key: cord-0028307-zde2yn62
authors: Wu, Shan-Shan; Hao, Li-Jun; Shi, Yuan-Yuan; Lu, Zhuo-Jian; Yu, Jia-Lin; Jiang, Si-Qi; Liu, Qing-Ling; Wang, Ting; Guo, Shi-Ying; Li, Ping; Li, Fei
title: Network Pharmacology-Based Analysis on the Effects and Mechanism of the Wang-Bi Capsule for Rheumatoid Arthritis and Osteoarthritis
date: 2022-02-25
journal: ACS Omega
DOI: 10.1021/acsomega.1c06729
sha: 1dfc5eb0c53d065e8dfd545925feaf5ba84ac131
doc_id: 28307
cord_uid: zde2yn62

[Image: see text] Wang-Bi capsule (WB) is a traditional Chinese medicine (TCM)-based herbal formula, and it has been used in the treatment of rheumatoid arthritis (RA) in China for many years. Additionally, WB is also used as a supplement to the treatment of osteoarthritis (OA) in clinical practice. Our research aimed to reveal the therapeutic effects and underling mechanism of WB on RA and OA through computational system pharmacology analysis and experimental study. Based on network pharmacology analysis, a total of 173 bioactive compounds interacted with 417 common gene targets related to WB, RA, and OA, which mainly involved the PI3K-Akt signaling pathway. In addition, the serine–threonine protein kinase 1 (AKT1) might be a core gene protein for the action of WB, which was further emphasized by molecular docking. Moreover, the anti-inflammatory activity of WB in vitro was confirmed by reducing NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells. The anti-RA and OA effects of WB in vivo were confirmed by ameliorating the disease symptoms of collagen II-induced RA (CIA) and monosodium iodoacetate-induced OA (MIA) in rats, respectively. Furthermore, the role of the PI3K-Akt pathway in the action of WB was preliminarily verified by western blot analysis. In conclusion, our study elucidated that WB is a potentially effective strategy for the treatment of RA and OA, which might be achieved by regulating the PI3K-Akt pathway. It provides us with systematic insights into the effects and mechanism of WB on RA and OA.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, mainly characterized by pain, continuum multiarticular synovitis, swollen joints, as well as cartilage and bone damage, which can seriously impair physical function and quality of life. 1, 2 Osteoarthritis (OA) is a degenerative disease, resulting in synovial inflammation, progressive destruction of articular cartilage, and formation of osteophyte. 3, 4 RA and OA are the common arthritis seen in many populations. 5 Additionally, RA and OA share commonalities of disorder, including inflammation, swelling, osteochondral destruction, and disabling symptoms. 6, 7 Moreover, RA and OA belong to the category of "Bi syndrome" in the theory of traditional Chinese medicine (TCM). The etiology of RA and OA is complex and still unclear, while generally believed to be related to genetic predisposition and environmental factors. So far, there is no cure for RA and OA. Anti-inflammatory drugs, such as nonsteroidal antiinflammatory drugs and corticosteroids, are commonly used to treat arthritis, but these drugs are associated with severe side effects and lack robust efficacy. 8, 9 TCMs have been shown to be effective and safe in clinical applications for thousands of years. 10 Moreover, increasing pieces of evidence have demonstrated that TCMs, which include TCM formulas, herbal medicines, and other natural products, are precious resources for curing chronic and complex diseases. 11 Wang-Bi capsule (WB), as a TCM-based herbal formula, has been used in the treatment of RA in China for many years and has shown positive therapeutic effects. 12, 13 In addition, WB is also used as a supplement to the treatment of OA. WB is composed of several medicinal materials, with the characteristics of multicomponents and multitargets. However, WB still lacks sufficient experimental research, and its mechanism of action remains unclear.

Network pharmacology, describes the complexity between ingredients, targets, and diseases from the perspective of network, which has been applied to understand and elucidate the potential mechanism of multicomponent and multitarget TCMs due to the similarity with the overall philosophy of TCMs. 14, 15 In the present study, we aimed to reveal the therapeutic effects and underling mechanism of WB on RA and OA. Network pharmacology analysis and molecular docking are performed to explore and clarify the potential mechanism of WB. The anti-inflammatory effect of WB was studied with lipopolysaccharide (LPS)-induced RAW264.7 cells in vitro. At the same time, the effects of anti-RA and anti-OA for WB were evaluated with collagen-induced arthritis (CIA) rats and monosodium iodoacetate-induced osteoarthritis (MIA) rats in vivo, respectively. Moreover, western blotting assays were carried to confirm the previously predicted mechanism of WB.

2.1. Network Pharmacology Analysis. In total, 173 bioactive compounds of medicinal materials, except for sheep bone in WB, were obtained from TCMSP and the literature (Table S1 ). Among the 173 compounds, 16 shared compounds were derived from two or more medicinal materials (Table 1) , which indicated their vital role in WB to a certain extent. In addition, 1145 compound-related targets were acquired from PharmMapper and Swiss Target Prediction databases. Moreover, a total of 5387 RA-related targets and 3199 RA-related targets were acquired from GeneCards, DisGeNET, and OMIM databases, respectively. All targets were standardized with the UniProt database. Furthermore, 417 common gene targets were obtained after integrating the targets of compounds, RA and OA ( Figure 1A ). These common targets were regarded as drugdisease gene targets.

In order to reveal the relationship among medicinal materials of WB, active compounds, and drug-disease targets, a medicinal material−component−target (M−C−T) network was constructed by employing Cytoscape software, as shown in Figure  1B . The M−C−T network, containing 605 nodes and 5657 edges, visually demonstrated the interaction between the drug, compounds, and targets.

As present in Figure 1C , a protein−protein interaction (PPI) network was established with 417 common targets to analyze the potential protein interactions. In the PPI network, the nodes and edges, respectively, represented the target proteins and their interactions. The number of connections was reflected in the degree value of the node, where the larger and redder the node meant the higher the degree value and the more important a certain node was in this network. 16 Similarly, the redder the node, the more important it was in this network, such as serine− threonine protein kinase 1 (AKT1) and tumor necrosis factor (TNF).

GO analysis revealed 760 terms, including 571 biological processes (BPs), 67 cellular components (CCs), and 122 molecular functions (MFs). The top 20 of them that were selected in the descending order of P value were analyzed and shown in Figure 1D . The BPs mainly involved negative regulation of the apoptotic process, inflammatory response, regulation of phosphatidylinositol 3-kinase signaling, and cellular response to LPS. The CCs were mainly associated with the cytosol, cytoplasm, and plasma membrane. The MFs were mainly involved in enzyme binding, protein binding, and protein kinase activity. The KEGG pathway enrichment analysis obtained 120 related pathways, and the top 20 KEGG pathways are shown in Figure 1E , which mainly involved pathways in cancer, hepatitis B, proteoglycans in cancer, and PI3K-Akt signaling pathway. Combined with previous findings, the PI3K-Akt signaling pathway attracted our attention, and then, we designed further experiments to verify whether WB could exert anti-RA and OA effects by regulating this pathway.

2.2. Molecular Docking Analysis. Molecular docking analysis was performed to verify the accuracy and reliability of interactions between crucial active compounds and key protein of the pathway. Based on previous data, 16 shared compounds and AKT1 were, respectively, selected as receptors and ligand to conduct molecular docking analysis. The docking results are listed in Table 2 .

The 16 compounds all showed good affinity with the AKT1 protein and the top five were β-sitosterol, oleanolic acid, luteolin, anhydroicaritin, and (−)-taxifolin, respectively. Additionally, the previous network pharmacological analysis found that among the 16 shared compounds, the top 5 compounds in degree ranking were anhydroicaritin, oleanolic acid, kaempferol, luteolin, and quercetin (Table 1) , respectively. Integrating the analysis of molecular docking and network pharmacology, the results reveal that three core compounds (oleanolic acid, luteolin, and anhydroicaritin) may play an extremely vital role in WB. Moreover, the docking results of three compounds with the AKT1 protein are visualized and shown vividly in Figure 2A .

