The prognostic value of whole-genome DNA methylation in response to Leflunomide in patients with Rheumatoid Arthritis.

Objective: Although Leflunomide (LEF) is effective in treating rheumatoid arthritis (RA), there are still a considerable number of patients who respond poorly to LEF treatment. Till date, few LEF efficacy-predicting biomarkers have been identified. Herein, we explored and developed a DNA methylation-based predictive model for LEF-treated RA patient prognosis. Methods: Two hundred forty-five RA patients were prospectively enrolled from four participating study centers. A whole-genome DNA methylation profiling was conducted to identify LEF-related response signatures via comparison of 40 samples using Illumina 850k methylation arrays. Furthermore, differentially methylated positions (DMPs) were validated in the 245 RA patients using a targeted bisulfite sequencing assay. Lastly, prognostic models were developed, which included clinical characteristics and DMPs scores, for the prediction of LEF treatment response using machine learning algorithms. Results: We recognized a seven-DMP signature consisting of cg17330251, cg19814518, cg20124410, cg21109666, cg22572476, cg23403192, and cg24432675, which was effective in predicting RA patient's LEF response status. In the five machine learning algorithms, the support vector machine (SVM) algorithm provided the best predictive model, with the largest discriminative ability, accuracy, and stability. Lastly, the AUC of the complex model(the 7-DMP scores with the lymphocyte and the diagnostic age) was higher than the simple model (the seven-DMP signature, AUC:0.74 vs 0.73 in the test set). Conclusion: In conclusion, we constructed a prognostic model integrating a 7-DMP scores with the clinical patient profile to predict responses to LEF treatment. Our model will be able to effectively guide clinicians in determining whether a patient is LEF treatment sensitive or not.

[Group 2 innate lymphoid cells (ILC2) relieves pathogenesis of rheumatoid arthritis by secreting IL-13].

Objective To study the changes of group 2 innate lymphoid cells (ILC2) in rheumatoid arthritis (RA) and its possible mechanism. Methods The 28 joint disease activity score (DAS28) and peripheral blood samples were collected from RA patients. Erythrocyto sedimentation rate (ESR) and C-reactive protein (CRP) were measured by an automatic erythrocyte sedimentation device and immune transmission turbidimetry, respectively. Rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (ACCP) were assessed by immunoturbidimetry and chemiluminescence assay, respectively. Flow cytometry was used to detect the proportion of ILC2 in the patients with RA. The correlations between ILC2 and the above disease indexes were analyzed by Spearman correlation analysis. RA patients were divided into two groups: low disease activity (DAS28<3.2) group and high disease activity (DAS28≥3.2) group. The expression of interleukin-13 (IL-13) in ILC2 of RA patients was detected by flow cytometry, and the differences of ILC2 expression were compared in RA patients with different disease activity of IL-13. Results Compared with healthy people, the proportion and absolute number of ILC2 in the peripheral blood of RA patients increased. ILC2 was negatively correlated with ESR, CRP and DAS28. IL-13 secreted by ILC2 in RA patients with high disease activity was significantly lower than that in the ones with low disease activity. Conclusion ILC2 inhibits RA disease activity by secreting IL-13.

[Lactobacillus casei ATCC334 inhibits the secretion of inflammatory factors and joint lesions in collagen-induced arthritis (CIA) rats].

Objective To study the effects of Lactobacillus casei ATCC334 on the secretion of inflammatory factors and joint destruction in collagen-induced arthritis (CIA) rats and explore the possible mechanism. Methods Female Wistar rats were randomly divided into normal control group, CIA group and ATCC334 group with 7 rats each. The CIA model was established by intradermal injection of bovine type 2 collagen into the tail root of rats in the CIA group and ATCC334 group. The rats in the control group and CIA group were intragastrically perfused with 500 µL normal saline every day, and simultaneously the rats in the ATCC334 group were intragastrically perfused with Lactobacillus casei ATCC334 solution [2×108 colony forming unit (CFU)/mL] for continuous 28 days. Arthritis index and increased hind paw volume were used to evaluate the severity of arthritis. HE staining was used to observe the joint lesions. Levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and IL-17 in the serum of rats were detected by ELISA. Results Compared with the CIA group, the indices of arthritis index, increased posterior paw volume, joint lesions and TNF-α, IL-6 and IL-17 levels were significantly reduced in the serum of the ATCC334 group. Conclusion Lactobacillus casei ATCC334 can significantly reduce the secretion of inflammatory factors and joint bone damage in CIA rats.

Hyperoside exerts anti-inflammatory and anti-arthritic effects in LPS-stimulated human fibroblast-like synoviocytes in vitro and in mice with collagen-induced arthritis.

AIM: Hyperoside is a flavonol glycoside mainly found in plants of the genera Hypericum and Crataegus, which has shown anti-oxidant, anti-cancer and anti-inflammatory activities. In this study, we investigated the effects of hyperoside on human rheumatoid fibroblast-like synoviocytes (FLSs) in vitro and on mouse collagen-induced arthritis (CIA) in vivo. METHODS: FLSs were isolated from primary synovial tissues obtained from rheumatoid arthritis (RA) patients and exposed to LPS (1 µg/mL). Cell viability and proliferation were measured with MTT and BrdU assay. Cell migration was assessed using wound-healing assay and Transwell assay. DNA binding of NF-κB was measured using a TransAM-NFkappaB kit. The localization of p65 subunit was detected with immunocytochemistry. CIA was induced in mice by primary immunization with Bovine Type II collagen (CII) emulsified in CFA, followed by a booster injection 3 weeks later. The arthritic mice were treated with hyperoside (25, 50 mg·kg(-1)·d(-1), ip) for 3 weeks, and the joint tissues were harvested for histological analysis. RESULTS: Hyperoside (10, 50, 100 µmol/L) dose-dependently inhibited LPS-induced proliferation and migration of human RA FLSs in vitro. Furthermore, hyperoside decreased LPS-stimulated production of TNF-α, IL-6, IL-1 and MMP-9 in the cells. Moreover, hyperoside inhibited LPS-induced phosphorylation of p65 and IκBα, and suppressed LPS-induced nuclear translocation of p65 and DNA biding of NF-κB in the cells. Three-week administration of hyperoside significantly decreased the clinical scores, and alleviated synovial hyperplasia, inflammatory cell infiltration and cartilage damage in mice with CIA. CONCLUSION: Hyperoside inhibits LPS-induced proliferation, migration and inflammatory responses in human RA FLSs in vitro by suppressing activation of the NF-κB signaling pathway, which contributes to the therapeutic effects observed in mice with CIA.

Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro.

AIM: Hyperoside (quercetin-3-O-ß-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro. METHODS: Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay. RESULTS: Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 µmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L). CONCLUSION: Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside.