Emodin promotes the recovery of rheumatoid arthritis by regulating the crosstalk between macrophage subsets and synovial fibroblast subsets.

BACKGROUND: To study the relationships among emodin, synovial fibroblasts (FLSs), and macrophages (STMs) to provide guidance for the use of emodin in rheumatoid arthritis (RA) treatment. METHODS: RA clinical samples from patients with different pathological processes were collected, and the correlations between the subsets of FLSs and STMs and pathological processes were analyzed via flow cytometry. In vitro experimental methods such as enzyme linked immunosorbent assay (ELISA), Western blotting, Transwell assays, CCK-8 assays and cell coculture were used to assess cell proliferation, migration and secretion of inflammatory factors. A collagen-induced arthritis mouse model was constructed to investigate the therapeutic potential of emodin in RA by flow cytometry, micro-CT and staining. RESULTS: Unique subsets of FLSs and STMs, namely, FAPα+ THY1- FLSs, FAPα+ THY1+ FLSs, and MerTKpos TREM2high STMs, were identified in synovial tissues from RA patients. The number of MerTKpos TREM2high STMs was negatively correlated with the degree of damage in RA, while the number of FAPα+ THY1- FLSs was positively correlated with damage. On the one hand, emodin promoted the aggregation of MerTKposTREM2high STMs. Moreover, MerTKpos TREM2high STM-mediated secretion of exosomes was promoted, which can inhibit the secretion of pro-inflammatory factors by FAPα+ THY1+ FLSs and promote the secretion of anti-inflammatory factors by FAPα+ THY1+ FLSs, thereby inhibiting FAPα+ THY1- FLS proliferation and migration, improving the local immune microenvironment, and inhibiting RA damage. CONCLUSION: Emodin was shown to regulate the aggregation of STM subsets and exosome secretion, affecting the secretion, proliferation and migration of inflammatory factors in FLS subsets, and ultimately achieving good therapeutic efficacy in RA patients, suggesting that it has important clinical value.

Kikuchi-Fujimoto disease evolves into lupus encephalopathy characterized by venous sinus thrombosis: a case report.

Kikuchi-Fujimoto disease (KFD) is a benign, self-limiting illness that can progress to systemic lupus erythematosus (SLE) in approximately 30% of cases. Neurological injuries can occur in both diseases, albeit with distinct presentations. Venous sinus thrombosis is a serious cerebrovascular complication in patients with neuropsychiatric SLE but is rarely observed in patients with KFD. The involvement of various antibodies, particularly antiphospholipid antibodies, can cause vascular endothelial cell injury, resulting in focal cerebral ischemia and intracranial vascular embolism in SLE. However, there are cases in which thrombotic pathology occurs without antiphospholipid antibody positivity, attributed to vascular lesions. In this report, we present a case of KFD and lupus encephalopathy featuring cerebral venous sinus thrombosis, despite the patient being negative for antiphospholipid antibody. We also conducted a comparative analysis of C3 and C4 levels in cerebrospinal fluid (CSF) and peripheral blood, along with the protein ratio in CSF and serum, to elucidate the pathological changes and characteristics of lupus encephalopathy.

Curative effect of heat-sensitive moxibustion on chronic persistent asthma: a multicenter randomized controlled trial.

OBJECTIVE: To compare the curative effects of heat-sensitive moxibustion with conventional drugs on chronic persistent asthma and seek a valuable therapy to replace Western Medicine. METHODS: The participants in this multi-center, randomized, and controlled study were randomly divided into two groups: group A (n=144), treated with heat-sensitive moxibustion (50 sessions) and group B (n=144), treated with Seretide (salmeterol 50 plg/fluticasone 250 pg, twice a day). The scores of asthma control test (ACT), forced expiratory volume in 1 second (FEV1), peak expiratory flow (PEF), and attack frequency were measured after 15, 30, 60, and 90 days of treatment. Patients followed up 3 and 6 months after treatment. RESULTS: There was a significant difference (P= 0.0002) in the ACT score and lung function between the two groups after 3 months of treatment and (P=0.000 03) during the follow-up visits. In addition, heat-sensitive moxibustion reduced attack frequency in the period from inclusion to the 6-month follow-up visit. CONCLUSION: This study shows that heat-sensitive moxibustion may have a comparable curative effect to Seretide (salmetero/fluticasone) on asthma.

The elucidation of the anti-inflammatory mechanism of EMO in rheumatoid arthritis through an integrative approach combining bioinformatics and experimental verification.

Introduction: Emodin (EMO), a natural derivative of the anthraquinone family mainly extracted from rhubarb (Rheum palmatum), has previously been demonstrated to possess superior anti-inflammatory properties from a single target or pathway. In order to explore the underlying mechanism of action of EMO against rheumatoid arthritis (RA), a network pharmacology approach was employed. Methods: A gene expression profile from GSE55457 available from the Gene Expression Omnibus (GEO) database was used to identify the targets of EMO action. Further, single cell RNA sequencing data from GEO database of RA patients (GSE159117) were downloaded and analysed. To further investigate the anti-RA effect of EMO on MH7A cells, the expression of IL-6 and IL-1ß were monitored. Finally, RNA-seq analyses were conducted on synovial fibroblasts from EMO-treated. Result: We screened the key targets of EMO against RA using network pharmacology methods, including HMGB1, STAT1, EGR1, NR3C1, EGFR, MAPK14, CASP3, CASP1, IL4, IL13, IKBKB and FN1, and their reliability was verified using ROC curve. Single-cell RNA sequencing data analysis showed that these core target proteins mainly played a role by modulating monocytes. The anti-RA effect of EMO was further verified with MH7A cells, which showed that EMO could block cell differentiation and reduce the expression of IL-6 and IL-1ß. WB experiments confirmed that EMO could affect the expression of COX2, HMBG1 and the phosphorylation of p38. Finally, sequencing of synovial fibroblasts from rats treated with EMO showed consistent results with those predicted and verified, further proving the anti-inflammatory effect of EMO. Conclusion: Our research shows that EMO inhibits inflammatory response of rheumatoid arthritis (RA) by targeting HMGB1, STAT1, EGR1, NR3C1, EGFR, MAPK14, CASP3, CASP1, IL4, IL13, IKBKB, FN1 and Monocytes/macrophages.

Recent advances of flowering locus T gene in higher plants.

Flowering Locus T (FT) can promote flowering in the plant photoperiod pathway and also facilitates vernalization flowering pathways and other ways to promote flowering. The expression of products of the FT gene is recognized as important parts of the flowering hormone and can induce flowering by long-distance transportation. In the present study, many FT-like genes were isolated, and the transgenic results show that FT gene can promote flowering in plants. This paper reviews the progress of the FT gene and its expression products to provide meaningful information for further studies of the functions of FT genes.

Molecular cloning, characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia.

Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

Mechanism of Emodin in the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic, and autoimmune disease, and its main pathological changes are inflammatory cell infiltration accompanied by the secretion and accumulation of a variety of related cytokines, which induce the destruction of cartilage and bone tissue. Therefore, the modulation of inflammatory cells and cytokines is a key therapeutic target for controlling inflammation in RA. This review details the effects of emodin on the differentiation and maturation of T lymphocytes, dendritic cells, and regulatory T cells. In addition, the systematic introduction of emodin directly or indirectly affects proinflammatory cytokines (TNF-α, IL-6, IL-1, IL-1ß, IL-17, IL-19, and M-CSF) and anti-inflammatory cytokines (the secretion of IL-4, IL-10, IL-13, and TGF-ß) through the coregulation of a variety of inflammatory cytokines to inhibit inflammation in RA and promote recovery. Understanding the potential mechanism of emodin in the treatment of RA in detail provides a systematic theoretical basis for the clinical application of emodin in the future.