2.3. Anti-Inflammatory Effect of WB on LPS-Induced RAW264.7 In Vitro. In vitro experiments, cell viability was first examined with the CCK8 kit to evaluate the cytotoxicity of WB, LPS, and BBR. The results showed that WB (50−800 μg/mL), LPS (1 μg/mL), and BBR (8 μmol/L) had no significant inhibition effect on cell viability, indicating the dose of which had no significant cytotoxicity ( Figure 2B ). Free radical, such as NO, which played vital role in various inflammatory disorders. 17 The anti-inflammatory effect of WB was evaluated on LPS (1 μg/mL)-induced RAW264.7 cells by using the Griess reagent to detect the level of NO in the supernatant. As shown in Figure  2C , the NO production in the model group (LPS, 1 μg/mL) was significantly increased compared with the control group (P < 0.001), indicating the successful establishment of the cellular inflammation model. Compared with the model group, the different concentrations of the WB group and positive control BBR group significantly inhibited the NO production (P < 0.001), demonstrating the anti-inflammatory effect of WB in vitro. In addition, there was significant difference of WB at the concentrations of 50 and 800 μg/mL (P < 0.05). On day 21 after model establishment (d21), the symptoms of rats, including obvious weight loss (P < 0.001), paw volume severe increase (P < 0.001), and high arthritis index score (greater than 4), indicated the successful establishment of the CIA model ( Figure 3A −C). Next, rats in each group received different treatments for 3 weeks. During the treatment, the above-mentioned symptoms of rats in the DF group and WB various dose groups gradually alleviated to a varying degree. After administration (d42), the weight of rats in the DF group increased significantly compared with the Mod group (P < 0.001), while there was no significant difference in WB groups, as presented in Figure 3A . In addition, the paw volume of rats in the DF group (P < 0.001), combined with WB-M and WB-H groups (P < 0.01) significantly decreased ( Figure 3B ). The AI score of rats also reduced significantly in DF, WB-M, and WB-H groups (P < 0.001) compared with the Mod group ( Figure 3C) .

Moreover, the levels of cytokines including TNF-α, IL-1β, and IL-10 in serum were detected and shown in Figure 3D . The levels of pro-inflammatory cytokines TNF-α and IL-1β were markedly increased (P < 0.001), while the level of antiinflammatory cytokine IL-10 was significantly reduced (P < 0.001) in the Mod group compared with the Con group. Meanwhile, the expression levels of TNF-α and IL-1β were obviously inhibited, and IL-10 was markedly raised in DF, WB-M, and WB-H groups compared to those of the Mod group. Furthermore, the histopathological changes of ankle joints in rats are shown in Figure 3E . Rats in the Con group showed an intact ankle joint with a smooth articular surface and normal synovial tissue. On the contrary, the ankle joint in the Mod group showed an uneven articular surface and jagged destruction, accompanied by severe proliferation of the synovial tissue, and a large number of inflammatory cells infiltrated the synovial tissue and articular cartilage. Interestingly, admin-istration of DF, WB-H, and WB-M could effectively alleviate inflammatory cell infiltration, synovial proliferation, and cartilage destruction to a certain extent. What is more, to detect the imaging changes of ankle joints in rats, the micro-CT analysis was performed, and the results are shown in Figure 3F . The ankle joint of rats in the Con group exhibited a clear ankle joint structure and a smooth articular bone surface. In contrast, rats in the Mod group showed typical imaging features of RA, such as severely damaged and blurred ankle joint structure, rough bone surface with serious bone erosion, the unclear structure of multiple metatarsal joints, and the widened space of digital joints. Importantly, DF and WB-H could evidently ameliorate the bone destruction of ankle joint and reduce bone damage in rats.

