovered that quercetin can regulate ER stress-related chondrocyte apoptosis and facilitate the reversal of mitochondrial dysfunction in chondrocytes by activating the SIRT1/AMPK signaling pathway [19].Hu et al. discovered that quercetin can regulate the polarization of synovial macrophages from M1 to M2, inhibit the inflammatory response of chondrocytes, and alleviate osteoarthritis in rats [20].In addition to inhibiting inflammation, Permatasari et al. found that quercetin prevented proteoglycan destruction by inhibiting the expression of MMP3, MMP9, MMP13, and ADAMTS-5 in OA model rats [21].Kaempferol has functions such as regulating inflammation and reducing oxidative stress.Zhuang et al. found that kaempferol could regulate IL-1β-induced inflammatory response in rat chondrocytes by inhibiting the expression of NF-κB signaling pathway [22].Lee et al. found that kaempferol inhibited IL-1β-stimulated NF-κB ligand-receptor activator mediated osteoclastogenesis by down-regulating MAPKs, c-Fos, and NFATc1 [23].Xiao et al. found that kaempferol could inhibit NF-κB activation and reduce inflammation by inhibiting the generation of reactive oxygen species and blocking the osteopontin related signaling pathway regulated by aldosterone [24].Kaempferol can regulate the polarization of M1/M2 macrophages, and M1 macrophages have pro-inflammatory and accelerated cartilage destruction functions [25,26].β-sitosterol has anti-inflammatory, immunomodulatory, and analgesic effects [27].Liao et al. found that β-sitosterol effectively inhibited the production of reactive oxygen species induced by lipopolysaccharide, inhibited the activation of NF-κB, and subsequently downregulated the expression of IL-6, IL-1β, and TNF-α in cells [28].Stigmasterol can bind to the chondrocyte membrane and inhibit the expression of MMP and PTGS2 by inhibiting the NFκB pathway [29,30].Stigmasterol has the properties of reducing the expression of inflammatory factors and slowing down the catabolism of the cartilage matrix.Fei et al. discovered that luteolin can inhibit IL-1β-induced inflammation in rat chondrocytes, increase the production of type II collagen, and slow down the progression of osteoarthritis [31].Through molecular docking analysis of key molecules and protein targets, it was found that quercetin, kaempferol, and β-sitosterol could target and regulate the expression of TNF-α, IL-1β, MMP9, PTGS2, TP53, VEGFA, and other proteins.This suggests that the active ingredients of JBJN have anti-inflammatory, antioxidant, immune-regulating, and cartilage matrix-protecting functions.
According to PPI network analysis, TNF, IL6, IL1β, MMP9, PTGS2, TP53, and VEGFA may be the critical targets of JBJN in the treatment of KOA.Studies have shown that the proinflammatory response induced by TNF-α, IL-1β, and IL-6 is closely related to M1 macrophages, which induces chondrocyte apoptosis and accelerated cartilage matrix degradation by leading to the production of pro-chondrocytic mediators [32,33].MMP9 is a component of the MMPs family, which can degrade most of the articular cartilage matrix, induce the apoptosis of articular chondrocytes, and eventually lead to severe destruction of knee cartilage.Studies have shown that MMP9 can promote the expression of inflammatory mediators such as TNF-α and IL-1β, aggravate local inflammation, destroy cartilage matrix, and cause subchondral bone microstructure disorder [34,35].PTGS2 is a prostaglandin-endoperoxide synthase, which is a potent mediator of inflammation and can promote the metabolism of arachidonic acid to prostaglandins and leukotrienes, which in turn leads to inflammation [36].Celecoxib and other selective non-steroidal anti-inflammatory drugs as PTGS2 inhibitors have been widely used in the clinical treatment of KOA [37,38].Chondrocytes in patients with advanced osteoarthritis are highly expressed and promote new blood vessel formation, and VEGFA expression is gradually increased [39,40].VEGFA can upregulate the expression of MMPs in chondrocytes and downregulate the expression of extracellular matrix components, including aggrecan and type II collagen.TP53 is a tumor suppressor gene associated with tumorigenesis and a key signal that inhibits cell growth and promotes apoptosis.Studies have shown that KOA chondrocytes with high expression of p53 exhibit apoptotic or senescent morphology.Therefore, JBJN can interfere with the target proteins of KOA through a variety of classic inflammation, apoptosis, and cartilage metabolism processes and affect the development of KOA.
The results of the functional enrichment analysis revealed a significant enrichment of molecular proteins related to KOA in GO function and KEGG pathway.The biological process accounted for the largest proportion of GO analysis, and the content was mainly manifested in inflammatory response, cell migration regulation, reactive oxygen metabolism, hormone stress, cell proliferation, and others.The KEGG pathways were primarily enriched in the AGE-RAGE, IL-17, TNF, NF-κB, and PI3K-Akt signaling pathway.Among them, AGE is an advanced glycation end product, and RAGE is its receptor.During aging, AGE accumulates in articular cartilage and participates in the activation of proinflammatory responses and various inflammatory gene-related signaling pathways [41].The AGE-RAGE signaling pathway can be activated downstream of chondrocytes NF-κB and MAPK signal pathway, stimulate endoplasmic reticulum stress, produce PTGS2 and NO cartilage cells, and induce inflammation of cartilage cells [42].IL-17 signaling pathway and TNF signaling pathway, as classical inflammatory pathways, can activate NF-κB and MAPK signaling pathways, and induce synovial fibroblasts and chondrocytes to produce IL-1, Tnf-α, MMPs, and other chemokines [43].NF-κB is a multifunctional transcription factor involved in a variety of biological processes and is associated with chondrocyte proliferation, apoptosis, and synovial inflammatory processes [44].Studies have found that the NF-κB signaling pathway is abnormally activated in osteoarthritis, which directly or indirectly induces the expression of proinflammatory mediators such as MMP9, PTGS2, PGE2, and iNOS [45].PI3K/AKT is a classical signaling pathway that regulates the autophagy response, which can maintain the homeostasis of chondrocytes, inhibit the apoptosis of chondrocytes, and promote the proliferation of chondrocytes, thereby slowing down the process of cartilage degeneration in KOA [46,47].
In summary, the pathogenesis of KOA is complex, involving multiple interconnected signaling pathways that are cross-linked and mutually modulation.The active ingredients of JBJN can simultaneously modulate a variety of targets and pathways, showing the multi-component-multi-target-multi-pathway action pattern of TCM.Here, we utilized network pharmacology and molecular docking methods to investigate the mechanism of JBJN in treating KOA.And our investigation revealed that JBJN could decrease the levels of TNF-α and IL-1β in serum and synovial fluid of KOA rats, and downregulate the expression levels of MMP9 and PTGS2 proteins in the articular cartilage.However, our article does have some limitations.We did not conduct thorough experimental validation, and future studies should further validate the role of other pathways enriched in KEGG analysis, as well as the targets of different active ingredients of JBJN in KOA.In addition, the effective active ingredients of the drugs in this study were the values predicted by computer simulation, did not fully account for the changes in pharmacokinetics that occur after the active ingredients enter the body's metabolism.Therefore, further research is needed to understand the deeper molecular mechanisms.

