Rehmannia alcohol extract inhibits neuropeptide secretion and alleviates osteoarthritis pain through cartilage protection.

Osteoarthritis (OA) is a common joint disease characterized by chronic pain, and the perception of pain is closely associated with brain function and neuropeptide regulation. Rehmannia is common plant herb with anti-inflammatory and analgesic properties that is used to treat OA. However, it is unclear whether Rehmannia alleviates OA-related pain via regulation of neuropeptides and brain function. We examined the pain relief regulatory pathway in OA after treatment with Rehmannia by verifying the therapeutic effect of Rehmannia alcohol extract in vivo and vitro and exploring of the potential mechanism underlying the analgesic effect of Rahmanian using functional magnetic resonance imaging and measuring neuropeptide secretion. Our results showed that Rehmannia alcohol extract and the related active ingredient, Rehmannioside D, can delay cartilage degradation and alleviate inflammation in OA rats. The Rehmannia alcohol extract can also relieve OA pain, reduce the secretion of calcitonin gene-related peptide (CGRP) and substance P (SP), and reverse the pathological changes in the cerebral cortex and hippocampus. Our research results demonstrate that Rehmannia alleviates OA pain by protecting cartilage, preventing the stimulation of inflammatory factors on neuropeptide secretion, and influencing the relevant functional areas of the brain.

Achyranthes bidentata polysaccharides alleviate endoplasmic reticulum stress in osteoarthritis via lncRNA NEAT1/miR-377-3p pathway.

Endoplasmic reticulum stress (ERS) has been identified to be an important factor leading to chondrocyte apoptosis in osteoarthritis (OA). Previous studies have confirmed that Achyranthes bidentata polysaccharides (ABPS) can inhibit chondrocyte apoptosis; however, the mechanism of action of ABPS on chondrocyte ERS remains unclear. Thus in this study, we aim to investigate whether ABPS could inhibit OA-associated chondrocyte apoptosis by regulating ERS, especially by observing the relationship between the lncRNA NEAT1 and miR-377-3p, to explore further the protective mechanism of ABPS in OA. In vitro and in vivo experiments showed that ABPS inhibited chondrocyte ERS by regulating the expression of lncRNA NEAT1 and miR-377-3p. Moreover, both lncRNA NEAT1 silencing and miR-377-3p inhibition could attenuate the therapeutic effect of ABPS on ERS. Dual-luciferase results indicated that miR-377-3p targets the lncRNA NEAT1 gene in mouse chondrocytes. Therefore, we concluded that ABPS could inhibit thapsigargin (TG)-induced chondrocyte ERS through the lncRNA NEAT1/miR-377-3p axis.

Protective role of Achyranthes bidentata polysaccharides against chondrocyte extracellular matrix degeneration through lncRNA GAS5 in osteoarthritis.

Achyranthes bidentata polysaccharides (ABPS) is an active ingredient of the flowering plant Achyranthes bidentata that has been previously reported to be effective for the treatment of osteoarthritis (OA). However, the underlying molecular mechanism remain to be fully clarified. Emerging studies have shown that the long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) is involved in the pathogenesis of OA. Therefore, the present study aimed to investigate the potential mechanism of ABPS by focusing on its effects on the regulation of chondrocyte extracellular matrix (ECM) homeostasis, with particular emphasis on lncRNA GAS5. In the present study, the modified Hulth method was used to construct OA rats, which were gavaged with 400 mg/kg ABPS for 8 weeks. Histopathological changes in cartilage and subchondral bone were evaluated by hematoxylin-eosin staining and Safranin O-fast green staining. In in vitro experiments, IL-1ß-treated chondrocytes were infected with Lenti-lncRNA GAS5. Fluorescence in situ hybridization assay was performed to measure the expression of the lncRNA GAS5 in chondrocytes. Moreover, the relative expression level of lncRNA GAS5 in cartilage tissue and chondrocytes was detected using reverse transcription-quantitative PCR. Western blot analysis was used to detect protein expression levels of MMP-9, MMP-13, TIMP-1, TIMP-3 and type II collagen in cartilage tissue and chondrocytes. The results indicated that ABPS delayed the degradation of the ECM by chondrocytes in addition to reducing lncRNA GAS5 expression both in vivo and in vitro. Furthermore, silencing of lncRNA GAS5 expression in IL-1ß-treated chondrocytes downregulated the protein expression of MMP-9 and MMP-13 whilst upregulating the expression of tissue inhibitor matrix metalloproteinase (TIMP)-1, TIMP-3 and type II collagen. To conclude, the present study provides evidence that ABPS can inhibit the expression of lncRNA GAS5 in chondrocytes to regulate the homeostasis of ECM, which in turn may delay the occurrence of cartilage degeneration during OA.

