Efficacy of adalimumab combined with Tripterygium wilfordii Hook F in the treatment of patient with rheumatoid arthritis: A multicenter, open-label, randomized-controlled trial.

OBJECTIVES: To evaluate the efficacy and safety of adalimumab (ADA) combined with Tripterygium wilfordii Hook F (TwHF) in the treatment of methotrexate (MTX)-inadequate response patients with rheumatoid arthritis (RA). METHODS: In this multicenter, open-label, randomized controlled clinical trial, 64 RA patients with inadequate response to MTX were 1:1 randomly assigned into treatment or control groups. The treatment group was treated with ADA in combination with TwHF, and the control group was treated with ADA in combination with MTX for 24 weeks. The primary endpoint was the percentage of patients having low disease activity (2.6 ≤ DAS28-ESR < 3.2) and remission rates (DAS28-ESR < 2.6) at week 24. RESULTS: In total, 53 of the 64 patients (82.8%) completed this 24-week clinical trial. By intent-to-treat (ITT) analysis, a comparable outcome was observed between the two groups. The percentage of patients achieving low disease activity in the treatment group and control group were 43.8% and 46.9% (95% CI, 21.28 to 27.48, p = .802). Percentage of patients achieving low disease activity rates were respectively 28.1% and 31.3% in the treatment group and control group (95% CI, 19.18 to 25.58, p = .784). In per-protocol (PP) analysis, the results were consistent with the ITT model. The incidence of adverse events was comparable between the two groups. CONCLUSIONS: There were no significant differences in efficacy and safety between ADA combined with TwHF versus ADA combined with MTX in the treatment of RA. TwHF might be an alternative treatment for RA patients who are intolerant to MTX.

Stigmasterol Depresses the Proliferation and Facilitates the Apoptosis of Fibroblast-Like Synoviocytes via the PI3K/AKT Signaling Pathway in Collagen-Induced Arthritis Rats.

Rheumatoid arthritis (RA) is a chronic disease characterized by persistent synovitis and angiogenesis. Its clinical manifestations are synovial hyperplasia and progressive destruction of bone and cartilage, eventually leading to joint deformation and even disability. The healing effect of monomer stigmasterol, the main active ingredient of the Jinwujiangu recipe the Chinese Herbal Compound, on RA has been confirmed in several studies. Fibroblast-like synoviocytes (FLS) are related to the occurrence and development of RA. This study aims to investigate the effects of stigmasterol on FLS cell proliferation and apoptosis, as well as its impact on FLS cell cycle proteins and key genes in the Phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) pathway, providing insights into the development of stigmasterol as an alternative therapeutic drug for RA. We administered 20 g/kg stigmasterol to rats continuously for 5 d to obtain stigmasterol-containing serum, and established rat models of osteoarthritis induced by ossein to obtain FLS. To explore the effects of stigmasterol on the viability, migration, proliferation and apoptosis of collagen-induced arthritis (CIA)-FLS cells, we selected 0% (control), 5% (low concentration), 10% (medium concentration) and 20% (high concentration) drug-containing serum to intervene cells and conducted Cell Counting Kit-8 (CCK-8), Transwell, 5-ethynyl-2' -deoxyuridine (EdU) staining and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) experiments, respectively. The results showed that compared with the control group, low, medium, and high serum significantly inhibited the activity, migration, and proliferation of FLS cells, and promoted their apoptosis, and high serum had the best effect. In addition, we investigated the mechanism of stigmasterol inhibiting FLS proliferation and promoting its apoptosis by qPCR, Western blot, and immunofluorescence assays. The results showed that stigmasterol significantly inhibited the expression of Cyclin D1, cyclin-dependent kinase 4 (CDK4), and Retinoblastoma (Rb), and decreased the expression of key genes kinase insert domain-containing receptor (KDR), PI3K, AKT, phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) in the KDR-mediated PI3K/AKT signaling pathway, thus inhibiting the proliferation of FLS and promoting the apoptosis of FLS. It was suggested that stigmasterol may be a potential alternative drug for RA treatment.

Characterizations of the multi-kingdom gut microbiota in Chinese patients with gouty arthritis.