Knockout of SLAMF8 attenuates collagen-induced rheumatoid arthritis in mice through inhibiting TLR4/NF-κB signaling pathway.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial hyperplasia, cartilage damage, and ultimate bone destruction. The signaling lymphocytic activation molecule family member 8 (SLAMF8) is a cell surface receptor expressed on various immune cells. This study aimed to investigate the role of SLAMF8 in the pathogenesis of RA. The SLAMF8 gene was identified as a differentially expressed gene in RA by analyzing the Gene Expression Omnibus database and synovial tissue samples collected from RA patients. Upregulation of SLAMF8 was associated with increased disease activity and inflammation in RA. Mice with collagen type II-induced arthritis (CIA) showed highly expressed SLAMF8, severe paw swelling, elevated inflammatory cytokine production, and excessive accumulation of immune cells. However, knockout of SLAMF8 alleviated collagen type II immunization-induced synovial hyperplasia and joint arthritis in mice. The in-vitro and in-vivo study showed that genetic deletion of SLAMF8 significantly inhibited upregulation of Toll-like receptor 4 (TLR4) and activation of the nuclear factor kappa B (NF-κB) pathway in fibroblast-like synoviocytes and bone marrow-derived macrophages derived from WT and SLAMF8 knockout mice under lipopolysaccharide stimulation. In conclusion, SLAMF8 was aberrantly expressed in RA patient and played an indispensable role in initiating inflammation and maintaining the pro-inflammatory environment in the inflamed joint. Targeted inhibition of SLAMF8 attenuated the severity of RA via blocking the TLR4/NF-κB signaling pathway. These data suggested that SLAMF8 may be a potential target for the treatment of RA.

Deciphering the action mechanism of paeoniflorin in suppressing pancreatic cancer: A network pharmacology study and experimental validation.

Background: Paeoniflorin (PF) is the main active component of Chinese herbaceous peony that has been shown to have an anti-tumor effect. However, there are few studies on the prevention and treatment of pancreatic cancer with PF. Methods: We gathered Microarray data pertaining to paeoniflorin intervention in pancreatic cancer by utilizing the GEO database (GSE97124). Then, the DEGs were filtered by the 33R program. RNA-seq data of pancreatic cancer and normal tissue samples were taken from the TCGA and GTEx databases, respectively, and the WGCNA technique was utilized to examine the pancreatic cancer-specific genes. Paeoniflorin target genes for the treatment of pancreatic cancer were determined based on the overlap between DEGs and WGCNA. GO and KEGG enrichment analyses were then performed on paeoniflorin target genes to discover which biological processes were impacted. Using the 3 hierarchical methods included in the Cytohubba plugin, we re-screened the hub genes in the target genes to find the genes most relevant to paeoniflorin treatment. The overall survival effects of hub genes were confirmed using the TCGA database. Finally, the paeoniflorin targets identified by the network pharmacology analysis were validated using PANC-1 and Capan-2 cells. Results: We identified 148 main potential PF targets, and gene enrichment analysis suggested that the aforementioned targets play a crucial role in the regulation of MAPK, PI3K-AKT, and other pathways. The further screening of the prospective targets resulted in the identification of 39 hub genes. Using the TCGA database, it was determined that around 33.33% of the hub gene's high expression was linked with a bad prognosis. Finally, we demonstrated that PF inhibits IL-6 and IL-10 expression and p38 phosphorylation in pancreatic cancer cells, thereby reducing inflammation. Conclusion: PF may regulate inflammatory factors mainly through the p38 MAPK signal pathway. These findings provide theoretical and experimental evidence suggesting the PF as a promising natural source of anti-tumor compounds for pancreatic cancer.