Identification of the Key Role of NF-κB Signaling Pathway in the Treatment of Osteoarthritis With Bushen Zhuangjin Decoction, a Verification Based on Network Pharmacology Approach.

This study aimed to identify whether the NF-κB signaling pathway plays a key role in the treatment of osteoarthritis (OA) with Bushen Zhuangjin Decoction (BZD) based on a typical network pharmacology approach (NPA). Four sequential experiments were performed: 1) conventional high-performance liquid chromatography (HPLC), 2) preliminary observation of the therapeutic effects of BZD, 3) NPA using three OA-related gene expression profiles, and 4) verification of the key pathway identified by NPA. Only one HPLC-verified compound (paeoniflorin) was identified from the candidate compounds discovered by NPA. The genes verified in the preliminary observation were also identified by NPA. NPA identified a key role for the NF-κB signaling pathway in the treatment of OA with BZD, which was confirmed by conventional western blot analysis. This study identified and verified NF-κB signaling pathway as the most important inflammatory signaling pathway involved in the mechanisms of BZD for treating OA by comparing the NPA results with conventional methods. Our findings also indicate that NPA is a powerful tool for exploring the molecular targets of complex herbal formulations, such as BZD.

Anti-inflammatory activity of Radix Angelicae biseratae in the treatment of osteoarthritis determined by systematic pharmacology and in vitro experiments.

Radix Angelicae biseratae is a widely used Chinese traditional herbal medicine for osteoarthritis (OA). Its therapeutic efficacy has been confirmed in clinical practice. However, its mechanisms of action in treating OA have remained elusive. The purpose of the present study was to identify active components with good oral bioavailability and drug-like properties from Radix Angelicae biseratae through systematic pharmacology and in vitro experiments to determine targets of Radix Angelicae biseratae in the treatment of OA. The functional components of Radix Angelicae biseratae were screened from the Traditional Chinese Medicine Systems Pharmacology database based on oral bioavailability and drug-like properties. Subsequently, the databases STITCH, Open Targets Platform and DrugBank were searched and microarray analysis was performed to screen the active components of Radix Angelicae biseratae to treat OA and predict its potential target proteins. The interaction network and protein interaction network were then generated and examined, molecular docking between active components and targets was performed and the enrichment of potential target proteins was analyzed. Finally, reverse transcription-quantitative (RT-q)PCR and western blot analyses were used to verify the therapeutic effect of Radix Angelicae biseratae extract on the expression of OA-associated target proteins. The results provided eight active components in Radix Angelicae biseratae, which were firmly linked to 20 targets of OA. In combination with molecular docking and the analysis of the interaction network between components and targets, it was suggested that sitosterol was a major active component of Radix Angelicae biseratae in the treatment of OA. Protein interaction network analysis suggested that prostaglandin-endoperoxide synthase 2 (PTGS2), nitric oxide synthase 3 and cytochrome P450 2B6 may be critical targets for Radix Angelicae biseratae in the treatment of OA. In addition, RT-qPCR and western blot analyses suggested that Radix Angelicae biseratae extract inhibited the mRNA and protein expression of PTGS2 in degenerative articular cartilage cells in vitro, whilst other targets remain to be verified. Functional enrichment analysis indicated that Radix Angelicae biseratae confers pharmacological efficacy towards OA through exerting anti-inflammatory effects and immune regulation.

Bioinformatics analysis to identify key genes and pathways influencing synovial inflammation in osteoarthritis.