2.5. Anti-OA Effects of WB on MIA Rats In Vivo. The anti-OA effects of WB in vivo were evaluated in MIA rats. For this purpose, the body weight, serum cytokines, and pathological changes of knee joints in rats were examined and analyzed. Two weeks after modeling, rats were given medication by gavage for 4 weeks. As shown in Figure 4A , the body weights of rats in the Mod group were significantly lower than that in the Con group (P < 0.001), while the body weights in the GH group and WB groups had no significant difference compared with the Con group after 4 weeks of administration. In addition, the results of serum cytokines in rats are presented in Figure 4B , and the levels of TNF-α and IL-1β markedly increased in the Mod group, while IL-10 significantly decreased. After treatment, the levels of pro-inflammatory factors TNF-α and IL-1β in GH, WB-M, and WB-H groups were markedly downregulated (P < 0.001), while the level of anti-inflammatory factor IL-10 was obviously upregulated (P < 0.001). Moreover, macroscopic observations of femoral condyles and tibial plateaus in knee joints for rats were carried out, and the results are shown in Figure 4C . The articular cartilage surface in the Con group was intact and smooth, while that of the model group was severely damaged and the subchondral bone was exposed. GH and WB groups could alleviate the cartilage destruction to varying degrees. Especially, the WB-M and WB-H groups only presented slight cartilage damage. Furthermore, hematoxylin and eosin (HE) pathological results showed serious cartilage extensive destruction and widened joint space in the Mod group compared with the Con group, as shown in Figure 4D . By contrast, cartilage destruction in the GH group and WB groups were relieved to varying degrees. Especially in the WB-M and WB-H groups, the knee joints of rats presented slight cartilage changes and the joint spaces almost returned to normal.

2.6. Effect of WB Regulation on RA and OA through the PI3K-Akt Signaling Pathway. The results of network pharmacology and molecular docking analysis revealed that WB may play a role in the treatment of RA and OA by regulating the PI3K-Akt signaling pathway, of which AKT was the key protein. Therefore, to evaluate the ability of WB in regulating the PI3K-Akt pathway, the expression levels of AKT and p-AKT proteins were determined by employing western blotting. As demonstrated in Figure 5A , the expression of p-AKT/AKT was significantly higher in the Mod group in comparison with the 

Con group, while WB could diminish the level of p-AKT/AKT. The results indicated that WB might exert the therapeutic effects through regulating the PI3K-Akt pathway ( Figure 5B ).

RA and OA are common arthritis diseases clinically, and they share common features, such as inflammation, cartilage destruction, and disability in the late stage of the disease. 18−21 In the theory of TCM, RA and OA belong to the category of "Bi syndrome", which is mostly attributed to the deficiency of the liver and kidney. The specific pathogenesis of RA and OA is still unclear, and there is no cure for RA and OA at present. Some existing medications, such as nonsteroidal anti-inflammatory drugs, can be used for the treatment of RA and OA; however, they are often accompanied by side effects and poor long-term therapeutic effects. In contrast, TCM has the characteristics of low side effects, strong overall effect, addressing both symptoms and root causes, and good long-term effects. Correspondingly, TCM has unique advantages in the treatment of chronic and complex diseases. WB is a TCM-based herbal formula and has been used in clinical practice for many years. In the present study, network pharmacology analysis combined with molecular docking and experimental verification was performed to investigate the therapeutic effects and decipher the mechanism of WB on RA and OA. Based on the network pharmacology analysis, a total of 173 bioactive compounds were screened from WB, and 417 common gene targets related to WB, RA, as well as OA were identified. After that, the M−C−T network was established. Among the 173 compounds, 16 shared compounds were considered to play a vital role in WB. Moreover, the top five in the M−C−T network according to the degree value were anhydroicaritin, oleanolic acid, kaempferol, luteolin, and quercetin, respectively. The higher the degree value, the more important it was in the network. In addition, the PI3K-Akt signaling pathway and the AKT1 protein were regarded as the potential mechanism and core gene target of WB against RA and OA. Based on molecular docking, 16 shared compounds all showed good affinity with the AKT1 protein, and the lower the value, the better the affinity. The top five in affinity were anhydroicaritin, oleanolic acid, kaempferol, luteolin, and quercetin, respectively. Therefore, three compounds (oleanolic acid, luteolin, and anhydroicaritin) and AKT1 protein attracted our attention, which were considered to play a vital role of WB in the treatment of RA and OA.

As reported in the literature, oleanolic acid showed antiinflammatory activity by inhibiting the release of the LPSmediated high-mobility group box 1 in human umbilical vein endothelial cells. 22 Luteolin could ameliorate the development of arthritis caused by the injection of collagen type II in mice 23 and attenuate OA progression in the MIA rat model. 8 Anhydroicaritin suppressed RANKL-induced osteoclast differentiation. 24 Additionally, accumulated evidence showed that the PI3K-Akt pathway is involved in various BPs, including the development of RA and OA. 25−28 The AKT is a pivotal downstream effector of phosphatidylinositol kinase PI3K.