Figure 1
Figure 1 Compounds of JBJN-chemical component-target network.The blue square part in the figure is candidate targets, and the surrounding circular part is chemical components of the JBJN compound.The darker the color and the larger the shape, the higher the degree value, and each edge represents the relationship between the compound and the target.JBJN, Juanbi capsule.

Figure 2
Figure 2 The overlapping targets of JBJN in KOA treatment.The Venn diagram demonstrates the number of intersected and specific targets between JBJN and KOA.JBJN, Juanbi capsule; KOA, knee osteoarthritis.

Figure 3 PPI
Figure 3 PPI network diagram of overlapping targets.Critical targets of JBJN in treating KOA were screened by degree values, in which the darker color and the closer nodes to the center have higher degree values.JBJN, Juanbi capsule; KOA, knee osteoarthritis; TNF, tumor necrosis factor; IL, interleukin.

Figure 4
Figure 4 Enriched Go and KEGG signaling pathways of overlapping targets.(A) The Bubble chart shows the top ten in the BP, CC, and MF categories in the GO enrichment analysis.(B) The Bar graph of the top 30 pathways extracted by KEGG analysis.BP, biological process; CC, cell component; MF, molecular function.

Figure 5
Figure 5 Molecular docking pattern diagram of critical core protein targets.The yellow circles represent the docking states between small molecule compounds and macromolecular proteins, and the short yellow lines represent hydrogen bonds.(A, B) The docking status of quercetin with TNF and MMP9 respectively.(C, D) The docking status of kaempferol with IL1B and VEGFA, respectively.(E, F) The docking status of β-sitosterol with VEGFA and TP53 respectively.IL, interleukin; VEGFA, vascular endothelial growth factor A; TNF, tumor necrosis factor.

Figure 6
Figure 6 Microstructural changes in the knee articular surface in KOA model rats.2D imaging of the structure of the subchondral bone of the tibia.The black arrows indicate the damaged region of the articular surface.KOA, knee osteoarthritis.

Table 2 Results of molecular docking
PDB ID, Protein Data Bank identification; TNF, tumor necrosis factor; IL, interleukin.