OBJECTIVE: The gut microbial composition has been linked to metabolic and autoimmune diseases, including arthritis. However, there is a dearth of knowledge on the gut bacteriome, mycobiome, and virome in patients with gouty arthritis (GA). METHODS: We conducted a comprehensive analysis of the multi-kingdom gut microbiome of 26 GA patients and 28 healthy controls, using whole-metagenome shotgun sequencing of their stool samples. RESULTS: Profound alterations were observed in the gut bacteriome, mycobiome, and virome of GA patients. We identified 1,117 differentially abundant bacterial species, 23 fungal species, and 4,115 viral operational taxonomic units (vOTUs). GA-enriched bacteria included Escherichia coli_D GENOME144544, Bifidobacterium infantis GENOME095938, Blautia_A wexlerae GENOME096067, and Klebsiella pneumoniae GENOME147598, while control-enriched bacteria comprised Faecalibacterium prausnitzii_G GENOME147678, Agathobacter rectalis GENOME143712, and Bacteroides_A plebeius_A GENOME239725. GA-enriched fungi included opportunistic pathogens like Cryptococcus neoformans GCA_011057565, Candida parapsilosis GCA_000182765, and Malassezia spp., while control-enriched fungi featured several Hortaea werneckii subclades and Aspergillus fumigatus GCA_000002655. GA-enriched vOTUs mainly attributed to Siphoviridae, Myoviridae, Podoviridae, and Microviridae, whereas control-enriched vOTUs spanned 13 families, including Siphoviridae, Myoviridae, Podoviridae, Quimbyviridae, Phycodnaviridae, and crAss-like. A co-abundance network revealed intricate interactions among these multi-kingdom signatures, signifying their collective influence on the disease. Furthermore, these microbial signatures demonstrated the potential to effectively discriminate between patients and controls, highlighting their diagnostic utility. CONCLUSIONS: This study yields crucial insights into the characteristics of the GA microbiota that may inform future mechanistic and therapeutic investigations.

miR-223 in exosomes from bone marrow mesenchymal stem cells ameliorates rheumatoid arthritis via downregulation of NLRP3 expression in macrophages.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with major clinical manifestations of human limb joint invasion, joint synovitis, and symmetrical lesions. In recent years, bone marrow mesenchymal stem cells (BMSCs) have been found to have low immunogenicity and immunomodulatory effects, which can regulate other types of cells through exosomes. However, the effect of BMSCs on immune response in the progression of RA has not been fully elucidated. AIMS: The current research aimed to investigate the therapeutic effect of microRNA (miR)-223 in exosomes secreted by BMSCs on immune response in the progression of RA. METHODS: Firstly, BMSCs were isolated and extracted, and then the influence of BMSCs on the level of inflammatory cytokines was detected by enzyme linked immunosorbent assay (ELISA). Exosomes from BMSCs were extracted and characterized. Some key autoimmune response genes and their protein products were detected in vivo and in vitro by real-time quantitative PCR, western blot and ELISA. Finally, the targeting relationship between miR-223 and NLR family pyrin domain-containing 3 (NLRP3) was predicted by bioanalytical software and verified by luciferase reporter assay and rescue experiments in vitro. RESULTS: Exosomes from BMSCs could inhibit the release of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interleukin-18 (IL-18), and NLRP3 activation in macrophages and RA rats. In addition, we predicted online that miR-223 could target NLRP3 and provided a possible regulation pathway for the anti-inflammatory effects of BMSCs-secreted exosomes. Furthermore, we further confirmed that miR-223 could target and inhibit the expression of NLRP3. CONCLUSION: Taken together, these findings suggest that miR-223 carried by BMSCs-derived exosomes targets NLRP3 to regulate the activation of inflammasomes, which therefore can be served as a possible therapy for RA.

Jin-Wu-Jian-Gu Formulation Attenuates Rheumatoid Arthritis by Inhibiting the IL33-ST2 Signaling Pathway.