Besides, current studies have demonstrated that AKT positively regulates chondrocyte proliferation as well as cell growth in skeletal development. 26, 29 For experimental verification, the anti-inflammatory effect of WB in vitro was demonstrated by reducing NO production in LPS-induced RAW264.7 cells. In vivo, the anti-RA effects of WB were confirmed by CIA rats. WB could ameliorate CIA rats, including reversing weight loss, reducing paw volume and arthritis index, reducing the level of inflammatory factors (TNFα, IL-1β) and raising the level of anti-inflammatory factor IL-10, and alleviating inflammatory cell infiltration in the synovial tissue and bone destruction of the ankle joint. Similarly, the anti-OA effects of WB were confirmed by MIA rats. WB could ameliorate MIA rats, involving in reversing weight loss, decreasing the level of TNF-α combined with IL-1β and increasing the level of IL-10, and relieving the cartilage destruction of the knee joint. Interestingly, WB effectively inhibited the ratio of p-AKT/AKT, indicating that WB could suppress the PI3K-AKT pathway, which was also consistent with previous prediction.

This work revealed that WB might exert anti-RA and anti-OA effects by regulating the PI3K-Akt pathway. It is the first time to report the mechanism of WB on treating RA and OA. Importantly, it has potential significance for understanding the active substances and pharmacological mechanism of TCMbased herbal formula on complex diseases.

In summary, a combination of computational system pharmacology analysis and experimental study was performed to explore the therapeutic effects and mechanism of WB on RA and OA in this study. Our research systematically indicated that WB might be a useful strategy for treating RA and OA by regulating the PI3K-Akt pathway. It provides not only scientific evidence for the clinical application of WB but also insights into our understanding of the action mechanism for WB on RA and OA. In particular, it is of great significance for understanding the pharmacological mechanism of TCMs in the treatment of complex diseases. 

Handling. RAW 264.7 murine macrophage cells were supplied by the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37°C with 5% CO 2 in a DMEM medium containing 10% FBS and 1% penicillin−streptomycin.

Grade SPF female Wistar rats (140 ± 10 g) and male Wistar rats (160 ± 10 g) were provided by Laboratory Animal Business Department of Shanghai Institute of Family Planning (Shanghai China). All animals were maintained in a room (temperature 25°C

, relative humidity 50−65%) under a 12:12 h light and dark cycle and allowed free to food and water. The animals were accommodated for a week before the start of the experiments. All studies were performed according to the guidelines of China Pharmaceutical University Pharmacy Animal Experiment Center and approved by the Animal Ethics Committee of this institution. 30 In addition, combined with the literature investigation, other compounds with pharmacological activities but not previously meeting the conditions were also included as bioactive compounds.

The related targets of bioactive compounds screened from WB were collected from PharmMapper (http://www.lilabecust.cn/pharmmapper/) and Swiss Target Prediction (http:// www.swisstargetprediction.ch/). The related targets of RA and OA were collected from the databases including GeneCards (https://www.genecards.org/), DisGeNET (https://www. disgenet.org/), and OMIM (https://omim.org/). All targets were standardized with the UniProt database (https://www. uniprot.org/) and selected species were limited to "Homo sapiens". 31 Intersecting the component-related targets and disease-related targets through the Venny 2.1.0 tool (https:// bioinfogp.cnb.csic.es/tools/venny/index.html), the common targets obtained were regarded as the potential targets of WB for the treatment of RA and OA.

The relationship of medicinal materials, multiple components, and the common targets, which are associated with WB, RA, and OA, were revealed according to the above results. Then, the M−C−T network was constructed and exhibited based on Cytoscape software (version 3.8.0). 5.3.3. PPI Analysis. PPI analysis for the common targets was carried out using the STRING tool (https://string-db.org/) and species were "H. sapiens". 32 In addition, the PPI network was visualized by employing Cytoscape software.