Effect of adjuvant therapy with electroacupuncture on bone turnover markers and interleukin 17 in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the effect of electroacupuncture as an adjuvant treatment with first-line medications on bone metabolism biomarkers and interleukin-17 (IL-17) in the peripheral blood of patients with rheumatoid arthritis (RA). METHODS: Sixty RA patients were randomized into three groups. The control group was treated with methotrexate plus leflunomide (MTX+LEF), the acupuncture group was treated with simple needling plus MTX + LEF, and the patients in the electroacupuncture (EA) group were treated with EA plus MTX + LEF. EA or acupuncture was applied every other day for a total of 10 times over a treatment period of 8 weeks. RESULTS: In all three treatment groups, serum levels of the bone metabolism markers PICP, N-MID, and B-ALP were elevated and the concentrations of the inflammatory markers ß-CTx, IL-17, CRP, and TRACP-5b were reduced after treatment. These differences were significant for the EA group but not the other groups (P < 0.05). CONCLUSION: EA could effectively reduce the suffering and improve the quality of life of RA patients. It is a promising adjuvant therapy for enhancing the effectiveness of clinical therapeutics.

Icariin inhibits MMP­1, MMP­3 and MMP­13 expression through MAPK pathways in IL­1ß­stimulated SW1353 chondrosarcoma cells.

Osteoarthritis (OA) is the most common type of arthritis and is a leading cause of disability worldwide, resulting in pain, reduced quality of life and socioeconomic burden. Current therapies for OA focus on mitigating the symptoms of advanced disease, but novel therapeutic agents are needed to inhibit the processes leading to OA. The present study aimed to investigate the effects of Icariin on matrix metalloproteinase (MMP)­1, MMP­3 and MMP­13 expression in interleukin (IL)­1ß­stimulated human SW1353 chondrosarcoma cells, and to investigate the possible mechanism underlying the chondroprotective effects of Icariin. In the present study, IL­1ß was applied on SW1353 chondrosarcoma cells to mimic the microenvironment of osteoarthritis. The cells were treated with Icariin and mitogen­activated protein kinase (MAPK) signaling pathway activators or inhibitors. MMP­1, MMP­3, MMP­13, phosphorylated (P)­p38, P­c­Jun N­terminal kinase (JNK) and P­extracellular signal­regulated kinase (ERK) expression was assessed using reverse transcription­quantitative polymerase chain reaction, ELISA and western blot analysis. The results of the present study demonstrated that Icariin inhibited the expression of MMP­1, MMP­3, MMP­13, P­p38, P­ERK and P­JNK. Furthermore, it was revealed that the inhibition of p38 and ERK contributed to the inhibition of MMP­1 and MMP­3 by Icariin, whereas the inhibition of p38 and JNK contributed to the inhibition of MMP­13. The present results suggested that Icariin may have a chondroprotective effect, exerted through the inhibition of MMP­1, MMP­3 and MMP­13 via MAPK pathways. Therefore, Icariin may have potential as a novel therapeutic strategy for the treatment of osteoarthritis.

miR-142 inhibits the migration and invasion of glioma by targeting Rac1.

Increasing evidence has shown that aberrant microRNAs (miRNAs) are implicated in tumorigenesis and tumor progression by regulating oncogenes or tumor suppressors. Dysregulation of miR-142 has been reported in multiple tumors. However, its clinical roles and underlying mechanism in glioma remain to be elucidated. In the present study, we found that the expression of miR-142 was significantly downregulated in both glioma tissues and cell lines by qRT-PCR. Clinical analysis revealed that decreased miR-142 was markedly associated with advanced World Health Organization (WHO) grade. Moreover, we disclosed that miR-142 was a novel independent prognostic marker in the prediction of the 5-year survival of glioma patients. The ectopic overexpression of miR-142 inhibited cell migration, invasion and invasion­related gene expression. Notably, miR-142 modulated Rac1 by directly binding to its 3'-untranslated (3'-UTR) region, leading to the suppression of the expression of matrix metalloproteinases (MMPs). In glioma clinical samples, miR-142 was inversely correlated with Rac1 expression, and played positive roles in glioma migration and invasion. Alteration of Rac1 expression at least partially abolished the migration, invasion and MMP expression of miR-142 in glioma cells. In the present study, we identified Rac1 as a functional target of miR-142 in glioma. In conclusion, our data indicated that miR-142 inhibited the migration, invasion and MMP expression of glioma by targeting Rac1, and may represent a novel potential therapeutic target and prognostic marker for glioma.