The present research attempted to investigate the molecular mechanism of Jin-Wu-Jian-Gu Formulation (JWJGF) in inhibiting rheumatoid arthritis (RA) in a pharmacological approach for analysis and experimental validation. First, the potential targets and pathways of JWJGF for RA were predicted by network pharmacology. Second, the effect of JWJGF on RA was observed by hematoxylin-eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA). Further, we observed the effects of JWJGF on the IL33-ST2 signaling pathway by Western blot and quantitative real-time PCR (qPCR) experiments, and finally, we studied the effects of Liquiritigenin on rheumatoid arthritis synovial fibroblast (RASF) cells and the IL33-ST2 signaling pathway. Network pharmacology results showed that the key component of JWJGF was Liquiritigenin and the core target of JWJGF was IL-33. The results of HE and ELISA showed that JWJGF could alleviate RA. Western blot and qPCR findings indicated that JWJGF could inhibit the IL33-ST2 signaling pathway. Furthermore, JWJGF could inhibit the proliferation of RASF cells and the IL33-ST2 signaling pathway. In conclusion, this study revealed that JWJGF attenuated RA by inhibiting the IL33-ST2 signaling pathway.

Jinwu Jiangu Capsule affects synovial cells in rheumatoid arthritis through PI3K/Akt/mTOR signaling pathway.

Jinwu Jiangu Capsule is a medicinal formula from the Chinese Miao nationality. Leflunomide is recommended in organizational guidelines for the treatment of rheumatoid arthritis (RA). To investigate the effect of Jinwu Jiangu Capsule on PI3K/Akt/mTOR signal pathway in cells taken from RA patients New Zealand rabbits were administrated with Jinwu Jiangu Capsule suspension to prepare serum containing medicine. Lyophilized powder was prepared from this serum for cell treatment. The expression of LC3-II and PI3K, AKT, mTOR were detected by IF and western blot. Moreover, the levels of Atg1, Atg5, Atg14 were detected by RT-qPCR. The results showed that the expression of LC3-II was increased, and fluorescence spot of LC3-II was obvious in high-dose of Jinwu Jiangu Capsule group. Jinwu Jiangu Capsule decreased the level of PI3k, Akt, and mTOR protein, and increased the levels of Atg1, Atg5 and Atg14. Specially, the high-dose of Jinwu Jiangu Capsule had the most obvious inhibitory and up-regulation effects. However, there was no significant difference in the expression of Akt, mTOR and Atg1 in the medium-dose of Jinwu Jiangu Capsule group compared with the leflunomide group. In conclusion, Jinwu Jiangu Capsule regulates autophagy by inhibiting the PI3K/AKT/mTOR pathway in RA.

Prediction and Verification of the Major Ingredients and Molecular Targets of Tripterygii Radix Against Rheumatoid Arthritis.

Tripterygii Radix exhibits good clinical efficacy and safety in rheumatoid arthritis (RA) patients, but its effective components and mechanism of action are still unclear. The purpose of this study was to explore and verify the major ingredients and molecular targets of Tripterygii Radix in RA using drug-compounds-biotargets-diseases network and protein-protein interaction (PPI) network analyses. The processes and pathways were derived from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The most important compounds and biotargets were determined based on the degree values. RA fibroblast-like synoviocytes (RA-FLS) were separated from RA patients and identified by hematoxylin and eosin (HE) staining and immunohistochemistry. The purity of RA-FLS was acquired by flow cytometry marked with CD90 or VCAM-1. RA-FLS were subjected to control, dimethyl sulfoxide (control), kaempferol, or lenalidomide treatment. Cell migration was evaluated by the transwell assay. The relative expression of biotarget proteins and cytokines was analyzed by western blotting and flow cytometry. In total, 144 chemical components were identified from Tripterygii Radix; kaempferol was the most active ingredient among 33 other components. Fourteen proteins were found to be affected in RA from 285 common biotargets. The tumor necrosis factor (TNF) signaling pathway was predicted to be one of the most latent treatment pathways. Migration of RA-FLS was inhibited and the expression of protein kinase B (AKT1), JUN, caspase 3 (CASP3), TNF receptor 1 and 2 (TNFR1 and TNFR2), interleukin-6 (IL-6), and TNF-α was significantly affected by kaempferol. Thus, this study confirmed kaempferol as the effective component of Tripterygii Radix against RA-FLS and TNF signaling pathway and its involvement in the regulation of AKT1, JUN, CASP3, TNFR1, TNFR2, IL-6, and TNF-α expression.