5.3.4. GO and KEGG Pathway Enrichment Analysis. The DAVID Bioinformatics Resources 6.8 database (https://david. ncifcrf.gov/) and OmicShare tool (https://www.omicshare. com/tools/) were employed to perform Kyoto Encyclopedia of Genes and Genes and Genomes (KEGG) pathway enrichment analysis and gene ontology enrichment analysis, involving BP, CC, and MFs. 30 5.3.5. Molecular Docking Analysis. Molecular docking analysis was used to confirm the interactions between core components and key target of WB for treating RA and OA and to verify the accuracy of predictions in network pharmacology. 10 The crystal structure of the candidate protein was downloaded from RCSB PDB (https://www.rcsb.org/) and was subsequently modified by PyMOL software, involving in removal of water, protonation, as well as energy minimization. 33, 34 The core components were also converted into the PDB format. Additionally, the target protein and compounds were converted into the PDBQ format using AutoDock. Furthermore, AutoDock Vina was applied to dock the receptor protein with the molecule ligands of core components. 35 5.4. Experimental Verification of WB for the Application in RA and OA Treatment. 5.4.1. Sample Preparation and Assessment of Cell Viability and NO Production. In an in vitro study, the preparation of WB samples was as follows: WB with a content of 20.03 g was ultrasonically extracted with 100 mL of water at room temperature for 3 h, and after centrifugation, the supernatant was freeze-dried to obtain 9.16 g of powder. Before use, the powder was dissolved in the medium.

For the cell viability assay, RAW 264.7 cells were seeded in a 96-well plate at a density of 5 × 10 3 cells per well and cultured for 24 h. After that, cells were incubated with the medium or with LPS (1 μg/mL) in the presence or absence of various concentrations of WB samples for 24 h. Then, CCK8 was added and incubated for 1 h. The absorbance was measured at the wavelength of 450 nm using a microplate reader (POLARstar).

For the nitric oxide (NO) production assay, RAW 264.7 cells (2 × 10 5 cells/well) were plated in a 96-well plate and incubated for 24 h. Then, cells were treated in the same way as mentioned above. After that, the supernatant of cells was obtained, and the level of NO production was measured using Griess reagents. 36 The absorbance was measured at 540 nm. 5.4.2. Establishment of Collagen-Induced RA and Evaluation of the Anti-RA Effect In Vivo. The CIA model was established based on previously reported methods with modification. 17,37 In brief, bovine collagen II was dissolved in 0.05 mol L −1 acetic acid overnight at 4°C to obtain the collagen solution (2 mg mL −1 ). Then, on day 0, rats were given a primary immunization, respectively, with an intradermal tail vein injection of emulsion (200 μL), obtained by emulsifying collagen solution and CFA at a ratio of 1:1 in an ice bath. On day 7, rats were intradermally rechallenged with collagen II emulsified in IFA. Wistar female rats were randomly divided into six groups: control group (Con), model group (Mod), positive control diclofenac sodium group (DF), WB low-dose group (WB-L), WB medium-dose group (WB-M), and WB high-dose group (WB-H), and each group consisted of eight rats. Except for the control group, other groups replicated the CIA model according to the above methods. On day 21, the successful establishment of the CIA model in rats was determined by the arthritis index (AI) scores greater than or equal to 4. The criteria of AI scores were as follows: (0) normal; (1) erythema and slight swelling of the ankle joint; (2) erythema and slight swelling of the ankle joint to the plantar joint or palmar joint; (3) erythema and moderate swelling of the ankle joint to the plantar joint or palmar joint; and (4) erythema and severe swelling. The total score of each paw for an individual rat was as an AI, with a maximum total score of 16. After that, WB-L, WB-M, and WB-H groups were intragastrically given corresponding doses of WB (247.5, 742.5, and 2227.5 mg kg d −1 ) once a day for 3 weeks, and the DF group was treated with diclofenac sodium (6.75 mg kg d −1 ) on the same schedule, and at the same time, Con and Mod group rats were orally given an equal volume of CMC-Na. After treatment, all rats were sacrificed, and then, the serum, synovium, paws, and ankle joints were obtained for further experimental analysis.