Xitong Wan attenuates inflammation development through inhibiting the activation of nuclear factor-κB in rats with adjuvant-induced arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Xitong Wan (XTW), a traditional Chinese herbs formula, has been used to treat "Bi Zheng" in the clinical practice of traditional Chinese medicine (TCM) for hundreds of years. However, no scientific validation is available on the anti-rheumatic effect of XTW. AIM OF STUDY: This study was carried out to investigate the effects of XTW on joints swelling, joints destruction, production of inflammatory mediators and nuclear factor-κB (NF-κB) activation in rats with adjuvant-induced arthritis (AIA). MATERIALS AND METHODS: AIA was induced by intradermal injection of Complete Freund's adjuvant in the footpad of Wistar rats. Paw volume was measured every 7 days during XTW treatment. Histological score was calculated by hematoxylin and eosin staining. Osteoclast number in articular tissues was counted by tartrate-resistant acid phosphatase staining. Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 in serum were detected by enzyme-linked immunosorbent assay. Levels of NF-κBp65 and inhibitor of NF-κB (IκB)α in synovium were analyzed by Western blot assay. RESULTS: Compared with AIA group rats, XTW significantly decreased the paw volume of AIA rats. Meanwhile, XTW significantly reduced the histological score and osteoclast number in articular tissues of AIA rats. In addition, XTW markedly abated the levels of TNF-α, IL-1ß and IL-6 in serum, as well as enhanced the level of IκBα in synovium of AIA rats. However, XTW did not show significant effect on the level of p65 in synovium of AIA rats. CONCLUSIONS: These results suggest that XTW attenuates the inflammation development through inhibiting the NF-κB-mediated proinflammatory cytokines production in AIA rats. Our study provides the scientific evidence of XTW on treatment of rheumatoid arthritis in the clinical practice of TCM.

Relationship between IL-27 gene polymorphism and susceptibility of rheumatoid arthritis in Chinese Han population.

OBJECTIVE: To investigate the relationship between IL-27 gene polymorphism and the susceptibility of rheumatoid arthritis (RA) in a Chinese Hans population. METHODS: 310 RA patients and 310 healthy controls were examined in this study. Polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) technique was used in the detection of the genotype in three loci of IL-27 gene (-964A/G, 2905T/G, and 4730T/C). We compared genotype and allele frequency and distribution of these two groups. RESULTS: The genotype distribution of the case group and the control group were all in accordance with Hardy-Weinberg equilibrium (P>0.05). The difference of genotype and allele frequencies of three loci between these two groups showed no statistically significant (P>0.05). But the frequencies of G-T-C haplotype was significantly higher in the case group than in the control group, the difference showed statistically significant (OR=2.001, 95% CI: 1.121~3.573; P=0.0170). G-T-T haplotype in case group was significantly lower than that in the control group, the difference showed statistically significant (OR=0.715, 95% CI: 0.527~0.970, P=0.030). CONCLUSION: In Chinese Hans population, IL-27 gene haplotypes were correlated with the risk of RA. G-T-C haplotype was the risk factors for the incidence of RA, but G-T-T haplotype maybe was the protective factor of RA.

Ginsenoside Rb1 inhibits matrix metalloproteinase 13 through down-regulating Notch signaling pathway in osteoarthritis.