[Effects of Jinwu Jiangu recipe on IL-17/STAT3 signals in rheumatoid arthritis synoviocytes].

This paper aimed to investigate the effects of Jinwu Jiangu recipe total extract on the IL-17/STAT3 signals in rheumatoid arthritis synovial fibroblasts(RASF). The primary RASFs were cultured by tissue piece method in vitro， and divided into blank control group， Jinwu Jiangu recipe low dose group， Jinwu Jiangu recipe middle dose group， Jinwu Jiangu recipe high dose group， and tripterygium glycosides control group. They were then treated with corresponding serum free medium， different doses of Jinwu Jiangu recipe total extract(0.06， 0.6， 6.0 g·L⁻¹)， and tripterygium glycosides(0.03 g·L⁻¹) respectively for 24 hours. The gene expression levels of RORα， RORγt， and STAT3 mRNA were detected by polymerase chain reaction(PCR)， and the protein activity of IL-17R and pSTAT3 were measured by Western blot assay. The results showed that as compared with blank control group， the expression levels of RORα， RORγt， IL-17R and STAT3 mRNA in RASF were significantly declined(P<0.01). As compared with tripterygium glycosides control group， Jinwu Jiangu recipe total extract middle dose group and high dose group can down-regulate the expression levels of RORα， RORγt， IL-17R and STAT3 mRNA(P<0.05)， and the effect was more obvious in high dose group(P<0.01). As compared with blank control group， the protein expression levels of IL-17R and pSTAT3 in each treatment group were obviously decreased(P<0.01). As compared with tripterygium glycosides control group， Jinwu Jiangu recipe high dose group had more obvious effect in down-regulating the protein expression of pSTAT3(P<0.01). Therefore， Miao medicine Jinwu Jiangu recipe total extract can down-regulate the expressions of RORα， RORγt， and STAT3 mRNA， and inhibit the protein activity of IL-17R and pSTAT3 in RASF.

[Effect of Jinwu Jiangu Recipe on Expressions of NF-kappaB and IL-17 in Collagen Induced Arthritis Model Rats].

OBJECTIVE: To explore the effect of Jinwu Jiangu Recipe (JJR) on the expression of synovial cells' nuclear factor-kappaB (NF-kappaB) and serum interleukin 17 (IL-17) in collagen induced arthritis (CIA) rats. METHODS: Totally 60 Wistar rats were randomly divided into 6 groups, i.e., the blank control group, the model group, high, middle, and low dose JJR treatment groups, and the tripterygium control group, 10 in each group. Except rats in the blank control group, CIA model was established in rats of the rest 5 groups. Then they were treated from the 7th day of modeling. After 4 weeks of medication they were sacrificed, serum collected, and synovium of joints were isolated. The expression of serum IL-17 was detected in synovium of joints by enzyme linked immunosorbent assay (ELISA). And the expression of NF-kappaB/P65, Ikappabetaalpha and NF-KappaB/P50 were detected by Western blot. RESULTS: Compared with the blank control group, the serum IL-17 level increased in the model group (P <0. 01). Compared with the model group, the serum IL-17 level obviously decreased in high and middle dose JJR groups and the tripterygium control group (P < 0.01). Results of Western blot showed, when compared with the blank control group, protein activities of NF-kappaB/P65 and NF-kappaB/P50 were significantly enhanced in the model group (P < 0.01). Compared with the model group, protein activities of NF-kappaB/P65 and NF-kappaB/P50 significantly decreased in high and middle dose JJR groups and the tripterygium control group (P < 0.05, P < 0.01). All indices mentioned above were higher in the low dose JJR group than in the tripterygium control group (P < 0.05, P < 0.01). CONCLUSION: JJR could lower the expression of serum IL-17 in CIA model rats, and inhibit protein activities of NF-kappaB/P65 and NF-kappaB/P50.