The body weights of all rats were monitored using an animal electronic scale (TANITA, Shanghai, China), and the paw volumes of rats were measured with a PV-200 Toe volume measuring instrument (TECHMAN, Chengdu, China) . The AI of all rats in groups was evaluated with the scoring criteria. The levels of serum cytokines in rats, including TNF-α, IL-1β, and IL-10, were determined with the ELISA kits following corresponding manufacturer's protocols. The right paws and ankle joints of rats were dissected and fixed in formalin for 3 days. Micro-computed tomography (Micro-CT) analysis of the right paws and ankle joints was performed to acquire the threedimensional (3-D) structure images based on the SKYSCAN 1176 micro-CT system (Bruker, Shanghai, China) and CTvox software. The scanning parameters were as follows: voltage 70 kV, current 142 μA, isotropic voxel size 18 μm, with a 0.5 mm aluminum filter. In addition, the right ankle joints were decalcified in 10% EDTA and embedded in paraffin. Then, the ankle joint tissue sections were stained with HE, which were subsequently analyzed by employing an intelligent digital slice scanning system NanoZoomer S60 (HAMAMATSU, Beijing, China) and the corresponding NDP. view 2 software. Additionally, the scanning mode of all slices was as follows: bright field, 20 times mirror, and fully automatic scanning mode. 5.4.3. Establishment of MIA and Evaluation of Anti-OA Effect In Vivo. The MIA model was performed as previously reported. 38, 39 In brief, rats were given an intra-articular injection of 3 mg of MIA diluted in 50 μL of saline. Wistar male rats were randomly divided into six groups: control group (Con, equal CMC-Na), model group (Mod, equal CMC-Na), positive control glucosamine hydrochloride capsule group (GH, 129.6 mg kg d −1 ), WB low-dose group (WB-L, 247.5 mg kg d −1 ), WB medium-dose group (WB-M, 742.5 mg kg d −1 ), and WB highdose group (WB-H, 2227.5 mg kg d −1 ). After that, Con group rats were injected with saline (50 μL) into the articular cavity of unilateral knee joints, while the other group rats were injected with MIA solution (3 mg/50 μL). Subsequently, after 2 weeks of the injection, 36 rats received the treatment once a day by gavage for 4 weeks. After treatment, rats in all experimental groups were sacrificed, and then, the serum, synovium, and knee joints were acquired for further analysis.

The body weights and the levels of serum cytokines (TNF-α, IL-1β, and IL-10) were measured as previously described. In addition, the knee joints in each group were randomly selected for macroscopic observations, which conducted by separating the femoral condyle and tibial plateau and then taking pictures. Moreover, the knee joint tissue sections were stained with HE similarly as previously mentioned, and subsequently analyzed with the intelligent digital slice scanning system NanoZoomer S60 and the NDP. view 2 software. 5.4.4. Western Blotting Assay. The total proteins of synovium were extracted with RIPA lysate freshly supplemented with 1% protease and 2% phosphatase inhibitor (Beyotime, Shanghai, China) before use. Then, the proteins were obtained by centrifugation of lysate at 4°C with 12,000 rpm for 15 min (Eppendorf, Hamburg, Germany). Subsequently, the protein concentrations were measured with the BCA protein assay kit (KeyGEN Bio TECH, Nanjing, China). Additionally, the protein samples were denatured at 99°C for 10 min and were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred from gel onto nitrocellulose (NC) membranes, which were blocked with 5% non-fat milk or bovine serum albumin for 2 h and then incubated at 4°C with the primary antibodies (1:1000) overnight. 40, 41 After that, the membranes were washed three times with TBST (Servicebio, Wuhan, China) and incubated with second antibodies (1:2000) for 1 h at room temperature. Then, the protein bands were visualized by the ECL kit (Tanon, Shanghai, China) and Tanon-5200 gel imaging system (Tanon, Shanghai, China). The gray value of the bands was quantified with ImageJ software.

5.5. Statistical Analysis. The statistical analysis was performed with GraphPad Prism 8.0 software. All data were presented as mean ± SD. The one-way analysis of variance was applied for comparisons between groups, and P < 0.05 was considered as statistically significant. Complete contact information is available at: https://pubs.acs.org/10.1021/acsomega.1c06729

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