Osteoarthritis (OA) is a chronic arthropathy that occurs in the middle­aged and elderly population. The present study aimed to identify gene signature differences between synovial cells from OA synovial membrane with and without inflammation, and to explain the potential mechanisms involved. The differentially expressed genes (DEGs) between 12 synovial membrane with inflammation and 12 synovial membrane without inflammation from the dataset GSE46750 were identified using the Gene Expression Omnibus 2R. The DEGs were subjected to enrichment analysis, protein­protein interaction (PPI) analysis and module analysis. The analysis results were compared with text­mining results. A total of 174 DEGs were identified. Gene Ontology enrichment results demonstrated that functional molecules encoded by the DEGs primarily had extracellular location, molecular functions predominantly involving 'chemokine activity' and 'cytokine activity', and were associated with biological processes, including 'inflammatory response' and 'immune response'. The Kyoto Encyclopedia of Genes and Genomes results demonstrated that DEGS may function through pathways associated with 'rheumatoid arthritis', 'chemokine signaling pathway', 'complement and coagulation cascades', 'TNF signaling pathway', 'intestinal immune networks for IgA production', 'cytokine­cytokine receptor interaction', 'allograft rejection', 'Toll­like receptor signaling pathway' and 'antigen processing and presentation'. The top 10 hub genes [interleukin (IL)6, IL8, matrix metallopeptidase (MMP)9, colony stimulating factor 1 receptor, FOS proto­oncogene, AP1 transcription factor subunit, insulin­like growth factor 1, TYRO protein tyrosine kinase binding protein, MMP3, cluster of differentiation (CD)14 and CD163] and four gene modules were identified from the PPI network using Cytoscape. In addition, text­mining was used to identify the commonly used drugs and their targets for the treatment of OA. It was initially verified whether the results of the present study were useful for the study of OA treatment targets and pathways. The present study provided insight for the molecular mechanisms of OA synovitis. The hub genes and associated pathways derived from analysis may be targets for OA treatment. IL8 and MMP9, which were validated by text­mining, may be used as molecular targets for the OA treatment, while other hub genes require further validation.

Electroacupuncture inhibits sodium nitroprusside­mediated chondrocyte apoptosis through the mitochondrial pathway.

In China, electroacupuncture (EA) is a therapeutic method that is extensively applied in the clinical treatment of osteoarthritis (OA); however, the underlying molecular mechanism remains unclear. Chondrocyte apoptosis may be observed in cartilage tissue in OA, and is often considered a key target for the treatment of this condition. Therefore, the present study aimed to determine the effects of EA on sodium nitroprusside (SNP)­induced chondrocyte apoptosis. Chondrocytes were obtained from the knee joints of Sprague Dawley rats by type II collagenase digestion. Following microscopic observation and authentication with type II collagen immunohistochemistry, articular cartilage cells were used in subsequent experiments. Using inverted phase contrast microscopy, DAPI staining and flow cytometry, it was revealed that chondrocytes treated with SNP became apoptotic, whereas EA inhibited SNP­induced chondrocyte apoptosis. Subsequently, JC­1 single staining, reverse transcription­quantitative polymerase chain reaction analysis, western blotting, colorimetric assays and immunofluorescence staining were performed for further investigation. The results demonstrated that, when compared with normal chondrocytes, the mitochondrial membrane potential of SNP­treated chondrocytes was markedly lowered, B­cell lymphoma 2 (Bcl­2) expression was reduced, and the expression levels of Bcl­2­associated X protein (Bax), cytochrome c, caspase­9 and caspase­3 were increased. Compared with in SNP­treated chondrocytes, the decrease in the mitochondrial membrane potential of chondrocytes treated with SNP and EA was smaller, Bcl­2 expression was increased, and the expression levels of Bax, cytochrome c, caspase­9 and caspase­3 were decreased following EA intervention. In conclusion, the present study demonstrated that EA modulated the mitochondrial pathway to suppress SNP­mediated chondrocyte apoptosis. Therefore, EA may be of value in the treatment of OA.

Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition.

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose­ and time­dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription­quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin­dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis.

Deciphering the underlying mechanisms of Diesun Miaofang in traumatic injury from a systems pharmacology perspective.

Diesun Miaofang (DSMF) is a traditional herbal formula, which has been reported to activate blood, remove stasis, promote qi circulation and relieve pain. DSMF holds a great promise for the treatment of traumatic injury in an integrative and holistic manner. However, its underlying mechanisms remain to be elucidated. In the present study, a systems pharmacology model, which integrated cluster ligands, human intestinal absorption and aqueous solution prediction, chemical space mapping, molecular docking and network pharmacology techniques were used. The compounds from DSMF were diverse in the clusters and chemical space. The majority of the compounds exhibited drug-like properties. A total of 59 compounds were identified to interact with 16 potential targets. In the herb-compound-target network, the majority of compounds acted on only one target; however, a small number of compounds acted on a large number of targets, up to a maximum of 12. The comparison of key topological properties in compound-target networks associated with the above efficacy intuitively demonstrated that potential active compounds possessed diverse functions. These results successfully explained the polypharmacological mechanism underlying the efficiency of DSMF for the treatment of traumatic injury as well as provided insight into potential novel therapeutic strategies for traumatic injury from herbal medicine.