Mounting evidence suggests that an excess of matrix metalloproteinase-13 (MMP-13) plays an important role in the breakdown of extracellular matrix in osteoarthritis (OA). Here, the effects of ginsenoside Rb1 (GRb1) on the expression of MMP-13 in IL-1ß-induced SW 1353 chondrosarcoma cells and an experimental rat model of OA induced by anterior cruciate ligament transection (ACLT) were investigated. SW1353 chondrosarcoma cells were pretreated with or without GRb1 and Notch signaling pathway inhibitor, DAPT, then were stimulated with IL-1ß. In rats, experimental OA was induced by ACLT. These rats then received intra-articular injections of vehicle, an inhibitor of γ-secretase, DAPT, and/or GRb1. Expression of MMP-13, collagen type II (CII), Notch1, and jagged 1 (JAG1) were verified by western blotting and immunohistochemistry. In addition, levels of MMP-13 mRNA were detected using quantitative real-time PCR. In histological analyses, treatment with DAPT reduced the number of cartilage lesions present and the expressions of MMP-13, CII, Notch1, and JAG1. In addition, treatment with GRb1 was associated with lower levels of Notch1 and JAG1 in both IL-1ß-induced SW1353 chondrosarcoma cells and in the rat OA model. Furthermore, the suppressive effect of GRb1 on MMP-13 was greater than that exhibited by the signaling pathway inhibitor. In conclusion, GRb1 inhibits MMP-13 through down-regulating Notch signaling pathway in OA.

Bufalin, a bioactive component of the Chinese medicine chansu, inhibits inflammation and invasion of human rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) contribute to the destruction of cartilage and bone by production of metalloproteinases (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM). Bufalin, a major component of Venenum Bufonis, can attenuate the invasion of various cancer cells. Here, we investigated the effects of bufalin on tumor necrosis factor-alpha (TNF-α)-induced invasion of RAFLSs. Western blot analysis and electrophoretic mobility shift assay were conducted to analyze the nuclear translocation of p65/nuclear factor-kappa B (NF-κB) and NF-κB DNA-binding activity. Semiquantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were performed to assess the expression of cytokines. Our results revealed that TNF-α significantly increased p65 translocation into nucleus (P < 0.01) and enhanced NF-κB DNA-binding activity, which were dose-dependently inhibited by bufalin. Furthermore, bufalin attenuated the TNF-α-induced interleukin-1beta (IL-1ß), IL-6, and IL-8 production in RAFLSs in a concentration-dependent manner. Interestingly, TNF-α-induced invasion of RAFLSs was dampened by the pretreatment of bufalin. Additionally, bufalin decreased the mRNA abundance and secretion of MMP-9 in TNF-α-treated RAFLSs. Our results reveal that bufalin can inhibit TNF-α-induced NF-κB activation, cytokine production, invasion, and MMP-9 expression in RAFLSs, indicating a therapeutic potential of bufalin on RA.

Chondroprotective effects and multi-target mechanisms of Icariin in IL-1 beta-induced human SW 1353 chondrosarcoma cells and a rat osteoarthritis model.

Cartilage degradation is the most predominant pathological change during osteoarthritis (OA). Furthermore, accumulating evidence suggests that an excess of matrix metalloproteinase-13 (MMP-13) plays a critical role in the breakdown of cartilage. Here, the effects of Icariin on the expression of MMP-13 in IL-1ß-induced SW 1353 chondrosarcoma cells were investigated. In addition, the in vivo effects of Icariin on an experimental rat model of OA induced by anterior cruciate ligament transection (ACLT) was examined. SW1353 chondrosarcoma cells were pretreated with or without Icariin and MAPK and Wnt/ß-catenin signaling pathway inhibitors, then were stimulated with IL-1ß. In rats, experimental OA was induced by ACLT. These rats then received intra-articular injections of vehicle, signaling pathway inhibitors, and/or Icariin. Expression of MMP-13, phosphorylated p38, phosphorylated JNK, and ß-catenin were verified by western blotting. In addition, levels of MMP-13 mRNA were detected using quantitative real-time PCR. In histological analyses, treatment with Icariin reduced the number of cartilage lesions present. In addition, treatment with Icariin was associated with lower levels of phosphorylated p38, phosphorylated JNK, and ß-catenin in both IL-1ß-induced SW1353 chondrosarcoma cells and in the rat OA model. Furthermore, the suppressive effect of Icariin on MMP-13 was greater than that exhibited by other signaling pathway inhibitors. Overall, these data suggest that Icariin has therapeutic potential for the treatment of OA.